EP1180164A4 - Methods using of fab i and compounds modulating fab i activity - Google Patents

Methods using of fab i and compounds modulating fab i activity

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Publication number
EP1180164A4
EP1180164A4 EP00935862A EP00935862A EP1180164A4 EP 1180164 A4 EP1180164 A4 EP 1180164A4 EP 00935862 A EP00935862 A EP 00935862A EP 00935862 A EP00935862 A EP 00935862A EP 1180164 A4 EP1180164 A4 EP 1180164A4
Authority
EP
European Patent Office
Prior art keywords
fab
binding
coa
nadh
nadph
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP00935862A
Other languages
German (de)
French (fr)
Other versions
EP1180164A2 (en
Inventor
Walter E Dewolf
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Affinium Pharmaceuticals Inc
Original Assignee
SmithKline Beecham Ltd
SmithKline Beecham Corp
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by SmithKline Beecham Ltd, SmithKline Beecham Corp filed Critical SmithKline Beecham Ltd
Publication of EP1180164A2 publication Critical patent/EP1180164A2/en
Publication of EP1180164A4 publication Critical patent/EP1180164A4/en
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/001—Oxidoreductases (1.) acting on the CH-CH group of donors (1.3)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/10—Antimycotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention relates, in part, to newly identified polynucleotides and polypeptides, vanants and de ⁇ vatives of these polynucleotides and polypeptides, processes for making these polynucleotides and these polypeptides, and their vanants and de ⁇ vatives, agonists and antagonists of the polypeptides, and uses of these polynucleotides, polypeptides, vanants, denvatives, agonists and antagonists
  • the invention relates to polynucleotides and polypeptides of staphylococcal Fab I enoyl-ACP reductase, hereinafter referred to as "FAB I" BACKGROUND OF THE INVENTION
  • FAS fatty acid synthase
  • Fab I functions as an enoyl-acyl earner protein (ACP) reductase (Bergler, et al, ( 1994), J Biol Chem 269, 5493-5496) in the final step of the four reactions involved in each cycle of bacterial fatty acid biosynthesis
  • the first step is catalysed by ⁇ -ketoacyl-ACP synthase, which condenses malonyl- ACP with acetyl-CoA (FabH, synthase III)
  • ⁇ -ketoacyl-ACP synthase which condenses malonyl- ACP with acetyl-CoA
  • FabH acetyl-CoA
  • FabB and FabF synthases I and II respectively
  • the second step in the elongation cycle is ketoester reduction by NADPH-dependent ⁇ -ketoacyl-ACP reductase (FabG)
  • ⁇ -hydroxyacyl- ACP dehydrase either FabA or FabZ
  • trans-2-enoyl-ACP which is in turn converted to acyl-ACP by NADH-dependent enoyl-ACP reductase (Fab I)
  • Fab I Further rounds of this cycle, adding two carbon atoms per cycle, eventually lead to palmitoyl-ACP (16C) where upon the cycle is stopped largely due to feedback inhibition of Fab I by palmitoyl- ACP (Heath, et al. ( 1996), J Biol Chem. 271. 1833- 1836)
  • Fab I is therefore a major biosynthetic enzyme which is also a key regulatory point in the overall synthetic pathway
  • Fab I is essential to the survival of the organism (Bergler, et al, ( 1994), J Biol Chem 269, 5493-5496) Laboratory generated point mutations in the Eab I gene lead to diazabonne resistant E colt Fab I is conserved in Gram negative organisms with 98% identity between E colt and S nphtmitriitm Fab I (Bergler. et al, ( 1992), J Gen Mtcrobiol 138.
  • FAB I of the invention shows 54% similanty to the mycobacte ⁇ al protein, InhA, which is highly conserved throughout mycobacte ⁇ a including WI tuberculosis
  • E colt Fab I was found to be 34% identical, 57% similar to Brassica napus (rape seed) enoyl-ACP reductase and S aureus FAB I of the present invention was also 34% identical, 57% similar
  • FAB I of the present invention was found to be 44% identical, 64% similar over 252 amino acids to E colt Fab I
  • FAB I of the present invention is only 27% identical, 48% similar to a mammalian 2,4-dienoyl-coenzyme A reductase This mammalian homolog differs from FAB I in that it is involved in the ⁇ -oxidation of polyunsaturated en
  • FABI Factors that may be used to screen compounds for antibiotic activity, such as a simple high through-put assay for screening inhibitors of FAS
  • factors may also be used to determine their roles in pathogenesis of infection, dysfunction and disease Identification and characte ⁇ zation of such factors, which can play a role in preventing, ameliorating or correcting infections, dysfunctions or diseases are critical steps in making important discovenes to improve human health SUMMARY OF THE INVENTION
  • FAB I agonists are molecules that mimic FAB I, that bind to FAB I-bindmg molecules or binding molecules, and that elicit or augment FAB I-mduced responses
  • preferred agonists are molecules that interact with FAB I, or with other modulators of FAB I activities, and thereby potentiate or augment an effect of FAB I or more than one effect of FAB I and are bactenostatic or bacte ⁇ ocidal
  • FAB I agonists are molecules that mimic FAB I, that bind to FAB I-bindmg molecules or binding molecules, and that elicit or augment FAB I-mduced responses
  • molecules that interact with FAB I, or with other modulators of FAB I activities and thereby potentiate or augment an effect of FAB I or more than one effect of FAB I and are bactenostatic or bacte ⁇ ocidal
  • FAB I antagonists include those which mimic FAB I so as to bind to FAB 1-b ⁇ nd ⁇ ng molecules but not elicit a FAB I-induced response or more than one FAB I- induced response
  • preferred antagonists are molecules that bind to or interact with FAB I so as to inhibit an effect of FAB I or more than one effect of FAB I or which prevent expression of FAB I
  • Further particularly preferred antagonists of FAB I lower or abolish a FABI enzymatic activity or activities.
  • compositions comp ⁇ se a FAB I polynucleotide for expression of a FAB I polypeptide in a host organism to raise an immunological response, preferably to raise immunity in such host against bacteria, preferably staphylocci or closely genetically related organisms
  • An antagonist that inhibits or an agonist that activates an activity a polypeptide selected from the group consisting of a polypeptide comp ⁇ sing an amino acid sequence which is at least 90% identical to the amino acid sequence of SEQ ID NO 2 or 4, and a polypeptide comprising an amino acid sequence as set forth in SEQ ID NO 2 or 4, wherein said activity is selected from the group consisting of uncompetitive inhibition by Apo-ACP versus NADH (K ⁇ (app), competitive inhibition by Apo-ACP versus crotonoyl CoA, induction of negative cooperativity with respect to CCA binding, use of NADH and NADPH as substrates by Fab I, binding of NADH and NADPH by Fabl, oxidation of NADH and NADPH by Fabl, ratio of Kmapp for NADH as compared to NADPH, use of NADH and crotonoyl CoA as substrates by Fab I in a sequential kinetic mechanism, sequential binding of NADH and
  • a method for the treatment of an individual infected with a bactena comprising the steps of administe ⁇ ng to the individual a antibacte ⁇ ally effective amount of an antagonist that inhibits or an agonist that activates an activity of a polypeptide selected from the group consisting of a polypeptide comp ⁇ sing an ammo acid sequence which is at least 90% identical to the amino acid sequence of SEQ ID NO: 1
  • Fab I polypeptide comprising the steps of administe ⁇ ng to the individual a antibacte ⁇ ally effective amount of an antagonist that inhibits or an agoinist that activates an activity of Fab I selected from the group consisting of uncompetitive inhibition by Apo-ACP versus NADH (K ⁇ (app), competitive inhibition by Apo-ACP versus crotonoyl CoA, induction of negative cooperativity with respect to CCA binding, use of NADH and NADPH as substrates by Fab I, binding of NADH and NADPH by Fabl, oxidation of NADH and NADPH by Fabl, ratio of Kmapp for NADH as compared to NADPH, use of NADH and crotonoyl CoA as substrates by Fab I in a sequential kinetic mechanism, sequential binding of NADH and crotonoyl CoA by Fab I, increasing inhibition of Fabl by saturated fatty acyl CoA's of increasing chain length, feedback regulatory mechanism of Fab I by saturated fatty
  • a method for the treatment of an individual infected with a bacte ⁇ a comprising the steps of administenng to the individual a antibacte ⁇ ally effective amount of an antagonist that inhibits or an agonist that activates that activates an activity of Fab I selected from the group consisting of uncompetitive inhibition by Apo-ACP versus NADH (K ⁇ (app), competitive inhibition by Apo-ACP versus crotonoyl CoA, induction of negative cooperativity with respect to CCA binding, use of NADH and NADPH as substrates by Fab I, binding of NADH and NADPH by Fabl, oxidation of NADH and NADPH by Fabl, ratio of Kmapp for NADH as compared to NADPH, use of NADH and crotonoyl CoA as substrates by Fab I in a sequential kinetic mechanism, sequential binding of NADH and crotonoyl CoA by Fab I, increasing inhibition of Fabl by saturated fatty acyl CoA's of increasing chain
  • a method for the treatment of an individual infected by Streptococcus pneumoniae comprising the steps of administering to the individual a antibacterially effective amount of an antagonist that inhibits or anagonist that activates an activity of Streptococcus pneumoniae Fab I selected from the group consisting of: uncompetitive inhibition by Apo-ACP versus NADH (Ki(app); competitive inhibition by Apo-ACP versus crotonoyl CoA; induction of negative cooperativity with respect to CCA binding; use of NADH and NADPH as substrates by Fab I; binding of NADH and NADPH by Fabl; oxidation of NADH and NADPH by Fabl; ratio of Kmapp for NADH as compared to NADPH; use of NADH and crotonoyl CoA as substrates by Fab I in a sequential kinetic mechanism; sequential binding of NADH and crotonoyl CoA by Fab I; increasing inhibition of Fabl by saturated fatty acyl CoA's of increasing
  • a method for the treatment of an individual having need to inhibit Fab I polypeptide comp ⁇ sing the steps of administering to the individual a antibactenall effective amount of an antagonist that inhibits an activity of a polypeptide selected from the group consisting of a polypeptide comp ⁇ sing an amino acid sequence which is at least 90% identical to the amino acid sequence of SEQ ID NO 2 or 4, and a polypeptide comp ⁇ sing an amino acid sequence as set forth in SEQ ID NO 2 or 4, , wherein said activity is selected from the group consisting of uncompetitive inhibition by Apo-ACP versus NADH (K ⁇ (app), competitive inhibition by Apo-ACP versus crotonoyl CoA, induction of negative cooperativity with respect to CCA binding, use of NADH and NADPH as substrates by Fab I, binding of NADH and NADPH by Fabl, oxidation of NADH and NADPH by Fabl, ratio of Kmapp for NADH as compared
  • a method for inhibiting an activity of Fab I polypeptide comprising the steps of contacting a composition comprising said polypeptide with an effective amount of an antagonist that inhibits an activbity of Fab I, wherein said activity is selected from the group consisting of uncompetitive inhibition by Apo-ACP versus NADH (K ⁇ (app), competitiv e inhibition by Apo-ACP versus crotonoyl CoA induction of negative cooperativity with respect to CCA binding, use of NADH and NADPH as substrates by Fab I binding of NADH and NADPH by Fabl, oxidation of NADH and NADPH by Fabl, ratio of Kmapp for NADH as compared to NADPH, use of NADH and crotonoyl CoA as substrates by Fab I in a sequential kinetic mechanism, sequential binding of NADH and crotonoyl CoA by Fab I increasing inhibition of Fabl by saturated fatty acyl CoA's of increasing chain length, feedback regulatory mechanism of
  • NADH binding to Fab I prior to or simultaneous with ACP binding A method for inhibiting an activity of Fab I, wherein said activity is selected from the group consisting of uncompetitive inhibition by Apo-ACP versus NADH (K ⁇ (app), competitive inhibition by Apo-ACP versus crotonoyl CoA, induction of negative cooperativity with respect to CCA binding, use of NADH and NADPH as substrates by Fab I, binding of NADH and NADPH by Fabl, oxidation of NADH and NADPH by Fabl, ratio of Kmapp for NADH as compared to NADPH, use of NADH and crotonoyl CoA as substrates by Fab I in a sequential kinetic mechanism, sequential binding of NADH and crotonoyl CoA by Fab I, increasing inhibition of Fabl by saturated fatty acyl CoA's of increasing chain length, feedback regulatory mechanism of Fab I by saturated fatty acyl CoA's, competitive inhibition by palmitoyl CoA
  • a method for inhibiting a growth of bacteria compnsmg the steps of contacting a composition comp ⁇ sing bacteria with an antibactenally effective amount of an antagonist that inhibits an activity of Fab I, wherein said activity is selected from the group consisting of uncompetitive inhibition by Apo-ACP versus NADH (K ⁇ (app), competitive inhibition by Apo-ACP versus crotonoyl CoA, induction of negative cooperativity with respect to CCA binding, use of NADH and NADPH as substrates by Fab I, binding of NADH and NADPH by Fabl, oxidation of NADH and NADPH by Fabl, ratio of Kmapp for NADH as compared to NADPH, use of NADH and crotonoyl CoA as substrates by Fab I in a sequential kinetic mechanism, sequential binding of NADH and crotonoyl CoA by Fab I, increasing inhibition of Fabl by saturated fatty acyl CoA's of increasing chain length, feedback regulatory mechanism of Fab I
  • a method for inhibiting a Fab I polypeptide compnsing the steps of contacting a composition compnsing bactena with an antibactenally effective amount of an antagonist that inhibits an activity of Fab I, wherein said activity is selected from the group consisting of uncompetitive inhibition by Apo-ACP versus NADH (K ⁇ (app), competitive inhibition by Apo-ACP versus crotonoyl CoA, induction of negative cooperativity with respect to CCA binding, use of NADH and NADPH as substrates by Fab I, binding of NADH and NADPH by Fabl, oxidation of NADH and NADPH by Fabl, ratio of Kmapp for NADH as compared to NADPH, use of NADH and crotonoyl CoA as substrates by Fab I in a sequential kinetic mechanism, sequential binding of NADH and crotonoyl CoA by Fab I, increasing inhibition of Fabl by saturated fatty
  • any bacte ⁇ a is selected from the group consisting of a member of the genus Staphvlococcus, Staphylococcus aureus, a member of the genus Streptococcus, and Streptococcus pneumoniae
  • Figure 1 shows the cycle of fatty acid elongation in bacte ⁇ a
  • Figure 2 shows a chemical synthesis of deuterated pyridine nucleotides
  • Figure 3 shows predicted and observed product structures and (M+H) + 's based on E colt Fab I
  • Figure 4 shows mass spectra of components from reaction 3 of Figure 3
  • Figure 5 shows a stereochemical course of 5 aureus Fab I
  • Figure 6 shows graphically that S aureus Fab I uses both NADPH and NADH as substrates
  • Figure 7 shows graphically that S auieus Fab I exhibits a sequential reaction mechanism
  • Figure 8 shows a pH profile of S aureus Fab I
  • Figure 9 shows inhibition by palmitoyl CoA
  • Figure 10 shows inhibition by beta-NADP +
  • Figure 1 1 shows a kinetic model for inhibition by beta-NADP +
  • Figure 12 shows graphically that inhibition by apo-ACP vs NADH
  • Figure 13 shows graphically the inhibition by apo-ACP vs CCA
  • Figure 14 shows minimal kinetic mechanism for inhibition by apo-ACP
  • Figure 15 shows induced cooperative inhibition by apo-ACP
  • Figure 16 shows the polypeptide of Staplnlococctts aureus FAB I deduced from the polynucleotide of Figure 17 [SEQ ID NO 1 ]
  • Figure 17 shows the polypnucleotide sequence of Staphylococcus aureus FAB I GLOSSARY
  • Binding molecule(s), refers to molecules or ions which bind or interact specifically with FAB I polypeptides or polynucleotides of the present invention, including, for example enzyme substrates and substrate and co-factor mimetics, as well as classical receptors (which also may be referred to as "binding molecules' and “interaction molecules,” respectively and as “FAB I binding molecules' and “FAB I interaction molecules”) Binding between polypeptides of the invention and such molecules, including binding or binding or interaction molecules may be exclusive to polypeptides of the invention, which is very highly preferred, or it may be highly specific for polypeptides of the invention, which is highly preferred, or it may be highly specific to a group of proteins that includes polypeptides of the invention, which is prefened, or it may be specific to several groups of proteins at least one of which includes a polypeptide of the invention Binding molecules also may be non-naturally occurring, such as antibodies and antibody-de ⁇ ved reagents that bind specifically to
  • “Digestion of DNA” refers to cleavage of DNA with a restriction enzyme that acts only at certain sequences in the DNA
  • the various rest ⁇ ction enzymes referred to herein are commercially available and their reaction conditions, cofactors and other requirements for use are well known and routine to the skilled artisan
  • plasmid or DNA fragment is digested with about 2 units of enzyme in about 20 ⁇ l of reaction buffer
  • isolating DNA fragments for plasmid construction typically 5 to 50 ⁇ g of DNA are digested with 20 to 250 units of enzyme in proportionately larger volumes
  • D ⁇ sease(s) means (l) bactenal infections, such as staphlococcal infections including, but not limited to infections of the upper respiratory tract (e g , otitis media, bactenal tracheitis, acute epiglottitis, thyroiditis), the lower respiratory tract (e g , empyema, lung abscess), the cardiac system (e g , infective endocarditis), the gastrointestinal tract (e g , secretory dia
  • Streptococcus Group A Streptococcus, Group B Streptococcus, Group C Streptococcus, Group D Streptococcus, Group G Streptococcus, Streptococcus pneumoniae Streptococcus pyogenes. Streptococcus agalactiae. Streptococcus faecalis, Streptococcus faecium. Streptococcus ditrans.
  • Neissena gonorrheae Neisseria meningitidis, Staphylococcus aureus, Staphylococcus epidermidis, Corynebacterium diptheriae, Gardnerella vaginalis, Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium ulcerans, Mycobacterium leprae, Actinomyctes israelii, Listeria monocytogenes, Bordetella pertusis, Bordatella parapertusis, Bordetella bronchiseptica, Escherichia coli, Shigella dysenteriae, Haemophilus influenzae.
  • Haemophilus aegyp ⁇ us Haemophilus parain ⁇ ttenzae, Haemophilus ducreyi, Bordetella, Salmonella typhi, Citrobacter frettndii, Proteus mirabilis, Proteus vulgaris, Yersinia pestis, Kleibsiella pneumoniae, Serratia marcessens, Serratia liquefaciens, Vibrio cholera, Shigella dysenterii, Shigella flexneri. Pseudomonas aerttginosa, Franscisella tttlarensis. Brucella abortis, Bacillus anthracis.
  • Genetic Element(s) means a polynucleotide comprising a region that encodes a polypeptide or a region that regulates transcription or translation or other processes important to expression of the polypeptide in a host cell, or a polynucleotide comprising both a region that encodes a polypeptide and a region operably linked thereto that regulates expression.
  • Genetic elements may be comprised within a vector that replicates as an episomal element; that is, as a molecule physically independent of the host cell genome. They may be comprised within plasmids. Genetic elements also may be comprised within a host cell genome; not in their natural state but, rather, following manipulation such as isolation, cloning and introduction into a host cell in the form of purified DNA or in a vector, among others.
  • “Host cell(s)” means a cell which has been transformed, transfected, infected, or entered by an exogenous polynucleotide sequence, or is capable of transformation, infection, transfection, or entry by an exogenous polyncleotide sequence.
  • Identity means a relationships between two polypeptide sequences or two polynucleotide sequences, as determined by comparing the sequences. In the art, identity also means the degree of sequence relatedness between two polypeptide or two polynucleotide sequences as determined by the match between two strings of such sequences. Both identity and similarity can be readily calculated (Computational Molecular Biology, Lesk, A.M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D.W., ed., Academic Press, New
  • Isolated means altered “by the hand of man” from its natural state, i e , if it occurs in nature, it has been changed or removed from its original environment, or both
  • a polynucleotide or a polypeptide naturally present in a living organism is not “isolated,” but the same polynucleotide or polypeptide separated from the coexisting mate ⁇ als of its natural state is “isolated”, as the term is employed herein
  • a polynucleotide or polypeptide that is introduced into an organism by transformation, genetic manipulation or by any other recombinant method is "isolated” even if it is still present in said organism, which organism may be living or non-living
  • polynucleotides can be joined to other polynucleotides, such as DNAs, for mutagenesis, to form fusion proteins, and for propagation or expression in a host, for instance
  • the isolated polynucleotides, alone or joined to other polynucleotides such as vectors can be introduced into host cells, in culture or in whole organisms Introduced into host cells in culture or in whole organisms, such DNAs still would be isolated, as the term is used herein, because they would not be in their naturally occurnng form or environment
  • the polynucleotides and polypeptides may occur in a composition, such as a media formulations, solutions for introduction of polynucleotides or polypeptides, for example, into cells, compositions or solutions for chemical or enzymatic reactions, for instance, which are not naturally occurnng compositions, and, therein remain isolated polynucleotides or polypeptides within the meaning of that term as it is employed herein "Li
  • Orthogonucelot ⁇ de(s) refers to relatively short polynucleotides Often the term refers to single-stranded deoxy ⁇ bonucleotides, but it can refer as well to single-or double-stranded ⁇ bonucleotides, RNA DNA hybrids and double-stranded DNAs, among others
  • Oligonucleotides such as single-stranded DNA probe oligonucleotides, often are synthesized by chemical methods, such as those implemented on automated oligonucleotide synthesizers However, oligonucleotides can be made by a variety of other methods, including in vitro recombinant DNA-mediated techniques and by expression of DNAs in cells and organisms
  • oligonucleotides typically are obtained without a 5' phosphate
  • the 5' ends of such oligonucleotides are not substrates for phosphodiester bond formation by ligation reactions that employ DNA gases typically used to form recombinant DNA molecules
  • a phosphate can be added by standard techniques, such as those that employ a kinase and ATP
  • the 3' end of a chemically synthesized oligonucleotide generally has a free hydroxyl group and, in the presence of a ligase, such as T4 DNA ligase, readily will form a phosphodiester bond with a 5' phosphate of another polynucleotide, such as another oligonucleotide.
  • a ligase such as T4 DNA ligase
  • this reaction can be prevented selectively, where desired, by removing the 5' phosphates of the other polynucleot ⁇ de(s) p ⁇ or to ligation
  • Organ ⁇ sm(s) when used herein with reference to a life form used for cloning or that causes (or is believed to cause) disease means (l) a prokaryote, including but not limited to, a member of the genus Streptococcus, Staphylococcus, Bordetella, Corynebacterium,
  • Enterobacter Enterobacter, Yersinia, Fancisella, Pasturella, Moraxella, Acinetobacter, Erysipelothrix, Branhamella, Actinobacillus, Streptobacillus, Listeria, Calymmatobacterium, Brucella, Bacillus, Clostridium, Treponema, Escherichia, Salmonella, Kleibsiella, Vibrio, Proteus, Erwinia, Borrelia, Leptospira.
  • Streptococcus dttrans Neissena gonorrheae, Neissena meningitidis, Staphylococcus aureus, Staphylococcus epidermidis, Corynebacterium diptheriae, Gardnerella vaginalis, Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium ttlcerans, Mycobacterium leprae, Actinomyctes israelii, Listeria monocytogenes, Bordetella pertusis, Bordatella parapertusis, Bordetella bronchiseptica, Escherichia coli, Shigella dysenteriae, Haemophilus influenzae, Haemophilus aegyptitis, Haemophilus parainfluenzae, Haemophilus ducreyi, Bordetella, Salmonella typhi, Citrobacter freundii, Proteus mirabilis, Proteus vulgaris, Y
  • plasmids disclosed herein are either commercially available, publicly available on an unrestricted basis, or can be constructed from available plasmids by routine application of well known, published procedures.
  • Many plasmids and other cloning and expression vectors that can be used in accordance with the present invention are well known and readily available to those of skill in the art.
  • those of skill readily may construct any number of other plasmids suitable for use in the invention. The properties, construction and use of such plasmids, as well as other vectors, in the present invention will be readily apparent to those of skill from the present disclosure.
  • Polynucleotide(s) means any polyribonucleotide or polydeoxribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA.
  • polynucleotides as used herein refers to, among others, single-and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions or single-, double- and t ⁇ ple- stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single- stranded or, more typically, double-stranded, or triple-stranded, or a mixture of single- and double-stranded regions
  • polynucleotide as used herein refers to t ⁇ ple-stranded regions comp ⁇ sing RNA or DNA or both RNA and DNA
  • the strands in such regions may be from the same molecule or from different molecules
  • the regions may include all of one or more of the molecules, but more typically involve only a region of some of the molecules
  • One of the molecules of a tnple-helical region often is an oligonucleotide
  • polynucleotide includes DNAs or RNAs as desc ⁇ bed above that contain one or more modified bases
  • DNAs or RNAs with backbones modified for stability or for other reasons are "polynucleotides" as that term is intended herein
  • DNAs or RNAs compnsing unusual bases, such as inosine, or modified bases, such as tntylated bases, to name just two examples are polynucleotides as the term is used herein
  • polynucleotide as it is employed herein embraces such chemically, enzymatically or metabohcally modified forms of polynucleotides, as well as the chemical forms of DNA and RNA characteristic of viruses and cells, including simple and complex cells, inlet alia
  • Polypept ⁇ de(s) includes all polypeptides as descnbed below The basic structure of polypeptides is well known and has been descnbed in innumerable textbooks and other publications in the art In this context, the term is used herein to refer to any peptide or protein comp ⁇ sing two or more amino acids joined to each other in a linear chain by peptide bonds As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, ohgopeptides and ohgomers, for example, and to longer chains, which generally are refe ⁇ ed to in the art as proteins, of which there are many types It will be appreciated that polypeptides often contain amino acids other than the 20 amino acids commonly referred to as the 20 naturally occurring amino acids, and that many amino acids, including the terminal amino acids, may be modified in a given polypeptide, either by natural processes, such as processing and other post-translational modifications, but
  • polypeptides are not always entirely linear
  • polypeptides may be branched as a result of ubiquitination, and they may be circular, with or without branching, generally as a result of posttranslation events, including natural processing event and events brought about by human manipulation which do not occur naturally Circular
  • branched and branched circular polypeptides may be synthesized by non-translation natural process and by entirely synthetic methods, as well Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini
  • blockage of the amino or carboxyl group in a polypeptide, or both, by a covalent modification is common in naturally occurnng and synthetic polypeptides and such modifications may be present m polypeptides of the present invention, as well
  • the amino terminal residue of polypeptides made in E coli or other cells, p ⁇ or to proteolytic processing almost inva ⁇ ably
  • glycosylation often does not occur bactenal hosts such as, for example, E colt Accordingly, when glycosylation is desired, a polypeptide should be expressed in a glycosylatmg host, generally a eukaryotic cell
  • polypeptide encompasses all such modifications, particularly those that are present in polypeptides synthesized by expressing a polynucleotide in a host cell
  • Transformation means the process by which a cell is transformed" by exogenous DNA when such exogenous DNA has been introduced inside the cell membrane
  • Exogenous DNA may or may not be integrated (covalently linked) into chromosomal DNA making up the genome of the cell
  • the exogenous DNA may be maintained on an episomal element, such as a plasmid
  • a stably transformed or transfected cell is often one in which the exogenous DNA has become integrated into the chromosome so that it is inhe ⁇ ted by daughter cells through chromosome replication This stability is demonstrated by the ability of the eukaryotic cell to establish cell lines or clones comprised of a population of daughter cell containing the exogenous DNA Nanant(s)' of polynucleotides or polypeptides, as the term is used herein, are polynucleotides or polypeptides that differ from a reference polynucleotide or polypeptide, respectively
  • changes in the nucleotide sequence of the vanant may be silent That is, they may not alter the amino acids encoded by the polynucleotide Where alterations are limited to silent changes of this type a vanant will encode a polypeptide with the same am o acid sequence as the reference Also as noted below changes in the nucleotide sequence of the vanant may alter the ammo acid sequence of a polypeptide encoded by the reference polynucleotide Such nucleotide changes may result in amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence, as discussed below
  • a vanant and reference polypeptide may differ in amino acid sequence by one or more substitutions, additions, deletions, fusions and truncations, which may be present in any combination DESCRIPTION OF THE INVENTION
  • Fab I functions as the final step of the elongation cycle of bacte ⁇ al fatty acid biosynthesis
  • the enzyme from S aureus has been cloned, overexpressed in E colt and purified to >95% purity
  • Kinetic studies revealed sequential addition of the substrates crotonoyl CoA and NADH over the broad optimal pH range of about or equal to 5 0-7 0
  • the lack of a demonstrable back reaction or product inhibition prevented discrimination between an ordered versus a random mechanism
  • the K m for crotonoyl CoA was virtually identical to that of the E coli Fab I (about or equal to 2 5 mM), but the K m for NADH was approximately 30-fold higher (about or equal to 410 ⁇ M versus 13 ⁇ M for E colt)
  • Studies using deuterated substrates revealed a stereochemical course of the Fab I reaction identical to that reported for the E colt enzyme the hydride is transferred from the B-face of the nicotinamide ring of NA
  • the present invention relates to novel FAB I polypeptides and polynucleotides, among other things, as described in greater detail below.
  • the invention relates to polypeptides and polynucleotides of a novel FAB I gene of Staphylococcus aureus, which is related by amino acid sequence homology to Fab I enzymes from Mycobacteria (InhA),
  • the invention relates especially to FAB I having the nucleotide and amino acid sequences set out in Figure 17 [S ⁇ Q ID NO: l ] and Figure 16 [S ⁇ Q ID NO:2] respectively, and to the FAB I nucleotide sequences in NCIMB Deposit No. 40771 and amino acid sequences encoded therefrom, which is herein referred to as "the deposited clone" or as the "DNA of the deposited clone.” It will be appreciated that the nucleotide and amino acid sequences set out in Figure 16 [S ⁇ Q ID NO:2] and Figure 17 [S ⁇ Q ID NO: l] were obtained by sequencing the FAB I DNA of the deposited clone.
  • isolated polynucleotides which encode the FAB I polypeptide having the deduced amino acid sequence offigure 16 [S ⁇ Q ID NO:2].
  • a polynucleotide of the present invention encoding FAB I polypeptide may be obtained using standard cloning and screening procedures, such as those for cloning genomic DNAs from bacteria using Staphylococcus aureus WCUH29 cells as starting material.
  • standard cloning and screening procedures such as those for cloning genomic DNAs from bacteria using Staphylococcus aureus WCUH29 cells as starting material.
  • the polynucleotide set out infigure 17 [S ⁇ Q ID NO: l ] was discovered in a genomic DNA library derived from Staphylococcus aureus WCUH29.
  • FAB I of the invention is structurally related to other proteins of the enoyl-ACP reductase family, as shown by the results of sequencing the genomic DNA encoding FAB I in the deposited clone.
  • the DNA sequence thus obtained is set out infigure 17 [S ⁇ Q ID NO: l].
  • Polynucleotides of the present invention may be in the form of RNA, such as mRNA, or in the form of DNA, including, for instance, cDNA and genomic DNA obtained by cloning or produced by chemical synthetic techniques or by a combination thereof
  • the DNA may be double-stranded or single-stranded Single-stranded DNA may be the coding strand, also known as the sense strand, or it may be the non-coding strand, also referred to as the anti- sense strand
  • the coding sequence which encodes the polypeptide may be identical to the coding sequence of the polynucleotide shown infigure 17 [SEQ ID NO 1] It also may be a polynucleotide with a different sequence, which, as a result of the redundancy (degeneracy) of the genetic code, encodes the polypeptide of the DNA offigure 16 [SEQ ID NO 2]
  • Polynucleotides of the present invention which encode the polypeptide offigure 16 [SEQ ID NO 2] may include, but are not limited to the coding sequence for the mature polypeptide, by itself, the coding sequence for the mature polypeptide and additional coding sequences, such as those encoding a leader or secretory sequence, such as a pre-, or pro- or prepro- protein sequence, the coding sequence of the mature polypeptide, with or without the aforementioned additional coding sequences, together with additional, non-coding sequences, including for example, but not limited to non-coding 5' and 3' sequences, such as the transc ⁇ bed, non-translated sequences that play a role in transc ⁇ ption (including termination signals, for example), ⁇ bosome binding, mRNA stability elements, and additional coding sequence which encode additional amino acids, such as those which provide additional functionalities
  • the polypeptide may be fused to a marker sequence, such as a peptide, which facilitates punfication of the fused poly
  • polynucleotide encoding a polypeptide as used herein encompasses polynucleotides which include a sequence encoding a polypeptide of the present invention, particularly staphylococcal FAB I ha ing the amino acid sequence set out infigure 16 [SEQ ID NO 2]
  • the term encompasses polynucleotides that include a single continuous region or discontinuous regions encoding the polypeptide (for example, interrupted by integrated phage, insertion sequence, recombination, or editing) together with additional regions, that also may contain coding and/or non-coding sequences
  • the present invention further relates to variants of the herein above desc ⁇ bed polynucleotides which encode for fragments, analogs and derivatives of the polypeptide having the deduced amino acid sequence offigure 16 [SEQ ID NO 2]
  • a vanant of the polynucleotide may be a naturally occur ⁇ ng vanant such as a naturally occurnng allehc vanant
  • vanants in this regard are vanants that differ from the aforementioned polynucleotides by nucleotide substitutions, deletions or additions
  • the substitutions, deletions or additions may involve one or more nucleotides
  • the variants may be altered in coding or non-coding regions or both Alterations in the coding regions may produce conservative or non-conservative amino acid substitutions, deletions or additions
  • polynucleotides encoding FAB I vanants, analogs, denvatives and fragments, and vanants, analogs and denvatives of the fragments which have the amino acid sequence of FAB I polypeptide offigure 16 [SEQ ID NO 2] in which several, a few, 5 to 10, 1 to 5, 1 to 3, 2, 1 or no amino acid residues are substituted, deleted or added, m any combination Especially preferred among these are silent substitutions, additions and deletions, which do not alter the properties and activities of FAB I Also especially preferred this regard are conservative substitutions Most highly preferred are polynucleotides encoding polypeptides having the amino acid sequence offigure 16 [SEQ ID NO;2]. without substitutions.
