WO2000078782A1 - Acetyl coenzyme a carboxylase from staphylococcus aureus - Google Patents

Acetyl coenzyme a carboxylase from staphylococcus aureus Download PDF

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Publication number
WO2000078782A1
WO2000078782A1 PCT/US2000/016726 US0016726W WO0078782A1 WO 2000078782 A1 WO2000078782 A1 WO 2000078782A1 US 0016726 W US0016726 W US 0016726W WO 0078782 A1 WO0078782 A1 WO 0078782A1
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Prior art keywords
polypeptide
seq
polynucleotide
sequence
isolated
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PCT/US2000/016726
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French (fr)
Inventor
Karen O'dwyer
Glenn S. Van Aller
Lei Wang
Richard L. Warren
Christopher M. Traini
Judith M. Ward
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Smithkline Beecham Corporation
Smithkline Beecham Plc
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Publication of WO2000078782A1 publication Critical patent/WO2000078782A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/93—Ligases (6)

Definitions

  • the invention relates to polynucleotides and pohpeptides of the acetyl coenzyme A carboxylase farniK. as well as their ⁇ a ⁇ ants. herein referred to as "accA. accB (fabE). accC. accD.” "accA. accB (fabE). accC. accD polynucleot ⁇ de(s).” and “accA. accB (fabE). accC. accD polypept ⁇ de(s)" as the case may be
  • Staphylococcal genes and gene products make up a medically important genera of microbes
  • the ⁇ are known to produce tv ⁇ o types of disease, invasive and toxigenic Invasive infections are characterized generalh by abscess formation effecting both skin surfaces and deep tissues S aureus is the second leading cause of bacteremia in cancer patients Osteomyelitis, septic arthritis, septic thrombophlebitis and acute bacterial endocarditis are also relatively common
  • There are at least three clinical conditions resulting from the toxigenic properties of Staphylococci The manifestation of these diseases result from the actions of exotoxins as opposed to tissue invasion and bacteremia These conditions include Staphylococcal food poisoning, scalded skin syndrome and toxic shock syndrome
  • the frequency of Staphylococcus aureus infections has nsen dramatically in the past few decades
  • the present invention relates to accA. accB (fabE). accC. accD. in particular accA. accB (fabE). accC. accD pohpeptides and accA. accB (fabE). accC. accD poKnucleotides. recombmant matenals and methods for their production
  • the invention relates to mediods for usmg such pohpeptides and poKnucleotides.
  • the invention relates to methods for identifying agonists and antagonists using the materials pro ⁇ ided by the invention, and for treating microbial infections and conditions associated with such infections with the identified agonist or antagonist compounds
  • the invention relates to diagnostic assays for detecting diseases associated with microbial infections and conditions associated with such infections, such as assays for detecting accA. accB (fabE). accC. accD expression or activity
  • the invention relates to accA. accB (fabE). accC. accD polypeptides and poKnucleotides as descnbed m greater detail below
  • the invention relates to pohpeptides and polynucleotides of a accA. accB (fabE).
  • accC. accD of Staphylococcus aureus. ti at is related by amino acid sequence homology to yqhW of Bacillus subtihs and accA. accB (fabE), accC. accD of E cob polypeptide
  • the invention relates especially to accA. accB (fab ⁇ ). accC.
  • sequences recited in the Sequence Listing below as "DNA” represent an exemplification of the invention, since those of ordinary skill will recognize that such sequences can be usefulK employed in polynucleotides in general including ⁇ bopolynucleotides
  • Staphylococcus aureus accB (fabE) polynucleotide sequence [SEQ ID NO:3J. 5 ' -
  • a deposit compnsing a Staphylococcus aureus WCUH 29 strain has been deposited with the National Collections of Indust ⁇ al and Marine Bactena Ltd (herein "NCIMB"), 23 St Machar D ⁇ ve, Aberdeen AB2 1RY. Scotland on 11 September 1 95 and assigned NCIMB Deposit No 40771. and referred to as Staphylococcus aureus WCUH29 on deposit
  • the Staphylococcus aureus strain deposit is referred to herein as "d e deposited stram” or as "the DNA of the deposited strain "
  • the deposited stram comp ⁇ ses a full length accA. accB (fabE). accC. accD gene
  • the sequence of the polynucleotides comp ⁇ sed in the deposited strain, as well as the amino acid sequence of any polypeptide encoded thereby, are controlling m the event of any conflict with any desc ⁇ ption of sequences herem
  • the deposit of the deposited stram has been made under the te ⁇ ns of die Budapest Treaty on the
  • deposited stram will be irrevocably and without restnction or condition released to the public upon the issuance of a patent
  • the deposited stram is provided merely as convenience to those of skill in the art and is not an admission tliat a deposit is required for enablement. such as that required under 35 U S C ⁇ 1 12
  • a license may be required to make, use or sell the deposited stram. and compounds den ⁇ ed therefrom, and no such license is hereby granted
  • an isolated nucleic acid molecule encoding a mature poKpeptide expressible by the Staphylococcus aureus WCUH 29 stram.
