Classifications

• • 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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