  • polynucleotides that are at least 70% identical to a polynucleotide encoding FAB I polypeptide having the amino acid sequence set out infigure 16 [SEQ ID NO:2], and polynucleotides which are complementary to such polynucleotides.
  • polynucleotides that comprise a region that is at least 80% identical to a polynucleotide encoding FAB I polypeptide encoded by the Staphylococcus aureus DNA of the deposited clone and polynucleotides complementary thereto or as set out infigure 17 [SEQ ID NO: l].
  • polynucleotides at least 90% identical to the same are particularly preferred, and among these particularly prefe ⁇ ed polynucleotides, those with at least 95% are especially preferred. Furthermore, those with at least 97% are highly prefened among those with at least 95%, and among these those with at least 98% and at least 99% are particularly highly preferred, with at least 99% being the more prefe ⁇ ed.
  • Particularly prefe ⁇ ed embodiments in this respect are polynucleotides which encode polypeptides which retain substantially the same biological function or activity as the mature polypeptide encoded by the DNA offigure 17 [SEQ ID NO: l].
  • the present invention further relates to polynucleotides that hybridize to the herein above-described sequences.
  • the present invention especially relates to polynucleotides which hybridize under stringent conditions to the herein above-described polynucleotides.
  • stringent conditions means hybridization will occur only if there is at least 95% and preferably at least 97% identity between the sequences.
  • polynucleotides of the invention may be used as a hybridization probe for RNA, cDNA and genomic DNA to isolate full-length cDNAs and genomic clones encoding FAB I and to isolate cDNA and genomic clones of other genes that have a high sequence similarity to the FAB I gene.
  • Such probes generally will comprise at least 15 bases.
  • such probes will have at least 30 bases and may have at least 50 bases.
  • Particularly prefe ⁇ ed probes will have at least 30 bases and will have 50 bases or less.
  • the coding region of the FAB I gene may be isolated by screening using the known DNA sequence to synthesize an oligonucleotide probe.
  • a labeled oligonucleotide having a sequence complementary to that of a gene of the present invention is then used to screen a library of cDNA, genomic DNA or mRNA to determine which members of the library the probe hybndizes to
  • polynucleotides and polypeptides of the present invention may be employed as research reagents and mate ⁇ als for discovery of treatments of and diagnostics for disease, particularly human disease, as further discussed herein relating to polynucleotide assays, inter alia
  • polynucleotides of the invention that are oligonucleotides, including SEQ ID NOS 3 and 4, derived from the sequence of SEQ ID NO 1 may be used as PCR primers in the processes herein desc ⁇ bed to determine whether or not the Staphylococcus aureus genes identified herein in whole or in part are transcribed in infected tissue
  • sequences may also have utility in diagnosis of the stage of infection and type of infection the pathogen has attained
  • the polynucleotides may encode a polypeptide which is the mature protein plus additional amino or carboxyl-terminal am o acids, or amino acids inte ⁇ or to the mature polypeptide (when the mature form has more than one polypeptide chain, for instance)
  • Such sequences may play a role in processing of a protein from precursor to a mature form, may allow protein transport, may lengthen or shorten protein half-life or may facilitate manipulation of a protein for assay or production, among other things
  • the additional amino acids may be processed away from the mature protein by cellular enzymes
  • a precursor protein, having the mature form of the polypeptide fused to one or more prosequences may be an inactive form of the polypeptide When prosequences are removed such inactive precursors generally are activated Some or all of the prosequences may be removed before activation Generally, such precursors are called proproteins
  • a polynucleotide of the present invention may encode a mature protein, a mature protein plus a leader sequence (which may be refe ⁇ ed to as a preprote ), a precursor of a mature protein having one or more prosequences which are not the leader sequences of a preprotem, or a preproprotem, which is a precursor to a proprotem, having a leader sequence and one or more prosequences, which generally are removed dunng processing steps that produce active and mature forms of the polypeptide Deposited materials
  • a deposit containing a Staphylococcus aureus WCUH 29 strain has been deposited with the National Collections of Industrial and Marine Bacteria Ltd (NCIMB), 23 St Machar Drive, Aberdeen AB2 1RY, Scotland on 1 1 September 1995 and assigned NCIMB Deposit No 40771
  • the FAB I clone deposit is referred to herein as the deposited clone" or as ' the DNA of the deposited clone "
  • the deposited matenal is a strain that contains the full length FAB I DNA, refe ⁇ ed to as "NCIMB 40771 " upon deposit
  • sequence of the polynucleotides contained in the deposited material, as well as the amino acid sequence of the polypeptide encoded thereby, are controlling in the event of any conflict with any descnption of sequences herein
  • polypeptides The present invention further relates to a prokaryotic FAB I polypeptide which has the deduced ammo acid sequence offigure 16 [SEQ ID NO 2]
  • the invention also relates to fragments, analogs and de ⁇ vatives of these polypeptides
  • fragment when refernng to the polypeptide offigure 16 [SEQ ID NO 2], means a polypeptide which retains essentially the same biological function or activity as such polypeptide
  • an analog includes a proprotem which can be activated by cleavage of the proprotem portion to produce an active mature polypeptide
  • the polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide or a synthetic polypeptide In certain prefe ⁇ ed embodiments it is a recombinant polypeptide
  • the fragment, derivative or analog of the polypeptide offigure 16 [SEQ ID NO 2] may be (l) one in which one or more of the amino acid residues are substituted with a conserved or non-conserved ammo acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code, or (n) one in which one or more of the amino acid residues includes a substituent group, or (in) one in which the mature polypeptide is fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol).
  • a conserved or non-conserved ammo acid residue preferably a conserved amino acid residue
  • substituted amino acid residue may or may not be one encoded by the genetic code
  • n one in which one or more of the amino acid residues includes a substituent group
  • the mature polypeptide is fused with another compound, such as a compound to increase the half-life of the polypeptide
  • particularly prefened embodiments of the invention in this regard are polypeptides having the amino acid sequence of FAB I set out infigure 16 [SEQ ID NO 2] vanants, analogs, derivatives and fragments thereof, and vanants.
  • analogs and de ⁇ vatives of the fragments are particularly prefe ⁇ ed embodiments of the invention in this regard. polypeptides having the amino acid sequence of the FAB I, variants, analogs, de ⁇ vatives and fragments thereof, and vanants, analogs and de ⁇ vatives of the fragments
  • prefe ⁇ ed vanants are those that vary from a reference by conservative amino acid substitutions Such substitutions are those that substitute a given amino acid in a polypeptide by another am o acid of like charactenstics Typically seen as conservative substitutions are the replacements, one for another, among the aliphatic amino acids Ala, Val, Leu and He, interchange of the hydroxyl residues Ser and Thr, exchange of the acidic residues Asp and Glu, substitution between the amide residues Asn and Gin, exchange of the basic residues Lys and Arg and replacements among the aromatic residues Phe, Tyr
  • vanants, analogs, denvatives and fragments, and vanants, analogs and de ⁇ vatives of the fragments having the amino acid sequence of the FAB I polypeptide offigure 16 [SEQ ID NO 2] in which several, a few, 5 to 10, 1 to 5, 1 to 3, 2, 1 or no amino acid residues are substituted, deleted or added, in any combination
  • polypeptides and polynucleotides of the present invention are preferably provided in an isolated form, and preferably are punfied to homogeneity
  • polypeptides of the present invention include the polypeptide of SEQ ID NO 2
  • polypeptides which have at least 80% identity to the polypeptide of SEQ ID NO 2 and more preferably at least 90% simila ⁇ ty (more preferably at least 90% identity) to the polypeptide of SEQ ID NO 2 and still more preferably at least 95% simila ⁇ ty (still more preferably at least 95% identity) to the polypeptide of SEQ ID NO 2 and also include portions of such polypeptides with such portion of the polypeptide generally containing at least 30 amino acids and more preferably at least 50 amino acids
  • Fragments or portions of the polypeptides of the present invention may be employed for producing the co ⁇ esponding full-length polypeptide by peptide synthesis, therefore, the fragments may be employed as intermediates for producing the full-length polypeptides Fragments or portions of the polynucleotides of the present invention may be used to synthesize full-length polynucleotides of the present invention Fragments Also among prefe ⁇ ed embodiments of this aspect of the present invention are polypeptides comprising fragments of FAB I, most particularly fragments of FAB I having the amino acid set out infigure 16 [SEQ ID NO 2] and fragments of vanants and de ⁇ vatives of the FAB I offigure 16 [SEQ ID NO 2]
  • a fragment is a polypeptide having an amino acid sequence that entirely is the same as part but not all of the ammo acid sequence of the aforementioned FAB I polypeptides and vanants or derivatives thereof
  • fragments may be "free-standing," ; e , not part of or fused to other amino acids or polypeptides, or they may be comprised within a larger polypeptide of which they form a part or region
  • the presently discussed fragments most preferably form a single continuous region
  • several fragments may be compnsed withm a single larger polypeptide
  • certain prefe ⁇ ed embodiments relate to a fragment of a FAB I polypeptide of the present compnsed within a precursor polypeptide designed for expression in a host and having heterologous pre and pro- polypeptide regions fused to the ammo terminus of the FAB I fragment and an additional region fused to the carboxyl terminus of the fragment Therefore, fragments in one aspect of the meaning intended herein, refers to the portion or portions of a fusion polypeptide or fusion protein denved from FAB I
  • Particularly preferred fragments include those encoded by amino acids 1-20, 21-40, 41-60, 61-80, 81-100, 101-120, 121-140, 141-160, 161-180, 181 -200, 201-220, 221-240, 241- 256 and contiguous combinations thereof
  • prefe ⁇ ed fragments include, for example, those with high homology to other sequenced structurally related elements These include, for example, regions homologous to fragments of E coli fabl including about 13 to about 25, about 32 to about 39, about 1 12 to about 125, about 156 to about 196 and about 2 1 to about 252
  • Truncation mutants include FAB I polypeptides having the amino acid sequence offigure 16 [SEQ ID NO 2] or of vanants or de ⁇ vatives thereof, except for deletion of a continuous se ⁇ es of residues (that is, a continuous region, part or portion) that includes the amino terminus, or a continuous senes of residues that includes the carboxyl terminus or, as in double truncation mutants, deletion of two continuous se ⁇ es of residues, one including the amino terminus and one including the carboxyl terminus Fragments having the size ranges set out about also are prefe ⁇ ed embodiments of truncation fragments, which are especially prefe ⁇ ed among fragments generally Degradation products of the polypeptides of the invention in a host cell, particularly a staphylococcus, are also prefe ⁇ ed polypeptides
  • fragments charactenzed by structural or functional att ⁇ butes of FAB I Prefe ⁇ ed embodiments of the invention in this regard include fragments that comp ⁇ se alpha-helix and alpha-helix forming regions ("alpha- regions"), beta-sheet and beta-sheet-forming regions ("beta-regions"), turn and turn-forming regions ("turn-regions”), coil and coil-formmg regions ("coil-regions”), hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions and high antigenic index regions of FAB I
  • the regions defined by the residues 1 to about 8, about 26 to about 34, about 39 to about 61, about 70 to about 80, about 95 to about 107, about 127 to about 139, about 162 to about 173 and about 207 to about 220 are charactenzed by amino acids believed to compnse alpha helix forming regions. Additionally, the regions defined by the residues about 9 to about 13, about 19 to about 25, about 62 to about 69, about 85 to about 95, about 141 to about 156.
  • about 173 to about 188 about 220 to about 226, about 232 to about 240 are charactenzed by residues believed to compnse beta-sheet regions
  • the regions defined by the residues about 80 to about 85, about 108 to about 1 12, about 120 to about 125, about 178 to about 183, and about 198 to about 203 are characterized by regions behved to comp ⁇ se turn regions.
  • the regions defined by the residues about 80 to about 85, about 105 to about 1 10. about 120 to about 125, about 138 to about 143, about 145 to about 153, about 175 to about 180, about 187 to about 192, and about 240 to about 245 are charactenzed by amino acid compositions believed to comp ⁇ se coil regions.
  • the regions defined by the residues about 9 to about 14, about 20 to about 36. about 86 to about 98. about 1 15 to about 129. about 138 to about 152, about 161 to about 179, about 185 to about 195, about 226 to about 240, about 249 to about 256 are charactenzed by hydrophobic amino acids Additionally, the regions defined by the residues about 2 to about 8, about 14 to about 19, about 36 to about 86, 98 to about 1 15, about 130 to about 138, about 152 to about 161, about 180 to about 186, about 206 to about 226, about 239 to and about 249 are characterized by hydrophilic amino acids Also, the regions defined by the residues about 20 to about 40, about 43 to about 62, about 72 to about 93, about 102 to about 117, about 128 to about 140, about 180 to about 220, and about 228 to about 240 are charactenzed by ammo acid residues believed to comprise an alpha amphipathic region and the regions defined by the residue
  • regions defined by residues about 34 to about 62, about 98 to about 112 and about 206 to about 222 are believed to comrpise surface forming regions Additionally, the regions defined by residues about 3 to about 9, about 14 to about 20, about 25 to about 33, about 38 to about 60, about 65 to about 88, about 98 to about 1 14, about 120 to about 124, about 130 to about 140, about 178 to about 185, about 195 to about 228, and about 236 to about 247 are believed to comp ⁇ se regions of high antigenicity Such regions set forth above may be compnsed within a larger polypeptide or may be by themselves a prefe ⁇ ed fragment of the present invention, as discussed above It will be appreciated that the term "about” as used in this paragraph has the meaning set out above regarding fragments in general.
  • Further prefe ⁇ ed regions are those that mediate activities of FAB I
  • Most highly prefe ⁇ ed in this regard are fragments that have a chemical, biological or other activity of FAB I, including those with a similar activity or an improved activity, or with a decreased undesirable activity
  • Highly prefe ⁇ ed m this regard are fragments that contain regions that are homologs in sequence, or in position, or in both sequence and to active regions of related polypeptides, such as the related polypeptides set out infigure 16 [SEQ ID NO.2], which include E coli Enoyl (ACP) reductase, Fab I
  • truncation mutants are particularly prefe ⁇ ed fragments in these regards.
  • Further prepfe ⁇ ed polynucelotide fragments are those that are antigenic or lmmunogenic in an animal, especially in a human
  • the invention also relates to, among others, polynucleotides encoding the aforementioned fragments, polynucleotides that hyb ⁇ dize to polynucleotides encoding the fragments, particularly those that hybndize under stnngent conditions, and polynucleotides, such as PCR p ⁇ mers, for amplifying polynucleotides that encode the fragments
  • prefe ⁇ ed polynucleotides are those that co ⁇ espondent to the prefe ⁇ ed fragments, as discussed above
  • the present invention also relates to vectors which comprise a polynucleotide or polynucleotides of the present invention, host cells which are genetically engineered with vectors of the invention and the production of polypeptides of the invention by recombinant techniques
  • Host cells can be genetically engineered to incorporate polynucleotides and express polypeptides of the present invention
  • polynucleotides may be introduced into host cells using well known techniques of infection, transduction, transfection, transvection and transformation
  • the polynucleotides may be introduced alone or with other polynucleotides
  • Such other polynucleotides may be introduced independently, co-introduced or introduced joined to the polynucleotides of the invention
  • the polynucleotides may be joined to a vector containing a selectable marker for propagation in a host
  • the vector construct may be introduced into host cells by the aforementioned techniques
  • a plasmid vector is introduced as DNA in a precipitate, such as a calcium phosphate precipitate, or in a complex with a charged hpid Electroporation also may be used to introduce polynucleotides into a host If the vector is a virus, it may be packaged m vitro or introduced into
  • vectors Prefe ⁇ ed among vectors, in certain respects, are those for expression of polynucleotides and polypeptides of the present invention
  • vectors comp ⁇ se cis-actmg control regions effective for expression in a host operatively linked to the polynucleotide to be expressed
  • Appropnate trans-acting factors either are supplied by the host, supplied by a complementing vector or supplied by the vector itself upon introduction
  • the vectors provide for specific expression
  • Such specific expression may be inducible expression or expression only in certain types of cells or both inducible and cell-specific
  • Particularly prefe ⁇ ed among inducible vectors are vectors that can be induced for expression by environmental factors that are easy to manipulate, such as temperature and nut ⁇ ent additives
  • a vanety of vectors suitable to this aspect of the invention, including constitutive and inducible expression vectors for use in prokaryotic and eukaryotic hosts, are well known and employed routinely by those of skill in the art
  • the engineered host cells can be cultured m conventional nutnent media, which may be modified as appropnate for, inter alia, activating promoters, selecting transformants or amplifying genes Culture conditions, such as temperature, pH and the like, previously used with the host cell selected for expression generally will be suitable for expression of polypeptides of the present invention as will be apparent to those of skill in the art
  • a great va ⁇ ety of expression vectors can be used to express
  • the appropnate DNA sequence may be inserted into the vector by any of a vanety of well-known and routine techniques
  • a DNA sequence for expression is joined to an expression vector by cleaving the DNA sequence and the expression vector with one or more restnction endonucleases and then joining the restriction fragments together using T4 DNA ligase Procedures for restnction and ligation that can be used to this end are well known and routine to those of skill Suitable procedures in this regard, and for constructing expression vectors using alternative techniques, which also are well known and routine to those skill, are set forth m great detail in Sambrook et al , (1989) MOLECULAR CLONING, A LABORATORY MANUAL, 2nd Ed , Cold Sp ⁇ ng Harbor Laboratory Press, Cold Spnng Harbor, New York
  • the DNA sequence in the expression vector is operatively linked to approp ⁇ ate expression control sequence(s), including, for instance, a promoter to direct mRNA transcnption
  • a promoter to direct mRNA transcnption Representatives of such promoters include the phage lambda PL promoter, the E coli lac, tip and tac promoters, the SV40 early and late promoters and promoters of retroviral LTRs, to name just a few of the well-known promoters It will be understood that numerous promoters not mentioned are suitable for use in this aspect of the invention are well known and readily may be employed by those of skill in the manner illustrated by the discussion and the examples herein
  • expression constructs will contain sites for transc ⁇ ption initiation and termination, and, in the transcribed region, a nbosome binding site for translation
  • the coding portion of the mature transc ⁇ pts expressed by the constructs will include a translation initiating AUG at the beginning and a termination codon approp ⁇ ately positioned at the end of the polypeptide to be translated
  • constructs may contain control regions that regulate as well as engender expression Generally, in accordance with many commonly practiced procedures, such regions will operate by controlling transcnption, such as transcription factors, repressor binding sites and termination, among others Vectors for propagation and expression generally will include selectable markers
  • the expression vectors preferably contain one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells
  • selectable marker genes include dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, and tetracychne or ampicilhn resistance genes for cultunng E coli and other prokaryotes
  • the vector containing the approp ⁇ ate DNA sequence as desc ⁇ bed elsewhere herein, as well as an approp ⁇ ate promoter, and other approp ⁇ ate control sequences, may be introduced into an appropnate host using a va ⁇ ety of well known techniques suitable to expression therein of a desired polypeptide
  • appropnate hosts include bacterial cells, such as streptocooi, staphylococci, £ colt, streptomyces and Salmonella typhtmurium cells, fungal cells, such as yeast cells and Aspergtllus cells, insect cells such as Drosophila S2 and Spodoptera Sf9 cells, animal cells such as CHO, COS and Bowes melanoma cells, and plant cells Hosts for of a great va ⁇ ety of expression constructs are well known, and those of skill will be enabled by the present disclosure readily to select a host for expressing a polypeptides in accordance with this aspect of the present invention
  • the present invention also includes recombinant constructs, such as expression constructs, comp ⁇ sing one or more of the sequences described above
  • the constructs compnse a vector, such as a plasmid or viral vector, into which such a sequence of the invention has been inserted
  • the sequence may be inserted in a forward or reverse onentation
  • the construct further compnses regulatory sequences, including, for example, a promoter, operably linked to the sequence
  • suitable vectors and promoters are known to those of skill in the art, and there are many commercially available vectors suitable for use in the present invention
  • vectors prefe ⁇ ed for use in bacte ⁇ a are pQE70, pQE60 and pQE-9, available from Qiagen, pBS vectors, Phagesc ⁇ pt vectors, Bluesc ⁇ pt vectors, pNH8A, pNHl ⁇ a, pNHl 8A, pNH46A, available from Stratagene, and ptrc99a, pKK223-3, pKK233-3, pDR540, pRTT5 available from Pharmacia
  • prefe ⁇ ed eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXTl and pSG available from Stratagene, and pSVK3, pBPV, pMSG and pSVL available from Pharmacia
  • These vectors are listed solely by way of illustration of the many commercially available and well known vectors that are available to those of skill
  • prokaryotic promoters suitable for expression of polynucleotides and polypeptides in accordance with the present invention are the E colt lad and lacZ and promoters, the T3 and T7 promoters, the gpt promoter, the lambda PR, PL promoters and the tip promoter
  • known eukaryotic promoters suitable in this regard are the CMV immediate early promoter, the HSV thymidine kinase promoter, the early and late SV40 promoters, the promoters of retroviral LTRs, such as those of the Rous sarcoma virus ("RSV"), and metallothionein promoters, such as the mouse metalloth ⁇ one ⁇ n-I promoter
  • the present invention also relates to host cells containing the above-descnbed constructs discussed above
  • the host cell can be a higher eukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell, such as a yeast cell, or the host cell can be a prokaryotic cell, such as a bactenal cell
  • Introduction of the construct into the host cell can be affected by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic hpid-mediated transfection, electroporation, transduction, scrape loading, ballistic introduction, infection or other methods
  • the polypeptides of the invention can be synthetically produced by conventional peptide synthesizers.
  • Mature proteins can be expressed in mammalian cells, yeast, bacteria, or other cells under the control of appropriate promoters. Cell-free translation systems can also be employed to produce such proteins using RNAs derived from the DNA constructs of the present invention. Appropriate cloning and expression vectors for use with prokaryotic and eukaryotic hosts are described by Sambrook et al., (1989) MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y..
  • recombinant expression vectors will include origins of replication, a promoter derived from a highly-expressed gene to direct transcription of a downstream structural sequence, and a selectable marker to permit isolation of vector containing cells after exposure to the vector.
  • suitable promoters are those derived from the genes that encode glycolytic enzymes such as 3-phosphogIycerate kinase ("PGK”), a-factor, acid phosphatase, and heat shock proteins, among others.
  • PGK 3-phosphogIycerate kinase
  • Selectable markers include the ampicillin resistance gene of E. coli and the trpl gene of 5. cerevisiae.
  • Enhancers are cw-acting elements of DNA, usually about from 10 to 300 bp that act to increase transcriptional activity of a promoter in a given host cell-type.
  • enhancers include the SV40 enhancer, which is located on the late side of the replication origin at bp 100 to 270, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovims enhancers.
  • Polynucleotides of the invention encoding the heterologous structural sequence of a polypeptide of the invention generally will be inserted into the vector using standard techniques so that it is operably linked to the promoter for expression.
  • the polynucleotide will be positioned so that the transcription start site is located appropriately 5' to a ribosome binding site.
  • the ribosome binding site will be 5' to the AUG that initiates translation of the polypeptide to be expressed.
  • secretion signals may be incorporated into the expressed polypeptide. These signals may be endogenous to the polypeptide or they may be heterologous signals.
  • the polypeptide may be expressed in a modified form, such as a fusion protein, and may include not only secretion signals but also additional heterologous functional regions.
  • a region of additional amino acids, particularly charged amino acids may be added to the N-terminus of the polypeptide to improve stability and persistence in the host cell, during purification or during subsequent handling and storage.
  • region also may be added to the polypeptide to facilitate purification. Such regions may be removed prior to final preparation of the polypeptide.
  • the addition of peptide moieties to polypeptides to engender secretion or excretion, to improve stability or to facilitate purification, among others, are familiar and routine techniques in the art.
  • a prefe ⁇ ed fusion protein comprises a heterologous region from immunolglobulin that is useful to solubilize or purify polypeptides.
  • EP-A-O 464 533 (Canadian counterpart 2045869) discloses fusion proteins comprising various portions of constant region of immunoglobin molecules together with another human protein or part thereof.
  • the Fc part in fusion protein is thoroughly advantageous for use in therapy and diagnosis and thus results, for example, in improved pharmacokinetic properties (see EP-A 0232 262).
  • Fc portion proves to be a hindrance to use in assays, therapy or diagnosis, for example when the fusion protein is to be used as antigen for immunizations.
  • human proteins such as, shIL5- has been fused with Fc portions for the purpose of high-throughput screening assays to identify antagonists of hIL-5. See, D. Bennett et al, (1995) Journal of Molecular Recognition, Vol. 8 52-58 and K. Johanson et al, ( 1995) The Journal of Biological Chemistry, Vol. 270, No. 16, pp 9459-9471.
  • Suitable prokaryotic hosts for propagation, maintenance or expression of polynucleotides and polypeptides in accordance with the invention include streptococci, Escherichia coli, Bacillus subtilis and Salmonella typhimurium. Various species of Pseudomonas, Streptomvces, and Staphylococcus are also suitable hosts in this regard Moreover, many other hosts also known to those of skill may be employed in this regard
  • useful expression vectors for bactenal use can comp ⁇ se a selectable marker and bacte ⁇ al o ⁇ gm of replication de ⁇ ved from commercially available plasmids comprising genetic elements of the well known cloning vector pBR322 (ATCC 37017)
  • Such commercial vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and GEM 1 (Promega Biotec, Madison, WI, USA)
  • pBR322 "backbone" sections are combined with an appropnate promoter and the structural sequence to be expressed Following transformation of a suitable host strain and growth of the host strain to an appropriate cell density, where the selected promoter is inducible it is induced by appropnate means (e -; , temperature shift or exposure to chemical inducer) and cells are cultured for an additional period
  • Microbial cells employed in expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents, such methods are well know to those skilled in the art
  • Va ⁇ ous mammalian cell culture systems can be employed for expression, as well Examples of mammalian expression systems include the COS-7 lines of monkey kidney fibroblast, described in Gluzman et al, ( 1981 ) Cell 23 175 Other cell lines capable of expressing a compatible vector include for example, the C127, 3T3, CHO, HeLa, human kidney 293 and BHK cell lines
  • Mammalian expression vectors may comprise an origin of replication, a suitable promoter and enhancer, and also any necessary ribosome binding sites, polyadenylation sites, splice donor and acceptor sites, transcnptional termination sequences, and 5' flanking non- transc ⁇ bed sequences that are necessary for expression
  • DNA sequences denved from the SV40 splice sites, and the SV40 polyadenylation sites are used for required non-transcribed genetic elements of these types
  • FAB I polypeptide can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography Most preferably, high performance liquid chromatography (“HPLC”) is employed for punfication Well known techniques for refolding protein may be employed
  • Polypeptides of the present invention include naturally purified products, products of chemical synthetic procedures, and products produced by recombinant techniques from a prokaryotic or eukaryotic host, including, for example, bacterial, yeast, higher plant insect and mammalian cells Depending upon the host employed in a recombinant production procedure, the polypeptides of the present invention may be glycosylated or may be non- glycosylated In addition, polypeptides of the invention may also include an initial modified methionine residue, in some cases as a result of host-mediated processes
  • FAB I polynucleotides and polypeptides may be used in accordance with the present invention for a vanety of applications, particularly those that make use of the chemical and biological properties of FAB I Additional applications relate to diagnosis and to treatment of disorders of cells, tissues and organisms These aspects of the invention are illustrated further by the following discussion
  • This invention is also related to the use of the FAB I polynucleotides to detect complementary polynucleotides such as, for example, as a diagnostic reagent Detection of FAB I in a eukaryote, particularly a mammal, and especially a human, will provide a diagnostic method that can add to, define or allow a diagnosis of a disease Eukaryotes (herein also " ⁇ nd ⁇ v ⁇ dual(s)”), particularly mammals, and especially humans, carrying a FAB I gene may be detected at the DNA level by a va ⁇ ety of techniques Nucleic acids for diagnosis may be obtained from an individual's cells and tissues, such as bone, blood, muscle, cartilage, and skin Tissue biopsy and autopsy matenal is also prefe ⁇ ed for samples from an individual to use in a diagnostic assay Genomic DNA may be used directly for detection or may be amplified enzymatically by using PCR p ⁇ or to analysis PCR (Saiki et al , (1986
  • Point mutations can be identified by hybridizing amplified DNA to radiolabeled FAB I RNA or alternatively, radiolabeled FAB I antisense DNA sequences. Perfectly matched sequences can be distinguished from mismatched duplexes by RNase A digestion or by differences in melting temperatures.
  • Sequence differences between a reference gene and genes having mutations also may be revealed by direct DNA sequencing.
  • cloned DNA segments may be employed as probes to detect specific DNA segments.
  • the sensitivity of such methods can be greatly enhanced by appropriate use of PCR or another amplification method.
  • a sequencing primer is used with double-stranded PCR product or a single-stranded template molecule generated by a modified PCR.
  • the sequence determination is performed by conventional procedures with radiolabeled nucleotide or by automatic sequencing procedures with fluorescent-tags.
  • DNA sequence differences may be achieved by detection of alteration in electrophoretic mobility of DNA fragments in gels, with or without denaturing agents. Small sequence deletions and insertions can be visualized by high resolution gel electrophoresis. DNA fragments of different sequences may be distinguished on denaturing formamide gradient gels in which the mobilities of different DNA fragments are retarded in the gel at different positions according to their specific melting or partial melting temperatures (see, e.g., Myers et al., (1985) Science, 230: 1242).
  • Sequence changes at specific locations also may be revealed by nuclease protection assays, such as RNase and S I protection or the chemical cleavage method (e.g.. Cotton et al., (1985) Proc. Natl. Acad. Sci., USA, 85: 4397-4401).
  • the detection of a specific DNA sequence may be achieved by methods such as hybridization, RNase protection, chemical cleavage, direct DNA sequencing or the use of restriction enzymes, (e.g., restriction fragment length polymorphisms ("RFLP”) and Southern blotting of genomic DNA.
  • restriction enzymes e.g., restriction fragment length polymorphisms ("RFLP") and Southern blotting of genomic DNA.
  • mutations also can be detected by in situ analysis.
  • a mutation may be ascertained for example, by a DNA sequencing assay.
  • Samples are processed by methods known in the art to capture the RNA.
  • First strand cDNA is synthesized from the RNA samples by adding an oligonucleotide primer consisting of sequences which hybridize to a region on the mRNA.