  • accA. accB (fabE).
  • accA. accB (fabE).
  • AccA. accB (fabE).
  • accC. accD poKpeptide of the mvention is substantially phylogenetically related to other proteins of the acety 1 coenzyme A carboxylase family
  • polypeptides of Staphylococcus aureus referred to herem as "accA. accB (fabE), accC. accD” and "accA. accB (fabE).
  • accC. accD polypeptides as well as biologically, diagnostically . prophylactically . clinically or therapeutically useful ⁇ anants thereof, and compositions compnsing the same
  • va ⁇ ants of accA. accB (fabE).
  • accC. accD gene The present mvention further provides for an isolated polypeptide that (a) comprises or consists of an ammo acid sequence that has at least 95% identity, most preferably at least 97-99% or exact identity , to that of SEQ ID NO 2. 4. 6. 8 over the entire length of SEQ ID NO 2. 4. 6. 8.
  • polypeptides of the mvention mclude a polypeptide of Table 1 [SEQ ID NO 2. 4 6. 8] (in particular a mature polypeptide) as well as polypeptides and fragments, particularly those that has a biological activity of accA. accB (fabE). accC. accD. and also those that ha ⁇ e at least 95% identity to a polypeptide of Table 1 [SEQ ID NO 2. 4. 6. 8] and also mclude portions of such polypeptides with such portion of the polypeptide generally comp ⁇ smg at least 30 ammo acids and more preferably at least 50 ammo acids
  • the invention also cludes a polypeptide consisting of or comp ⁇ smg a poKpeptide of the fomiula
  • X-(Rl) m -(R 2 )-(R3)n-Y w herem at d e amino terminus X is hydrogen, a metal or any other moiet ⁇ desc ⁇ bed herem for modified polypeptides and at die carboxyl terminus.
  • Y is hydrogen, a metal or any other moiety desc ⁇ bed herem for modified polypeptides.
  • Rj and R3 are any ammo acid residue or modified ammo acid residue, m is an integer between 1 and 1000 or zero, n is an integer between 1 and 1000 or zero, and R 2 is an ammo acid sequence of the mvention. particularly an ammo acid sequence selected from Table 1 or modified forms thereof In the formula abo ⁇ e.
  • R 2 is o ⁇ ented so that its ammo terminal ammo acid residue is at the left, covalently bound to Ri and its terminal ammo acid residue is at the ⁇ ght. covalently bound to R3 Any stretch of ammo acid residues denoted by either R ] or R3.
  • m and/or n is greater than 1. may be either a heteropolymer or a homopohmer. preferably a heteropolymer Other preferred embodiments of the mvention are provided where m is an mteger between 1 and 50. 100 or 500. and n is an mteger between 1 and 50. 100, or 500
  • a polypeptide of die mvention is demed from Staphylococcus aureus however it may preferably be obtained from other organisms of die same taxonomic genus
  • a poKpeptide of the mvention also be obtained, for example, from organisms of die same taxonomic family or order
  • a fragment is a va ⁇ ant polypeptide having an ammo acid sequence that is entirely the same as part but not all of an ⁇ ammo acid sequence of any poKpeptide of the mvention As with accA. accB (fabE). accC. accD polypeptides. fragments may be "free-standing.” or comp ⁇ sed within a larger polypeptide of which they form a part or region, most preferably as a smgle cont uous region m a smgle larger polypeptide Preferred fragments mclude, for example, truncation polypeptides havmg a portion of an ammo acid sequence of Table 1 [SEQ ID NO 2. 4.
  • va ⁇ ants thereof such as a contmuous se ⁇ es of residues that mcludes an ammo- and/or carboxyl-termmal amino acid sequence Degradation forms of the polypeptides of the mvention produced by or in a host cell, particularly a Staphylococcus aureus.
  • fragments characterized by structural or functional attributes such as fragments that comp ⁇ se alpha-helix and alpha-helix formmg regions, beta-sheet and beta-sheet-formmg regions, turn and turn-formmg regions, coil and coil-forming regions, hydrophilic regions, hydrophobic regions alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions, substrate binding region, and high antigenic index regions
  • fragments include an isolated polypeptide comprising an amino acid sequence having at least 15, 20, 30, 40, 50 or 100 contiguous amino acids from the amino acid sequence of SEQ ID NO 2, 4, 6, 8, or an isolated polypeptide comprising an amino acid sequence having at least 15, 20, 30, 40, 50 or 100 contiguous amino acids truncated or deleted from the amino acid sequence of SEQ ID NO.2, 4, 6, 8.
  • Fragments of die polypeptides of the may be employ ed for producmg the corresponding full-length polypeptide by peptide synthesis, therefore, these ⁇ a ⁇ ants may be emplo ⁇ ed as intermediates for producmg d e full-lengdi polypeptides of the mvention Polynucleotides It is an object of the mvention to provide polynucleotides that encode accA. accB (fabE). accC. accD polypeptides. particularly polynucleotides that encode a polypeptide herem designated accA. accB (fabE). accC. accD
  • the polvnucleotide comp ⁇ ses a region encoding accA. accB (fabE).
  • accC. accD polypeptides compnsmg a sequence set out m Table 1 [SEQ ID NO 1. 3. 5. 7] that mcludes a full length gene, or a variant thereof The Applicants believe that this fiiU length gene is essential to the growth and/or survival of an organism that possesses it. such as Staphylococcus aureus
  • isolated nucleic acid molecules encoding and/or expressmg accA. accB (fabE).
  • accC. accD polypeptides and polynucleotides particularly Staphylococcus aureus accA. accB (fabE).
  • accC. accD polypeptides and polynucleotides mcludmg. for example, unprocessed RNAs. ⁇ bozyme RNAs. mRNAs. cDNAs. genomic DNAs.
  • B- and Z-DNAs Further embodiments of the mvention mclude biologically, diagnostically. prophylactically. clinically or therapeutically useful polynucleotides and polypeptides.
  • mcludmg at least one full length gene, that encodes a accA. accB (fabE).
  • accC. accD poKpeptide havmg a deduced ammo acid sequence of Table 1 [SEQ ID NO 2. 4. 6. 8] and polynucleotides closely related thereto and va ⁇ ants thereof
  • accA. accB (fabE).
  • accC. accD polypeptide from Staphylococcus aureus comprising or consisting of an ammo acid sequence of Table 1 [SEQ ID NO 2. 4, 6. 8]. or a va ⁇ ant thereof
  • Usmg die mformation provided herem. such as a polynucleotide sequence set out m Table 1 [SEQ ID NO 1. 3. 5. 7J. a polynucleotide of die mvention encoding accA. accB (fabE). accC. accD polypeptide may be obtained usmg standard cloning and screening methods, such as those for cloning and sequencmg chromosomal DNA fragments from bacte ⁇ a using Staphylococcus aureus WCUH 29 cells as starting mate ⁇ al.
  • a polynucleotide sequence of the invention such as a polynucleotide sequence given m Table 1 [SEQ ID NO 1. 3. 5. 7]
  • a library of clones of chromosomal DNA of Staphylococcus aureus WCUH 29 in E cob or some other suitable host is probed with a radiolabeled ohgonucleotide.
  • Clones carry ing DNA identical to that of the probe can then be distinguished usmg stringent hy b ⁇ dization conditions
  • sequencing the indiv ldual clones thus identified by hy b ⁇ dization with sequencing primers designed from the original polypeptide or poly nucleotide sequence it is then possible to extend the polynucleotide sequence in both directions to determine a full length gene sequence
  • sequencmg is performed, for example, using denatured double stranded DNA prepared from a plasmid clone Suitable techniques are described by Mamatis. T . F ⁇ tsch. E F and Sambrook et al .
  • each DNA sequence set out in Table 1 [SEQ ID NO 1. 3. 5. 7] contains an open reading frame encoding a protem having about the number of ammo acid residues set forth in Table 1 [SEQ ID NO 2. 4, 6, 8] witii a deduced molecular weight that can be calculated usmg ammo acid residue molecular weight values well known to those skilled m the art
  • the present mvention provides for an isolated polynucleotide comprising or consisting of (a) a polynucleotide sequence that has at least 95% identity , even more preferably at least 97-99% or exact identity to SEQ ID NO 1. 3. 5. 7 over the entire length of SEQ ID NO 1. 3. 5. 7. or the entire length of that portion of SEQ ID NO 1. 3. 5. 7 which encodes SEQ ID NO 2. 4. 6. 8. (b) a polynucleotide sequence encoding a polypeptide that has at least 95% identity . even more preferably at least 97-99% or 100% exact, to the ammo acid sequence of SEQ ID NO 2. 4. 6. 8. over the entire length of SEQ ID NO 2. 4. 6, 8
  • a polynucleotide encoding a polypeptide of the present invention, mcludmg homologs and orthologs from species odier than Staphylococcus aureus. may be obtained by a process tiiat comp ⁇ ses the steps of screening an appropnate library under stringent hyb ⁇ dization conditions w th a labeled or detectable probe consisting of or compnsmg the sequence of SEQ ID NO 1. 3. 5. 7 or a fragment thereof, and isolating a full- length gene and/or genomic clones compnsmg said polynucleotide sequence
  • the mvention provides a polynucleotide sequence identical over its entire length to a coding sequence (open reading frame) in Table 1 [SEQ ID NO 1. 3, 5. 7] Also provided by the mvention is a coding sequence for a mature polypeptide or a fragment thereof, by itself as well as a coding sequence for a mature polypeptide or a fragment m reading frame with another coding sequence, such as a sequence encoding a leader or secretory sequence, a pre-, or pro- or prepro-protem sequence
  • the polynucleotide of the mvention may also comp ⁇ se at least one non-coding sequence, mcludmg for example, but not limited to at least one non-coding 5 ' and 3 ' sequence, such as the transc ⁇ bed but non-translated sequences, termination signals (such as rho- dependent and rho-independent termination signals), ⁇ bosome binding sites.