  • Reverse transcriptase and deoxv nucleotides are added to allow synthesis of the first strand cDNA P ⁇ mer sequences are synthesized based on the DNA sequence of the FAB I protein of the invention
  • the primer sequence is generally comprised of at least 15 consecutive bases and may contain at least 30 or even 50 consecutive bases
  • Cells carrying mutations in the gene of the present invention may also be detected at the DNA level by a va ⁇ ety of techniques, to allow for serotyping, for example Nucleic acids for diagnosis may be obtained from an individual s cells or bodily fluids, including but not limited to blood, urine, saliva, tissue biopsy and autopsy material
  • the genomic DNA may be used directly for detection or may be amplified enzymatically by using PCR (Saiki et al , ( 1986) Nature, 324 163-166) prior to analysis RT-PCR can also be used to detect mutations It is particularly prefe ⁇ ed to used RT-PCR in conjunction with automated detection systems, such as, for example, GeneScan
  • the above p ⁇ mers may be used for amplifying FAB I DNA isolated from a sample de ⁇ ved from an individual
  • the invention also provides the primers of Table 1 with 1, 2, 3 or 4 nucleotides removed from the 5' and/or the 3' end
  • the p ⁇ mers may be used to amplify the gene isolated from the individual such that the gene may then be subject to va ⁇ ous techniques for elucidation of the DNA sequence In this way, mutations in the DNA sequence may be determined, for example, to ascertain the serotype of the organism
  • Sequence differences between the reference gene and genes having mutations may be revealed by the direct DNA sequencing method
  • cloned DNA segments may be employed as probes to detect specific DNA segments
  • the sensitivity of this method is greatly enhanced when combined with PCR
  • a sequencing p ⁇ mer is used with double-stranded PCR product or a single-stranded template molecule generated by a modified PCR
  • the sequence determination is performed by conventional procedures with radiolabeled nucleotide or by automatic sequencing procedures with fluorescent-tags
  • Genetyping based on DNA sequence differences may be achieved by detection of alteration in electrophoretic mobility of DNA fragments in gels with or without denatu ⁇ ng agents
  • Small sequence deletions and insertions can be visualized by high resolution gel electrophoresis
  • DNA fragments of different sequences may be distinguished on denatunng formamide gradient gels in which the mobilities of different DNA fragments are retarded in the gel at different positions according to their specific melting or partial melting temperatures (see, e g Myers et al , (
  • Sequence changes at specific locations may also be revealed by nuclease protection assays, such as RNase and S 1 protection or the chemical cleavage method (e g , Cotton et al ,
  • the detection of a specific DNA sequence and/or quantitation of the level of the sequence may be achieved by methods such as hybridization, RNase protection, chemical cleavage, direct DNA sequencing or the use of restnction enzymes, (e g , Restnction Fragment
  • the invention provides a process for diagnosing disease, particularly bacterial infections, and most particularly staphylococcal infections, comprising determining from a sample derived from an individual an increased level of expression of polynucleotide having the sequence offigure 17 [SEQ ID NO 1 ] Increased expression of polynucleotide can be measured using any on of the methods well known in the art for the quantation of polynucleotides, such as, for example, PCR, RT-PCR, RNase protection. Northern blotting and other hybridization methods
  • mutations can also be detected by in situ analysis
  • the present invention also relates to a diagnostic assays such as quantitative and diagnostic assays for detecting levels of FAB I protein in cells and tissues, including determination of normal and abnormal levels
  • a diagnostic assay in accordance with the invention for detecting a pathological expression level of FAB I protein compared to normal control tissue samples may be used to detect the presence of bacte ⁇ al infection, for example
  • Assay techniques that can be used to determine levels of a protein, such as an FAB I protein of the present invention, in a sample de ⁇ ved from a host are well- known to those of skill in the art
  • Such assay methods include radioimmunoassays, competitive-binding assays Western Blot analysis and ELISA assavs Among these ELISAs frequently are prefe ⁇ ed
  • An ELISA assay initially comprises prepa ⁇ ng an antibody specific to FAB I, preferably a monoclonal antibody
  • a reporter antibody generally is prepared which binds to the monoclonal antibody The reporter antibody is attached
  • a competition assay may be employed wherein antibodies specific to FAB I attached to a solid support and labeled FAB I and a sample derived from form an individual are passed over the solid support and the amount of label detected attached to the solid support can be co ⁇ elated to a quantity of FAB I in the sample Antibodies
  • the polypeptides, their fragments or other derivatives, or analogs thereof, or cells expressing them can be used as an immunogen to produce antibodies thereto
  • These antibodies can be, for example, polyclonal or monoclonal antibodies
  • the present invention also includes chimeric, single chain, and humanized antibodies, as well as Fab fragments, or the product of an Fab expression library Vanous procedures known in the art may be used for the production of such antibodies and fragments
  • Antibodies generated against the polypeptides co ⁇ esponding to a sequence of the present invention can be obtained by direct injection of the polypeptides into an individual or by administe ⁇ ng the polypeptides to an individual, preferably a nonhuman The antibody so obtained will then bind the polypeptides itself In this manner, even a sequence encoding only a fragment of the polypeptides can be used to generate antibodies binding the whole native polypeptides Such antibodies can then be used to isolate the polypeptide from tissue expressing that polypeptide
  • any technique which provides antibodies produced by continuous cell line cultures can be used Examples include the hybndoma technique (Kohler, G et al , (19 '5) Nature 256 495-497, the trioma technique, the human B- cell hybndoma technique (Kozbor et al , ( 1983) Immunology Today 4 72 and the EBV- hyb ⁇ doma technique to produce human monoclonal antibodies (Cole et al, (1985) in Monoclonal Antibodies And Cancer Therapy , Alan R Liss, Inc pp 77-96
  • Polypeptide derivatives include antigemcally or immunologically equivalent derivatives which form a particular aspect of this invention
  • anigemcally equivalent derivative encompasses a polypeptide or its equivalent which will be specifically recognised by certain antibodies which, when raised to the protein or polypeptide according to the present invention, interfere with the immediate physical interaction between pathogen and mammalian host
  • immunologically equivalent derivative encompasses a peptide or its equivalent which when used in a suitable formulation to raise antibodies in a vertebrate, the antibodies act to interfere with the immediate physical interaction between pathogen and mammalian host
  • the polypeptide such as an antigemcally or immunologically equivalent derivative or a fusion protein thereof is used as an antigen to immunize a mouse or other animal such as a rat or chicken
  • the fusion protein may provide stability to the polypeptide
  • the antigen may be associated, for example by conjugation , with an immunogenic earner protein for example bovine serum albumin (BSA) or keyhole limpet haemocyanin (KLH)
  • BSA bovine serum albumin
  • KLH keyhole limpet haemocyanin
  • a multiple antigemc peptide comprising multiple copies of the protein or polypeptide, or an antigemcally or immunologically equivalent polypeptide thereof may be sufficiently antigemc to improve immunogenicity so as to obviate the use of a earner
  • antibody- containing cells from the immunised mammal are fused with myeloma cells to create hybndoma cells secreting monoclonal antibodies
  • the hyb ⁇ domas are screened to select a cell line with high binding affinity and favorable cross reaction with other staphylococcal species using one or more of the original polypeptide and/or the fusion protein
  • the selected cell line is cultured to obtain the desired Mab
  • Hybndoma cell lines secreting the monoclonal antibody are another aspect of this invention.
  • phage display technology could be utilised to select antibody genes with binding activities towards the polypeptide either from repertoires of PCR amplified v- genes of lymphocytes from humans screened for possessing anti-Fbp or from naive libraries (McCafferty, J et al , (1990), Nature 348, 552-554, Marks, J et al , ( 1992) Biotechnology 10, 779-783)
  • the affinity of these antibodies can also be improved by chain shuffling (Clackson, T et al , ( 1991) Nature 352, 624-628)
  • the antibody should be screened again for high affinity to the polypeptide and/or fusion protein
  • the above-described antibodies may be employed to isolate or to identify clones expressing the polypeptide or purify the polypeptide of the present invention by attachment of the antibody to a solid support for isolation and/or purification by affinity chromatography
  • the polypeptide may be used as an antigen for vaccination of a host to produce specific antibodies which protect against invasion of bacteria, for example by blocking adherence of bacteria to damaged tissue Examples of tissue damage include wounds in skin or connective tissue caused, e g , by mechanical, traumatic, chemical or thermal damage or by implantation of indwelling devices, or wounds in the mucous membranes, such as the mouth, mammary glands, urethra or vagina
  • the polypeptides or cells expressing them can be used as an immunogen to produce antibodies thereto
  • These antibodies can be, for example, polyclonal or monoclonal antibodies
  • the term antibodies also includes chimeric, single chain, and humanized antibodies, as well as Fab fragments, or the product of an Fab expression library Various procedures known in the art may be used for the production of such antibodies and fragments
  • Antibodies generated against the polypeptides of the present invention can be obtained by direct injection of the polypeptides into an individual or by administering the polypeptides to an individual, preferably a nonhuman The antibody so obtained will then bind the polypeptides itself In this manner, even a sequence encoding only a fragment of the polypeptides can be used to generate antibodies binding the whole native polypeptides Such antibodies can then be used to isolate the polypeptide from tissue expressing that polypeptide
  • a fragment of the final antibody may be prepared
  • the antibody may be either intact antibody of M r approx 150,000 or a derivative of it, for example a Fab fragment or a Fv fragment as described in Ske ⁇ a, A and Pluckthun, A ( 1988) Science 240 1038- 1040 If two antigen binding domains are present each domain may be directed against a different epitope - termed T-uspecific' antibodies
  • the antibody of the invention may be prepared by conventional means for example by established monoclonal antibody technology (Kohler, G et al (1975), Nature 256, 495- 497) or using recombinant means e g combinatorial libraries, for example as described in Huse, W D et al , ( 1989) Science 246, 1275- 1281
  • the antibody is prepared by expression of a DNA polymer encoding said antibody in an appropriate expression system such as described above for the expression of polypeptides of the invention
  • an appropriate expression system such as described above for the expression of polypeptides of the invention
  • the choice of vector for the expression system will be determined in part by the host, which may be a prokaryotic cell, such as E colt (preferably strain B) or Streptomyces sp or a eukaryotic cell, such as a mouse C127, mouse myeloma, human HeLa, Chinese hamster ovary, filamentous or unicellular fungi or insect cell
  • the host may also be a transgenic animal or a transgenic plant, for example as described in Hiatt, A et al, ( 1989) Nature 34, 76-78
  • Suitable vectors include plasmids, bacte ⁇ ophages, cosmids and recombinant viruses, derived from, for example, baculoviruses and vaccinia
  • the modification need not be restricted to one of Tiumanisation' other primate sequences (for example Newman, R et al , 1992, Biotechnology 10, 1455- 1460) may also be used.
  • the humanised monoclonal antibody, or its fragment having binding activity, form a particular aspect of this invention
  • This invention provides a method of screening drugs to identify those which interfere with the interaction of the FAB I protein or active fragment to mammalian cells, the method comprising incubating a mammalian cell or membrane preparation with labeled polypeptide in the presence of the drug and measuring the ability of the drug to block this interaction
  • a polynucleotide of the invention in genetic immunization will preferably employ a suitable delivery method such as direct injection of plasmid DNA into muscles (Wolff et al , ( 1992) Hum Mol Genet, 1 363, Manthorpe et al , (1963) Hum Gene Ther 4, 419), delivery of DNA complexed with specific protein carriers ( Wu, et al, (1989) J Biol Chem 264, 16985), coprecipitation of DNA with calcium phosphate (Benvenisty & Reshef, PNAS, 1986 83,9551), encapsulation of DNA in various forms of hposomes (Kaneda et al , (1989) Science 243, 375), particle bombardment (Tang et al , (1992) Nature 356 152, Eisenbraun et al , (1993) DNA Cell Biol 72 791 ) and in vivo infection using cloned retroviral vectors (Seeger et al
  • FAB I antibodies against FAB I may be employed to inhibit FAS or FAB I enzymatic activity or FAB I expression
  • FAB I may also be employed to inhibit infections including, but not limited to infections of upper respiratory tract (e g otitis media, bacte ⁇ al tracheitis, acute epiglottitis, thyroiditis), lower respiratory (e g empyema, lung abscess),card ⁇ ac (e g infective endocarditis), gastrointestinal (e g secretory dia ⁇ hoea, splenic abscess, retrope ⁇ toneal abscess), CNS (e g cerebral abscess), eye (e g blephantis, conjunctivitis, keratitis, endophthalmitis, preseptal and orbital cellulitis, darcryocystitis), kidney and u ⁇ nary tract (e g epididymitis, intrarenal and pennephnc abscess, toxic shock syndrome
  • FAB I may also be employed to treat bacterial infection, such as, for example, infections of upper respiratory tract (e g otitis media, bacterial tracheitis, acute epiglottitis, thyroiditis), lower respiratory (e g empyema, lung abscess),card ⁇ ac (e g infective endocarditis), gastrointestinal (e g secretory dia ⁇ hoea, splenic abscess, retrope ⁇ toneal abscess), CNS (e g cerebral abscess), eye (e g blephantis, conjunctivitis, keratitis, endophthalmitis, preseptal and orbital cellulitis, darcryocystitis), kidney and urinary tract (e g epididymitis, intrarenal and pennephnc abscess, toxic shock syndrome), skin (e g impetigo, follicuhtis, cutaneous abscesses, cellulitis, wound infection, bacterial
  • This invention also provides a method for identification of molecules, such as binding molecules, that bind FAB I
  • Genes encoding proteins that bind FAB I, such as binding proteins, can be identified by numerous methods known to those of skill in the art Examples of such methods are descnbed in many laboratory manuals such as, for instance,
  • FAB I polyadenylated RNA
  • a cDNA library is created from this RNA, the library is divided into pools and the pools are transfected individually into cells that are not responsive to FAB I
  • the transfected cells then are exposed to labeled FAB I (FAB I can be labeled by a vanety of well-known techniques including standard methods of radio-iodination or inclusion of a recognition site for a site-specific protein kinase ) Following exposure, the cells are fixed and binding of FAB I is determined
  • a labeled ligand can be photoaffinity linked to a cell extract, such as a membrane or a membrane extract, prepared from cells that express a molecule that it binds, such as a binding molecule
  • Cross-linked mate ⁇ al is resolved by polyacrylamide gel electrophoresis ("PAGE") and exposed to X-ray film
  • PAGE polyacrylamide gel electrophoresis
  • the labeled complex containing the hgand-binding can be excised, resolved into peptide fragments, and subjected to protein microsequencing
  • the amino acid sequence obtained from microsequencing can be used to design unique or degenerate oligonucleotide probes to screen cDNA branes to identify genes encoding the putative binding molecule
  • Polypeptides of the invention also can be used to assess FAB I binding capacity of FAB I binding molecules, such as binding molecules, in cells or in cell-free preparations
  • Polypeptides and polynucleotides of the invention may also be used to assess the binding of small molecule substrates and ligands in, for example, cells, cell-free preparations, chemical libraries, and natural product mixtures
  • substrates and ligands may be natural substrates and ligands or may be stmctural or functional mimetics See, e g , Co gan et al ,
  • Polypeptides and polynucleotides of the present invention are responsible for many biological functions, including many disease states, in particular the Diseases hereinbefore mentioned It is therefore desirable to devise screening methods to identify compounds which stimulate or which inhibit the function of the polypeptide or polynucleotide Accordingly, in a further aspect, the present invention provides for a method of screening compounds to identify those which stimulate or which inhibit the function of a polypeptide or polynucleotide of the invention, as well as related polypeptides and polynucleotides
  • agonists or antagonists may be employed for therapeutic and prophylactic purposes for such Diseases as hereinbefore mentioned
  • Compounds may be identified from a vanety of sources, for example, cells, cell-free preparations, chemical hbranes, and natural product mixtures Such agonists, antagonists or inhibitors so-identified may be natural or modified substrates, ligands, receptors, enzymes, etc , as the case may be, of FAB I polypeptides and polynucle
  • the screening methods may simply measure the binding of a candidate compound to the polypeptide or polynucleotide, or to cells or membranes bearing the polypeptide or polynucleotide, or a fusion protein of the polypeptide by means of a label directly or indirectly associated with the candidate compound Alternatively, the screening method may involve competition with a labeled competitor Further, these screening methods may test whether the candidate compound results in a signal generated by activation or inhibition of the polypeptide or polynucleotide, using detection systems appropriate to the cells comprising the polypeptide or polynucleotide Inhibitors of activation are generally assayed in the presence of a known agonist and the effect on activation by the agonist by the presence of the candidate compound is observed Constitutively active polypeptide and/or constitutively expressed polypeptides and polynucleotides may be employed in screening methods for inverse agonists or inhibitors, in the absence of an agonist or inhibitor, by testing whether the candidate compound results in inhibition of activation of the polypeptide
  • polypeptides and antibodies that bind to and/or interact with a polypeptide of the present invention may also be used to configure screening methods for detecting the effect of added compounds on the production of mRNA and/or polypeptide in cells
  • an ELISA assay may be constmcted for measuring secreted or cell associated levels of polypeptide using monoclonal and polyclonal antibodies by standard methods known in the art This can be used to discover agents which may inhibit or enhance the production of polypeptide (also called antagonist or agonist, respectively) from suitably manipulated cells or tissues
  • the invention also provides a method of screening compounds to identify those which enhance (agonist) or block (antagonist) the action of FAB I polypeptides or polynucleotides, particularly those compounds that are bacte ⁇ static and/or bacte ⁇ cidal
  • the method of screening may involve high-throughput techniques
  • a synthetic reaction mix for agonists or antagonists, a synthetic reaction mix, a cellular compartment, such as a membrane, cell envelope or cell wall, or a preparation of any thereof, comp ⁇ sing FAB I polypeptide and a labeled substrate or ligand of such polypeptide is incubated in the absence or the presence of a candidate molecule that may be a FAB I agonist or antagonist
  • the ability of the candidate molecule to agonize or antagonize the FAB I polypeptide is reflected in decreased binding of the labeled ligand or decreased production of product from such substrate Molecules that bind gratuitously, i e .
  • Molecules that bind well and. as the case may be, increase the rate of product production from substrate, increase signal transduction, or increase chemical channel activity are agonists Detection of the rate or level of, as the case may be, production of product from substrate, signal transduction, or chemical channel activity may be enhanced by using a reporter system Reporter systems that may be useful in this regard include but are not limited to colo ⁇ met ⁇ c, labeled substrate converted into product, a reporter gene that is responsive to changes in FAB I polynucleotide or polypeptide activity, and binding assays known in the art
  • Polypeptides of the invention may be used to identify membrane bound or soluble receptors, if any, for such polypeptide, through standard receptor binding techniques known in the art These techniques include, but are not limited to, ligand binding and crosshnking assays in which the polypeptide is labeled with a radioactive isotope (for instance, ' ->I), chemically modified (for instance, biotmylated), or fused to a peptide sequence suitable for detection or purification, and incubated with a source of the putative receptor (e g , cells, cell membranes, cell supernatants, tissue extracts, bodily mate ⁇ als) Other methods include biophysical techniques such as surface plasmon resonance and spectroscopy These screening methods may also be used to identify agonists and antagonists of the polypeptide which compete with the binding of the polypeptide to its receptor(s), if any Standard methods for conducting such assays are well understood in the art
  • the fluorescence polarization value for a fluorescently-tagged molecule depends on the rotational conelation time or tumbling rate Protein complexes, such as formed by FAB I polypeptide associating with another FAB I polypeptide or other polypeptide, labeled to comprise a fluorescently-labeled molecule will have higher polarization values than a fluorescently labeled monome ⁇ c protein It is prefe ⁇ ed that this method be used to characterize small molecules that disrupt polypeptide complexes Fluorescence energy transfer may also be used characterize small molecules that interfere with the formation of FAB I polypeptide dimers, t ⁇ mers, tetramers or higher order stmctures, or stmctures formed by FAB I polypeptide bound to another polypeptide FAB I polypeptide can be labeled with both a donor and acceptor fluorophore Upon mixing of the two labeled species and excitation of the donor fluorophore. fluorescence energy transfer can be detected by observing fluorescence of the
  • FAB I polypeptide self-association as well as an association of FAB I polypeptide and another polypeptide or small molecule FAB I polypeptide can be coupled to a sensor chip at low site density such that covalently bound molecules will be monome ⁇ c Solution protein can then passed over the FAB I polypeptide -coated surface and specific binding can be detected in real-time by monitoring the change in resonance angle caused by a change in local refractive index
  • This technique can be used to characterize the effect of small molecules on kinetic rates and equilibrium binding constants for FAB I polypeptide self-association as well as an association of FAB I polypeptide and another polypeptide or small molecule
  • a scintillation proximity assay may be used to characterize the interaction between an association of FAB I polypeptide with another FAB I polypeptide or a different polypeptide FAB I polypeptide can be coupled to a scintillation-filled bead Addition of radio-labeled FAB I polypeptide results in binding where the radioactive source molecule is in
  • ICS biosensors have been described by AMBRI (Australian Membrane Biotechnology Research Institute) They couple the self-association of macromolecules to the closing of gramacidin-faci tated ion channels in suspended membrane bilayers and hence to a measurable change in the admittance (similar to impedence) of the biosensor
  • methods for identifying compounds which bind to or otherwise interact with and inhibit or activate an activity or expression of a polypeptide and/or polynucleotide of the invention compnsing contacting a polypeptide and/or polynucleotide of the invention with a compound to be screened under conditions to permit binding to or other interaction between the compound and the polypeptide and/or polynucleotide to assess the binding to or other interaction with the compound, such binding or interaction preferably being associated with a second component capable of providing a detectable signal in response to the binding or interaction of the polypeptide and/or polynucleotide with the compound, and determining whether the compound binds to or otherwise interacts with and activates or inhibits an activity or expression of the polypeptide and/or polynucleotide by detecting the presence or absence of a signal generated from the binding or interaction of the compound with the polypeptide and or polynucleotide
  • an assay for FAB I agonists is a competitive assay that combines FAB I and a potential agonist with FAB 1-b ⁇ nd ⁇ ng molecules, recombinant FAB I binding molecules, natural substrates or ligands, or substrate or ligand mimetics, under approp ⁇ ate conditions for a competitive inhibition assay
  • FAB I can be labeled, such as by radioactivity or a colo ⁇ met ⁇ c compound, such that the number of FAB I molecules bound to a binding molecule or converted to product can be determined accurately to assess the effectiveness of the potential antagonist
  • a further example of a screen is a detect the reduction of crotonyl-CoA by measuring the consumption of NADH Crotonyl-ACP may also be used in place of crotonyl-CoA in such a screen Test compound are added to the reaction mix to determine the effect on the reduction of crotonyl-CoA or crotonyl-ACP Agonists can be identified if the level of reduction is increased and
  • Apo-ACP is uncompetitive versus NADH (K ⁇ (app) and is proportional to the square of [CCA])
  • Apo-ACP is competitive versus crotonoyl CoA and induces negative cooperativity with respect to CCA binding
  • Palmitoyl CoA appears to be a competitive inhibitor with respect to crotonoyl CoA Slope replots are proportional to [I] , suggesting that inhibition is effected through the binding of multiple palmitoyl CoA molecules to Fab I
  • Apo-ACP is uncompetitive versus NADH and competitive versus crotonoyl CoA while inducing negative cooperativity in the binding of CCA This indicates an oligomeric quaternary stmcture, particularly, but not limited to, a dimer or tetramer 1 1
  • Studies using apo-ACP and the pseudo-product inhibitors beta-NADP + and palmitoyl CoA indicate an ordered addition of substrates (NADH binding first) 12.
  • the uncompetitive inhibition of apo-ACP versus NADH suggests that prior binding of the pyridine nucelotide is required for ACP binding
  • the S. aureus enzyme had similar affinity to the E.coh enzyme for the crotonoyl CoA substrate, yet the E.coh enzyme possessed up to 70 fold greater affinity for
  • Potential antagonists of the invention include, among others, small organic molecules, peptides, polypeptides and antibodies that bind to a polynucleotide and/or polypeptide of the invention and thereby inhibit or extinguish its activity or expression
  • Potential antagonists also may be small organic molecules, a peptide, a polypeptide such as a closely related protein or antibody that binds the same sites on a binding molecule, such as a binding molecule, without inducing FAB I-induced activities, thereby preventing the action or expression of FAB I polypeptides and/or polynucleotides by excluding FAB I polypeptides and/or polynucleotides from binding
  • Potential antagonists include a small molecule that binds to and occupies the binding site of the polypeptide thereby preventing binding to cellular binding molecules, such that normal biological activity is prevented Examples of small molecules include but are not limited to small organic molecules, peptides or peptide- like molecules Other potential antagonists include antisense molecules (
  • polypeptide antagonists include antibodies or, in some cases, oligonucleotides or proteins which are closely related to the hgands, substrates, receptors, enzymes, etc , as the case may be, of the polypeptide, e g , a fragment of the ligands, substrates, receptors, enzymes, etc , or small molecules which bind to the polypeptide of the present invention but do not elicit a response, so that the activity of the polypeptide is prevented
  • polypeptides of the invention are biomimetics, functional mimetics of the natural FAB I polypeptide These functional mimetics may be used for, among other things, antagonizing the activity of FAB I polypeptide or as a antigen or immunogen in a manner described elsewhere herein
  • Functional mimetics of the polypeptides of the invention include but are not limited to tmncated polypeptides
  • prefe ⁇ ed functional mimetics include, a polypeptide comprising the polypeptide sequence set forth in SEQ ID NO 2 lacking 20, 30, 40, 50, 60, 70 or 80 amino- or carboxy-terminal amino acid residues, including fusion proteins compnsing one or more of these tmncated sequences
  • Polynucleotides encoding each of these functional mimetics may be used as expression cassettes to express each mimetic polypeptide It is prefe ⁇ ed that these cassettes comp ⁇ se 5' and 3 restriction sites to allow for a convenient means to hgate the cassettes together when
  • the present invention relates to a screening kit for identifying agonists, antagonists, ligands, receptors, substrates, enzymes, etc for a polypeptide and/or polynucleotide of the present in ention or compounds which decrease or enhance the production of such polypeptides and/or polynucleotides , which comprises (a) a polypeptide and/or a polynucleotide of the present invention, (b) a recombinant cell expressing a polypeptide and/or polynucleotide of the present invention, (c) a cell membrane expressing a polypeptide and/or polynucleotide of the present invention, or (d) antibody to a polypeptide and/or polynucleotide of the present invention, which polypeptide is preferably that of SEQ ID NO 2, and which polynucleotide is preferably that of SEQ ID NO 1
  • a polypeptide and/or polynucleotide of the present invention may also be used in a method for the structure-based design of an agonist, antagonist or inhibitor of the polypeptide and/or polynucleotide, by (a) determining in the first instance the three-dimensional stmcture of the polypeptide and/or polynucleotide, or complexes thereof, (b) deducing the three-dimensional stmcture for the likely reactive s ⁇ te(s), binding s ⁇ te(s) or mot ⁇ f(s) of an agonist antagonist or inhibitor, (c) synthesizing candidate compounds that are predicted to bind to or react with the deduced binding s ⁇ te(s), reactive s ⁇ te(s), and/or mot ⁇ f(s), and
  • the present invention provides methods of treating abnormal conditions such as, for instance, a Disease, related to either an excess of, an under-expression of, an elevated activity of, or a decreased activity of FAB I polypeptide and/or polynucleotide
  • the present invention relates to genetically engineered soluble fusion proteins comprising a polypeptide of the present invention, or a fragment thereof, and various portions of the constant regions of heavy or light chains of immunoglobulins of various subclasses (IgG, IgM, IgA, IgE) Preferred as an immunoglobulin is the constant part of the heavy chain of human IgG, particularly IgG l , where fusion takes place at the hinge region
  • the Fc part can be removed simply by incorporation of a cleavage sequence which can be cleaved with blood clotting factor Xa
  • this invention relates to processes for the preparation of these fusion proteins by genetic engineering, and to the use thereof for dmg screening, diagnosis and therapy
  • a further aspect of the invention also relates to polynucleotides encoding such fusion proteins Examples of fusion protein technology can be found in International Patent Application Nos W094/29458 and W094/22914 In still another approach, expression of
  • each of the polynucleotide sequences provided herein may be used in the discovery and development of antibacterial compounds
  • the encoded protein upon expression, can be used as a target for the screening of antibacterial dmgs
  • the polynucleotide sequences encoding the amino terminal regions of the encoded protein or Shine-Delgarno or other translation facilitating sequences of the respective mRNA can be used to constmct antisense sequences to control the expression of the coding sequence of interest
  • the invention also provides the use of the polypeptide, polynucleotide, agonist or antagonist of the invention to interfere with the initial physical interaction between a pathogen or pathogens and a eukaryotic, preferably mammalian, host responsible for sequelae of infection
  • the molecules of the invention may be used in the prevention of adhesion of bacteria, in particular gram positive and/or gram negative bacteria, to eukaryotic, preferably mammalian, extracellular matrix proteins on in-dwelling devices or to
  • FAB I agonists and antagonists preferably bacte ⁇ static or bactericidal agonists and antagonists
  • the antagonists and agonists of the invention may be employed, for instance, to prevent, inhibit and/or treat diseases
  • the antagonists may be employed in a composition with a pharmaceutically acceptable earner, e g , as hereinafter desc ⁇ bed
  • the antagonists may be employed for instance to inhibit diseases including, but not limited to infections of upper respiratory tract (e g otitis media, bacterial tracheitis, acute epiglottitis, thyroiditis), lower respiratory (e g empyema, lung abscess),card ⁇ ac (e g infective endocarditis), gastrointestinal (e g secretory dia ⁇ hoea, splenic abscess, retrope ⁇ toneal abscess), CNS (e g cerebral abscess), eye (e g blepha ⁇ tis, conjunctivitis, keratitis, endophthalmitis, preseptal and orbital cellulitis, darcryocystitis), kidney and u ⁇ nary tract (e g epididymitis, intrarenal and pennephnc abscess, toxic shock syndrome), skin (e g impetigo, follicuhtis , cutaneous abscesses, cellulitis, wound infection
  • the antagonists may be employed in a composition with a pharmaceutically acceptable ca ⁇ ier, e g , as hereinafter described
  • each of the DNA sequences provided herein may be used in the discovery and development of antibacterial compounds
  • the encoded protein upon expression can be used as a target for the screening of antibacterial dmgs
  • the DNA sequences encoding the amino terminal regions of the encoded protein or Shine-Delgarno or other translation facilitating sequences of the respective mRNA can be used to constmct antisense sequences to control the expression of the coding sequence of interest
  • an antibacterial targeted at FAB I agonist and antagonist compounds provided by the invention are believed to be active against a wide variety of Gram negative and positive organisms
  • a Fab I homolog (InhA) has been identified in Mycobacterium tuberculosis Therefore, an antibacterial provided by the invention, targeted at FAB I, may also have anti -mycobacte ⁇ al activity Further, since Diazabonne de ⁇ vatives inhibit LPS biosynthesis
  • the invention also relates to compositions comp ⁇ sing the polynucleotide or the polypeptides discussed above or the agonists or antagonists
  • the polypeptides of the present invention may be employed combination with a non-ste ⁇ le or ste ⁇ le earner or earners for use with cells, tissues or organisms, such as a pharmaceutical earner suitable for administration to a subject
  • Such compositions comprise, for instance, a media additive or a therapeutically effective amount of a polypeptide of the invention and a pharmaceutically acceptable earner or excipient
  • Such earners may include, but are not limited to. saline, buffered saline, dextrose, water, glycerol, ethanol and combinations thereof
  • the formulation should suit the mode of administration Kits
  • the invention further relates to pharmaceutical packs and kits comp ⁇ sing one or more containers filled with one or more of the ingredients of the aforementioned compositions of the invention Associated with such contamer(s) can be a notice in the form prescnbed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, reflecting approval by the agency of the manufacture, use or sale of the product for human administration
  • kits for the FAB I gene and homologs will enable the directed therapy of a FAB I directed antibactenal DNA hyb ⁇ disation or protein (monoclonal antibody) based kits for the identification of Fab I and homologs of FAB
  • Polypeptides and other compounds of the present invention may be employed alone or in conjunction with other compounds, such as therapeutic compounds
  • compositions may be administered in any effective, convenient manner including, for instance, administration by topical, oral, anal, vaginal, intravenous, lntrape ⁇ toneal, intramuscular, subcutaneous, mtranasal or intradermal routes among others
  • compositions generally are administered in an amount effective for treatment or prophylaxis of a specific indication or indications
  • the compositions are administered in an amount of at least about 10 ⁇ g/kg body weight In most cases they will be administered in an amount not in excess of about 8 mg/kg body weight per day
  • dose is from about 10 ⁇ g/kg to about 1 mg/kg body weight, daily It will be appreciated that optimum dosage will be determined by standard methods for each treatment modality and indication, taking into account the indication, its seventy, route of administration, complicating conditions and the like
  • the active agent may be administered to a individual as an injectable composition, for example as a sterile aqueous dispersion, preferably lsotomc
  • the composition may be formulated for topical application for example in the form of ointments, creams, lotions, eye ointments, eye drops, ear drops, mouthwash, impregnated dressings and sutures and aerosols, and may contain appropriate conventional additives, including, for example, preservatives, solvents to assist dmg penetration, and emollients in ointments and creams
  • Such topical formulations may also contain compatible conventional earners, for example cream or ointment bases, and ethanol or oleyl alcohol for lotions
  • Such earners may constitute from about 1 % to about 98% by weight of the formulation, more usually they will constitute up to about 80% by weight of the formulation
  • the daily dosage level of the active agent will be from 0 01 to 10 mg/kg, typically around 1 mg/kg
  • the physician in any event will determine the actual dosage which will be most suitable for an individual and will vary with the age weight and response of the particular individual
  • the above dosages are exemplary of the average case There can, of course, be individual instances where higher or lower dosage ranges are merited, and such are within the scope of this invention
  • In-dwelling devices include surgical implants, prosthetic devices and catheters, l e , devices that are introduced to the body of an individual and remain in position for an extended time
  • Such devices include, for example, artificial joints, heart valves, pacemakers, vascular grafts, vascular catheters, cerebrospinal fluid shunts, urinary catheters, continuous ambulatory peritoneal dialysis (CAPD) catheters, etc
  • composition of the invention may be administered by injection to achieve a systemic effect against relevant bacteria shortly before insertion of an in-dwelling device Treatment may be continued after surgery during the in-body time of the device
  • composition could also be used to broaden pe ⁇ operative cover for any surgical technique to prevent staphylococcal wound infections
  • compositions of this invention may be used generally as a wound treatment agent to prevent adhesion of bacteria to matrix proteins exposed in wound tissue and for prophylactic use in dental treatment as an alternative to, or in conjunction with, antibiotic prophylaxis
  • the composition of the invention may be used to bathe an indwelling device immediately before insertion
  • the active agent will preferably be present at a concentration of 1 ⁇ g/ml to lOmg/ml for bathing of wounds or indwelling devices
  • a vaccine composition is conveniently in injectable form Conventional adjuvants may be employed to enhance the immune response
  • a suitable unit dose for vaccination is 0 5-5ug/kg of antigen, and such dose is preferably administered 1 -3 times and with an interval of 1 -3 weeks
  • aureus FAB I gene The polynucleotide having the DNA sequence given in SEQ ID NO 1 was obtained from the sequencing of a library of clones of chromosomal DNA of 5 aureus WCUH 29 ⁇ n E coli It has been demonstrated by the process herein described that it is transcribed m vivo in an established infection of 5 aureus WCUH 29 ⁇ n a mouse model of infection
  • a library of clones of chromosomal DNA of S aureus WCUH 29m E colt or some other suitable host is probed with a radiolabelled oligonucleotide, preferably a 17mer or longer, de ⁇ ved from the partial sequence
  • Clones ca ⁇ ying DNA identical to that of the probe can then be distinguished using high st ⁇ ngency washes
  • sequencing the individual clones thus identified with sequencing p ⁇ mers designed from the original sequence it is then possible to extend the sequence in both directions to determine the full gene sequence Conveniently such sequencing is performed using denatured double stranded DNA prepared from a plasmid clone Suitable techniques are desc ⁇ bed by Mamatis, T , F ⁇ tsch, E F and Sambrook et al , (1989) MOLECULAR CLONING, A LABORATORY MANUAL, 2nd Ed , Cold Spring Harbor Laboratory Press, Cold Sp ⁇
  • sequencing data from two or more clones containing overlapping S aureusWCMH 29 DNA was used to constmct the contiguous DNA sequence in SEQ ID NO 1 Libraries may be prepared Libraries may be prepared by routine methods, for example Methods 1 and 2
  • Total cellular DNA is isolated from Staphylococcus aureus strain WCUH 29(NCIMB 40771 ) according to standard procedures and size-fractionated by either of two methods
  • Method 1 Total cellular DNA is mechanically sheared by passage through a needle in order to size-fractionate according to standard procedures
  • DNA fragments of up to 1 lkbp in size are rendered blunt by treatment with exonuclease and DNA polymerase, and EcoRI linkers added Fragments are ligated into the vector Lambda ZapII that has been cut with EcoRI, the library packaged by standard procedures and E colt infected with the packaged library
  • the library is amplified by standard procedures
  • Total cellular DNA is partially hydrolsed with a combination of four restriction enzymes (Rsal, Pall, Alul and Bsh 12351) and size-fractionated according to standard procedures EcoRI linkers are ligated to the DNA and the fragments then ligated into the vector Lambda ZapII that have been cut with EcoRI, the library packaged by standard procedures, and E colt infected with the packaged library
  • EcoRI linkers are ligated to the DNA and the fragments then ligated into the vector Lambda ZapII that have been cut with EcoRI, the library packaged by standard procedures, and E colt infected with the packaged library
  • the library is amplified by standard procedures
  • the activity of FAB I protein may be measured using either crotonoyl-CoA or crotonoyl -ACP as a substrate (Bergler, et al, (1994), J Biol Chem 269, 5493-5496) and monitoring the decrease in absorbance at 340nm due to the consumption of NADH Crotonoyl-ACP (Km 22uM) is a better substrate than crotonoyl-CoA (Km 2 7mM), crotonoyl-CoA is available from Sigma (C6146) A diazabonne derivative may be used as a positive control, this should be readily available via a 2 step synthesis with publicly available starting materials using methods known in the art Test compounds may be added to this assay to determine whether they agonize or antagonize enzymatic activity Example 3 Kinetic Characterization of Fab I Crotonoyl CoA was used as the substrate for Fab I and all activity measurements were based on a decrease in absorbance at 340 nm due to
  • Native S aureus Fab I exhibited a flat pH optimum between 5 and 7 over which the Vm and the Vm/Km's for the substrates crotonoyl CoA (CCA) and NADH were relatively constant

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Abstract

Prokayrotic FAB I polypeptides and DNA (RNA) encoding such FAB I and a procedure for producing such polypeptides by recombinant techniques is disclosed. Also disclosed are methods for utilizing such FAB I for the treatment of infection, such as bacterial infections. Antagonists against such FAB I and their use as a therapeutic to treat /infections, such as staphylococcal infections are also disclosed. Also disclosed are diagnostic assays for detecting diseases related to the presence of FAB I nucleic acid sequences and the polypeptides in a host. Also disclosed are diagnostic assays for detecting polynucleotides encoding FAB I and for detecting the polypeptide in a host.

Description

METHODS USING OF FAB I AND COMPOUNDS MODULATING FAB I ACTIVITY
This invention relates, in part, to newly identified polynucleotides and polypeptides, vanants and deπvatives of these polynucleotides and polypeptides, processes for making these polynucleotides and these polypeptides, and their vanants and deπvatives, agonists and antagonists of the polypeptides, and uses of these polynucleotides, polypeptides, vanants, denvatives, agonists and antagonists In particular, in these and in other regards, the invention relates to polynucleotides and polypeptides of staphylococcal Fab I enoyl-ACP reductase, hereinafter referred to as "FAB I" BACKGROUND OF THE INVENTION
Although the overall pathway of saturated fatty acid biosynthesis is similar in all organisms, the fatty acid synthase (FAS) systems vary considerably with respect to their structural organization Thus in Type I FAS systems, found in vertebrates and yeasts, the necessary enzymes required for fatty acid synthesis are present on one or two polypeptide chains respectively In contrast, in Type II systems found in most bacteria and plants, each step the pathway is catalysed by a separate mono-functional enzyme It would therefore appear that significant selectivity of inhibition of the bacterial and mammalian enzymes is possible
Fab I (previously designated EnvM) functions as an enoyl-acyl earner protein (ACP) reductase (Bergler, et al, ( 1994), J Biol Chem 269, 5493-5496) in the final step of the four reactions involved in each cycle of bacterial fatty acid biosynthesis
The first step is catalysed by β-ketoacyl-ACP synthase, which condenses malonyl- ACP with acetyl-CoA (FabH, synthase III) In subsequent rounds malonyl-ACP is condensed with the growing-chain acyl-ACP (FabB and FabF, synthases I and II respectively)
The second step in the elongation cycle is ketoester reduction by NADPH- dependent β-ketoacyl-ACP reductase (FabG) Subsequent dehydration by β-hydroxyacyl- ACP dehydrase (either FabA or FabZ) leads to trans-2-enoyl-ACP which is in turn converted to acyl-ACP by NADH-dependent enoyl-ACP reductase (Fab I) Further rounds of this cycle, adding two carbon atoms per cycle, eventually lead to palmitoyl-ACP (16C) where upon the cycle is stopped largely due to feedback inhibition of Fab I by palmitoyl- ACP (Heath, et al. ( 1996), J Biol Chem. 271. 1833- 1836) Fab I is therefore a major biosynthetic enzyme which is also a key regulatory point in the overall synthetic pathway
Early data suggested that there were two enoyl-ACP reductases in E coli, one
NADPH dependent and the other NADH dependent However, more recent work has found no evidence for the NADPH dependent enzyme and Fab I is the only enoyl ACP reductase identified in E colt (Heath, et al, ( 1995), J Biol Chem 270. 26538 - 26542, Bergler, et al,
( 1994), J Biol Chem 269, 5493-5496)
It has been shown that diazabonne antibiotics inhibit fatty acid, phosphohpid and hpopolysacchaπde (LPS) biosynthesis and it has also been shown that the antibacterial target of these compounds is Fab I For example derivative 2b l 8 from Grassberger, et al ( 1984) J Med Chem 27 947-953 has been shown to be a non-competitive inhibitor of Fab I having a Ki = 0 2mM (Bergler, et al, ( 1994), J Biol Chem 269, 5493-5496) The antibacterial activity of diazabonne derivatives against Gram-negatives and Gram positive organisms is well documented (Grassberger et al , J Med Chem 1984 27, 947-953, Gronowitz et al , Acta Pharm Suecica, 1971 8 377, Wersch et al U S Patent, 2,533,918, Lam er a/ , J Antimirob Chemother 1987 20 37-45)
Conditionally lethal Fab I mutants have been constructed in E colt and the Fab I gene from Salmonella typhimunum complements this mutation In addition, plasmids containing the Eab 1 gene from diazabonne resistant 5 t phtmurium conferred diazabonne resistance in E colt (Tumowsky, et al, ( 1989), J Bactenol , 171, 6555-6565) confirming
Fab I as the antibacterial target of diazaboπnes
Inhibition of Fab I either by diazabonne or by raising the temperature in an Fab I temperature sensitive mutant to non-permissive conditions is lethal, thus demonstrating that
Fab I is essential to the survival of the organism (Bergler, et al, ( 1994), J Biol Chem 269, 5493-5496) Laboratory generated point mutations in the Eab I gene lead to diazabonne resistant E colt Fab I is conserved in Gram negative organisms with 98% identity between E colt and S nphtmitriitm Fab I (Bergler. et al, ( 1992), J Gen Mtcrobiol 138. 2093-2100) and 75% identity between these proteins and H tnfliienzae Fab I Staphylococcus aureus FAB I of the invention shows 54% similanty to the mycobacteπal protein, InhA, which is highly conserved throughout mycobacteπa including WI tuberculosis E colt Fab I was found to be 34% identical, 57% similar to Brassica napus (rape seed) enoyl-ACP reductase and S aureus FAB I of the present invention was also 34% identical, 57% similar Moreover, FAB I of the present invention was found to be 44% identical, 64% similar over 252 amino acids to E colt Fab I FAB I of the present invention is only 27% identical, 48% similar to a mammalian 2,4-dienoyl-coenzyme A reductase This mammalian homolog differs from FAB I in that it is involved in the β-oxidation of polyunsaturated enoyl-CoAs and utilizes NADPH as cofactor rather than NADH Therefore, there is significant potential for selective inhibition of FABI Since there are no marketed antibiotics targeted against fatty acid biosynthesis it is likely that inhibitors of FAB I will not be susceptible to current antibiotic resistance mechanisms Moreover, this is a potentially broad spectrum target
There is an unmet need for developing new classes of antibiotic compounds Clearly, there is also a need for factors, such as FABI, that may be used to screen compounds for antibiotic activity, such as a simple high through-put assay for screening inhibitors of FAS Such factors may also be used to determine their roles in pathogenesis of infection, dysfunction and disease Identification and characteπzation of such factors, which can play a role in preventing, ameliorating or correcting infections, dysfunctions or diseases are critical steps in making important discovenes to improve human health SUMMARY OF THE INVENTION
It is an object of the present invention, there are provided FAB I agonists Among preferred agonists are molecules that mimic FAB I, that bind to FAB I-bindmg molecules or binding molecules, and that elicit or augment FAB I-mduced responses Also among preferred agonists are molecules that interact with FAB I, or with other modulators of FAB I activities, and thereby potentiate or augment an effect of FAB I or more than one effect of FAB I and are bactenostatic or bacteπocidal In accordance with yet another aspect of the present invention, there are provided
FAB I antagonists Among preferred antagonists are those which mimic FAB I so as to bind to FAB 1-bιndιng molecules but not elicit a FAB I-induced response or more than one FAB I- induced response Also among preferred antagonists are molecules that bind to or interact with FAB I so as to inhibit an effect of FAB I or more than one effect of FAB I or which prevent expression of FAB I Further particularly preferred antagonists of FAB I lower or abolish a FABI enzymatic activity or activities In a further aspect of the invention there are provided compositions compnsing a FAB
I polynucleotide or a FAB I polypeptide for administration to cells in vitro, to cells ex vno and to cells in vivo, or to a multicellular organism In certain particularly preferred embodiments of this aspect of the invention, the compositions compπse a FAB I polynucleotide for expression of a FAB I polypeptide in a host organism to raise an immunological response, preferably to raise immunity in such host against bacteria, preferably staphylocci or closely genetically related organisms
The following further embodiments are also provided by the invention An antagonist that inhibits or an agonist that activates an activity a polypeptide selected from the group consisting of a polypeptide compπsing an amino acid sequence which is at least 90% identical to the amino acid sequence of SEQ ID NO 2 or 4, and a polypeptide comprising an amino acid sequence as set forth in SEQ ID NO 2 or 4, wherein said activity is selected from the group consisting of uncompetitive inhibition by Apo-ACP versus NADH (Kι(app), competitive inhibition by Apo-ACP versus crotonoyl CoA, induction of negative cooperativity with respect to CCA binding, use of NADH and NADPH as substrates by Fab I, binding of NADH and NADPH by Fabl, oxidation of NADH and NADPH by Fabl, ratio of Kmapp for NADH as compared to NADPH, use of NADH and crotonoyl CoA as substrates by Fab I in a sequential kinetic mechanism, sequential binding of NADH and crotonoyl CoA by Fab I, increasing inhibition of Fabl by saturated fatty acyl CoA's of increasing chain length, feedback regulatory mechanism of Fab I by saturated fatty acyl CoA's, competitive inhibition by palmitoyl CoA versus crotonoyl CoA, competitive inhibition by palmitoyl CoA versus crotonoyl CoA modulation through binding of multiple palmitoyl CoA molecules to Fab I, binding of multiple palmitoyl CoA molecules to Fab I, negative cooperativity in the binding of CCA, formation of an dimeπc quaternary structure, formation of an tetrameπc quaternary structure, formation of an oligomeπc quaternary structure, binding of Fab I by pseudo-product inhibitors beta-NADP+ or palmitoyl coA, and NADH binding to Fab I prior to or simultaneous with ACP binding
A method for the treatment of an individual having need to inhibit or activate
Fab I polypeptide compπsing the steps of administeπng to the individual a antibacteπally effective amount of an antagonist that inhibits or an agonist that activates an activity of a polypeptide selected from the group consisting of a polypeptide compπsing an amino acid sequence which is at least 90% identical to the amino acid sequence of SEQ ID NO 2 or 4, and a polypeptide compπsing an ammo acid sequence as set forth in SEQ ID NO 2 or 4, wherein said activity is selected from the group consisting of uncompetitive inhibition by Apo-ACP versus NADH (Kι(app), competitive inhibition by Apo-ACP versus crotonoyl CoA, induction of negative cooperativity with respect to CCA binding, use of NADH and NADPH as substrates by Fab I, binding of NADH and NADPH by Fabl, oxidation of NADH and NADPH by Fabl, ratio of Kmapp for NADH as compared to NADPH, use of NADH and crotonoyl CoA as substrates by Fab I in a sequential kinetic mechanism, sequential binding of NADH and crotonoyl CoA by Fab I, increasing inhibition of Fabl by saturated fatty acyl CoA's of increasing chain length, feedback regulatory mechanism of Fab I by saturated fatty acyl CoA's, competitive inhibition by palmitoyl CoA versus crotonoyl CoA, competitive inhibition by palmitoyl CoA versus crotonoyl CoA modulation through binding of multiple palmitoyl CoA molecules to Fab I, binding of multiple palmitoyl CoA molecules to Fab I, negative cooperativity in the binding of CCA, formation of an dimeπc quaternary structure, formation of an tetrameπc quaternary structure, formation of an oligomeπc quaternary structure, binding of Fab I by pseudo-product inhibitors beta-NADP+ or palmitoyl coA, and NADH binding to Fab I prior to or simultaneous with ACP binding
A method for the treatment of an individual infected with a bactena comprising the steps of administeπng to the individual a antibacteπally effective amount of an antagonist that inhibits or an agonist that activates an activity of a polypeptide selected from the group consisting of a polypeptide compπsing an ammo acid sequence which is at least 90% identical to the amino acid sequence of SEQ ID
NO 2 or 4, and a polypeptide compπsing an amino acid sequence as set forth in SEQ ID NO 2 or 4, wherein said activity is selected from the group consisting of uncompetitive inhibition by Apo-ACP versus NADH (Kι(app), competitive inhibition by Apo-ACP versus crotonoyl CoA, induction of negative cooperativity with respect to CCA binding, use of NADH and NADPH as substrates by Fab I, binding of NADH and NADPH by Fabl, oxidation of NADH and NADPH by Fabl, ratio of Kmapp for NADH as compared to NADPH, use of NADH and crotonoyl CoA as substrates by Fab I in a sequential kinetic mechanism, sequential binding of NADH and crotonoyl CoA by Fab I, increasing inhibition of Fabl by saturated fatty acyl CoA's of increasing chain length, feedback regulatory mechanism of Fab I by saturated fatty acyl CoA's, competitive inhibition by palmitoyl CoA versus crotonoyl CoA, competitive inhibition by palmitoyl CoA versus crotonoyl CoA modulation through binding of multiple palmitoyl CoA molecules to Fab I binding of multiple palmitoyl CoA molecules to Fab I, negative cooperativity in the binding of CCA, formation of an dimeπc quaternary structure, formation of an tetrameπc quaternary structure, formation of an ohgomeπc quaternary structure, binding of Fab I by pseudo-product inhibitors beta-NADP-t- or palmitoyl coA, and NADH binding to Fab I pπor to or simultaneous with ACP binding
A method for the treatment of an individual having need to inhibit or activate
Fab I polypeptide comprising the steps of administeπng to the individual a antibacteπally effective amount of an antagonist that inhibits or an agoinist that activates an activity of Fab I selected from the group consisting of uncompetitive inhibition by Apo-ACP versus NADH (Kι(app), competitive inhibition by Apo-ACP versus crotonoyl CoA, induction of negative cooperativity with respect to CCA binding, use of NADH and NADPH as substrates by Fab I, binding of NADH and NADPH by Fabl, oxidation of NADH and NADPH by Fabl, ratio of Kmapp for NADH as compared to NADPH, use of NADH and crotonoyl CoA as substrates by Fab I in a sequential kinetic mechanism, sequential binding of NADH and crotonoyl CoA by Fab I, increasing inhibition of Fabl by saturated fatty acyl CoA's of increasing chain length, feedback regulatory mechanism of Fab I by saturated fatty acyl CoA's, competitive inhibition by palmitoyl CoA versus crotonoyl CoA, competitive inhibition by palmitoyl CoA versus crotonoyl CoA modulation through binding of multiple palmitoyl CoA molecules to Fab I, binding of multiple palmitoyl CoA molecules to Fab I, negative cooperativity in the binding of CCA, formation of an dimeπc quaternary structure, formation of an tetramenc quaternary structure, formation of an oligomeπc quaternary structure, binding of Fab I by pseudo-product inhibitors beta-NADP+ or palmitoyl coA, and
NADH binding to Fab I pπor to or simultaneous with ACP binding
A method for the treatment of an individual infected with a bacteπa comprising the steps of administenng to the individual a antibacteπally effective amount of an antagonist that inhibits or an agonist that activates that activates an activity of Fab I selected from the group consisting of uncompetitive inhibition by Apo-ACP versus NADH (Kι(app), competitive inhibition by Apo-ACP versus crotonoyl CoA, induction of negative cooperativity with respect to CCA binding, use of NADH and NADPH as substrates by Fab I, binding of NADH and NADPH by Fabl, oxidation of NADH and NADPH by Fabl, ratio of Kmapp for NADH as compared to NADPH, use of NADH and crotonoyl CoA as substrates by Fab I in a sequential kinetic mechanism, sequential binding of NADH and crotonoyl CoA by Fab I, increasing inhibition of Fabl by saturated fatty acyl CoA's of increasing chain length, feedback regulatory mechanism of Fab I by saturated fatty acyl CoA's, competitive inhibition by palmitoyl CoA versus crotonoyl CoA, competitive inhibition by palmitoyl CoA versus crotonoyl CoA modulation through binding of multiple palmitoyl CoA molecules to Fab I, binding of multiple palmitoyl CoA molecules to Fab I, negative cooperativity in the binding of CCA, formation of an dimeπc quaternary structure, formation of an tetrameπc quaternary structure, formation of an oligomeπc quaternary structure, binding of Fab I by pseudo-product inhibitors beta-NADP-- or palmitoyl coA; and
NADH binding to Fab I prior to or simultaneous with ACP binding.
A method for the treatment of an individual infected by Streptococcus pneumoniae comprising the steps of administering to the individual a antibacterially effective amount of an antagonist that inhibits or anagonist that activates an activity of Streptococcus pneumoniae Fab I selected from the group consisting of: uncompetitive inhibition by Apo-ACP versus NADH (Ki(app); competitive inhibition by Apo-ACP versus crotonoyl CoA; induction of negative cooperativity with respect to CCA binding; use of NADH and NADPH as substrates by Fab I; binding of NADH and NADPH by Fabl; oxidation of NADH and NADPH by Fabl; ratio of Kmapp for NADH as compared to NADPH; use of NADH and crotonoyl CoA as substrates by Fab I in a sequential kinetic mechanism; sequential binding of NADH and crotonoyl CoA by Fab I; increasing inhibition of Fabl by saturated fatty acyl CoA's of increasing chain length; feedback regulatory mechanism of Fab I by saturated fatty acyl CoA's; competitive inhibition by palmitoyl CoA versus crotonoyl CoA; competitive inhibition by palmitoyl CoA versus crotonoyl CoA modulation through binding of multiple palmitoyl CoA molecules to Fab I; binding of multiple palmitoyl CoA molecules to Fab I; negative cooperativity in the binding of CCA; formation of an dimeric quaternary structure; formation of an tetrameric quaternary structure; formation of an oligomeric quaternary structure; binding of Fab I by pseudo-product inhibitors beta-NADP+ or palmitoyl coA; and
NADH binding to Fab I prior to or simultaneous with ACP binding. An antagonist that inhibits an activity of a polypeptide selected from the group consisting of a polypeptide comprising an amino acid sequence which is at least 90% identical to the amino acid sequence of SEQ ID NO 2 or 4, and a polypeptide compπsing an amino acid sequence as set forth in SEQ ID NO 2 or 4, , wherein said activity is selected from the group consisting of uncompetitive inhibition by Apo-ACP versus NADH (Kι(app), competitive inhibition by Apo-ACP versus crotonoyl CoA, induction of negative cooperativity with respect to CCA binding, use of NADH and NADPH as substrates by Fab I, binding of NADH and NADPH by Fabl, oxidation of NADH and NADPH by Fabl, ratio of Kmapp for NADH as compared to NADPH, use of NADH and crotonoyl CoA as substrates by Fab I in a sequential kinetic mechanism, sequential binding of NADH and crotonoyl CoA by Fab I, increasing inhibition of Fabl by saturated fatty acyl CoA's of increasing chain length, feedback regulatory mechanism of Fab I by saturated fatty acyl CoA's, competitive inhibition by palmitoyl CoA versus crotonoyl CoA, competitive inhibition by palmitoyl CoA versus crotonoyl CoA modulation through binding of multiple palmitoyl CoA molecules to Fab I, binding of multiple palmitoyl CoA molecules to Fab I, negative cooperativity in the binding of CCA, formation of an dimeric quaternary structure, formation of an tetrameric quaternary structure, formation of an oligomeric quaternary structure, binding of Fab I by pseudo-product inhibitors beta-NADP+ or palmitoyl coA, and
NADH binding to Fab I prior to or simultaneous with ACP binding A method for the treatment of an individual having need to inhibit Fab I polypeptide compπsing the steps of administering to the individual a antibactenall effective amount of an antagonist that inhibits an activity of a polypeptide selected from the group consisting of a polypeptide compπsing an amino acid sequence which is at least 90% identical to the amino acid sequence of SEQ ID NO 2 or 4, and a polypeptide compπsing an amino acid sequence as set forth in SEQ ID NO 2 or 4, , wherein said activity is selected from the group consisting of uncompetitive inhibition by Apo-ACP versus NADH (Kι(app), competitive inhibition by Apo-ACP versus crotonoyl CoA, induction of negative cooperativity with respect to CCA binding, use of NADH and NADPH as substrates by Fab I, binding of NADH and NADPH by Fabl, oxidation of NADH and NADPH by Fabl, ratio of Kmapp for NADH as compared to NADPH, use of NADH and crotonoyl CoA as substrates by Fab I in a sequential kinetic mechanism, sequential binding of NADH and crotonoyl CoA by Fab I, increasing inhibition of Fabl by saturated fatty acyl CoA's of increasing chain length, feedback regulatory mechanism of Fab I by saturated fatty acyl CoA's, competitive inhibition by palmitoyl CoA versus crotonoyl CoA, competitive inhibition by palmitoyl CoA versus crotonoyl CoA modulation through binding of multiple palmitoyl CoA molecules to Fab I, binding of multiple palmitoyl CoA molecules to Fab I, negative cooperativity in the binding of CCA, formation of an dimeric quaternary structure, formation of an tetrameric quaternary structure, formation of an oligomeric quaternary structure, binding of Fab I by pseudo-product inhibitors beta-NADP+ or palmitoyl coA, and
NADH binding to Fab I prior to or simultaneous with ACP binding
A method for inhibiting an activity of Fab I polypeptide comprising the steps of contacting a composition comprising said polypeptide with an effective amount of an antagonist that inhibits an activbity of Fab I, wherein said activity is selected from the group consisting of uncompetitive inhibition by Apo-ACP versus NADH (Kι(app), competitiv e inhibition by Apo-ACP versus crotonoyl CoA induction of negative cooperativity with respect to CCA binding, use of NADH and NADPH as substrates by Fab I binding of NADH and NADPH by Fabl, oxidation of NADH and NADPH by Fabl, ratio of Kmapp for NADH as compared to NADPH, use of NADH and crotonoyl CoA as substrates by Fab I in a sequential kinetic mechanism, sequential binding of NADH and crotonoyl CoA by Fab I increasing inhibition of Fabl by saturated fatty acyl CoA's of increasing chain length, feedback regulatory mechanism of Fab I by saturated fatty acyl CoA's, competitive inhibition by palmitoyl CoA versus crotonoyl CoA, competitive inhibition by palmitoyl CoA versus crotonoyl CoA modulation through binding of multiple palmitoyl CoA molecules to Fab I, binding of multiple palmitoyl CoA molecules to Fab I, negative cooperativity in the binding of CCA, formation of an dimeric quaternary structure, formation of an tetrameric quaternary structure, formation of an oligomeric quaternary structure, binding of Fab I by pseudo-product inhibitors beta-NADP+ or palmitoyl co A, and
NADH binding to Fab I prior to or simultaneous with ACP binding A method for inhibiting an activity of Fab I, wherein said activity is selected from the group consisting of uncompetitive inhibition by Apo-ACP versus NADH (Kι(app), competitive inhibition by Apo-ACP versus crotonoyl CoA, induction of negative cooperativity with respect to CCA binding, use of NADH and NADPH as substrates by Fab I, binding of NADH and NADPH by Fabl, oxidation of NADH and NADPH by Fabl, ratio of Kmapp for NADH as compared to NADPH, use of NADH and crotonoyl CoA as substrates by Fab I in a sequential kinetic mechanism, sequential binding of NADH and crotonoyl CoA by Fab I, increasing inhibition of Fabl by saturated fatty acyl CoA's of increasing chain length, feedback regulatory mechanism of Fab I by saturated fatty acyl CoA's, competitive inhibition by palmitoyl CoA versus crotonoyl CoA, competitive inhibition by palmitoyl CoA versus crotonoyl CoA modulation through binding of multiple palmitoyl CoA molecules to Fab I, binding of multiple palmitoyl CoA molecules to Fab I, negative cooperativity in the binding of CCA, formation of an dimeric quaternary structure, formation of an tetrameric quaternary structure, formation of an oligomeric quaternary structure, binding of Fab I by pseudo-product inhibitors beta-NADP+ or palmitoyl coA, and
NADH binding to Fab I prior to or simultaneous with ACP binding
A method for inhibiting a growth of bacteria compnsmg the steps of contacting a composition compπsing bacteria with an antibactenally effective amount of an antagonist that inhibits an activity of Fab I, wherein said activity is selected from the group consisting of uncompetitive inhibition by Apo-ACP versus NADH (Kι(app), competitive inhibition by Apo-ACP versus crotonoyl CoA, induction of negative cooperativity with respect to CCA binding, use of NADH and NADPH as substrates by Fab I, binding of NADH and NADPH by Fabl, oxidation of NADH and NADPH by Fabl, ratio of Kmapp for NADH as compared to NADPH, use of NADH and crotonoyl CoA as substrates by Fab I in a sequential kinetic mechanism, sequential binding of NADH and crotonoyl CoA by Fab I, increasing inhibition of Fabl by saturated fatty acyl CoA's of increasing chain length, feedback regulatory mechanism of Fab I by saturated fatty acyl CoA's, competitive inhibition by palmitoyl CoA versus crotonoyl CoA. competitive inhibition by palmitoyl CoA versus crotonoyl CoA modulation through binding of multiple palmitoyl CoA molecules to Fab I, binding of multiple palmitoyl CoA molecules to Fab I, negative cooperativity in the binding of CCA, formation of an dimeric quaternary structure, formation of an tetrameric quaternary structure, formation of an oligomeric quaternary structure, binding of Fab I by pseudo-product inhibitors beta-NADP+ or palmitoyl coA, and
NADH binding to Fab I prior to or simultaneous with ACP binding A method for inhibiting a Fab I polypeptide compnsing the steps of contacting a composition compnsing bactena with an antibactenally effective amount of an antagonist that inhibits an activity of Fab I, wherein said activity is selected from the group consisting of uncompetitive inhibition by Apo-ACP versus NADH (Kι(app), competitive inhibition by Apo-ACP versus crotonoyl CoA, induction of negative cooperativity with respect to CCA binding, use of NADH and NADPH as substrates by Fab I, binding of NADH and NADPH by Fabl, oxidation of NADH and NADPH by Fabl, ratio of Kmapp for NADH as compared to NADPH, use of NADH and crotonoyl CoA as substrates by Fab I in a sequential kinetic mechanism, sequential binding of NADH and crotonoyl CoA by Fab I, increasing inhibition of Fabl by saturated fatty acyl CoA's of increasing chain length, feedback regulatory mechanism of Fab I by saturated fatty acyl CoA's, competitive inhibition by palmitoyl CoA versus crotonoyl CoA, competitiv e inhibition by palmitoyl CoA versus crotonoyl CoA modulation through binding of multiple palmitoyl CoA molecules to Fab I, binding of multiple palmitoyl CoA molecules to Fab I, negative cooperativity in the binding of CCA, formation of an dimeric quaternary structure, formation of an tetrameπc quaternary structure, formation of an oligomeric quaternary structure, binding of Fab I by pseudo-product inhibitors beta-NADP+ or palmitoyl co A, and NADH binding to Fab I prior to or simultaneous with ACP binding
It is preferred in the methods of the invention that any bacteπa is selected from the group consisting of a member of the genus Staphvlococcus, Staphylococcus aureus, a member of the genus Streptococcus, and Streptococcus pneumoniae
Other objects, features, advantages and aspects of the present invention will become apparent to those of skill from the following descnption It should be understood, however, that the following descnption and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only Vanous changes and modifications within the spint and scope of the disclosed invention will become readily apparent to those skilled in the art from reading the following description and from reading the other parts of the present disclosure.