  • the polynucleotide sequence may also comp ⁇ se additional coding sequence encoding additional ammo acids
  • a marker sequence that facilitates punfication of a fused poKpeptide can be encoded in certain embodiments of the mvention.
  • the marker sequence is a hexa-histidine peptide. as provided m the pQE vector (Qiagen. Inc ) and desc ⁇ bed m Gentz et al . Proc Natl Acad Sa USA 86 821-824 (1989). or an HA peptide tag (Wilson et al . Cell 37 767 (1984).
  • Polynucleotides of the mvention also mclude. but are not limited to. polynucleotides compnsmg a structural gene and its naturally associated sequences diat control gene expression
  • a preferred embodiment of the mvention is a polynucleotide of consistmg of or compnsmg nucleotide
  • the mvention also mcludes a polynucleotide consistmg of or compnsmg a polynucleotide of the formula
  • X-(R ⁇ ) m -(R 2 )-(R 3 ) n -Y wherein, at the 5' end of the molecule.
  • X is hydrogen, a metal or a modified nucleotide residue, or together with Y defines a covalent bond, and at the 3' end of the molecule.
  • Y is hy drogen, a metal, or a modified nucleotide residue, or together with X defines the covalent bond
  • each occurrence of Rj and R3 is mdependently any nucleic acid residue or modified nucleic acid residue
  • m is an integer between 1 and 3000 or zero .
  • n is an integer between 1 and 3000 or zero
  • R 2 is a nucleic acid sequence or modified nucleic acid sequence of the invention, particularly a nucleic acid sequence selected from Table 1 or a modified nucleic acid sequence thereof In the polynucleotide formula above.
  • R? is oriented so that its 5' end nucleic acid residue is at the left, bound to Rj and its 3' end nucleic acid residue is at the right, bound to R3 Any stretch of nucleic acid residues denoted by either Rj and/or R 2 .
  • m and/or n is greater than 1.
  • the polynucleotide of the above formula is a closed, circular polynucleotide. that can be a double-stranded polynucleotide wherein the formula shows a first strand to which the second strand is complementary
  • m and/or n is an integer between 1 and 1000.
  • Other prefened embodiments of the mvention are provided where m is an mteger between 1 and 50. 100 or 500. and n is an mteger between 1 and 50. 100. or 500 It is most prefened that a polynucleotide of the mvention is de ⁇ ved from Staphylococcus aureus.
  • a polynucleotide of the invention may also be obtamed. for example, from organisms of the same taxonomic family or order
  • polynucleotide encoding a polypeptide encompasses polynucleotides that mclude a sequence encoding a polypeptide of the mvention. particularly a bactenal polypeptide and more particularly a polypeptide of the Staphvlococais aureus accA. accB (fabE). accC. accD having an ammo acid sequence set out in Table 1 [SEQ ID NO 2. 4. 6. 8]
  • the term also encompasses polynucleotides that mclude a smgle contmuous region or discontinuous regions encoding the polypeptide (for example, polynucleotides interrupted by integrated phage.
  • an mtegrated insertion sequence an mtegrated vector sequence, an mtegrated transposon sequence, or due to RNA editing or genomic DNA reorganization) together witii additional regions, that also may compnse coding and/or non-coding sequences
  • the mvention further relates to va ⁇ ants of the polynucleotides descnbed herem that encode va ⁇ ants of a polypeptide havmg a deduced am o acid sequence of Table 1 [SEQ ID NO 2. 4, 6. 8] Fragments of polynucleotides of the mvention may be used, for example, to synthesize full-lengui polynucleotides of the mvention Further particularly preferred embodiments are polynucleotides encoding accA. accB (fabE). accC. accD va ⁇ ants. that have the ammo acid sequence of accA. accB (fabE). accC. accD polypeptide of Table 1 [SEQ ID NO 2. 4. 6.
  • Prefened isolated polynucleotide embodiments also mclude polynucleotide fragments, such as a polynucleotide comprising a nuclic acid sequence having at least 15, 20, 30, 40, 50 or 100 contiguous nucleic acids from the polynucleotide sequence of SEQ ID NO: l , 3, 5, 7, or an polynucleotide comprising a nucleic acid sequence having at least 15, 20, 30, 40, 50 or 100 contiguous nucleic acids truncated or deleted from the 5' and/or 3' end of the polynucleotide sequence of SEQ ID NO: l, 3, 5, 7.
  • polynucleotide fragments such as a polynucleotide comprising a nuclic acid sequence having at least 15, 20, 30, 40, 50 or 100 contiguous nucleic acids from the polynucleotide sequence of SEQ ID NO: l , 3, 5, 7, or an polynucleotide comprising a nucleic acid sequence having
  • polynucleotides that are at least 95% or 97% identical over their entire length to a polynucleotide encoding accA. accB (fabE).
  • accC. accD polypeptide having an ammo acid sequence set out m Table 1 [SEQ ID NO 2. 4. 6. 8].