BRIEF DESCRIPTION OF THE DRAWINGS
The following drawing depicts certain embodiments of the invention It is illustrative only and does not limit the invention otherwise disclosed herein
Figure 1 shows the cycle of fatty acid elongation in bacteπa Figure 2 shows a chemical synthesis of deuterated pyridine nucleotides
Figure 3 shows predicted and observed product structures and (M+H)+'s based on E colt Fab I
Figure 4 (A, B, and C) shows mass spectra of components from reaction 3 of Figure 3 Figure 5 shows a stereochemical course of 5 aureus Fab I
Figure 6 shows graphically that S aureus Fab I uses both NADPH and NADH as substrates Figure 7 shows graphically that S auieus Fab I exhibits a sequential reaction mechanism
Figure 8 shows a pH profile of S aureus Fab I
Figure 9 shows inhibition by palmitoyl CoA Figure 10 shows inhibition by beta-NADP+
Figure 1 1 shows a kinetic model for inhibition by beta-NADP+
Figure 12 shows graphically that inhibition by apo-ACP vs NADH
Figure 13 shows graphically the inhibition by apo-ACP vs CCA
Figure 14 shows minimal kinetic mechanism for inhibition by apo-ACP Figure 15 shows induced cooperative inhibition by apo-ACP
Figure 16 [SEQ ID NO 2] shows the polypeptide of Staplnlococctts aureus FAB I deduced from the polynucleotide of Figure 17 [SEQ ID NO 1 ]
Figure 17 [SEQ ID NO 1 ] shows the polypnucleotide sequence of Staphylococcus aureus FAB I GLOSSARY
The following illustrative explanations are provided to facilitate understanding of certain terms used frequently herein, particularly in the Examples The explanations are provided as a convenience and are not limitative of the invention
"Binding molecule(s)," as used herein, refers to molecules or ions which bind or interact specifically with FAB I polypeptides or polynucleotides of the present invention, including, for example enzyme substrates and substrate and co-factor mimetics, as well as classical receptors (which also may be referred to as "binding molecules' and "interaction molecules," respectively and as "FAB I binding molecules' and "FAB I interaction molecules") Binding between polypeptides of the invention and such molecules, including binding or binding or interaction molecules may be exclusive to polypeptides of the invention, which is very highly preferred, or it may be highly specific for polypeptides of the invention, which is highly preferred, or it may be highly specific to a group of proteins that includes polypeptides of the invention, which is prefened, or it may be specific to several groups of proteins at least one of which includes a polypeptide of the invention Binding molecules also may be non-naturally occurring, such as antibodies and antibody-deπved reagents that bind specifically to polypeptides of the invention
"Digestion of DNA" refers to cleavage of DNA with a restriction enzyme that acts only at certain sequences in the DNA The various restπction enzymes referred to herein are commercially available and their reaction conditions, cofactors and other requirements for use are well known and routine to the skilled artisan
For analytical purposes, typically, 1 μg of plasmid or DNA fragment is digested with about 2 units of enzyme in about 20 μl of reaction buffer For the purpose of isolating DNA fragments for plasmid construction, typically 5 to 50 μg of DNA are digested with 20 to 250 units of enzyme in proportionately larger volumes
Appropriate buffers and substrate amounts for particular restπction enzymes are described in standard laboratory manuals, such as those referenced below, and they are specified by commercial suppliers Incubation times of about 1 hour at 37°C are ordinaπly used, but conditions may vary in accordance with standard procedures, the supplier's instructions and the particulars of the reaction After digestion, reactions may be analyzed, and fragments may be punfied by electrophoresis through an agarose or polyacrylamide gel, using well known methods that are routine for those skilled in the art "Dιsease(s)" means (l) bactenal infections, such as staphlococcal infections including, but not limited to infections of the upper respiratory tract (e g , otitis media, bactenal tracheitis, acute epiglottitis, thyroiditis), the lower respiratory tract (e g , empyema, lung abscess), the cardiac system (e g , infective endocarditis), the gastrointestinal tract (e g , secretory diaπhoea, splenic abscess, retropeπtoneal abscess), the CNS (e g , cerebral abscess), eye (e g , blephaπtis, conjunctivitis, keratitis, endophthalmitis, preseptal and orbital cellulitis, darcryocystitis), the kidney or unnary tract (e g , epididymitis, mtrarenal and peπnephnc abscess, toxic shock syndrome), the skin (e g , impetigo, follicuhtis, cutaneous abscesses, cellulitis, wound infection, bacterial myositis), and the bones and joints (e g septic arthntis, osteomyelitis) and/or (n) an infection caused by or related to a member of the genus Streptococcus, Staphylococcus, Bordetella, Corynebacterium, Mycobacterium, Neissena, Haemophtlus, Acttnomycetes, Streptomycetes, Nocardta, Enterobacter, Yersinia, Eancisella, Pasturella, Moraxella, Actnetobacter, Erysφelothrix, Branhamella, Acttnobacillus, Streptobacillus, Listeria, Calymmatobactertum, Brucella, Bacillus, Clostridium, Treponema, Escherichia, Salmonella, Kletbsiella, Vibrio, Proteus, Envinia Borreha, Leptosptra, Spirillum, Campylobacter, Shigella, Legionella, Pseudomonas, Aeromonas, Rickettsta, Chlamvdia, Borreha and Mycoplasma, and further including, but not limited to, a member of the species or group. Group A Streptococcus, Group B Streptococcus, Group C Streptococcus, Group D Streptococcus, Group G Streptococcus, Streptococcus pneumoniae Streptococcus pyogenes. Streptococcus agalactiae. Streptococcus faecalis, Streptococcus faecium. Streptococcus ditrans. Neissena gonorrheae, Neisseria meningitidis, Staphylococcus aureus, Staphylococcus epidermidis, Corynebacterium diptheriae, Gardnerella vaginalis, Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium ulcerans, Mycobacterium leprae, Actinomyctes israelii, Listeria monocytogenes, Bordetella pertusis, Bordatella parapertusis, Bordetella bronchiseptica, Escherichia coli, Shigella dysenteriae, Haemophilus influenzae. Haemophilus aegypύus, Haemophilus parainβttenzae, Haemophilus ducreyi, Bordetella, Salmonella typhi, Citrobacter frettndii, Proteus mirabilis, Proteus vulgaris, Yersinia pestis, Kleibsiella pneumoniae, Serratia marcessens, Serratia liquefaciens, Vibrio cholera, Shigella dysenterii, Shigella flexneri. Pseudomonas aerttginosa, Franscisella tttlarensis. Brucella abortis, Bacillus anthracis. Bacillus cereus, Clostridium perfringens, Clostridium tetani, Clostridium botulinum, Treponema pallidttin, Rickettsia rickettsii and Chlamydia trachomitis.
"Genetic Element(s)" means a polynucleotide comprising a region that encodes a polypeptide or a region that regulates transcription or translation or other processes important to expression of the polypeptide in a host cell, or a polynucleotide comprising both a region that encodes a polypeptide and a region operably linked thereto that regulates expression.
Genetic elements may be comprised within a vector that replicates as an episomal element; that is, as a molecule physically independent of the host cell genome. They may be comprised within plasmids. Genetic elements also may be comprised within a host cell genome; not in their natural state but, rather, following manipulation such as isolation, cloning and introduction into a host cell in the form of purified DNA or in a vector, among others.
"Host cell(s)" means a cell which has been transformed, transfected, infected, or entered by an exogenous polynucleotide sequence, or is capable of transformation, infection, transfection, or entry by an exogenous polyncleotide sequence.
"Identity", as known in the art, means a relationships between two polypeptide sequences or two polynucleotide sequences, as determined by comparing the sequences. In the art, identity also means the degree of sequence relatedness between two polypeptide or two polynucleotide sequences as determined by the match between two strings of such sequences. Both identity and similarity can be readily calculated (Computational Molecular Biology, Lesk, A.M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D.W., ed., Academic Press, New
l i York, 1993, Computer Analysis of Sequence Data, Part I, Griffin, A M , and Griffin, H G , eds , Humana Press, New Jersey, 1994, Sequence Analysis in Molecular Biology , von Heinje, G , Academic Press, 1987, and Sequence Analysis Primer. Gπbskov, M and Devereux, J , eds , M Stockton Press, New York, 1991 ) While there exist a number of methods to measure identity and similarity between two polynucleotide or two polypeptide sequences, both terms are well known to skilled artisans (Sequence Analysis in Molecular Biology , von Heinje, G , Academic Press, 1987, Sequence Analysts Primer. Gπbskov, M and Devereux, J , eds , M Stockton Press, New York, 1991 , and Caπllo, H , and Lipman, D , SIAM J Applied Math , 48 1073 ( 1988) Methods commonly employed to determine identity or similarity between two sequences include, but are not limited to those disclosed in Caπllo, H , and Lipman, D , SIAM J Applied Math , 48 1073 ( 1988) Preferred methods to determine identity are designed to give the largest match between the two sequences tested Methods to determine identity and similarity are codified in computer programs Preferred computer program methods to determine identity and similarity between two sequences include, but are not limited to, GCG program package (Devereux, J , et al , ( 1984) Nucleic Acids Research 12(1) 387), BLASTP, BLASTN, and FASTA (Atschul, S F et al , ( 1990) J Molec Biol 215 403)
"Isolated" means altered "by the hand of man" from its natural state, i e , if it occurs in nature, it has been changed or removed from its original environment, or both For example, a polynucleotide or a polypeptide naturally present in a living organism is not "isolated," but the same polynucleotide or polypeptide separated from the coexisting mateπals of its natural state is "isolated", as the term is employed herein Moreover, a polynucleotide or polypeptide that is introduced into an organism by transformation, genetic manipulation or by any other recombinant method is "isolated" even if it is still present in said organism, which organism may be living or non-living
As part of or following isolation, such polynucleotides can be joined to other polynucleotides, such as DNAs, for mutagenesis, to form fusion proteins, and for propagation or expression in a host, for instance The isolated polynucleotides, alone or joined to other polynucleotides such as vectors, can be introduced into host cells, in culture or in whole organisms Introduced into host cells in culture or in whole organisms, such DNAs still would be isolated, as the term is used herein, because they would not be in their naturally occurnng form or environment Similarly, the polynucleotides and polypeptides may occur in a composition, such as a media formulations, solutions for introduction of polynucleotides or polypeptides, for example, into cells, compositions or solutions for chemical or enzymatic reactions, for instance, which are not naturally occurnng compositions, and, therein remain isolated polynucleotides or polypeptides within the meaning of that term as it is employed herein "Ligation" means a process of forming phosphodiester bonds between two or more polynucleotides, which most often are double stranded DNAs Techniques for ligation are well known to the art and protocols for ligation are described in standard laboratory manuals and references, such as, for instance, Sambrook et al , MOLECULAR CLONING, A LABORATORY MANUAL, 2nd Ed , Cold Spring Harbor Laboratory Press, Cold Spπng Harbor, New York ( 1989) and Maniatis et al , pg 146, as cited below
"Olιgonucelotιde(s)" refers to relatively short polynucleotides Often the term refers to single-stranded deoxyπbonucleotides, but it can refer as well to single-or double-stranded πbonucleotides, RNA DNA hybrids and double-stranded DNAs, among others
Oligonucleotides, such as single-stranded DNA probe oligonucleotides, often are synthesized by chemical methods, such as those implemented on automated oligonucleotide synthesizers However, oligonucleotides can be made by a variety of other methods, including in vitro recombinant DNA-mediated techniques and by expression of DNAs in cells and organisms
Initially, chemically synthesized DNAs typically are obtained without a 5' phosphate The 5' ends of such oligonucleotides are not substrates for phosphodiester bond formation by ligation reactions that employ DNA gases typically used to form recombinant DNA molecules Where ligation of such oligonucleotides is desired, a phosphate can be added by standard techniques, such as those that employ a kinase and ATP
The 3' end of a chemically synthesized oligonucleotide generally has a free hydroxyl group and, in the presence of a ligase, such as T4 DNA ligase, readily will form a phosphodiester bond with a 5' phosphate of another polynucleotide, such as another oligonucleotide. As is well known, this reaction can be prevented selectively, where desired, by removing the 5' phosphates of the other polynucleotιde(s) pπor to ligation
"Organιsm(s)" when used herein with reference to a life form used for cloning or that causes (or is believed to cause) disease means (l) a prokaryote, including but not limited to, a member of the genus Streptococcus, Staphylococcus, Bordetella, Corynebacterium,
Mycobacterium, Neissena, Haemophilus, Actinomvcetes, Streptomvcetes, Nocardia,
Enterobacter, Yersinia, Fancisella, Pasturella, Moraxella, Acinetobacter, Erysipelothrix, Branhamella, Actinobacillus, Streptobacillus, Listeria, Calymmatobacterium, Brucella, Bacillus, Clostridium, Treponema, Escherichia, Salmonella, Kleibsiella, Vibrio, Proteus, Erwinia, Borrelia, Leptospira. Spirillum, Campylobacter, Shigella, Legionella, Pseudomonas, Aeromonas, Rickettsia, Chlamydia, Borrelia and Mycoplasma, and further including, but not limited to, a member of the species or group. Group A Streptococcus, Group B Streptococcus, Group C Streptococcus, Group D Streptococcus, Group G Streptococcus, Streptococcus pneumoniae. Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus faecalis. Streptococcus faecium. Streptococcus dttrans, Neissena gonorrheae, Neissena meningitidis, Staphylococcus aureus, Staphylococcus epidermidis, Corynebacterium diptheriae, Gardnerella vaginalis, Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium ttlcerans, Mycobacterium leprae, Actinomyctes israelii, Listeria monocytogenes, Bordetella pertusis, Bordatella parapertusis, Bordetella bronchiseptica, Escherichia coli, Shigella dysenteriae, Haemophilus influenzae, Haemophilus aegyptitis, Haemophilus parainfluenzae, Haemophilus ducreyi, Bordetella, Salmonella typhi, Citrobacter freundii, Proteus mirabilis, Proteus vulgaris, Yersinia pestis, Kleibsiella pneumoniae, Serratia marcessens, Serratia liquefaciens, Vibrio cholera, Shigella dysenterii, Shigella flexneri, Pseudomonas aeruginosa, Franscisella tularensis, Brucella abortis, Bacillus anthracis, Bacillus cereus, Clostridium perfringens, Clostridium tetani, Clostridium botulinum, Treponema pallidum, Rickettsia rickettsii and Chlamydia trachomitis, (ii) an archaeon, including but not limited to Archaebacter. "Plasmid(s)" generally are designated herein by a lower case p preceded and/or followed by capital letters and/or numbers, in accordance with standard naming conventions that are familiar to those of skill in the art.
Starting plasmids disclosed herein are either commercially available, publicly available on an unrestricted basis, or can be constructed from available plasmids by routine application of well known, published procedures. Many plasmids and other cloning and expression vectors that can be used in accordance with the present invention are well known and readily available to those of skill in the art. Moreover, those of skill readily may construct any number of other plasmids suitable for use in the invention. The properties, construction and use of such plasmids, as well as other vectors, in the present invention will be readily apparent to those of skill from the present disclosure.
"Polynucleotide(s)" means any polyribonucleotide or polydeoxribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. Thus, for instance, polynucleotides as used herein refers to, among others, single-and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions or single-, double- and tπple- stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single- stranded or, more typically, double-stranded, or triple-stranded, or a mixture of single- and double-stranded regions
In addition, polynucleotide as used herein refers to tπple-stranded regions compπsing RNA or DNA or both RNA and DNA The strands in such regions may be from the same molecule or from different molecules The regions may include all of one or more of the molecules, but more typically involve only a region of some of the molecules One of the molecules of a tnple-helical region often is an oligonucleotide
As used herein, the term polynucleotide includes DNAs or RNAs as descπbed above that contain one or more modified bases Thus, DNAs or RNAs with backbones modified for stability or for other reasons are "polynucleotides" as that term is intended herein Moreover, DNAs or RNAs compnsing unusual bases, such as inosine, or modified bases, such as tntylated bases, to name just two examples, are polynucleotides as the term is used herein
It will be appreciated that a great vanety of modifications have been made to DNA and RNA that serve many useful purposes known to those of skill in the art The term polynucleotide as it is employed herein embraces such chemically, enzymatically or metabohcally modified forms of polynucleotides, as well as the chemical forms of DNA and RNA characteristic of viruses and cells, including simple and complex cells, inlet alia
"Polypeptιde(s)", as used herein, includes all polypeptides as descnbed below The basic structure of polypeptides is well known and has been descnbed in innumerable textbooks and other publications in the art In this context, the term is used herein to refer to any peptide or protein compπsing two or more amino acids joined to each other in a linear chain by peptide bonds As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, ohgopeptides and ohgomers, for example, and to longer chains, which generally are refeπed to in the art as proteins, of which there are many types It will be appreciated that polypeptides often contain amino acids other than the 20 amino acids commonly referred to as the 20 naturally occurring amino acids, and that many amino acids, including the terminal amino acids, may be modified in a given polypeptide, either by natural processes, such as processing and other post-translational modifications, but
9? also by chemical modification techniques which are well known to the art Even the common modifications that occur naturally in polypeptides are too numerous to list exhaustively here, but they are well descnbed in basic texts and in more detailed monographs, as well as in a voluminous research literature, and they are well known to those of skill in the art Among the known modifications which may be present in polypeptides of the present are, to name an illustrative few, acetylation, acylation, ADP-πbosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety covalent attachment of a nucleotide or nucleotide denvative, covalent attachment of a hpid or hpid derivative, covalent attachment of phosphotidy nositol, cross-linking, cyc zation, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cystine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation lodination, methylation, myπstoylation oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination Such modifications are well known to those of skill and have been descnbed in great detail in the scientific literature Several particularly common modifications, glycosylation, hpid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP-nbosylation, for instance, are descnbed in most basic texts, such as, for instance PROTEINS - STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed , T E Creighton, W H Freeman and Company, New York (1993) Many detailed reviews are available on this subject, such as, for example, those provided by Wold, F , Posttranslational Protein Modifications Perspectives and Prospects, pgs 1-12 in POSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B C Johnson, Ed , Academic Press, New York (1983), Seifter et al , ( 1990) Meth Enzvmol 182 626-646 and Rattan et al , (1992) Protein Synthesis Posttranslational Modifications and Aging, Ann N Y Acad Sci 663 48-62
It will be appreciated, as is well known and as noted above, that polypeptides are not always entirely linear For instance, polypeptides may be branched as a result of ubiquitination, and they may be circular, with or without branching, generally as a result of posttranslation events, including natural processing event and events brought about by human manipulation which do not occur naturally Circular, branched and branched circular polypeptides may be synthesized by non-translation natural process and by entirely synthetic methods, as well Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini In fact blockage of the amino or carboxyl group in a polypeptide, or both, by a covalent modification, is common in naturally occurnng and synthetic polypeptides and such modifications may be present m polypeptides of the present invention, as well For instance, the amino terminal residue of polypeptides made in E coli or other cells, pπor to proteolytic processing, almost invaπably will be N-formylmethionine During post-translational modification of the peptide, a methionine residue at the NH-.-termιnus may be deleted Accordingly, this invention contemplates the use of both the methionine-containing and the methiomneless amino terminal variants of the protein of the invention
The modifications that occur in a polypeptide often will be a function of how it is made For polypeptides made by expressing a cloned gene in a host for instance, the nature and extent of the modifications in large part will be determined by the host cell posttranslational modification capacity and the modification signals present in the polypeptide amino acid sequence For instance, as is well known, glycosylation often does not occur bactenal hosts such as, for example, E colt Accordingly, when glycosylation is desired, a polypeptide should be expressed in a glycosylatmg host, generally a eukaryotic cell
It will be appreciated that the same type of modification mav be present in the same or varying degree at several sites in a given polypeptide Also, a given polypeptide may contain many types of modifications
In general, as used herein, the term polypeptide encompasses all such modifications, particularly those that are present in polypeptides synthesized by expressing a polynucleotide in a host cell
"Transformation" means the process by which a cell is transformed" by exogenous DNA when such exogenous DNA has been introduced inside the cell membrane Exogenous DNA may or may not be integrated (covalently linked) into chromosomal DNA making up the genome of the cell In prokaryotes and yeasts, for example, the exogenous DNA may be maintained on an episomal element, such as a plasmid With respect to eukaryotic cells, a stably transformed or transfected cell is often one in which the exogenous DNA has become integrated into the chromosome so that it is inheπted by daughter cells through chromosome replication This stability is demonstrated by the ability of the eukaryotic cell to establish cell lines or clones comprised of a population of daughter cell containing the exogenous DNA Nanant(s)' of polynucleotides or polypeptides, as the term is used herein, are polynucleotides or polypeptides that differ from a reference polynucleotide or polypeptide, respectively Vanants in this sense are descπbed below and elsewhere in the present disclosure m greater detail (1 ) A polynucleotide that differs in nucleotide sequence from another, reference polynucleotide Generally, differences are limited so that the nucleotide sequences of the reference and the vanant are closely similar overall and, in many regions, identical
As noted below, changes in the nucleotide sequence of the vanant may be silent That is, they may not alter the amino acids encoded by the polynucleotide Where alterations are limited to silent changes of this type a vanant will encode a polypeptide with the same am o acid sequence as the reference Also as noted below changes in the nucleotide sequence of the vanant may alter the ammo acid sequence of a polypeptide encoded by the reference polynucleotide Such nucleotide changes may result in amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence, as discussed below
(2) A polypeptide that differs in amino acid sequence from another, reference polypeptide Generally, differences are limited so that the sequences of the reference and the vanant are closely similar overall and, in many region, identical
A vanant and reference polypeptide may differ in amino acid sequence by one or more substitutions, additions, deletions, fusions and truncations, which may be present in any combination DESCRIPTION OF THE INVENTION
Fab I functions as the final step of the elongation cycle of bacteπal fatty acid biosynthesis The enzyme from S aureus, has been cloned, overexpressed in E colt and purified to >95% purity Kinetic studies revealed sequential addition of the substrates crotonoyl CoA and NADH over the broad optimal pH range of about or equal to 5 0-7 0 The lack of a demonstrable back reaction or product inhibition prevented discrimination between an ordered versus a random mechanism The Km for crotonoyl CoA was virtually identical to that of the E coli Fab I (about or equal to 2 5 mM), but the Km for NADH was approximately 30-fold higher (about or equal to 410 μM versus 13 μM for E colt) Studies using deuterated substrates revealed a stereochemical course of the Fab I reaction identical to that reported for the E colt enzyme the hydride is transferred from the B-face of the nicotinamide ring of NADH, and reduction occurs by a svn addition of hydrogen at the pro-
2R and pro-3S positions of the double bond of crotonoyl CoA Inhibition of S aureus Fab I by a series of eight fatty acyl CoA's from acetyl to palmitoyl increases with the chain length of the acyl group, suggesting that, like E. coli Fab I, S. aureus Fab I is subject to feedback inhibition by long chain acyl-ACP's. This characteristic presumably plays a key regulatory role in vivo. The present invention relates to novel FAB I polypeptides and polynucleotides, among other things, as described in greater detail below. In particular, the invention relates to polypeptides and polynucleotides of a novel FAB I gene of Staphylococcus aureus, which is related by amino acid sequence homology to Fab I enzymes from Mycobacteria (InhA),
H.influenzae, Brassica napus (rape seed) enoyl-ACP reductase, and to the E.coli Fab I protein.
The invention relates especially to FAB I having the nucleotide and amino acid sequences set out in Figure 17 [SΕQ ID NO: l ] and Figure 16 [SΕQ ID NO:2] respectively, and to the FAB I nucleotide sequences in NCIMB Deposit No. 40771 and amino acid sequences encoded therefrom, which is herein referred to as "the deposited clone" or as the "DNA of the deposited clone." It will be appreciated that the nucleotide and amino acid sequences set out in Figure 16 [SΕQ ID NO:2] and Figure 17 [SΕQ ID NO: l] were obtained by sequencing the FAB I DNA of the deposited clone. Hence, the sequence of the deposited clone is controlling as to any discrepancies between it (and the sequence it encodes) and the sequences of Figure 16 [SΕQ ID NO:2] or Figure 17 [SΕQ ID NO:l]. Polynucleotides
In accordance with one aspect of the present invention, there are provided isolated polynucleotides which encode the FAB I polypeptide having the deduced amino acid sequence offigure 16 [SΕQ ID NO:2].
Using the information provided herein, such as the polynucleotide sequence set out infigure 17 [SΕQ ID NO: l], a polynucleotide of the present invention encoding FAB I polypeptide may be obtained using standard cloning and screening procedures, such as those for cloning genomic DNAs from bacteria using Staphylococcus aureus WCUH29 cells as starting material. Illustrative of the invention, the polynucleotide set out infigure 17 [SΕQ ID NO: l ] was discovered in a genomic DNA library derived from Staphylococcus aureus WCUH29.
FAB I of the invention is structurally related to other proteins of the enoyl-ACP reductase family, as shown by the results of sequencing the genomic DNA encoding FAB I in the deposited clone. The DNA sequence thus obtained is set out infigure 17 [SΕQ ID NO: l]. It contains an open reading frame encoding a protein of about 256 amino acid residues with a deduced molecular weight of about 27 99 kDa The protein exhibits greatest homology to the E colt Fab I protein among known proteins FAB I offigure 16 [SEQ ID NO 2] has about 44% identity and about 64% similarity with the amino acid sequence of E coli enoyl (ACP) reductase (Fab I), SwissProt Acession Number P29132
Polynucleotides of the present invention may be in the form of RNA, such as mRNA, or in the form of DNA, including, for instance, cDNA and genomic DNA obtained by cloning or produced by chemical synthetic techniques or by a combination thereof The DNA may be double-stranded or single-stranded Single-stranded DNA may be the coding strand, also known as the sense strand, or it may be the non-coding strand, also referred to as the anti- sense strand
The coding sequence which encodes the polypeptide may be identical to the coding sequence of the polynucleotide shown infigure 17 [SEQ ID NO 1] It also may be a polynucleotide with a different sequence, which, as a result of the redundancy (degeneracy) of the genetic code, encodes the polypeptide of the DNA offigure 16 [SEQ ID NO 2]
Polynucleotides of the present invention which encode the polypeptide offigure 16 [SEQ ID NO 2] may include, but are not limited to the coding sequence for the mature polypeptide, by itself, the coding sequence for the mature polypeptide and additional coding sequences, such as those encoding a leader or secretory sequence, such as a pre-, or pro- or prepro- protein sequence, the coding sequence of the mature polypeptide, with or without the aforementioned additional coding sequences, together with additional, non-coding sequences, including for example, but not limited to non-coding 5' and 3' sequences, such as the transcπbed, non-translated sequences that play a role in transcπption (including termination signals, for example), πbosome binding, mRNA stability elements, and additional coding sequence which encode additional amino acids, such as those which provide additional functionalities Thus, for instance, the polypeptide may be fused to a marker sequence, such as a peptide, which facilitates punfication of the fused polypeptide In certain embodiments of this aspect of the invention, the marker sequence is a hexa-histidme as discussed, this should include other tagging approaches peptide, such as the tag provided in the pQE vector (Qiagen, Inc ), among others, many of which are commercially available As described in Gentz et al , (1989) Proc Natl Acad Sci , USA 86 821-824, for instance, hexa-histidme provides for convenient puπfication of the fusion protein The HA tag may also be used to create fusion proteins and conesponds to an epitope derived of influenza hemagglutinin protein, which has been described by Wilson et al , ( 1984) Cell 37 161 , for instance
In accordance with the foregoing, the term polynucleotide encoding a polypeptide as used herein encompasses polynucleotides which include a sequence encoding a polypeptide of the present invention, particularly staphylococcal FAB I ha ing the amino acid sequence set out infigure 16 [SEQ ID NO 2] The term encompasses polynucleotides that include a single continuous region or discontinuous regions encoding the polypeptide (for example, interrupted by integrated phage, insertion sequence, recombination, or editing) together with additional regions, that also may contain coding and/or non-coding sequences The present invention further relates to variants of the herein above descπbed polynucleotides which encode for fragments, analogs and derivatives of the polypeptide having the deduced amino acid sequence offigure 16 [SEQ ID NO 2] A vanant of the polynucleotide may be a naturally occurπng vanant such as a naturally occurnng allehc vanant, or it may be a variant that is not known to occur naturally Such non-naturally occurnng variants of the polynucleotide may be made by mutagenesis techniques, including those applied to polynucleotides, cells or organisms
Among vanants in this regard are vanants that differ from the aforementioned polynucleotides by nucleotide substitutions, deletions or additions The substitutions, deletions or additions may involve one or more nucleotides The variants may be altered in coding or non-coding regions or both Alterations in the coding regions may produce conservative or non-conservative amino acid substitutions, deletions or additions
Among the particularly preferred embodiments of the invention in this regard are polynucleotides encoding polypeptides having the amino acid sequence of FAB I set out infigure 16 [SEQ ID NO 2], vanants, analogs, deπvatives and fragments thereof, and fragments of the vanants, analogs and denvatives
Further particularly preferred in this regard are polynucleotides encoding FAB I vanants, analogs, denvatives and fragments, and vanants, analogs and denvatives of the fragments, which have the amino acid sequence of FAB I polypeptide offigure 16 [SEQ ID NO 2] in which several, a few, 5 to 10, 1 to 5, 1 to 3, 2, 1 or no amino acid residues are substituted, deleted or added, m any combination Especially preferred among these are silent substitutions, additions and deletions, which do not alter the properties and activities of FAB I Also especially preferred this regard are conservative substitutions Most highly preferred are polynucleotides encoding polypeptides having the amino acid sequence offigure 16 [SEQ ID NO;2]. without substitutions.
Further preferred embodiments of the invention are polynucleotides that are at least 70% identical to a polynucleotide encoding FAB I polypeptide having the amino acid sequence set out infigure 16 [SEQ ID NO:2], and polynucleotides which are complementary to such polynucleotides. Alternatively, most highly preferred are polynucleotides that comprise a region that is at least 80% identical to a polynucleotide encoding FAB I polypeptide encoded by the Staphylococcus aureus DNA of the deposited clone and polynucleotides complementary thereto or as set out infigure 17 [SEQ ID NO: l]. In this regard, polynucleotides at least 90% identical to the same are particularly preferred, and among these particularly prefeπed polynucleotides, those with at least 95% are especially preferred. Furthermore, those with at least 97% are highly prefened among those with at least 95%, and among these those with at least 98% and at least 99% are particularly highly preferred, with at least 99% being the more prefeπed. Particularly prefeπed embodiments in this respect, moreover, are polynucleotides which encode polypeptides which retain substantially the same biological function or activity as the mature polypeptide encoded by the DNA offigure 17 [SEQ ID NO: l].
The present invention further relates to polynucleotides that hybridize to the herein above-described sequences. In this regard, the present invention especially relates to polynucleotides which hybridize under stringent conditions to the herein above-described polynucleotides. As herein used, the term "stringent conditions" means hybridization will occur only if there is at least 95% and preferably at least 97% identity between the sequences.