  • polynucleotides that are complementary to such polynucleotides Most highly prefened are polynucleotides that comp ⁇ se a region that is at least 95% are especially prefened 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 prefened with at least 99% being the more prefened
  • Prefened embodiments are polynucleotides encoding polypeptides that retain substantially the same biological function or activity as a mature polypeptide encoded by a DNA of Table 1 [SEQ ID NO 1. 3. 5. 7] hi accordance with certain prefened embodiments of this mvention there are provided polynucleotides that hybndize. particularly under stringent conditions, to accA. accB (fabE). accC. accD polynucleotide sequences, such as diose polynucleotides m Table 1
  • the mvention further relates to polynucleotides that hybndize to die polynucleotide sequences provided herem
  • the mvention especially relates to polynucleotides that hybndize under stringent conditions to the polvnucleotides desc ⁇ bed herem
  • stringent conditions and “stringent hybndization conditions” mean hvbndization occurring only if there is at least 95% and preferably at least 97% identity between the sequences
  • a specific example of stringent hybridization conditions is overnight incubation at 42°C in a solution comprising 50% formamide. 5x SSC (150mM NaCl. 15mM t ⁇ sodium citrate).
  • the invention also provides a polynucleotide consisting of or comprising a polynucleotide sequence obtained by screening an appropriate library compnsmg a complete gene for a polynucleotide sequence set forth in SEQ ID NO 1. 3, 5. 7 under stringent hybridization conditions with a probe having the sequence of said polynucleotide sequence set forth in SEQ ID NO 1. 3. 5. 7 or a fragment thereof, and isolating said polynucleotide sequence Fragments useful for obtaining such a polynucleotide include, for example, probes and primers fully descnbed elsewhere herein
  • the polynucleotides of the mvention may be used as a hvbndization probe for RNA.
  • cDNA and genomic DNA to isolate full-length cDNAs and genomic clones encoding accA. accB (fabE).
  • accC. accD and to isolate cDNA and genomic clones of other genes that have a high identity . particularly high sequence identity, to a accA. accB (fabE). accC.
  • Such probes generally will compnse at least 15 nucleotide residues or base pairs Preferably such probes will have at least 30 nucleotide residues or base pairs and may have at least 50 nucleotide residues or base pairs Particularly preferred probes will have at least 20 nucleotide residues or base pairs and will have lee than 30 nucleotide residues or base pairs
  • a coding region of a accA. accB (fabE). accC. accD gene mav be isolated by screening usmg a DNA sequence provided m Table 1 [SEQ ID NO 1. 3. 5. 7] to svnthesize an oligonucleotide probe A labeled oligonucleotide havmg a sequence complementary to that of a gene of the v ention is then used to screen a library of cDNA genomic DNA or rnRNA to determine which members of the librarv the probe h bndizes to
  • cDNAs have been prepared from mRNA extracted from a chosen tissue and an 'adaptor' sequence hgated onto each end Nucleic acid amplification (PCR) is then carried out to amplify the "missing" 5' end of the DNA using a combination of gene specific and adaptor specific oligonucleotide primers
  • the PCR reaction is then repeated using "nested" primers, that is, primers designed to anneal within the amplified product (typically an adaptor specific pnmer that anneals further 3' in the adaptor sequence and a gene specific primer that anneals further 5' in the selected gene sequence)
  • the products of this reaction can then be analyzed by DNA sequencing and a full-length DNA constructed either by joining the product directly to the existing DNA to giv e a complete sequence, or carry ing out a separate full- length PCR using the new sequence information for the design of the 5' primer
  • polynucleotides and polypeptides of the mvention mav be employ ed. for example, as research reagents and matenals for discovery of treatments of and diagnostics for diseases, particularly human diseases, as further discussed herem relating to polynucleotide assays
  • the polvnucleotides of the invention that are ohgonucleotides de ⁇ v ed from a sequence of Table 1 [SEQ ID NOS 1 or 2] may be used in the processes herein as described, but preferably for PCR. to determine whether or not the polynucleotides identified herein in whole or in part are transcribed in bacteria in infected tissue It is recognized that such sequences w ill also hav e utility in diagnosis of the stage of infection and type of infection the pathogen has attained
  • the mvention also provides polynucleotides that encode a polypeptide that is a mature protein plus additional ammo or carboxyl-terrninal ammo acids, or ammo acids uitenor to a mature polypeptide (when a mature form has more than one polypeptide chain, for instance) Such sequences may play a role m processing of a protem from precursor to a mature form may allow protein transport, mav lengthen or shorten protem half-life or may facilitate manipulation of a protem for assav or production, among other things As generally is the case in vivo, the additional ammo acids may be processed away from a mature protem b ⁇ cellular enzymes For each and every polynucleotide of the mvention there is provided a polynucleotide complementary to it It is prefened that these complementary polynucleotides are fully complementary to each polynucleotide with which uiey are complementary
  • a precursor protem. having a mature form of the polypeptide fused to one or more prosequences may be an mactiv e form of the polypeptide When prosequences are removed such mactiv e precursors generally are activated Some or all of the prosequences may be removed before activation Generally . such precursors are called proproteins
  • the entire polypeptide encoded by an open reading frame is often not required for activity Accordingly . it has become routme m molecular biology to map the boundanes of the primary structure required for activity with N-termmal and C-terminal deletion experiments
  • These experiments utilize exonuclease digestion or convenient restnction sites to cleave coding nucleic acid sequence
  • Promega (Madison, WI) sell an Erase-a-baseTM system that uses Exonuclease III designed to facilitate analysis of the deletion products (protocol available at www promega com)
  • the digested endpoints can be repaired (e g . by hgation to synthetic linkers) to the extent necessary to preserve an open reading frame In this way.