As discussed additionally herein regarding polynucleotide assays of the invention, for instance, polynucleotides of the invention as discussed above, may be used as a hybridization probe for RNA, cDNA and genomic DNA to isolate full-length cDNAs and genomic clones encoding FAB I and to isolate cDNA and genomic clones of other genes that have a high sequence similarity to the FAB I gene. Such probes generally will comprise at least 15 bases. Preferably, such probes will have at least 30 bases and may have at least 50 bases. Particularly prefeπed probes will have at least 30 bases and will have 50 bases or less. For example, the coding region of the FAB I gene may be isolated by screening using the known DNA sequence to synthesize an oligonucleotide probe. A labeled oligonucleotide having a sequence complementary to that of a gene of the present invention is then used to screen a library of cDNA, genomic DNA or mRNA to determine which members of the library the probe hybndizes to
The polynucleotides and polypeptides of the present invention may be employed as research reagents and mateπals for discovery of treatments of and diagnostics for disease, particularly human disease, as further discussed herein relating to polynucleotide assays, inter alia
The polynucleotides of the invention that are oligonucleotides, including SEQ ID NOS 3 and 4, derived from the sequence of SEQ ID NO 1 may be used as PCR primers in the processes herein descπbed to determine whether or not the Staphylococcus aureus genes identified herein in whole or in part are transcribed in infected tissue The invention also provides that such sequences may also have utility in diagnosis of the stage of infection and type of infection the pathogen has attained
The polynucleotides may encode a polypeptide which is the mature protein plus additional amino or carboxyl-terminal am o acids, or amino acids inteπor to the mature polypeptide (when the mature form has more than one polypeptide chain, for instance) Such sequences may play a role in processing of a protein from precursor to a mature form, may allow protein transport, may lengthen or shorten protein half-life or may facilitate manipulation of a protein for assay or production, among other things As generally is the case in vivo, the additional amino acids may be processed away from the mature protein by cellular enzymes
A precursor protein, having the mature form of the polypeptide fused to one or more prosequences may be an inactive form of the polypeptide When prosequences are removed such inactive precursors generally are activated Some or all of the prosequences may be removed before activation Generally, such precursors are called proproteins In sum, a polynucleotide of the present invention may encode a mature protein, a mature protein plus a leader sequence (which may be refeπed to as a preprote ), a precursor of a mature protein having one or more prosequences which are not the leader sequences of a preprotem, or a preproprotem, which is a precursor to a proprotem, having a leader sequence and one or more prosequences, which generally are removed dunng processing steps that produce active and mature forms of the polypeptide Deposited materials
A deposit containing a Staphylococcus aureus WCUH 29 strain has been deposited with the National Collections of Industrial and Marine Bacteria Ltd (NCIMB), 23 St Machar Drive, Aberdeen AB2 1RY, Scotland on 1 1 September 1995 and assigned NCIMB Deposit No 40771 The FAB I clone deposit is referred to herein as the deposited clone" or as ' the DNA of the deposited clone "
The deposited matenal is a strain that contains the full length FAB I DNA, refeπed to as "NCIMB 40771 " upon deposit
The sequence of the polynucleotides contained in the deposited material, as well as the amino acid sequence of the polypeptide encoded thereby, are controlling in the event of any conflict with any descnption of sequences herein
The deposit has been made under the terms of the Budapest Treaty on the International Recognition of the Deposit of Micro-organisms for Purposes of Patent Procedure The strain will be lπevocably and without restnction or condition released to the public upon the issuance of a patent The deposit is provided merely as convenience to those of skill in the art and is not an admission that a deposit is required for enablement, such as that required under 35 U S C § 1 12 A license may be required to make, use or sell the deposited matenals, and no such license is hereby granted
Polypeptides The present invention further relates to a prokaryotic FAB I polypeptide which has the deduced ammo acid sequence offigure 16 [SEQ ID NO 2] The invention also relates to fragments, analogs and deπvatives of these polypeptides
The terms "fragment," "deπvative" and "analog" when refernng to the polypeptide offigure 16 [SEQ ID NO 2], means a polypeptide which retains essentially the same biological function or activity as such polypeptide Thus, an analog includes a proprotem which can be activated by cleavage of the proprotem portion to produce an active mature polypeptide The polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide or a synthetic polypeptide In certain prefeπed embodiments it is a recombinant polypeptide
The fragment, derivative or analog of the polypeptide offigure 16 [SEQ ID NO 2] may be (l) one in which one or more of the amino acid residues are substituted with a conserved or non-conserved ammo acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code, or (n) one in which one or more of the amino acid residues includes a substituent group, or (in) one in which the mature polypeptide is fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol). or (IV) one in which the additional amino acids are fused to the mature polypeptide, such as a leader or secretory sequence or a sequence which is employed for puπfication of the mature polypeptide or a proprotem sequence Such fragments, derivatives and analogs are deemed to be within the scope of those skilled in the art from the teachings herein
Among the particularly prefened embodiments of the invention in this regard are polypeptides having the amino acid sequence of FAB I set out infigure 16 [SEQ ID NO 2] vanants, analogs, derivatives and fragments thereof, and vanants. analogs and deπvatives of the fragments Alternatively, particularly prefeπed embodiments of the invention in this regard are polypeptides having the amino acid sequence of the FAB I, variants, analogs, deπvatives and fragments thereof, and vanants, analogs and deπvatives of the fragments
Among prefeπed vanants are those that vary from a reference by conservative amino acid substitutions Such substitutions are those that substitute a given amino acid in a polypeptide by another am o acid of like charactenstics Typically seen as conservative substitutions are the replacements, one for another, among the aliphatic amino acids Ala, Val, Leu and He, interchange of the hydroxyl residues Ser and Thr, exchange of the acidic residues Asp and Glu, substitution between the amide residues Asn and Gin, exchange of the basic residues Lys and Arg and replacements among the aromatic residues Phe, Tyr Further particularly prefeπed in this regard are vanants, analogs, denvatives and fragments, and vanants, analogs and deπvatives of the fragments, having the amino acid sequence of the FAB I polypeptide offigure 16 [SEQ ID NO 2] in which several, a few, 5 to 10, 1 to 5, 1 to 3, 2, 1 or no amino acid residues are substituted, deleted or added, in any combination Especially prefeπed among these are silent substitutions, additions and deletions, which do not alter the properties and activities of the FAB I Also especially prefeπed m this regard are conservative substitutions Most highly prefeπed are polypeptides having the amino acid sequence offigure 16 [SEQ ID NO 2] without substitutions
The polypeptides and polynucleotides of the present invention are preferably provided in an isolated form, and preferably are punfied to homogeneity The polypeptides of the present invention include the polypeptide of SEQ ID NO 2
(in particular the mature polypeptide) as well as polypeptides which have at least 80% identity to the polypeptide of SEQ ID NO 2 and more preferably at least 90% similaπty (more preferably at least 90% identity) to the polypeptide of SEQ ID NO 2 and still more preferably at least 95% similaπty (still more preferably at least 95% identity) to the polypeptide of SEQ ID NO 2 and also include portions of such polypeptides with such portion of the polypeptide generally containing at least 30 amino acids and more preferably at least 50 amino acids
Fragments or portions of the polypeptides of the present invention may be employed for producing the coπesponding full-length polypeptide by peptide synthesis, therefore, the fragments may be employed as intermediates for producing the full-length polypeptides Fragments or portions of the polynucleotides of the present invention may be used to synthesize full-length polynucleotides of the present invention Fragments Also among prefeπed embodiments of this aspect of the present invention are polypeptides comprising fragments of FAB I, most particularly fragments of FAB I having the amino acid set out infigure 16 [SEQ ID NO 2] and fragments of vanants and deπvatives of the FAB I offigure 16 [SEQ ID NO 2]
In this regard a fragment is a polypeptide having an amino acid sequence that entirely is the same as part but not all of the ammo acid sequence of the aforementioned FAB I polypeptides and vanants or derivatives thereof
Such fragments may be "free-standing," ; e , not part of or fused to other amino acids or polypeptides, or they may be comprised within a larger polypeptide of which they form a part or region When compnsed within a larger polypeptide, the presently discussed fragments most preferably form a single continuous region However, several fragments may be compnsed withm a single larger polypeptide For instance, certain prefeπed embodiments relate to a fragment of a FAB I polypeptide of the present compnsed within a precursor polypeptide designed for expression in a host and having heterologous pre and pro- polypeptide regions fused to the ammo terminus of the FAB I fragment and an additional region fused to the carboxyl terminus of the fragment Therefore, fragments in one aspect of the meaning intended herein, refers to the portion or portions of a fusion polypeptide or fusion protein denved from FAB I
Particularly preferred fragments include those encoded by amino acids 1-20, 21-40, 41-60, 61-80, 81-100, 101-120, 121-140, 141-160, 161-180, 181 -200, 201-220, 221-240, 241- 256 and contiguous combinations thereof
More particularly prefeπed fragments include, for example, those with high homology to other sequenced structurally related elements These include, for example, regions homologous to fragments of E coli fabl including about 13 to about 25, about 32 to about 39, about 1 12 to about 125, about 156 to about 196 and about 2 1 to about 252
Among especially prefeπed fragments of the invention are truncation mutants of FAB I Truncation mutants include FAB I polypeptides having the amino acid sequence offigure 16 [SEQ ID NO 2] or of vanants or deπvatives thereof, except for deletion of a continuous seπes of residues (that is, a continuous region, part or portion) that includes the amino terminus, or a continuous senes of residues that includes the carboxyl terminus or, as in double truncation mutants, deletion of two continuous seπes of residues, one including the amino terminus and one including the carboxyl terminus Fragments having the size ranges set out about also are prefeπed embodiments of truncation fragments, which are especially prefeπed among fragments generally Degradation products of the polypeptides of the invention in a host cell, particularly a staphylococcus, are also prefeπed polypeptides
Also prefeπed in this aspect of the invention are fragments charactenzed by structural or functional attπbutes of FAB I Prefeπed embodiments of the invention in this regard include fragments that compπse alpha-helix and alpha-helix forming regions ("alpha- regions"), beta-sheet and beta-sheet-forming regions ("beta-regions"), turn and turn-forming regions ("turn-regions"), coil and coil-formmg regions ("coil-regions"), hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions and high antigenic index regions of FAB I
Among the prefeπed fragments in this regard are those that compnse regions of FAB I that contain structural features, such as the features set out above. In this regard, the regions defined by the residues 1 to about 8, about 26 to about 34, about 39 to about 61, about 70 to about 80, about 95 to about 107, about 127 to about 139, about 162 to about 173 and about 207 to about 220 are charactenzed by amino acids believed to compnse alpha helix forming regions. Additionally, the regions defined by the residues about 9 to about 13, about 19 to about 25, about 62 to about 69, about 85 to about 95, about 141 to about 156. about 173 to about 188 about 220 to about 226, about 232 to about 240 are charactenzed by residues believed to compnse beta-sheet regions Also, the regions defined by the residues about 80 to about 85, about 108 to about 1 12, about 120 to about 125, about 178 to about 183, and about 198 to about 203 are characterized by regions behved to compπse turn regions. Also, the regions defined by the residues about 80 to about 85, about 105 to about 1 10. about 120 to about 125, about 138 to about 143, about 145 to about 153, about 175 to about 180, about 187 to about 192, and about 240 to about 245 are charactenzed by amino acid compositions believed to compπse coil regions. The regions defined by the residues about 9 to about 14, about 20 to about 36. about 86 to about 98. about 1 15 to about 129. about 138 to about 152, about 161 to about 179, about 185 to about 195, about 226 to about 240, about 249 to about 256 are charactenzed by hydrophobic amino acids Additionally, the regions defined by the residues about 2 to about 8, about 14 to about 19, about 36 to about 86, 98 to about 1 15, about 130 to about 138, about 152 to about 161, about 180 to about 186, about 206 to about 226, about 239 to and about 249 are characterized by hydrophilic amino acids Also, the regions defined by the residues about 20 to about 40, about 43 to about 62, about 72 to about 93, about 102 to about 117, about 128 to about 140, about 180 to about 220, and about 228 to about 240 are charactenzed by ammo acid residues believed to comprise an alpha amphipathic region and the regions defined by the residues about 1 to about 10, about 27 to about 50, about 60 to about 72, about 97 to about 1 10, about 158 to about 191, about 208 to about 220, and about 240 to about 252 are charactenzed by ammo acid residues believed to compnse a beta amphipathic region Additionally, the regions defined by the residues about 38 to 58, about 77 to about 89, about 98 to about 1 14, about 130 to about 142, about 190 to about 204, about 208 to about 230, and about 234 to about 246 are characteπstic of am o acid residues believed to compnse flexible regions. The regions defined by residues about 34 to about 62, about 98 to about 112 and about 206 to about 222 are believed to comrpise surface forming regions Additionally, the regions defined by residues about 3 to about 9, about 14 to about 20, about 25 to about 33, about 38 to about 60, about 65 to about 88, about 98 to about 1 14, about 120 to about 124, about 130 to about 140, about 178 to about 185, about 195 to about 228, and about 236 to about 247 are believed to compπse regions of high antigenicity Such regions set forth above may be compnsed within a larger polypeptide or may be by themselves a prefeπed fragment of the present invention, as discussed above It will be appreciated that the term "about" as used in this paragraph has the meaning set out above regarding fragments in general.
Further prefeπed regions are those that mediate activities of FAB I Most highly prefeπed in this regard are fragments that have a chemical, biological or other activity of FAB I, including those with a similar activity or an improved activity, or with a decreased undesirable activity Highly prefeπed m this regard are fragments that contain regions that are homologs in sequence, or in position, or in both sequence and to active regions of related polypeptides, such as the related polypeptides set out infigure 16 [SEQ ID NO.2], which include E coli Enoyl (ACP) reductase, Fab I Among particularly prefeπed fragments in these regards are truncation mutants, as discussed above Further prepfeπed polynucelotide fragments are those that are antigenic or lmmunogenic in an animal, especially in a human
It will be appreciated that the invention also relates to, among others, polynucleotides encoding the aforementioned fragments, polynucleotides that hybπdize to polynucleotides encoding the fragments, particularly those that hybndize under stnngent conditions, and polynucleotides, such as PCR pπmers, for amplifying polynucleotides that encode the fragments In these regards, prefeπed polynucleotides are those that coπespondent to the prefeπed fragments, as discussed above Vectors, host cells, expression The present invention also relates to vectors which comprise a polynucleotide or polynucleotides of the present invention, host cells which are genetically engineered with vectors of the invention and the production of polypeptides of the invention by recombinant techniques
Host cells can be genetically engineered to incorporate polynucleotides and express polypeptides of the present invention For instance, polynucleotides may be introduced into host cells using well known techniques of infection, transduction, transfection, transvection and transformation The polynucleotides may be introduced alone or with other polynucleotides Such other polynucleotides may be introduced independently, co-introduced or introduced joined to the polynucleotides of the invention Thus for instance, the polynucleotides may be joined to a vector containing a selectable marker for propagation in a host The vector construct may be introduced into host cells by the aforementioned techniques Generally, a plasmid vector is introduced as DNA in a precipitate, such as a calcium phosphate precipitate, or in a complex with a charged hpid Electroporation also may be used to introduce polynucleotides into a host If the vector is a virus, it may be packaged m vitro or introduced into a packaging cell and the packaged virus may be transduced into cells A wide vanety of techniques suitable for making polynucleotides and for introducing polynucleotides into cells in accordance with this aspect of the invention are well known and routine to those of skill the art Such techniques are reviewed at length in Sambrook et al cited above, which is illustrative of the many laboratory manuals that detail these techniques In accordance with this aspect of the invention the vector may be, for example, a plasmid vector, a single or double-stranded phage vector, a single or double-stranded RNA or DNA viral vector Such vectors may be introduced into cells as polynucleotides, preferably DNA, by well known techniques for introducing DNA and RNA into cells The vectors, in the case of phage and viral vectors also may be and preferably are introduced into cells as packaged or encapsidated virus by well known techniques for infection and transduction Viral vectors may be replication competent or replication defective In the latter case viral propagation generally will occur only in complementing host cells
Prefeπed among vectors, in certain respects, are those for expression of polynucleotides and polypeptides of the present invention Generally, such vectors compπse cis-actmg control regions effective for expression in a host operatively linked to the polynucleotide to be expressed Appropnate trans-acting factors either are supplied by the host, supplied by a complementing vector or supplied by the vector itself upon introduction
In certain prefeπed embodiments in this regard, the vectors provide for specific expression Such specific expression may be inducible expression or expression only in certain types of cells or both inducible and cell-specific Particularly prefeπed among inducible vectors are vectors that can be induced for expression by environmental factors that are easy to manipulate, such as temperature and nutπent additives A vanety of vectors suitable to this aspect of the invention, including constitutive and inducible expression vectors for use in prokaryotic and eukaryotic hosts, are well known and employed routinely by those of skill in the art The engineered host cells can be cultured m conventional nutnent media, which may be modified as appropnate for, inter alia, activating promoters, selecting transformants or amplifying genes Culture conditions, such as temperature, pH and the like, previously used with the host cell selected for expression generally will be suitable for expression of polypeptides of the present invention as will be apparent to those of skill in the art A great vaπety of expression vectors can be used to express a polypeptide of the invention Such vectors include, among others, chromosomal, episomal and virus-deπved vectors, e g , vectors denved from bacteπal plasmids, from bacteπophage, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses such as baculoviruses, papova viruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors deπved from combinations thereof, such as those deπved from plasmid and bacteπophage genetic elements, such as cosmids and phagemids, all may be used for expression in accordance with this aspect of the present invention Generally, any vector suitable to maintain, propagate or express polynucleotides to express a polypeptide in a host may be used for expression in this regard
The appropnate DNA sequence may be inserted into the vector by any of a vanety of well-known and routine techniques In general, a DNA sequence for expression is joined to an expression vector by cleaving the DNA sequence and the expression vector with one or more restnction endonucleases and then joining the restriction fragments together using T4 DNA ligase Procedures for restnction and ligation that can be used to this end are well known and routine to those of skill Suitable procedures in this regard, and for constructing expression vectors using alternative techniques, which also are well known and routine to those skill, are set forth m great detail in Sambrook et al , (1989) MOLECULAR CLONING, A LABORATORY MANUAL, 2nd Ed , Cold Spπng Harbor Laboratory Press, Cold Spnng Harbor, New York
The DNA sequence in the expression vector is operatively linked to appropπate expression control sequence(s), including, for instance, a promoter to direct mRNA transcnption Representatives of such promoters include the phage lambda PL promoter, the E coli lac, tip and tac promoters, the SV40 early and late promoters and promoters of retroviral LTRs, to name just a few of the well-known promoters It will be understood that numerous promoters not mentioned are suitable for use in this aspect of the invention are well known and readily may be employed by those of skill in the manner illustrated by the discussion and the examples herein
In general, expression constructs will contain sites for transcπption initiation and termination, and, in the transcribed region, a nbosome binding site for translation The coding portion of the mature transcπpts expressed by the constructs will include a translation initiating AUG at the beginning and a termination codon appropπately positioned at the end of the polypeptide to be translated
In addition, the constructs may contain control regions that regulate as well as engender expression Generally, in accordance with many commonly practiced procedures, such regions will operate by controlling transcnption, such as transcription factors, repressor binding sites and termination, among others Vectors for propagation and expression generally will include selectable markers
Such markers also may be suitable for amplification or the vectors may contain additional markers for this purpose In this regard, the expression vectors preferably contain one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells Prefeπed markers include dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, and tetracychne or ampicilhn resistance genes for cultunng E coli and other prokaryotes
The vector containing the appropπate DNA sequence as descπbed elsewhere herein, as well as an appropπate promoter, and other appropπate control sequences, may be introduced into an appropnate host using a vaπety of well known techniques suitable to expression therein of a desired polypeptide Representative examples of appropnate hosts include bacterial cells, such as streptocooi, staphylococci, £ colt, streptomyces and Salmonella typhtmurium cells, fungal cells, such as yeast cells and Aspergtllus cells, insect cells such as Drosophila S2 and Spodoptera Sf9 cells, animal cells such as CHO, COS and Bowes melanoma cells, and plant cells Hosts for of a great vaπety of expression constructs are well known, and those of skill will be enabled by the present disclosure readily to select a host for expressing a polypeptides in accordance with this aspect of the present invention
More particularly, the present invention also includes recombinant constructs, such as expression constructs, compπsing one or more of the sequences described above The constructs compnse a vector, such as a plasmid or viral vector, into which such a sequence of the invention has been inserted The sequence may be inserted in a forward or reverse onentation In certain prefeπed embodiments in this regard, the construct further compnses regulatory sequences, including, for example, a promoter, operably linked to the sequence Large numbers of suitable vectors and promoters are known to those of skill in the art, and there are many commercially available vectors suitable for use in the present invention
The following vectors, which are commercially available, are provided by way of example Among vectors prefeπed for use in bacteπa are pQE70, pQE60 and pQE-9, available from Qiagen, pBS vectors, Phagescπpt vectors, Bluescπpt vectors, pNH8A, pNHlόa, pNHl 8A, pNH46A, available from Stratagene, and ptrc99a, pKK223-3, pKK233-3, pDR540, pRTT5 available from Pharmacia Among prefeπed eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXTl and pSG available from Stratagene, and pSVK3, pBPV, pMSG and pSVL available from Pharmacia These vectors are listed solely by way of illustration of the many commercially available and well known vectors that are available to those of skill in the art for use in accordance with this aspect of the present invention It will be appreciated that any other plasmid or vector suitable for, for example, introduction, maintenance, propagation or expression of a polynucleotide or polypeptide of the invention in a host may be used in this aspect of the invention Promoter regions can be selected from any desired gene using vectors that contain a reporter transcription unit lacking a promoter region, such as a chloramphenicol acetyl transferase ("CAT") transcπption unit, downstream of restnction site or sites for introducing a candidate promoter fragment, i e , a fragment that may contain a promoter As is well known, introduction into the vector of a promoter-containing fragment at the restnction site upstream of the cat gene engenders production of CAT activity, which can be detected by standard CAT assays Vectors suitable to this end are well known and readily available Two such vectors are pKK232-8 and pCM7 Thus, promoters for expression of polynucleotides of the present invention include not only well known and readily available promoters, but also promoters that readily may be obtained by the foregoing technique, using a reporter gene
Among known prokaryotic promoters suitable for expression of polynucleotides and polypeptides in accordance with the present invention are the E colt lad and lacZ and promoters, the T3 and T7 promoters, the gpt promoter, the lambda PR, PL promoters and the tip promoter Among known eukaryotic promoters suitable in this regard are the CMV immediate early promoter, the HSV thymidine kinase promoter, the early and late SV40 promoters, the promoters of retroviral LTRs, such as those of the Rous sarcoma virus ("RSV"), and metallothionein promoters, such as the mouse metallothιoneιn-I promoter
Selection of appropnate vectors and promoters for expression in a host cell is a well known procedure and the requisite techniques for expression vector construction, introduction of the vector into the host and expression in the host are routine skills in the art
The present invention also relates to host cells containing the above-descnbed constructs discussed above The host cell can be a higher eukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell, such as a yeast cell, or the host cell can be a prokaryotic cell, such as a bactenal cell
Introduction of the construct into the host cell can be affected by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic hpid-mediated transfection, electroporation, transduction, scrape loading, ballistic introduction, infection or other methods Such methods are descnbed in many standard laboratory manuals, such as Davis et al, (1986) BASIC METHODS IN MOLECULAR BIOLOGY, and Sambrook et al , (1989) MOLECULAR CLONING A LABORATORY MANUAL, 2nd Ed , Cold Spring Harbor Laboratory Press, Cold Spπng Harbor, N Y Constructs in host cells can be used in a conventional manner to produce the gene product encoded by the recombinant sequence. Alternatively, the polypeptides of the invention can be synthetically produced by conventional peptide synthesizers.
Mature proteins can be expressed in mammalian cells, yeast, bacteria, or other cells under the control of appropriate promoters. Cell-free translation systems can also be employed to produce such proteins using RNAs derived from the DNA constructs of the present invention. Appropriate cloning and expression vectors for use with prokaryotic and eukaryotic hosts are described by Sambrook et al., (1989) MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y..
Generally, recombinant expression vectors will include origins of replication, a promoter derived from a highly-expressed gene to direct transcription of a downstream structural sequence, and a selectable marker to permit isolation of vector containing cells after exposure to the vector. Among suitable promoters are those derived from the genes that encode glycolytic enzymes such as 3-phosphogIycerate kinase ("PGK"), a-factor, acid phosphatase, and heat shock proteins, among others. Selectable markers include the ampicillin resistance gene of E. coli and the trpl gene of 5. cerevisiae.
Transcription of the DNA encoding the polypeptides of the present invention by higher eukaryotes may be increased by inserting an enhancer sequence into the vector. Enhancers are cw-acting elements of DNA, usually about from 10 to 300 bp that act to increase transcriptional activity of a promoter in a given host cell-type. Examples of enhancers include the SV40 enhancer, which is located on the late side of the replication origin at bp 100 to 270, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovims enhancers. Polynucleotides of the invention, encoding the heterologous structural sequence of a polypeptide of the invention generally will be inserted into the vector using standard techniques so that it is operably linked to the promoter for expression. The polynucleotide will be positioned so that the transcription start site is located appropriately 5' to a ribosome binding site. The ribosome binding site will be 5' to the AUG that initiates translation of the polypeptide to be expressed. Generally, there will be no other open reading frames that begin with an initiation codon, usually AUG, and lie between the ribosome binding site and the initiating AUG. Also, generally, there will be a translation stop codon at the end of the polypeptide and there will be a polyadenylation signal and a transcription termination signal appropriately disposed at the 3' end of the transcribed region.
For secretion of the translated protein into the lumen of the endoplasmic reticulum, into the periplasmic space or into the extracellular environment, appropriate secretion signals may be incorporated into the expressed polypeptide. These signals may be endogenous to the polypeptide or they may be heterologous signals.
The polypeptide may be expressed in a modified form, such as a fusion protein, and may include not only secretion signals but also additional heterologous functional regions. Thus, for instance, a region of additional amino acids, particularly charged amino acids, may be added to the N-terminus of the polypeptide to improve stability and persistence in the host cell, during purification or during subsequent handling and storage. Also, region also may be added to the polypeptide to facilitate purification. Such regions may be removed prior to final preparation of the polypeptide. The addition of peptide moieties to polypeptides to engender secretion or excretion, to improve stability or to facilitate purification, among others, are familiar and routine techniques in the art. A prefeπed fusion protein comprises a heterologous region from immunolglobulin that is useful to solubilize or purify polypeptides. For example, EP-A-O 464 533 (Canadian counterpart 2045869) discloses fusion proteins comprising various portions of constant region of immunoglobin molecules together with another human protein or part thereof. In many cases, the Fc part in fusion protein is thoroughly advantageous for use in therapy and diagnosis and thus results, for example, in improved pharmacokinetic properties (see EP-A 0232 262). On the other hand, for some uses it would be desirable to be able to delete the Fc part after the fusion protein has been expressed, detected and purified in the advantageous manner described. This is the case when Fc portion proves to be a hindrance to use in assays, therapy or diagnosis, for example when the fusion protein is to be used as antigen for immunizations. In drug discovery, for example, human proteins, such as, shIL5- has been fused with Fc portions for the purpose of high-throughput screening assays to identify antagonists of hIL-5. See, D. Bennett et al, (1995) Journal of Molecular Recognition, Vol. 8 52-58 and K. Johanson et al, ( 1995) The Journal of Biological Chemistry, Vol. 270, No. 16, pp 9459-9471. Suitable prokaryotic hosts for propagation, maintenance or expression of polynucleotides and polypeptides in accordance with the invention include streptococci, Escherichia coli, Bacillus subtilis and Salmonella typhimurium. Various species of Pseudomonas, Streptomvces, and Staphylococcus are also suitable hosts in this regard Moreover, many other hosts also known to those of skill may be employed in this regard
As a representative but non-limiting example, useful expression vectors for bactenal use can compπse a selectable marker and bacteπal oπgm of replication deπved from commercially available plasmids comprising genetic elements of the well known cloning vector pBR322 (ATCC 37017) Such commercial vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and GEM 1 (Promega Biotec, Madison, WI, USA) These pBR322 "backbone" sections are combined with an appropnate promoter and the structural sequence to be expressed Following transformation of a suitable host strain and growth of the host strain to an appropriate cell density, where the selected promoter is inducible it is induced by appropnate means (e -; , temperature shift or exposure to chemical inducer) and cells are cultured for an additional period
Cells typically then are harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification
Microbial cells employed in expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents, such methods are well know to those skilled in the art
Vaπous mammalian cell culture systems can be employed for expression, as well Examples of mammalian expression systems include the COS-7 lines of monkey kidney fibroblast, described in Gluzman et al, ( 1981 ) Cell 23 175 Other cell lines capable of expressing a compatible vector include for example, the C127, 3T3, CHO, HeLa, human kidney 293 and BHK cell lines
Mammalian expression vectors may comprise an origin of replication, a suitable promoter and enhancer, and also any necessary ribosome binding sites, polyadenylation sites, splice donor and acceptor sites, transcnptional termination sequences, and 5' flanking non- transcπbed sequences that are necessary for expression In certain prefeπed embodiments in this regard DNA sequences denved from the SV40 splice sites, and the SV40 polyadenylation sites are used for required non-transcribed genetic elements of these types FAB I polypeptide can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography Most preferably, high performance liquid chromatography ("HPLC") is employed for punfication Well known techniques for refolding protein may be employed to regenerate active conformation when the polypeptide is denatured during isolation and or purification this invention also provides the use of anti-FAB I antibodies for detection of FAB I duπng punfication
Polypeptides of the present invention include naturally purified products, products of chemical synthetic procedures, and products produced by recombinant techniques from a prokaryotic or eukaryotic host, including, for example, bacterial, yeast, higher plant insect and mammalian cells Depending upon the host employed in a recombinant production procedure, the polypeptides of the present invention may be glycosylated or may be non- glycosylated In addition, polypeptides of the invention may also include an initial modified methionine residue, in some cases as a result of host-mediated processes
FAB I polynucleotides and polypeptides may be used in accordance with the present invention for a vanety of applications, particularly those that make use of the chemical and biological properties of FAB I Additional applications relate to diagnosis and to treatment of disorders of cells, tissues and organisms These aspects of the invention are illustrated further by the following discussion
Polynucleotide assays
This invention is also related to the use of the FAB I polynucleotides to detect complementary polynucleotides such as, for example, as a diagnostic reagent Detection of FAB I in a eukaryote, particularly a mammal, and especially a human, will provide a diagnostic method that can add to, define or allow a diagnosis of a disease Eukaryotes (herein also "ιndιvιdual(s)"), particularly mammals, and especially humans, carrying a FAB I gene may be detected at the DNA level by a vaπety of techniques Nucleic acids for diagnosis may be obtained from an individual's cells and tissues, such as bone, blood, muscle, cartilage, and skin Tissue biopsy and autopsy matenal is also prefeπed for samples from an individual to use in a diagnostic assay Genomic DNA may be used directly for detection or may be amplified enzymatically by using PCR pπor to analysis PCR (Saiki et al , (1986) Nature, 324 163-166) RNA or cDNA may also be used in the same ways As an example, PCR primers complementary to the nucleic acid encoding FAB I can be used to identify and analyze FAB I presence and expression Using PCR, characteπzation of the strain of prokaryote present in a eukaryote, particularly a mammal, and especially a human, may be made by an analysis of the genotype of the prokaryote gene For example, deletions and insertions can be detected by a change in size of the amplified product in comparison to the genotype of a reference sequence. Point mutations can be identified by hybridizing amplified DNA to radiolabeled FAB I RNA or alternatively, radiolabeled FAB I antisense DNA sequences. Perfectly matched sequences can be distinguished from mismatched duplexes by RNase A digestion or by differences in melting temperatures.
Sequence differences between a reference gene and genes having mutations also may be revealed by direct DNA sequencing. In addition, cloned DNA segments may be employed as probes to detect specific DNA segments. The sensitivity of such methods can be greatly enhanced by appropriate use of PCR or another amplification method. For example, a sequencing primer is used with double-stranded PCR product or a single-stranded template molecule generated by a modified PCR. The sequence determination is performed by conventional procedures with radiolabeled nucleotide or by automatic sequencing procedures with fluorescent-tags.
Genetic testing based on DNA sequence differences may be achieved by detection of alteration in electrophoretic mobility of DNA fragments in gels, with or without denaturing agents. Small sequence deletions and insertions can be visualized by high resolution gel electrophoresis. DNA fragments of different sequences may be distinguished on denaturing formamide gradient gels in which the mobilities of different DNA fragments are retarded in the gel at different positions according to their specific melting or partial melting temperatures (see, e.g., Myers et al., (1985) Science, 230: 1242).
Sequence changes at specific locations also may be revealed by nuclease protection assays, such as RNase and S I protection or the chemical cleavage method (e.g.. Cotton et al., (1985) Proc. Natl. Acad. Sci., USA, 85: 4397-4401).
Thus, the detection of a specific DNA sequence may be achieved by methods such as hybridization, RNase protection, chemical cleavage, direct DNA sequencing or the use of restriction enzymes, (e.g., restriction fragment length polymorphisms ("RFLP") and Southern blotting of genomic DNA.