  • nucleic acid of SEQ ID NO 1, 3. 5. 7 readily provides contiguous fragments of SEQ ID NO 2, 4. 6, 8 sufficient to provide an activity, such as an enzymatic, bmdmg or antibody -mducmg activity
  • Nucleic acid sequences encoding such fragments of SEQ ID NO 2. 4. 6, 8 and vanants thereof as desc ⁇ bed herem are within the mvention. as are polypeptides so encoded
  • a polynucleotide of the mvention may encode a mature protem. a mature protem plus a leader sequence (which may be refened to as a preprotein), a precursor of a mature protem having one or more prosequences that are not the leader sequences of a preprotein. or a preproprotein. that is a precursor to a proprotem. havmg a leader sequence and one or more prosequences. that generally are removed during process g steps that produce active and mature forms of the polypeptide Vectors, Host Cells, Expression Systems
  • the mvention also relates to vectors that compnse a polynucleotide or polynucleotides of the mvention.
  • the present mvention relates to expression systems that compnse a polynucleotide or polynucleotides of the present mvention. to host cells that are genetically engmeered with such expression systems, and to the production of polypeptides of the mvention by recombmant techniques
  • host cells can be genetically engmeered to incorporate expression systems or portions thereof or polynucleotides of the invention
  • Introduction of a polynucleotide mto the host cell can be effected by methods desc ⁇ bed m many standard laboratory manuals, such as Davis, et al . BASIC METHODS IN MOLECULAR BIOLOGY. (1986) and Sambrook. et al . MOLECULAR CLONING A LABORATORY MANUAL. 2nd Ed . Cold Sp ⁇ ng Harbor Laboratory Press. Cold Spring Harbor, N Y (1989). such as. calcium phosphate transfection. DEAE-dextran mediated transfection. transvection. microinjection. cationic hpid-mediated transfection. electroporation. transduction. scrape loading, ballistic introduction and infection
  • bacte ⁇ al cells such as cells of streptococci. staphylococci. enterococci E cob. streptomyces. cyanobactena. Bacillus subttbs, and Staphylococcus aureus. fungal cells, such as cells of a yeast. Kluveromyces . Saccharomyces . a basidiomycete. Candida albicans and Aspergillus. insect cells such as cells of Drosophila S2 and Spodoptera Sf9. animal cells such as CHO. COS. HeLa, C127, 3T3. BHK.
  • vectors mclude. among others, chromosomal-, episomal- and virus-de ⁇ ved vectors, for example, vectors denved from bactenal plasmids. from bactenophage. from transposons. from yeast episomes. from insertion elements, from yeast chromosomal elements, from viruses such as baculoviruses. papova viruses, such as SV40.
  • the expression system constructs may comp ⁇ se control regions diat regulate as well as engender expression
  • any system or vector suitable to maintain, propagate or express polynucleotides and/or to express a polypeptide in a host may be used for expression m diis regard
  • the appropnate DNA sequence may be inserted mto the expression system by any of a vanety of well-known and routme techniques, such as. for example, those set forth in Sambrook et al . MOLECULAR CLONING, A LABORATORY MANUAL, (supra)
  • Polypeptides of the mvention can be recovered and punfied from recombmant cell cultures by well-known methods mcludmg ammomum sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatograph . phosphocellulose chromatography . hydrophobic mteraction chromatography. affinity' chromatography . hydroxylapatite chromatography . and lectm chromatography Most preferably . high performance liquid chromatography is employed for pu ⁇ fication Well known techniques for refolding protem may be emplov ed to regenerate active conformation when the polypeptide is denatured during isolation and or punfication Diagnostic, Prognostic, Serotyping and Mutation Assays
  • This mvention is also related to the use of accA. accB (fabE).
  • accC. accD polynucleotides and polypeptides of die mvention for use as diagnostic reagents Detection of ace A. accB (fabE).
  • accC. accD polynucleotides and/or polypeptides m a eukaryote. particularly a mammal, and especially a human, will provide a diagnostic method for diagnosis of disease, staging of disease or response of an infectious orgamsm to drugs Eukarvotes, particularly mammals, and especially humans, particularly those infected or suspected to be infected with an orgamsm compnsmg the accA. accB (fabE).