In addition to more conventional gel-electrophoresis and DNA sequencing, mutations also can be detected by in situ analysis. A mutation may be ascertained for example, by a DNA sequencing assay. Samples are processed by methods known in the art to capture the RNA. First strand cDNA is synthesized from the RNA samples by adding an oligonucleotide primer consisting of sequences which hybridize to a region on the mRNA. Reverse transcriptase and deoxv nucleotides are added to allow synthesis of the first strand cDNA Pπmer sequences are synthesized based on the DNA sequence of the FAB I protein of the invention The primer sequence is generally comprised of at least 15 consecutive bases and may contain at least 30 or even 50 consecutive bases Cells carrying mutations in the gene of the present invention may also be detected at the DNA level by a vaπety of techniques, to allow for serotyping, for example Nucleic acids for diagnosis may be obtained from an individual s cells or bodily fluids, including but not limited to blood, urine, saliva, tissue biopsy and autopsy material The genomic DNA may be used directly for detection or may be amplified enzymatically by using PCR (Saiki et al , ( 1986) Nature, 324 163-166) prior to analysis RT-PCR can also be used to detect mutations It is particularly prefeπed to used RT-PCR in conjunction with automated detection systems, such as, for example, GeneScan RNA or cDNA may also be used for the same purpose, PCR or RT-PCR As an example, PCR pπmers complementary to the nucleic acid encoding FAB I can be used to identify and analyze mutations Examples of representative pnmers are shown below in Table 1 For example, deletions and insertions can be detected by a change in size of the amplified product in companson to the normal genotype Point mutations can be identified by hybridizing amplified DNA to radiolabeled RNA or alternatively, radiolabeled antisense DNA sequences Perfectly matched sequences can be distinguished from mismatched duplexes by RNase A digestion or by differences in melting temperatures Table 1
Primers used for detection of mutations in FAB I gene SEQ ID NO: SEQUENCE
3 5-CGCCTCGAGATGTTAAATCTTGAAAACAAAACATATGTC-3'
4 5 -CGCGGATCCAATCAAGTCAGGTTGAAATATCCA-3' The above pπmers may be used for amplifying FAB I DNA isolated from a sample deπved from an individual The invention also provides the primers of Table 1 with 1, 2, 3 or 4 nucleotides removed from the 5' and/or the 3' end The pπmers may be used to amplify the gene isolated from the individual such that the gene may then be subject to vaπous techniques for elucidation of the DNA sequence In this way, mutations in the DNA sequence may be determined, for example, to ascertain the serotype of the organism
Sequence differences between the reference gene and genes having mutations may be revealed by the direct DNA sequencing method In addition, cloned DNA segments may be employed as probes to detect specific DNA segments The sensitivity of this method is greatly enhanced when combined with PCR For example, a sequencing pπmer is used with double-stranded PCR product or a single-stranded template molecule generated by a modified PCR The sequence determination is performed by conventional procedures with radiolabeled nucleotide or by automatic sequencing procedures with fluorescent-tags Genetyping based on DNA sequence differences may be achieved by detection of alteration in electrophoretic mobility of DNA fragments in gels with or without denatuπng agents Small sequence deletions and insertions can be visualized by high resolution gel electrophoresis DNA fragments of different sequences may be distinguished on denatunng formamide gradient gels in which the mobilities of different DNA fragments are retarded in the gel at different positions according to their specific melting or partial melting temperatures (see, e g Myers et al , (\ 985) Science, 230 1242)
Sequence changes at specific locations may also be revealed by nuclease protection assays, such as RNase and S 1 protection or the chemical cleavage method (e g , Cotton et al ,
(1985) PNAS USA, 85 4397-4401 ) Thus, the detection of a specific DNA sequence and/or quantitation of the level of the sequence may be achieved by methods such as hybridization, RNase protection, chemical cleavage, direct DNA sequencing or the use of restnction enzymes, (e g , Restnction Fragment
Length Polymorphisms (RFLP)) and Southern blotting of genomic DNA The invention provides a process for diagnosing disease, particularly bacterial infections, and most particularly staphylococcal infections, comprising determining from a sample derived from an individual an increased level of expression of polynucleotide having the sequence offigure 17 [SEQ ID NO 1 ] Increased expression of polynucleotide can be measured using any on of the methods well known in the art for the quantation of polynucleotides, such as, for example, PCR, RT-PCR, RNase protection. Northern blotting and other hybridization methods
In addition to more conventional gel-electrophoresis and DNA sequencing, mutations can also be detected by in situ analysis
Polypeptide assays
The present invention also relates to a diagnostic assays such as quantitative and diagnostic assays for detecting levels of FAB I protein in cells and tissues, including determination of normal and abnormal levels Thus, for instance, a diagnostic assay in accordance with the invention for detecting a pathological expression level of FAB I protein compared to normal control tissue samples may be used to detect the presence of bacteπal infection, for example Assay techniques that can be used to determine levels of a protein, such as an FAB I protein of the present invention, in a sample deπved from a host are well- known to those of skill in the art Such assay methods include radioimmunoassays, competitive-binding assays Western Blot analysis and ELISA assavs Among these ELISAs frequently are prefeπed An ELISA assay initially comprises prepaπng an antibody specific to FAB I, preferably a monoclonal antibody In addition a reporter antibody generally is prepared which binds to the monoclonal antibody The reporter antibody is attached a detectable reagent such as radioactive, fluorescent or enzvmatic reagent, in this example horseradish peroxidase enzyme To carry out an ELISA a sample is removed from a host and incubated on a solid support, e g , a polystyrene dish, that binds the proteins in the sample Any free protein binding sites on the dish are then covered by incubating with a non-specific protein such as bovine serum albumin Next, the monoclonal antibody is incubated in the dish duπng which time the monoclonal antibodies attach to any FAB I proteins attached to the polystyrene dish Unbound monoclonal antibody is washed out with buffer The reporter antibody linked to horseradish peroxidase is placed in the dish resulting in binding of the reporter antibody to any monoclonal antibody bound to FAB I Unattached reporter antibody is then washed out Reagents for peroxidase activity, including a colonmetπc substrate are then added to the dish Immobilized peroxidase, linked to FAB I through the pnmary and secondary antibodies, produces a colored reaction product The amount of color developed in a given time penod indicates the amount of FAB I protein present in the sample Quantitative results typically are obtained by reference to a standard curve
A competition assay may be employed wherein antibodies specific to FAB I attached to a solid support and labeled FAB I and a sample derived from form an individual are passed over the solid support and the amount of label detected attached to the solid support can be coπelated to a quantity of FAB I in the sample Antibodies
The polypeptides, their fragments or other derivatives, or analogs thereof, or cells expressing them can be used as an immunogen to produce antibodies thereto These antibodies can be, for example, polyclonal or monoclonal antibodies The present invention also includes chimeric, single chain, and humanized antibodies, as well as Fab fragments, or the product of an Fab expression library Vanous procedures known in the art may be used for the production of such antibodies and fragments Antibodies generated against the polypeptides coπesponding to a sequence of the present invention can be obtained by direct injection of the polypeptides into an individual or by administeπng the polypeptides to an individual, preferably a nonhuman The antibody so obtained will then bind the polypeptides itself In this manner, even a sequence encoding only a fragment of the polypeptides can be used to generate antibodies binding the whole native polypeptides Such antibodies can then be used to isolate the polypeptide from tissue expressing that polypeptide
For preparation of monoclonal antibodies, any technique which provides antibodies produced by continuous cell line cultures can be used Examples include the hybndoma technique (Kohler, G et al , (19 '5) Nature 256 495-497, the trioma technique, the human B- cell hybndoma technique (Kozbor et al , ( 1983) Immunology Today 4 72 and the EBV- hybπdoma technique to produce human monoclonal antibodies (Cole et al, (1985) in Monoclonal Antibodies And Cancer Therapy , Alan R Liss, Inc pp 77-96
Techniques described for the production of single chain antibodies (U S Patent No 4,946,778) can be adapted to produce single chain antibodies to immunogenic polypeptide products of this invention Also, transgenic mice, or other organisms such as other mammals, may be used to express humanized antibodies to immunogenic polypeptide products of this invention
Polypeptide derivatives include antigemcally or immunologically equivalent derivatives which form a particular aspect of this invention
The term "antigemcally equivalent derivative" as used herein encompasses a polypeptide or its equivalent which will be specifically recognised by certain antibodies which, when raised to the protein or polypeptide according to the present invention, interfere with the immediate physical interaction between pathogen and mammalian host The term "immunologically equivalent derivative" as used herein encompasses a peptide or its equivalent which when used in a suitable formulation to raise antibodies in a vertebrate, the antibodies act to interfere with the immediate physical interaction between pathogen and mammalian host
The polypeptide, such as an antigemcally or immunologically equivalent derivative or a fusion protein thereof is used as an antigen to immunize a mouse or other animal such as a rat or chicken The fusion protein may provide stability to the polypeptide The antigen may be associated, for example by conjugation , with an immunogenic earner protein for example bovine serum albumin (BSA) or keyhole limpet haemocyanin (KLH) Alternatively a multiple antigemc peptide comprising multiple copies of the the protein or polypeptide, or an antigemcally or immunologically equivalent polypeptide thereof may be sufficiently antigemc to improve immunogenicity so as to obviate the use of a earner Using the procedure of Kohler et al, ( 1975) Nature 256 , 495-497), antibody- containing cells from the immunised mammal are fused with myeloma cells to create hybndoma cells secreting monoclonal antibodies
The hybπdomas are screened to select a cell line with high binding affinity and favorable cross reaction with other staphylococcal species using one or more of the original polypeptide and/or the fusion protein The selected cell line is cultured to obtain the desired Mab
Hybndoma cell lines secreting the monoclonal antibody are another aspect of this invention
Alternatively phage display technology could be utilised to select antibody genes with binding activities towards the polypeptide either from repertoires of PCR amplified v- genes of lymphocytes from humans screened for possessing anti-Fbp or from naive libraries (McCafferty, J et al , (1990), Nature 348, 552-554, Marks, J et al , ( 1992) Biotechnology 10, 779-783) The affinity of these antibodies can also be improved by chain shuffling (Clackson, T et al , ( 1991) Nature 352, 624-628) The antibody should be screened again for high affinity to the polypeptide and/or fusion protein
The above-described antibodies may be employed to isolate or to identify clones expressing the polypeptide or purify the polypeptide of the present invention by attachment of the antibody to a solid support for isolation and/or purification by affinity chromatography The polypeptide may be used as an antigen for vaccination of a host to produce specific antibodies which protect against invasion of bacteria, for example by blocking adherence of bacteria to damaged tissue Examples of tissue damage include wounds in skin or connective tissue caused, e g , by mechanical, traumatic, chemical or thermal damage or by implantation of indwelling devices, or wounds in the mucous membranes, such as the mouth, mammary glands, urethra or vagina
The polypeptides or cells expressing them can be used as an immunogen to produce antibodies thereto These antibodies can be, for example, polyclonal or monoclonal antibodies The term antibodies also includes chimeric, single chain, and humanized antibodies, as well as Fab fragments, or the product of an Fab expression library Various procedures known in the art may be used for the production of such antibodies and fragments Antibodies generated against the polypeptides of the present invention can be obtained by direct injection of the polypeptides into an individual or by administering the polypeptides to an individual, preferably a nonhuman The antibody so obtained will then bind the polypeptides itself In this manner, even a sequence encoding only a fragment of the polypeptides can be used to generate antibodies binding the whole native polypeptides Such antibodies can then be used to isolate the polypeptide from tissue expressing that polypeptide
As mentioned above, a fragment of the final antibody may be prepared
The antibody may be either intact antibody of Mr approx 150,000 or a derivative of it, for example a Fab fragment or a Fv fragment as described in Skeπa, A and Pluckthun, A ( 1988) Science 240 1038- 1040 If two antigen binding domains are present each domain may be directed against a different epitope - termed T-uspecific' antibodies
The antibody of the invention may be prepared by conventional means for example by established monoclonal antibody technology (Kohler, G et al (1975), Nature 256, 495- 497) or using recombinant means e g combinatorial libraries, for example as described in Huse, W D et al , ( 1989) Science 246, 1275- 1281
Preferably the antibody is prepared by expression of a DNA polymer encoding said antibody in an appropriate expression system such as described above for the expression of polypeptides of the invention The choice of vector for the expression system will be determined in part by the host, which may be a prokaryotic cell, such as E colt (preferably strain B) or Streptomyces sp or a eukaryotic cell, such as a mouse C127, mouse myeloma, human HeLa, Chinese hamster ovary, filamentous or unicellular fungi or insect cell The host may also be a transgenic animal or a transgenic plant, for example as described in Hiatt, A et al, ( 1989) Nature 34, 76-78 Suitable vectors include plasmids, bacteπophages, cosmids and recombinant viruses, derived from, for example, baculoviruses and vaccinia The Fab fragment may also be prepared from its parent monoclonal antibody by enzyme treatment, for example using papain to cleave the Fab portion from the Fc portion Preferably the antibody or derivative thereof is modified to make it less immunogenic in the individual For example, if the individual is human the antibody may most preferably be "humanised' , where the complimentaπty determining regιon(s) of the hybπdoma-deπved antibody has been transplanted into a human monoclonal antibody , for example as described in Jones, P et al (1986), Nature 321, 522-525 or Tempest et al , ( 1991 ) Biotechnology 9, 266-273
The modification need not be restricted to one of Tiumanisation' other primate sequences (for example Newman, R et al , 1992, Biotechnology 10, 1455- 1460) may also be used The humanised monoclonal antibody, or its fragment having binding activity, form a particular aspect of this invention
This invention provides a method of screening drugs to identify those which interfere with the interaction of the FAB I protein or active fragment to mammalian cells, the method comprising incubating a mammalian cell or membrane preparation with labeled polypeptide in the presence of the drug and measuring the ability of the drug to block this interaction
The use of a polynucleotide of the invention in genetic immunization will preferably employ a suitable delivery method such as direct injection of plasmid DNA into muscles (Wolff et al , ( 1992) Hum Mol Genet, 1 363, Manthorpe et al , (1963) Hum Gene Ther 4, 419), delivery of DNA complexed with specific protein carriers ( Wu, et al, (1989) J Biol Chem 264, 16985), coprecipitation of DNA with calcium phosphate (Benvenisty & Reshef, PNAS, 1986 83,9551), encapsulation of DNA in various forms of hposomes (Kaneda et al , (1989) Science 243, 375), particle bombardment (Tang et al , (1992) Nature 356 152, Eisenbraun et al , (1993) DNA Cell Biol 72 791 ) and in vivo infection using cloned retroviral vectors (Seeger et al , (1984) PNAS 81, 5849) Suitable promoters for muscle transfection include CMV, RSV, SRa, actin, MCK, alpha globin, adenovims and dihydrofolate reductase
Thus, among others, antibodies against FAB I may be employed to inhibit FAS or FAB I enzymatic activity or FAB I expression FAB I may also be employed to inhibit infections including, but not limited to infections of upper respiratory tract (e g otitis media, bacteπal tracheitis, acute epiglottitis, thyroiditis), lower respiratory (e g empyema, lung abscess),cardιac (e g infective endocarditis), gastrointestinal (e g secretory diaπhoea, splenic abscess, retropeπtoneal abscess), CNS (e g cerebral abscess), eye (e g blephantis, conjunctivitis, keratitis, endophthalmitis, preseptal and orbital cellulitis, darcryocystitis), kidney and uπnary tract (e g epididymitis, intrarenal and pennephnc abscess, toxic shock syndrome), skin (e g impetigo, follicuhtis, cutaneous abscesses, cellulitis, wound infection, bacterial myositis), and bone and joint (e g septic arthπtis, osteomyelitis)
FAB I may also be employed to treat bacterial infection, such as, for example, infections of upper respiratory tract (e g otitis media, bacterial tracheitis, acute epiglottitis, thyroiditis), lower respiratory (e g empyema, lung abscess),cardιac (e g infective endocarditis), gastrointestinal (e g secretory diaπhoea, splenic abscess, retropeπtoneal abscess), CNS (e g cerebral abscess), eye (e g blephantis, conjunctivitis, keratitis, endophthalmitis, preseptal and orbital cellulitis, darcryocystitis), kidney and urinary tract (e g epididymitis, intrarenal and pennephnc abscess, toxic shock syndrome), skin (e g impetigo, follicuhtis, cutaneous abscesses, cellulitis, wound infection, bacterial myositis), and bone and joint (e g septic arthπtis, osteomyelitis)
FAB I binding molecules and assays
This invention also provides a method for identification of molecules, such as binding molecules, that bind FAB I Genes encoding proteins that bind FAB I, such as binding proteins, can be identified by numerous methods known to those of skill in the art Examples of such methods are descnbed in many laboratory manuals such as, for instance,
Co gan et al , (1991) Current Protocols in Immunology 1(2) Chapter 5
For instance, expression cloning may be employed for this purpose To this end, to isolate genes in an individual which are responsive to FAB I, polyadenylated RNA is prepared from a cell responsive to FAB I, a cDNA library is created from this RNA, the library is divided into pools and the pools are transfected individually into cells that are not responsive to FAB I The transfected cells then are exposed to labeled FAB I (FAB I can be labeled by a vanety of well-known techniques including standard methods of radio-iodination or inclusion of a recognition site for a site-specific protein kinase ) Following exposure, the cells are fixed and binding of FAB I is determined These procedures conveniently are carried out on glass slides
Alternatively a labeled ligand can be photoaffinity linked to a cell extract, such as a membrane or a membrane extract, prepared from cells that express a molecule that it binds, such as a binding molecule Cross-linked mateπal is resolved by polyacrylamide gel electrophoresis ("PAGE") and exposed to X-ray film The labeled complex containing the hgand-binding can be excised, resolved into peptide fragments, and subjected to protein microsequencing The amino acid sequence obtained from microsequencing can be used to design unique or degenerate oligonucleotide probes to screen cDNA branes to identify genes encoding the putative binding molecule
Polypeptides of the invention also can be used to assess FAB I binding capacity of FAB I binding molecules, such as binding molecules, in cells or in cell-free preparations
Antagonists and Agonists - Assays and Molecules
Polypeptides and polynucleotides of the invention may also be used to assess the binding of small molecule substrates and ligands in, for example, cells, cell-free preparations, chemical libraries, and natural product mixtures These substrates and ligands may be natural substrates and ligands or may be stmctural or functional mimetics See, e g , Co gan et al ,
Current Protocols m Immunology 1(2) Chapter 5 ( 1991 )
Polypeptides and polynucleotides of the present invention are responsible for many biological functions, including many disease states, in particular the Diseases hereinbefore mentioned It is therefore desirable to devise screening methods to identify compounds which stimulate or which inhibit the function of the polypeptide or polynucleotide Accordingly, in a further aspect, the present invention provides for a method of screening compounds to identify those which stimulate or which inhibit the function of a polypeptide or polynucleotide of the invention, as well as related polypeptides and polynucleotides In general, agonists or antagonists may be employed for therapeutic and prophylactic purposes for such Diseases as hereinbefore mentioned Compounds may be identified from a vanety of sources, for example, cells, cell-free preparations, chemical hbranes, and natural product mixtures Such agonists, antagonists or inhibitors so-identified may be natural or modified substrates, ligands, receptors, enzymes, etc , as the case may be, of FAB I polypeptides and polynucleotides, or may be stmctural or functional mimetics thereof (see Cohgan et al , Current Protocols in Immunology 1(2) Chapter 5 (1991))
The screening methods may simply measure the binding of a candidate compound to the polypeptide or polynucleotide, or to cells or membranes bearing the polypeptide or polynucleotide, or a fusion protein of the polypeptide by means of a label directly or indirectly associated with the candidate compound Alternatively, the screening method may involve competition with a labeled competitor Further, these screening methods may test whether the candidate compound results in a signal generated by activation or inhibition of the polypeptide or polynucleotide, using detection systems appropriate to the cells comprising the polypeptide or polynucleotide Inhibitors of activation are generally assayed in the presence of a known agonist and the effect on activation by the agonist by the presence of the candidate compound is observed Constitutively active polypeptide and/or constitutively expressed polypeptides and polynucleotides may be employed in screening methods for inverse agonists or inhibitors, in the absence of an agonist or inhibitor, by testing whether the candidate compound results in inhibition of activation of the polypeptide or polynucleotide, as the case may be Further, the screening methods may simply comprise the steps of mixing a candidate compound with a solution containing a polypeptide or polynucleotide of the present invention, to form a mixture, measuring FAB I polypeptide and/or polynucleotide activity in the mixture, and comparing the FAB I polypeptide and/or polynucleotide activity of the mixture to a standard Fusion proteins, such as those made from Fc portion and FAB I polypeptide, as hereinbefore described, can also be used for high-throughput screening assays to identify antagonists of the polypeptide of the present invention, as well as of phylogenetically and and/or functionally related polypeptides (see D Bennett et al , J Mol Recognition, 8 52-58 (1995), and K Johanson et al , 1 Biol Chem. 270( 16) 9459-9471 (1995))
The polynucleotides, polypeptides and antibodies that bind to and/or interact with a polypeptide of the present invention may also be used to configure screening methods for detecting the effect of added compounds on the production of mRNA and/or polypeptide in cells For example, an ELISA assay may be constmcted for measuring secreted or cell associated levels of polypeptide using monoclonal and polyclonal antibodies by standard methods known in the art This can be used to discover agents which may inhibit or enhance the production of polypeptide (also called antagonist or agonist, respectively) from suitably manipulated cells or tissues
The invention also provides a method of screening compounds to identify those which enhance (agonist) or block (antagonist) the action of FAB I polypeptides or polynucleotides, particularly those compounds that are bacteπstatic and/or bacteπcidal The method of screening may involve high-throughput techniques For example, to screen for agonists or antagonists, a synthetic reaction mix, a cellular compartment, such as a membrane, cell envelope or cell wall, or a preparation of any thereof, compπsing FAB I polypeptide and a labeled substrate or ligand of such polypeptide is incubated in the absence or the presence of a candidate molecule that may be a FAB I agonist or antagonist The ability of the candidate molecule to agonize or antagonize the FAB I polypeptide is reflected in decreased binding of the labeled ligand or decreased production of product from such substrate Molecules that bind gratuitously, i e . without inducing the effects of FAB I polypeptide are most likely to be good antagonists Molecules that bind well and. as the case may be, increase the rate of product production from substrate, increase signal transduction, or increase chemical channel activity are agonists Detection of the rate or level of, as the case may be, production of product from substrate, signal transduction, or chemical channel activity may be enhanced by using a reporter system Reporter systems that may be useful in this regard include but are not limited to coloπmetπc, labeled substrate converted into product, a reporter gene that is responsive to changes in FAB I polynucleotide or polypeptide activity, and binding assays known in the art
Polypeptides of the invention may be used to identify membrane bound or soluble receptors, if any, for such polypeptide, through standard receptor binding techniques known in the art These techniques include, but are not limited to, ligand binding and crosshnking assays in which the polypeptide is labeled with a radioactive isotope (for instance, ' ->I), chemically modified (for instance, biotmylated), or fused to a peptide sequence suitable for detection or purification, and incubated with a source of the putative receptor (e g , cells, cell membranes, cell supernatants, tissue extracts, bodily mateπals) Other methods include biophysical techniques such as surface plasmon resonance and spectroscopy These screening methods may also be used to identify agonists and antagonists of the polypeptide which compete with the binding of the polypeptide to its receptor(s), if any Standard methods for conducting such assays are well understood in the art
The fluorescence polarization value for a fluorescently-tagged molecule depends on the rotational conelation time or tumbling rate Protein complexes, such as formed by FAB I polypeptide associating with another FAB I polypeptide or other polypeptide, labeled to comprise a fluorescently-labeled molecule will have higher polarization values than a fluorescently labeled monomeπc protein It is prefeπed that this method be used to characterize small molecules that disrupt polypeptide complexes Fluorescence energy transfer may also be used characterize small molecules that interfere with the formation of FAB I polypeptide dimers, tπmers, tetramers or higher order stmctures, or stmctures formed by FAB I polypeptide bound to another polypeptide FAB I polypeptide can be labeled with both a donor and acceptor fluorophore Upon mixing of the two labeled species and excitation of the donor fluorophore. fluorescence energy transfer can be detected by observing fluorescence of the acceptor Compounds that block dimeπzation will inhibit fluorescence energy transfer Surface plasmon resonance can be used to monitor the effect of small molecules on
FAB I polypeptide self-association as well as an association of FAB I polypeptide and another polypeptide or small molecule FAB I polypeptide can be coupled to a sensor chip at low site density such that covalently bound molecules will be monomeπc Solution protein can then passed over the FAB I polypeptide -coated surface and specific binding can be detected in real-time by monitoring the change in resonance angle caused by a change in local refractive index This technique can be used to characterize the effect of small molecules on kinetic rates and equilibrium binding constants for FAB I polypeptide self-association as well as an association of FAB I polypeptide and another polypeptide or small molecule A scintillation proximity assay may be used to characterize the interaction between an association of FAB I polypeptide with another FAB I polypeptide or a different polypeptide FAB I polypeptide can be coupled to a scintillation-filled bead Addition of radio-labeled FAB I polypeptide results in binding where the radioactive source molecule is in close proximity to the scintillation fluid Thus, signal is emitted upon FAB I polypeptide binding and compounds that prevent FAB I polypeptide self-association or an association of FAB I polypeptide and another polypeptide or small molecule will diminish signal
ICS biosensors have been described by AMBRI (Australian Membrane Biotechnology Research Institute) They couple the self-association of macromolecules to the closing of gramacidin-faci tated ion channels in suspended membrane bilayers and hence to a measurable change in the admittance (similar to impedence) of the biosensor
This approach is linear over six decades of admittance change and is ideally suited for large scale, high through-put screening of small molecule combinatorial libraries
In other embodiments of the invention there are provided methods for identifying compounds which bind to or otherwise interact with and inhibit or activate an activity or expression of a polypeptide and/or polynucleotide of the invention compnsing contacting a polypeptide and/or polynucleotide of the invention with a compound to be screened under conditions to permit binding to or other interaction between the compound and the polypeptide and/or polynucleotide to assess the binding to or other interaction with the compound, such binding or interaction preferably being associated with a second component capable of providing a detectable signal in response to the binding or interaction of the polypeptide and/or polynucleotide with the compound, and determining whether the compound binds to or otherwise interacts with and activates or inhibits an activity or expression of the polypeptide and/or polynucleotide by detecting the presence or absence of a signal generated from the binding or interaction of the compound with the polypeptide and or polynucleotide
Another example of an assay for FAB I agonists is a competitive assay that combines FAB I and a potential agonist with FAB 1-bιndιng molecules, recombinant FAB I binding molecules, natural substrates or ligands, or substrate or ligand mimetics, under appropπate conditions for a competitive inhibition assay FAB I can be labeled, such as by radioactivity or a coloπmetπc compound, such that the number of FAB I molecules bound to a binding molecule or converted to product can be determined accurately to assess the effectiveness of the potential antagonist A further example of a screen is a detect the reduction of crotonyl-CoA by measuring the consumption of NADH Crotonyl-ACP may also be used in place of crotonyl-CoA in such a screen Test compound are added to the reaction mix to determine the effect on the reduction of crotonyl-CoA or crotonyl-ACP Agonists can be identified if the level of reduction is increased and antagonists can be identified if the level of reduction is decreased Diazabonne or palmitoyl CoA may be used as a positive control for antagonism It is prefeπed that these screens be used in a highthrouput assay using a microtitre plate format and a plate reader
The following observations relating to Fab I enzyme kinetics are useful in the methods of the inventions and in other aspects of the invention 1 Apo-ACP is uncompetitive versus NADH (Kι(app) and is proportional to the square of [CCA])
2 Apo-ACP is competitive versus crotonoyl CoA and induces negative cooperativity with respect to CCA binding
3 Stereochemical and steady-state kinetic studies have been earned out on purified 5 aureus Fab I
4 The stereochemical course for the S aureus Fab I is identical to that reported for the E colt enzyme 5 In contrast to the E colt enzyme which uses only NADH. 5 aureus Fab I uses both NADH and NADPH as substrates However, the Kmapp for NADH is approximately 30-fold higher than for NADPH
6 With NADH and crotonoyl CoA as substrates, Fab I displays a sequential kinetic mechanism
7 Kinetic parameters vary little over the pH range of 50 -7 0
8 Increasing inhibition is observed with saturated fatty acyl CoA's of increasing chain length suggestive of a possible feedback regulatory mechanism in vivo
9 Palmitoyl CoA appears to be a competitive inhibitor with respect to crotonoyl CoA Slope replots are proportional to [I] , suggesting that inhibition is effected through the binding of multiple palmitoyl CoA molecules to Fab I
10 Apo-ACP is uncompetitive versus NADH and competitive versus crotonoyl CoA while inducing negative cooperativity in the binding of CCA This indicates an oligomeric quaternary stmcture, particularly, but not limited to, a dimer or tetramer 1 1 Studies using apo-ACP and the pseudo-product inhibitors beta-NADP+ and palmitoyl CoA indicate an ordered addition of substrates (NADH binding first) 12. The uncompetitive inhibition of apo-ACP versus NADH suggests that prior binding of the pyridine nucelotide is required for ACP binding
13 The S. aureus enzyme had similar affinity to the E.coh enzyme for the crotonoyl CoA substrate, yet the E.coh enzyme possessed up to 70 fold greater affinity for
NADH, which was supπsing
14 Hιsl0 tagged S. aureus Fab I displays distinct kinetic characteristics as compared to native Fab Ienzyme
15 S. aureus Fab I is inhibited by fatty acyl CoAs indicating that this enzyme is subject to feed back inhibition by long chain acyl-acyl carrier proteins in vivo.