  • accC. accD gene or protein may be detected at the nucleic acid or ammo acid level by a vanety of well known techniques as well as by methods provided herem
  • Polypeptides and polynucleotides for prognosis, diagnosis or other analy sis may be obtamed from a putatively infected and/or infected mdividual's bodily matenals
  • Polynucleotides from any of these sources, particularly DNA or RNA. may be used directly for detection or may be amplified enzymatically by usmg PCR or any other amplification technique pnor to analysis RNA. particularly rnRNA.
  • cDNA and genomic DNA may also be used m die same way s Usmg amplification, characte ⁇ zation of the species and stram of infectious or resident organism present m an individual, may be made by an analysis of the genotype of a selected polynucleotide of the orgamsm Deletions and insertions can be detected by a change m size of the amplified product m companson to a genotype of a reference sequence selected from a related organism, preferably a different species of the same genus or a different stram of the same species Pomt mutations can be identified by hybndizing amplified DNA to labeled accA. accB (fabE). accC.
  • Polynucleotide sequence differences may also be detected by alterations the electrophoretic mobility of polynucleotide fragments m gels as compared to a reference sequence This may be earned out with or without denaturing agents Polynucleotide differences may also be detected by direct DNA or RNA sequencing See. for example. My ers et al .
  • nuclease protection assay s such as RNase. VI and SI protection assay or a chemical cleavage method See. for example. Cotton et al . Proc Natl Acad Sci USA. 85 4397-4401 (1985)
  • an array of oligonucleotides probes compnsmg accA. accB (fabE). accC. accD nucleotide sequence or fragments thereof can be constructed to conduct efficient screening of. for example, genetic mutations, serotype.
  • taxonomic classification or identification Array technology methods are vv ell known and hav e general applicability and can be used to address a v anety of questions m molecular genetics mcludmg gene expression, genetic linkage, and genetic vanabihty (see. for example. Chee et al . Science. 274 670 (1996))
  • the present invention relates to a diagnostic kit that comprises (a) a polynucleotide of the present invention, preferably the nucleotide sequence of SEQ ID NO 1. 3. 5, 7. or a fragment thereof, (b) a nucleotide sequence complementary to that of (a), (c) a polypeptide of the present invention, preferably the polypeptide of SEQ ID NO 2. 4. 6. 8 or a fragment thereof, or (d) an antibody to a polypeptide of the present mvention. preferably to the polypeptide of SEQ ID NO 2. 4. 6, 8 It will be appreciated that in any such kit, (a), (b). (c) or (d) may comprise a substantial component Such a kit ill be of use in diagnosing a disease or susceptibility to a Disease, among others
  • This mvention also relates to the use of polynucleotides of the present mvention as diagnostic reagents Detection of a mutated form of a polynucleotide of the mvention. preferable.
  • SEQ ID NO 1. 3. 5. 7. that is associated with a disease or pathogenicity will provide a diagnostic tool that can add to. or define, a diagnosis of a disease, a prognosis of a course of disease, a determination of a stage of disease, or a susceptibility to a disease, that results from under-expression.
  • Organisms, particularly infectious organisms, carrying mutations m such polynucleotide may be detected at the polynucleotide level by a vanety of techniques, such as those desc ⁇ bed elsewhere herem
  • the differences m a polynucleotide and/or polypeptide sequence between organisms possessing a first phenotype and organisms possessing a different, second different phenotype can also be determined If a mutation is observed in some or all organisms possessing the first phenotype but not in any organisms possessing the second phenotype.
  • a polynucleotide and/or polypeptide of the invention may also be detected at the polynucleotide or polypeptide level by a vanetv of techniques, to allow for serotyping.
  • RT-PCR can be used to detect mutations in the RNA It is particularly prefened to use RT-PCR m conjunction with automated detection systems, such as. for example. GeneScan RNA- cDNA or genomic DNA may also be used for the same purpose.
  • PCR As an example. PCR primers complementary to a polynucleotide encoding accA.
  • accB (fabE).
  • accC. accD polypeptide can be used to identify and analy ze mutations
  • the mvention further prov ides these primers with 1. 2. 3 or 4 nucleotides removed from the 5' and/or the 3' end
  • These p ⁇ mers may be used for. among other thmgs. amplify mg accA. accB (fabE). accC.
  • the p ⁇ mers may be used to amplify a polynucleotide isolated from an infected indi idual, such diat die polynucleotide mav then be subject to vanous techniques for elucidation of the polynucleotide sequence In this way. mutations m die polvnucleotide sequence may be detected and used to diagnose and/or prognose the infection or its stage or course, or to serotype and/or classify the infectious agent
  • the mvention further provides a process for diagnosing, disease, preferably bacterial infections, more preferably infections caused by Staphylococcus aureus. comprising determining from a sample derived from an individual, such as a bodily material, an increased level of expression of polynucleotide having a sequence of Table 1 [SEQ ID NO 1. 3. 5. 7] Increased or decreased expression of a accA. accB (fabE). accC. accD polynucleotide can be measured using any on of the methods well known in the art for the quantitation of polynucleotides, such as. for example, amplification. PCR. RT-PCR. RNase protection. Northern blotting, spectrometry and other hybridization methods
  • accC. accD polypeptide compared to normal control tissue samples may be used to detect die presence of an infection, for example Assay techniques that can be used to determine levels of a accA. accB (fabE).