Potential antagonists of the invention include, among others, small organic molecules, peptides, polypeptides and antibodies that bind to a polynucleotide and/or polypeptide of the invention and thereby inhibit or extinguish its activity or expression Potential antagonists also may be small organic molecules, a peptide, a polypeptide such as a closely related protein or antibody that binds the same sites on a binding molecule, such as a binding molecule, without inducing FAB I-induced activities, thereby preventing the action or expression of FAB I polypeptides and/or polynucleotides by excluding FAB I polypeptides and/or polynucleotides from binding Potential antagonists include a small molecule that binds to and occupies the binding site of the polypeptide thereby preventing binding to cellular binding molecules, such that normal biological activity is prevented Examples of small molecules include but are not limited to small organic molecules, peptides or peptide- like molecules Other potential antagonists include antisense molecules (see Okano, J Neurochem 56 560 ( 1991 ), OLIGODEOXYNUCLEOTIDES AS ANTISENSE INHIBITORS OF GENE EXPRESSION, CRC Press, Boca Raton, FL ( 1988), for a descπption of these molecules) Preferred potential antagonists include compounds related to and variants of FAB I Small organic molecules of the invention preferably have a molecular weight below
2,000 daltons, more preferably between 300 and 1,000 daltons, and most preferably between 400 and 700 daltons An embodiment provides compounds that were not published prior to the filing date of this application Other examples of potential polypeptide antagonists include antibodies or, in some cases, oligonucleotides or proteins which are closely related to the hgands, substrates, receptors, enzymes, etc , as the case may be, of the polypeptide, e g , a fragment of the ligands, substrates, receptors, enzymes, etc , or small molecules which bind to the polypeptide of the present invention but do not elicit a response, so that the activity of the polypeptide is prevented
Certain of the polypeptides of the invention are biomimetics, functional mimetics of the natural FAB I polypeptide These functional mimetics may be used for, among other things, antagonizing the activity of FAB I polypeptide or as a antigen or immunogen in a manner described elsewhere herein Functional mimetics of the polypeptides of the invention include but are not limited to tmncated polypeptides For example, prefeπed functional mimetics include, a polypeptide comprising the polypeptide sequence set forth in SEQ ID NO 2 lacking 20, 30, 40, 50, 60, 70 or 80 amino- or carboxy-terminal amino acid residues, including fusion proteins compnsing one or more of these tmncated sequences Polynucleotides encoding each of these functional mimetics may be used as expression cassettes to express each mimetic polypeptide It is prefeπed that these cassettes compπse 5' and 3 restriction sites to allow for a convenient means to hgate the cassettes together when desired It is further prefeπed that these cassettes compπse gene expression signals known in the art or descπbed elsewhere herein
Thus, in another aspect, the present invention relates to a screening kit for identifying agonists, antagonists, ligands, receptors, substrates, enzymes, etc for a polypeptide and/or polynucleotide of the present in ention or compounds which decrease or enhance the production of such polypeptides and/or polynucleotides , which comprises (a) a polypeptide and/or a polynucleotide of the present invention, (b) a recombinant cell expressing a polypeptide and/or polynucleotide of the present invention, (c) a cell membrane expressing a polypeptide and/or polynucleotide of the present invention, or (d) antibody to a polypeptide and/or polynucleotide of the present invention, which polypeptide is preferably that of SEQ ID NO 2, and which polynucleotide is preferably that of SEQ ID NO 1
It will be appreciated that in any such kit, (a), (b), (c) or (d) may comprise a substantial component It will be readily appreciated by the skilled artisan that a polypeptide and/or polynucleotide of the present invention may also be used in a method for the structure-based design of an agonist, antagonist or inhibitor of the polypeptide and/or polynucleotide, by (a) determining in the first instance the three-dimensional stmcture of the polypeptide and/or polynucleotide, or complexes thereof, (b) deducing the three-dimensional stmcture for the likely reactive sιte(s), binding sιte(s) or motιf(s) of an agonist antagonist or inhibitor, (c) synthesizing candidate compounds that are predicted to bind to or react with the deduced binding sιte(s), reactive sιte(s), and/or motιf(s), and
(d) testing whether the candidate compounds are indeed agonists antagonists or inhibitors It will be further appreciated that this will normally be an iterative process, and this iterative process may be performed using automated and computer-controlled steps
In a further aspect, the present invention provides methods of treating abnormal conditions such as, for instance, a Disease, related to either an excess of, an under-expression of, an elevated activity of, or a decreased activity of FAB I polypeptide and/or polynucleotide
If the expression and/or activity of the polypeptide and/or polynucleotide is in excess, several approaches are available One approach compnses administering to an individual in need thereof an inhibitor compound (antagonist) as herein descπbed, optionally in combination with a pharmaceutically acceptable carrier, in an amount effective to inhibit the function and/or expression of the polypeptide and/or polynucleotide, such as, for example, by blocking the binding of ligands, substrates, receptors, enzymes, etc , or by inhibiting a second signal, and thereby alleviating the abnormal condition In another approach soluble forms of the polypeptides still capable of binding the ligand, substrate, enzymes receptors, etc in competition with endogenous polypeptide and/or polynucleotide may be administered Typical examples of such competitors include fragments of the FAB I polypeptide and/or polypeptide
In a further aspect, the present invention relates to genetically engineered soluble fusion proteins comprising a polypeptide of the present invention, or a fragment thereof, and various portions of the constant regions of heavy or light chains of immunoglobulins of various subclasses (IgG, IgM, IgA, IgE) Preferred as an immunoglobulin is the constant part of the heavy chain of human IgG, particularly IgG l , where fusion takes place at the hinge region In a particular embodiment, the Fc part can be removed simply by incorporation of a cleavage sequence which can be cleaved with blood clotting factor Xa Furthermore, this invention relates to processes for the preparation of these fusion proteins by genetic engineering, and to the use thereof for dmg screening, diagnosis and therapy A further aspect of the invention also relates to polynucleotides encoding such fusion proteins Examples of fusion protein technology can be found in International Patent Application Nos W094/29458 and W094/22914 In still another approach, expression of the gene encoding endogenous FAB I polypeptide can be inhibited using expression blocking techniques This blocking may be targeted against any step in gene expression, but is preferably targeted against transcription and/or translation An examples of a known technique of this sort involve the use of antisense sequences, either internally generated or separately administered (see, for example, O'Connor, J Neurochem (1991 ) 56 560 in Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, FL ( 1988)) Alternatively, oligonucleotides which form triple helices with the gene can be supplied (see, for example, Lee et al , Nucleic Acids Res ( 1979) 6 3073, Cooney et al , Science ( 1988) 241 456, Dervan et al , Science ( 1991 ) 251 1360) These ohgomers can be administered per se or the relevant ohgomers can be expressed in vivo
Each of the polynucleotide sequences provided herein may be used in the discovery and development of antibacterial compounds The encoded protein, upon expression, can be used as a target for the screening of antibacterial dmgs Additionally, the polynucleotide sequences encoding the amino terminal regions of the encoded protein or Shine-Delgarno or other translation facilitating sequences of the respective mRNA can be used to constmct antisense sequences to control the expression of the coding sequence of interest The invention also provides the use of the polypeptide, polynucleotide, agonist or antagonist of the invention to interfere with the initial physical interaction between a pathogen or pathogens and a eukaryotic, preferably mammalian, host responsible for sequelae of infection In particular, the molecules of the invention may be used in the prevention of adhesion of bacteria, in particular gram positive and/or gram negative bacteria, to eukaryotic, preferably mammalian, extracellular matrix proteins on in-dwelling devices or to extracellular matnx proteins in wounds, to block bacterial adhesion between eukaryotic, preferably mammalian, extracellular matrix proteins and bacterial FAB I proteins that mediate tissue damage and/or, to block the normal progression of pathogenesis in infections initiated other than by the implantation of in-dwelling devices or by other surgical techniques
In accordance with yet another aspect of the invention, there are provided FAB I agonists and antagonists, preferably bacteπstatic or bactericidal agonists and antagonists
The antagonists and agonists of the invention may be employed, for instance, to prevent, inhibit and/or treat diseases The antagonists may be employed in a composition with a pharmaceutically acceptable earner, e g , as hereinafter descπbed
The antagonists may be employed for instance to inhibit diseases including, but not limited to infections of upper respiratory tract (e g otitis media, bacterial tracheitis, acute epiglottitis, thyroiditis), lower respiratory (e g empyema, lung abscess),cardιac (e g infective endocarditis), gastrointestinal (e g secretory diaπhoea, splenic abscess, retropeπtoneal abscess), CNS (e g cerebral abscess), eye (e g blephaπtis, conjunctivitis, keratitis, endophthalmitis, preseptal and orbital cellulitis, darcryocystitis), kidney and uπnary tract (e g epididymitis, intrarenal and pennephnc abscess, toxic shock syndrome), skin (e g impetigo, follicuhtis , cutaneous abscesses, cellulitis, wound infection, bacteπal myositis), and bone and joint (e g septic arthπtis, osteomyelitis)
The antagonists may be employed in a composition with a pharmaceutically acceptable caπier, e g , as hereinafter described
Each of the DNA sequences provided herein may be used in the discovery and development of antibacterial compounds The encoded protein upon expression can be used as a target for the screening of antibacterial dmgs Additionally, the DNA sequences encoding the amino terminal regions of the encoded protein or Shine-Delgarno or other translation facilitating sequences of the respective mRNA can be used to constmct antisense sequences to control the expression of the coding sequence of interest In view of the conservation in the amino acid sequence of FAB I with known enzymes in H influenzae, E colt and S typhtmuriitm an antibacterial targeted at FAB I agonist and antagonist compounds provided by the invention are believed to be active against a wide variety of Gram negative and positive organisms In addition, a Fab I homolog (InhA) has been identified in Mycobacterium tuberculosis Therefore, an antibacterial provided by the invention, targeted at FAB I, may also have anti -mycobacteπal activity Further, since Diazabonne deπvatives inhibit LPS biosynthesis (Lam et al ( 1987) J Anttmicrob Chemother 20 37-45) and LPS is a known virulence factor, the invention provides antibacterial compounds targeted at FAB I that have enhanced activity in vivo Heltcobacter pylon (herein "H pylon") bacteria infect the stomachs of over one-third of the world's population causing stomach cancer, ulcers, and gastritis (International Agency for Research on Cancer ( 1994) Schtstosomes Ltx er Flukes and Heltcobacter Pylori (International Agency for Research on Cancer, Lyon, France, http //www uicc ch/ecp/ecp2904 htm) Moreover, the International Agency for Research on Cancer recently recognized a cause-and-effect relationship between H pylon and gastric adenocarcinoma, classifying the bacterium as a Group I (definite) carcinogen Prefeπed antimicrobial compounds of the invention (agonists and antagonists of FAB I polypeptides and/or polynucleotides) found using screens provided by the invention, or known in the art, particularly naπow-spectmm antibiotics, should be useful in the treatment of H pylori infection Such treatment should decrease the advent of H /n/oπ-induced cancers, such as gastrointestinal carcinoma Such treatment should also prevent, inhibit and/or cure gastric ulcers and gastritis
Compositions
The invention also relates to compositions compπsing the polynucleotide or the polypeptides discussed above or the agonists or antagonists Thus, the polypeptides of the present invention may be employed combination with a non-steπle or steπle earner or earners for use with cells, tissues or organisms, such as a pharmaceutical earner suitable for administration to a subject Such compositions comprise, for instance, a media additive or a therapeutically effective amount of a polypeptide of the invention and a pharmaceutically acceptable earner or excipient Such earners may include, but are not limited to. saline, buffered saline, dextrose, water, glycerol, ethanol and combinations thereof The formulation should suit the mode of administration Kits
The invention further relates to pharmaceutical packs and kits compπsing one or more containers filled with one or more of the ingredients of the aforementioned compositions of the invention Associated with such contamer(s) can be a notice in the form prescnbed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, reflecting approval by the agency of the manufacture, use or sale of the product for human administration
Also provided by the invention are diagnostic kits for the FAB I gene and homologs will enable the directed therapy of a FAB I directed antibactenal DNA hybπdisation or protein (monoclonal antibody) based kits for the identification of Fab I and homologs of FAB
I from different species Such a kit could also detect mutations in the FAB I gene /protein and its homologs
Administration
Polypeptides and other compounds of the present invention may be employed alone or in conjunction with other compounds, such as therapeutic compounds
The pharmaceutical compositions may be administered in any effective, convenient manner including, for instance, administration by topical, oral, anal, vaginal, intravenous, lntrapeπtoneal, intramuscular, subcutaneous, mtranasal or intradermal routes among others
The pharmaceutical compositions generally are administered in an amount effective for treatment or prophylaxis of a specific indication or indications In general, the compositions are administered in an amount of at least about 10 μg/kg body weight In most cases they will be administered in an amount not in excess of about 8 mg/kg body weight per day Preferably, in most cases, dose is from about 10 μg/kg to about 1 mg/kg body weight, daily It will be appreciated that optimum dosage will be determined by standard methods for each treatment modality and indication, taking into account the indication, its seventy, route of administration, complicating conditions and the like
In therapy or as a prophylactic, the active agent may be administered to a individual as an injectable composition, for example as a sterile aqueous dispersion, preferably lsotomc Alternatively the composition may be formulated for topical application for example in the form of ointments, creams, lotions, eye ointments, eye drops, ear drops, mouthwash, impregnated dressings and sutures and aerosols, and may contain appropriate conventional additives, including, for example, preservatives, solvents to assist dmg penetration, and emollients in ointments and creams Such topical formulations may also contain compatible conventional earners, for example cream or ointment bases, and ethanol or oleyl alcohol for lotions Such earners may constitute from about 1 % to about 98% by weight of the formulation, more usually they will constitute up to about 80% by weight of the formulation
For administration to human individuals, it is expected that the daily dosage level of the active agent will be from 0 01 to 10 mg/kg, typically around 1 mg/kg The physician in any event will determine the actual dosage which will be most suitable for an individual and will vary with the age weight and response of the particular individual The above dosages are exemplary of the average case There can, of course, be individual instances where higher or lower dosage ranges are merited, and such are within the scope of this invention
In-dwelling devices include surgical implants, prosthetic devices and catheters, l e , devices that are introduced to the body of an individual and remain in position for an extended time Such devices include, for example, artificial joints, heart valves, pacemakers, vascular grafts, vascular catheters, cerebrospinal fluid shunts, urinary catheters, continuous ambulatory peritoneal dialysis (CAPD) catheters, etc
The composition of the invention may be administered by injection to achieve a systemic effect against relevant bacteria shortly before insertion of an in-dwelling device Treatment may be continued after surgery during the in-body time of the device In addition, the composition could also be used to broaden peπoperative cover for any surgical technique to prevent staphylococcal wound infections
Many orthopaedic surgeons consider that individuals with prosthetic joints should be considered for antibiotic prophylaxis before dental treatment that could produce a bacteraemia Late deep infection is a serious complication sometimes leading to loss of the prosthetic joint and is accompanied by significant morbidity and mortality It may therefore be possible to extend the use of the active agent as a replacement for prophylactic antibiotics in this situation
In addition to the therapy described above, the compositions of this invention may be used generally as a wound treatment agent to prevent adhesion of bacteria to matrix proteins exposed in wound tissue and for prophylactic use in dental treatment as an alternative to, or in conjunction with, antibiotic prophylaxis Alternatively, the composition of the invention may be used to bathe an indwelling device immediately before insertion The active agent will preferably be present at a concentration of 1 μg/ml to lOmg/ml for bathing of wounds or indwelling devices
A vaccine composition is conveniently in injectable form Conventional adjuvants may be employed to enhance the immune response A suitable unit dose for vaccination is 0 5-5ug/kg of antigen, and such dose is preferably administered 1 -3 times and with an interval of 1 -3 weeks
With the indicated dose range, no adverse toxicological effects will be observed with the compounds of the invention which would preclude their administration to suitable individuals The antibodies described above may also be used as diagnostic reagents to detect the presence of bacteria containing the FAB I protein
In order to facilitate understanding of the following example certain frequently occuπing methods and/or terms will be described EXAMPLES The present invention is further described by the following examples The examples are provided solely to illustrate the invention by reference to specific embodiments These exemplification's, while illustrating certain specific aspects of the invention, do not portray the limitations or circumscribe the scope of the disclosed invention
Certain terms used herein are explained in the foregoing glossary All examples were earned out using standard techniques, which are well known and routine to those of skill in the art, except where otherwise descnbed in detail Routine molecular biology techniques of the following examples can be earned out as descnbed in standard laboratory manuals, such as Sambrook et al , ( 1989) MOLECULAR CLONING A LABORATORY MANUAL, 2nd Ed , Cold Spnng Harbor Laboratory Press, Cold Spπng Harbor, N Y
All parts or amounts set out in the following examples are by weight, unless otherwise specified
Unless otherwise stated size separation of fragments in the examples below was earned out using standard techniques of agarose and polyacrylamide gel electrophoresis ("PAGE") in Sambrook et al , MOLECULAR CLONING A LABORATORY MANUAL, 2nd
Ed , Cold Spnng Harbor Laboratory Press, Cold Spring Harbor, N Y ( 1989) and numerous other references such as, for instance, by Goeddel et al , ( 1980) Nucleic Acids Res 8 4057 Unless descnbed otherwise, hgations were accomplished using standard buffers, incubation temperatures and times, approximately equimolar amounts of the DNA fragments to be ligated and approximately 10 units of T4 DNA ligase ("ligase ) per 0 5 μg of DNA Example 1 Isolation and sequencing of 5. aureus FAB I gene The polynucleotide having the DNA sequence given in SEQ ID NO 1 was obtained from the sequencing of a library of clones of chromosomal DNA of 5 aureus WCUH 29ιn E coli It has been demonstrated by the process herein described that it is transcribed m vivo in an established infection of 5 aureus WCUH 29ιn a mouse model of infection
To obtain the polynucleotide encoding FAB I protein using the DNA sequence given in SEQ ID NO 1 typically a library of clones of chromosomal DNA of S aureus WCUH 29m E colt or some other suitable host is probed with a radiolabelled oligonucleotide, preferably a 17mer or longer, deπved from the partial sequence Clones caπying DNA identical to that of the probe can then be distinguished using high stπngency washes By sequencing the individual clones thus identified with sequencing pπmers designed from the original sequence it is then possible to extend the sequence in both directions to determine the full gene sequence Conveniently such sequencing is performed using denatured double stranded DNA prepared from a plasmid clone Suitable techniques are descπbed by Mamatis, T , Fπtsch, E F and Sambrook et al , (1989) MOLECULAR CLONING, A LABORATORY MANUAL, 2nd Ed , Cold Spring Harbor Laboratory Press, Cold Spπng Harbor, New York (see Screening By Hybridization 1 90 and Sequencing Denatured Double-Stranded DNA Templates 13 70)
In some cases the sequencing data from two or more clones containing overlapping S aureusWCMH 29 DNA was used to constmct the contiguous DNA sequence in SEQ ID NO 1 Libraries may be prepared Libraries may be prepared by routine methods, for example Methods 1 and 2
Total cellular DNA is isolated from Staphylococcus aureus strain WCUH 29(NCIMB 40771 ) according to standard procedures and size-fractionated by either of two methods
Method 1 Total cellular DNA is mechanically sheared by passage through a needle in order to size-fractionate according to standard procedures DNA fragments of up to 1 lkbp in size are rendered blunt by treatment with exonuclease and DNA polymerase, and EcoRI linkers added Fragments are ligated into the vector Lambda ZapII that has been cut with EcoRI, the library packaged by standard procedures and E colt infected with the packaged library The library is amplified by standard procedures
Method 2
Total cellular DNA is partially hydrolsed with a combination of four restriction enzymes (Rsal, Pall, Alul and Bsh 12351) and size-fractionated according to standard procedures EcoRI linkers are ligated to the DNA and the fragments then ligated into the vector Lambda ZapII that have been cut with EcoRI, the library packaged by standard procedures, and E colt infected with the packaged library The library is amplified by standard procedures Example 2 FAB I enzyme activity analysis
The activity of FAB I protein may be measured using either crotonoyl-CoA or crotonoyl -ACP as a substrate (Bergler, et al, (1994), J Biol Chem 269, 5493-5496) and monitoring the decrease in absorbance at 340nm due to the consumption of NADH Crotonoyl-ACP (Km 22uM) is a better substrate than crotonoyl-CoA (Km 2 7mM), crotonoyl-CoA is available from Sigma (C6146) A diazabonne derivative may be used as a positive control, this should be readily available via a 2 step synthesis with publicly available starting materials using methods known in the art Test compounds may be added to this assay to determine whether they agonize or antagonize enzymatic activity Example 3 Kinetic Characterization of Fab I Crotonoyl CoA was used as the substrate for Fab I and all activity measurements were based on a decrease in absorbance at 340 nm due to the utilization of NADH (Figure
1 ) All assays were performed with 100 mM NaADA, pH 6 5 buffer Acyl-CoA inhibition data was performed in the presence of ImM crotonoyl CoA and ImM NADH Example 4 Kinetic characterization of Fab I from S.aureus Detailed kinetic analysis of the HIS J ΓJ tagged enzyme demonstrated significant evidence for substrate inhibition (by both substrates) and a drastic change from a sequential to and apparent ping-pong mechanism between pH 6 5 and 6 25 However, studies performed at pH 6 0-7 0 with the native enzyme illustrated a sequential mechanistic pattern and kinetic parameters resulting from these studies indicated no overt change in either magnitude or kinetic mechanism Thus the HIS J Q tagged Fab I behaved distinctly from native Fab I enzyme
The kinetic parameters for native S aureus Fab I are shown in Table 2 TABLE 2. Kinetic constants for S.aureus Fab I
* Bergler, H , et al ( 1994) J Biol Chem 269, 5493-6
Native S aureus Fab I exhibited a flat pH optimum between 5 and 7 over which the Vm and the Vm/Km's for the substrates crotonoyl CoA (CCA) and NADH were relatively constant
Native S aureus Fab I was susceptible to inhibition by fatty acyl CoAs The potency of inhibition increased with chain length (Table 3)
TABLE 3. Inhibition of non his tagged Fab I by saturated fatty acyl CoA's
It will be clear that the invention may be practiced otherwise than as particularly described in the foregoing descnption and examples
Numerous modifications and vanations of the present invention are possible in light of the above teachings and, therefore, are within the scope of the appended claims
Each reference cited herein is hereby incorporated by reference in its entirety
Moreover, each patent application to which this application claims prioπty is hereby incorporated by reference in its entirety

Claims

What is claimed is:
1. An antagonist that inhibits or an agonist that activates an activity a polypeptide selected from the group consisting of a polypeptide compπsing an amino acid sequence which is at least 90% identical to the amino acid sequence of SEQ ID NO 2 or 4, and a polypeptide compnsing an amino acid sequence as set forth in SEQ ID NO.2 or 4, wherein said activity is selected from the group consisting of uncompetitive inhibition by Apo-ACP versus NADH (Kι(app), competitive inhibition by Apo-ACP versus crotonoyl CoA, induction of negative cooperativity with respect to CCA binding, use of NADH and NADPH as substrates by Fab I, binding of NADH and NADPH by Fabl, oxidation of NADH and NADPH by Fabl, ratio of Kmapp for NADH as compared to NADPH, use of NADH and crotonoyl CoA as substrates by Fab I in a sequential kinetic mechanism, sequential binding of NADH and crotonoyl CoA by Fab I, increasing inhibition of Fabl by saturated fatty acyl CoA's of increasing chain length, feedback regulatory mechanism of Fab I by saturated fatty acyl CoA's, competitive inhibition by palmitoyl CoA versus crotonoyl CoA, competitive inhibition by palmitoyl CoA versus crotonoyl CoA modulation through binding of multiple palmitoyl CoA molecules to Fab I, binding of multiple palmitoyl CoA molecules to Fab I, negative cooperativity in the binding of CCA, formation of an dimeπc quaternary stmcture, formation of an tetrameπc quaternary stmcture; formation of an oligomeric quaternary stmcture, binding of Fab I by pseudo-product inhibitors beta-NADP+ or palmitoyl coA; and NADH binding to Fab I prior to or simultaneous with ACP binding
2 A method for the treatment of an individual having need to inhibit or activate
Fab I polypeptide compπsing the steps of administering to the individual a antibactenally effective amount of an antagonist that inhibits or an agonist that activates an activity of a polypeptide selected from the group consisting of a polypeptide compnsing an amino acid sequence which is at least 90% identical to the amino acid sequence of SEQ ID NO 2 or 4, and a polypeptide compnsing an amino acid sequence as set forth in SEQ ID NO 2 or 4, wherein said activity is selected from the group consisting of uncompetitive inhibition by Apo-ACP versus NADH (Kι(app), competitive inhibition by Apo-ACP versus crotonoyl CoA induction of negative cooperativity with respect to CCA binding, use of NADH and NADPH as substrates by Fab I, binding of NADH and NADPH by Fabl, oxidation of NADH and NADPH by Fabl, ratio of Kmapp for NADH as compared to NADPH, use of NADH and crotonoyl CoA as substrates by Fab I in a sequential kinetic mechanism, sequential binding of NADH and crotonoyl CoA by Fab I increasing inhibition of Fabl by saturated fatty acyl CoA's of increasing chain length, feedback regulatory mechanism of Fab I by saturated fatty acyl CoA's, competitive inhibition by palmitoyl CoA versus crotonoy l CoA, competitive inhibition by palmitoyl CoA versus crotonoy l CoA modulation through binding of multiple palmitoyl CoA molecules to Fab I, binding of multiple palmitoyl CoA molecules to Fab I negative cooperativity in the binding of CCA, formation of an dimeric quaternary stmcture, formation of an tetrameric quaternary stmcture, formation of an oligomeric quaternary stmcture, binding of Fab I by pseudo-product inhibitors beta-NADP+ or palmitoyl coA, and
NADH binding to Fab I prior to or simultaneous with ACP binding 3 A method for the treatment of an individual infected with a bactena compnsing the steps of administering to the individual a antibactenally effective amount of an antagonist that inhibits or an agonist that activates an activity of a polypeptide selected from the group consisting of a polypeptide compπsing an amino acid sequence which is at least 90% identical to the amino acid sequence of SEQ ID NO 2 or 4, and a polypeptide compπsing an amino acid sequence as set forth in SEQ ID NO 2 or 4, wherein said activity is selected from the group consisting of uncompetitive inhibition by Apo-ACP versus NADH (Kι(app), competitive inhibition by Apo-ACP versus crotonoyl CoA, induction of negative cooperativity with respect to CCA binding, use of NADH and NADPH as substrates by Fab I, binding of NADH and NADPH by Fabl, oxidation of NADH and NADPH by Fabl , ratio of Kmapp for NADH as compared to NADPH, use of NADH and crotonoyl CoA as substrates by Fab I in a sequential kinetic mechanism, sequential binding of NADH and crotonoyl CoA by Fab I, increasing inhibition of Fabl by saturated fatty acyl CoA's of increasing chain length, feedback regulatory mechanism of Fab I by saturated fatty acyl CoA's, competitive inhibition by palmitoyl CoA versus crotonoyl CoA, competitive inhibition by palmitoyl CoA versus crotonoyl CoA modulation through binding of multiple palmitoyl CoA molecules to Fab I, binding of multiple palmitoyl CoA molecules to Fab I, negative cooperativity in the binding of CCA, formation of an dimeric quaternary stmcture, formation of an tetrameric quaternary stmcture, formation of an oligomeric quaternary stmcture, binding of Fab I by pseudo-product inhibitors beta-NADP+ or palmitoyl co A, and
NADH binding to Fab I pπor to or simultaneous with ACP binding
4 The method of claim 3 wherein said bacteria is selected from the group consisting of a member of the genus Staphylococcus, Staphylococcus aureus, a member of the genus Streptococcus, and Streptococcus pneumoniae 5 A method for the treatment of an individual having need to inhibit or activate Fab I polypeptide compnsing the steps of administering to the individual a antibactenally effective amount of an antagonist that inhibits or an agoinist that activates an activity of Fab I selected from the group consisting of uncompetitive inhibition by Apo-ACP versus NADH (Kι(app), competitive inhibition by Apo-ACP versus crotonoyl CoA, induction of negativ e cooperativity with respect to CCA binding, use of NADH and NADPH as substrates by Fab I, binding of NADH and NADPH by Fabl, oxidation of NADH and NADPH by Fabl, ratio of Kmapp for NADH as compared to NADPH, use of NADH and crotonoyl CoA as substrates by Fab I in a sequential kinetic mechanism, sequential binding of NADH and crotonoyl CoA by Fab I, increasing inhibition of Fabl by saturated fatty acyl CoA's of increasing chain length, feedback regulatory mechanism of Fab I by saturated fatty acyl CoA's, competitive inhibition by palmitoyl CoA versus crotonoyl CoA, competitive inhibition by palmitoyl CoA versus crotonoyl CoA modulation through binding of multiple palmitoyl CoA molecules to Fab I, binding of multiple palmitoyl CoA molecules to Fab I, negative cooperativity in the binding of CCA, formation of an dimeric quaternary stmcture, formation of an tetrameric quaternary stmcture, formation of an oligomeric quaternary stmcture, binding of Fab I by pseudo-product inhibitors beta-NADP+ or palmitoyl coA, and
NADH binding to Fab I prior to or simultaneous with ACP binding
6 A method for the treatment of an individual infected with a bacteria comprising the steps of administenng to the individual a antibactenally effective amount of an antagonist that inhibits or an agonist that activates that activates an activity of Fab I selected from the group consisting of uncompetitive inhibition by Apo-ACP versus NADH (Kι(app), competitive inhibition by Apo-ACP versus crotonoyl CoA, induction of negative cooperativity with respect to CCA binding, use of NADH and NADPH as substrates by Fab I, binding of NADH and NADPH by Fabl, oxidation of NADH and NADPH by Fabl, ratio of Kmapp for NADH as compared to NADPH, use of NADH and crotonoyl CoA as substrates by Fab I in a sequential kinetic mechanism, sequential binding of NADH and crotonoyl CoA by Fab I, increasing inhibition of Fabl by saturated fatty ac l CoA's of increasing chain length, feedback regulatory mechanism of Fab I by saturated fatty acyl CoA's, competitive inhibition by palmitoyl CoA versus crotonoyl CoA, competitive inhibition by palmitoyl CoA versus crotonoyl CoA modulation through binding of multiple palmitoyl CoA molecules to Fab I, binding of multiple palmitoyl CoA molecules to Fab I, negative cooperativity in the binding of CCA, formation of an dimeπc quaternary stmcture, formation of an tetrameric quaternary stmcture, formation of an oligomeric quaternary stmcture, binding of Fab I by pseudo-product inhibitors beta-NADP+ or palmitoyl coA, and
NADH binding to Fab I prior to or simultaneous with ACP binding 7 The method of claim 6 wherein said bactena is selected from the group consisting of a member of the genus Staphylococcus, Staphylococcus aureus, a member of the genus Streptococcus, and Streptococcus pneumoniae
8 A method for the treatment of an individual infected by Streptococcus pneumoniae comprising the steps of administeπng to the individual a antibactenally effective amount of an antagonist that inhibits or anagonist that activates an activity of Streptococcus pneumoniae Fab I selected from the group consisting of uncompetitive inhibition by Apo-ACP versus NADH (Kι(app), competitive inhibition by Apo-ACP versus crotonoyl CoA, induction of negative cooperativity with respect to CCA binding, use of NADH and NADPH as substrates by Fab I, binding of NADH and NADPH by Fabl, oxidation of NADH and NADPH by Fabl, ratio of Kmapp for NADH as compared to NADPH, use of NADH and crotonoyl CoA as substrates by Fab I in a sequential kinetic mechanism, sequential binding of NADH and crotonoyl CoA by Fab I, increasing inhibition of Fabl by saturated fatty acyl CoA's of increasing chain length, feedback regulatory mechanism of Fab I by saturated fatty acyl CoA's, competitive inhibition by palmitoyl CoA versus crotonoy l CoA, competitive inhibition by palmitoyl CoA versus crotonoyl CoA modulation through binding of multiple palmitoyl CoA molecules to Fab I, binding of multiple palmitoyl CoA molecules to Fab I, negative cooperativity in the binding of CCA, formation of an dimeπc quaternary stmcture, formation of an tetrameric quaternary stmcture, formation of an oligomeric quaternary stmcture, binding of Fab I by pseudo-product inhibitors beta-NADP+ or palmitoyl coA, and
NADH binding to Fab I prior to or simultaneous with ACP binding
9 An antagonist that inhibits an activity of a polypeptide selected from the group consisting of a polypeptide compπsing an am o acid sequence which is at least 90% identical to the amino acid sequence of SEQ ID NO 2 or 4, and a polypeptide compπsing an amino acid sequence as set forth in SEQ ID NO 2 or 4, , wherein said activity is selected from the group consisting of uncompetitive inhibition by Apo-ACP versus NADH (Kι(app), competitive inhibition by Apo-ACP versus crotonoyl CoA, induction of negative cooperativity with respect to CCA binding, use of NADH and NADPH as substrates by Fab I, binding of NADH and NADPH by Fabl, oxidation of NADH and NADPH by Fabl, ratio of Kmapp for NADH as compared to NADPH, use of NADH and crotonoyl CoA as substrates by Fab I in a sequential kinetic mechanism sequential binding of NADH and crotonoyl CoA by Fab I, increasing inhibition of Fabl by saturated fatty acyl CoA's of increasing chain length, feedback regulatory mechanism of Fab I by saturated fatty acyl CoA's, competitive inhibition by palmitoyl CoA versus crotonoyl CoA, competitive inhibition by palmitoyl CoA versus crotonoyl CoA modulation through binding of multiple palmitoyl CoA molecules to Fab I, binding of multiple palmitoyl CoA molecules to Fab I, negative cooperativity in the binding of CCA, formation of an dimeric quaternary stmcture, formation of an tetrameric quaternary stmcture, formation of an oligomeric quaternary stmcture, binding of Fab I by pseudo-product inhibitors beta-NADP+ or palmitoyl coA, and NADH binding to Fab I prior to or simultaneous with ACP binding
10 A method for the treatment of an individual having need to inhibit Fab I polypeptide comprising the steps of administenng to the individual a antibactenally effective amount of an antagonist that inhibits an activity of a polypeptide selected from the group consisting of a polypeptide compnsing an amino acid sequence which is at least 90% identical to the amino acid sequence of SEQ ID NO 2 or 4, and a polypeptide comprising an amino acid sequence as set forth in SEQ ID NO 2 or 4, , wherein said activity is selected from the group consisting of uncompetitive inhibition by Apo-ACP versus NADH (Kι(app), competitive inhibition by Apo-ACP versus crotonoyl CoA, induction of negative cooperativity with respect to CCA binding, use of NADH and NADPH as substrates by Fab I, binding of NADH and NADPH by Fabl, oxidation of NADH and NADPH by Fabl, ratio of Kmapp for NADH as compared to NADPH, use of NADH and crotonoyl CoA as substrates by Fab I in a sequential kinetic mechanism, sequential binding of NADH and crotonoyl CoA by Fab I, increasing inhibition of Fabl by saturated fatty acyl CoA's of increasing chain length, feedback regulatory mechanism of Fab I by saturated fatty acyl CoA's, competitive inhibition by palmitoyl CoA versus crotonoyl CoA, competitive inhibition by palmitoyl CoA versus crotonoyl CoA modulation through binding of multiple palmitoyl CoA molecules to Fab I. binding of multiple palmitoyl CoA molecules to Fab I, negative cooperativity in the binding of CCA, formation of an dimeric quaternary stmcture, formation of an tetrameric quaternary stmcture, formation of an oligomeric quaternary stmcture, binding of Fab I by pseudo-product inhibitors beta-NADP+ or palmitoyl coA, and
NADH binding to Fab I pπor to or simultaneous with ACP binding 1 1 A method for inhibiting an activity of Fab I polypeptide compπsing the steps of contacting a composition compπsing said polypeptide with an effective amount of an antagonist that inhibits an activbity of Fab I, wherein said activity is selected from the group consisting of uncompetitive inhibition by Apo-ACP versus NADH (Kι(app), competitive inhibition by Apo-ACP versus crotonoyl CoA, induction of negative cooperativity with respect to CCA binding, use of NADH and NADPH as substrates by Fab I, binding of NADH and NADPH by Fabl, oxidation of NADH and NADPH by Fabl, ratio of Kmapp for NADH as compared to NADPH, use of NADH and crotonoyl CoA as substrates by Fab I in a sequential kinetic mechanism, sequential binding of NADH and crotonoyl CoA by Fab I, increasing inhibition of Fabl by saturated fatty acyl CoA's of increasing chain length, feedback regulatory mechanism of Fab I by saturated fatty acyl CoA's, competitive inhibition by palmitoyl CoA versus crotonoyl CoA, competitive inhibition by palmitoyl CoA versus crotonoyl CoA modulation through binding of multiple palmitoyl CoA molecules to Fab I, binding of multiple palmitoyl CoA molecules to Fab I, negative cooperativity in the binding of CCA, formation of an dimeric quaternary stmcture, formation of an tetrameric quaternary stmcture, formation of an oligomeric quaternary stmcture, binding of Fab I by pseudo-product inhibitors beta-NADP+ or palmitoyl coA, and NADH binding to Fab I prior to or simultaneous with ACP binding
12 A method for inhibiting an activity of Fab I, wherein said activity is selected from the group consisting of uncompetitive inhibition by Apo-ACP versus NADH (Kι(app), competitive inhibition by Apo-ACP versus crotonoyl CoA, induction of negative cooperativity with respect to CCA binding, use of NADH and NADPH as substrates by Fab I, binding of NADH and NADPH by Fabl, oxidation of NADH and NADPH by Fabl, ratio of Kmapp for NADH as compared to NADPH, use of NADH and crotonoyl CoA as substrates by Fab I in a sequential kinetic mechanism, sequential binding of NADH and crotonoyl CoA by Fab I, increasing inhibition of Fabl by saturated fatty acyl CoA's of increasing chain length, feedback regulatory mechanism of Fab I by saturated fatty acyl CoA's, competitive inhibition by palmitoyl CoA versus crotonoyl CoA, competitive inhibition by palmitoyl CoA versus crotonoyl CoA modulation through binding of multiple palmitoyl CoA molecules to Fab I, binding of multiple palmitoyl CoA molecules to Fab I, negative cooperativity in the binding of CCA, formation of an dimeric quaternary stmcture, formation of an tetrameric quaternary stmcture, formation of an oligomeric quaternary stmcture binding of Fab I by pseudo-product inhibitors beta-NADP+ or palmitoyl coA, and NADH binding to Fab I prior to or simultaneous with ACP binding
13 The method of claim 12 wherein said bacteria is selected from the group consisting of a member of the genus Staphylococcus Staphylococcus aureus, a member of the genus Streptococcus, and Streptococcus pneumoniae
14 A method for inhibiting a growth of bacteria compπsing the steps of contacting a composition compπsing bacteria with an antibactenally effective amount of an antagonist that inhibits an activity of Fab I, wherein said activity is selected from the group consisting of uncompetitive inhibition by Apo-ACP versus NADH (Kι(app), competitive inhibition by Apo-ACP versus crotonoyl CoA, induction of negative cooperativity with respect to CCA binding, use of NADH and NADPH as substrates by Fab I, binding of NADH and NADPH by Fabl, oxidation of NADH and NADPH by Fabl, ratio of Kmapp for NADH as compared to NADPH, use of NADH and crotonoyl CoA as substrates by Fab I in a sequential kinetic mechanism, sequential binding of NADH and crotonoyl CoA by Fab I, increasing inhibition of Fabl by saturated fatty acyl CoA's of increasing chain length, feedback regulatory mechanism of Fab I by saturated fatty acyl CoA's, competitive inhibition by palmitoyl CoA versus crotonoyl CoA, competitive inhibition by palmitoyl CoA versus crotonoyl CoA modulation through binding of multiple palmitoyl CoA molecules to Fab I, binding of multiple palmitoyl CoA molecules to Fab I, negative cooperativity in the binding of CCA, formation of an dimeric quaternary stmcture, formation of an tetrameric quaternary stmcture, formation of an oligomeric quaternary stmcture, binding of Fab I by pseudo-product inhibitors beta-NADP+ or palmitoyl coA, and NADH binding to Fab I prior to or simultaneous with ACP binding
15 The method of claim 14 wherein said bacteria is selected from the group consisting of a member of the genus Staphylococcus, Staphylococcus aureus a member of the genus Streptococcus, and Streptococcus pneumoniae
16 A method for inhibiting a Fab I polypeptide compπsing the steps of contacting a composition compnsing bacteria with an antibactenally effective amount of an antagonist that inhibits an activity of Fab I, wherein said activity is selected from the group consisting of uncompetitive inhibition by Apo-ACP versus NADH (Kι(app), competitive inhibition by Apo-ACP versus crotonoyl CoA, induction of negative cooperativity with respect to CCA binding, use of NADH and NADPH as substrates by Fab I, binding of NADH and NADPH by Fabl, oxidation of NADH and NADPH by Fabl, ratio of Kmapp for NADH as compared to NADPH, use of NADH and crotonoyl CoA as substrates by Fab I in a sequential kinetic mechanism, sequential binding of NADH and crotonoyl CoA by Fab I, increasing inhibition of Fabl by saturated fatty acyl CoA's of increasing chain length, feedback regulatory mechanism of Fab I by saturated fatty acyl CoA's, competitive inhibition by palmitoyl CoA versus crotonoyl CoA, competitiv e inhibition by palmitoyl CoA versus crotonoyl CoA modulation through binding of multiple palmitoyl CoA molecules to Fab I, binding of multiple palmitoyl CoA molecules to Fab I, negativ e cooperativity in the binding of CCA, formation of an dimeric quaternary stmcture, formation of an tetrameric quaternary stmcture, formation of an oligomeric quaternary stmcture, binding of Fab I by pseudo-product inhibitors beta-NADP-t- or palmitoyl coA, and NADH binding to Fab I prior to or simultaneous with ACP binding
17 The method of claim 16 wherein said bacteria is selected from the group consisting of a member of the genus Staphylococcus Staphy lococcus aureus a member of the genus Streptococcus, and Streptococcus pneumoniae
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US6995254B1 (en) 1996-08-28 2006-02-07 Affinium Pharmaceuticals, Inc. Polynucleotide encoding the enoyl-acyl carrier protein reductase of Staphylococcus aureus, FAB I
US6951729B1 (en) 1999-10-27 2005-10-04 Affinium Pharmaceuticals, Inc. High throughput screening method for biological agents affecting fatty acid biosynthesis
US7048926B2 (en) 2000-10-06 2006-05-23 Affinium Pharmaceuticals, Inc. Methods of agonizing and antagonizing FabK
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