  • accC. accD polypeptide. in a sample denved from a host, such as a bodily mate ⁇ al. are well- known to those of skill m the art
  • Such assav methods mclude radioimmunoassav s. competitive-binding assays.
  • Polypeptides and polynucleotides of the mvention may also be used to assess the bmdmg of small molecule substrates and hgands m, for example, cells, cell-free preparations, chemical hbranes. and natural product mixtures
  • substrates and hgands may be natural substrates and ligands or may be structural or functional mimetics See. e g , Cohgan et al .
  • the present mvention provides for a method of screening compounds to identify those that agonize or that antagonize the function of a polypeptide or polynucleotide of the mvention.
  • agonists or antagonists may be employ ed for therapeutic and prophy lactic purposes for such Diseases as herem mentioned
  • Compounds may be identified from a v anety of sources, for example, cells, cell-free preparations, chemical hbranes. and natural product mixtures
  • Such agonists and antagonists so-identified may be natural or modified substrates, hgands. receptors, enzymes, etc , as the case may be. of accA. accB (fabE).
  • the screening methods may simply measure the bmdmg of a candidate compound to the polypeptide or poly nucleotide. or to cells or membranes bearing the poly peptide 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 w ith 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.
  • Inhibitors of activ ation 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, in the absence of an agonist or antagomst, by testing whether the candidate compound results m inhibition of activation of the polypeptide or polynucleotide.
  • the screenmg methods may simply comprise the steps of mixing a candidate compound with a solution comprising a polypeptide or polynucleotide of the present invention, to form a mixture, measuring accA. accB (fabE). accC. accD polypeptide and/or polynucleotide activity in the mixture, and comparing the accA. accB (fabE). accC, accD polypeptide and/or polynucleotide activity of the mixture to a standard Fusion proteins, such as those made from Fc portion and accA. accB (fabE). accC, accD 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 constructed for measuring secreted or cell associated levels of poKpeptide usmg monoclonal and polyclonal antibodies by standard methods known in the art This can be used to discov er agents that may inhibit or enhance the production of poKpeptide (also called antagonist or agonist, respectiv ely ) from suitablv manipulated cells or tissues
  • the mv ention also provides a method of screening compounds to identifv those that enhance (agomst) or block (antagonist) the action of accA. accB (fabE).
  • accC. accD polypeptides or polynucleotides. particularly those compounds that are bacte ⁇ static and/or bacte ⁇ cidal
  • the mediod of screening may involve higli-throughput techniques For example, to screen for agomsts or antagonists, a synthetic reaction mix. a cellular compartment, such as a membrane, cell envelope or cell wall, or a preparation of any thereof, compnsmg accA. accB (fabE). accC.
  • accD poKpeptide and a labeled substrate or ligand of such polypeptide is mcubated m die absence or the presence of a candidate molecule that may be a accA. accB (fabE).
  • accC. accD agomst or antagonist The ability of the candidate molecule to agomze or antagomze the accA. accB (fabE).
  • accC. accD polypeptide is reflected m decreased bmdmg of die labeled ligand or decreased production of product from such substrate Molecules that bmd gratuitously . i e . without mducmg the effects of accA. accB (fabE). accC.
  • accD polypeptide are most likely to be good antagonists Molecules that bmd well and. as the case may be. mcrease die rate of product production from substrate, mcrease signal transduction. or mcrease chemical channel activity are agomsts Detection of the rate or lev el of. as the case may be, production of product from substrate, signal transduction. or chemical channel activity may be enhanced by usmg a reporter sy stem Reporter systems that may be useful in this regard mclude but are not limited to colonmetnc. labeled substrate converted mto product, a reporter gene that is responsive to changes m accA. accB (fabE). accC.
  • Poly peptides 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 crosshnkmg assays in which the poKpeptide is labeled with a radioactive isotope (for instance. l ⁇ I). chemically modified (for instance, biotinylated).
  • a radioactive isotope for instance. l ⁇ I
  • chemically modified for instance, biotinylated
  • a source of the putative receptor e g , cells, cell membranes, cell supematants, tissue extracts, bodily materials
  • Other methods include biophysical techniques such as surface plasmon resonance and spectroscopv These screening methods may also be used to identify agonists and antagonists of the polypeptide that 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 correlation time or tumbling rate Protein complexes, such as formed by accA. accB (fabE). accC.
  • accD polypeptide associating with another accA. accB (fabE).
  • Fluorescence energy transfer may also be used characterize small molecules that interfere with the formation of accA. accB (fabE).
  • accC. accD 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 bv obser ing fluorescence of the acceptor Compounds that block dime ⁇ zation will inhibit fluorescence energv transfer
  • Surface plasmon resonance can be used to monitor the effect of small molecules on accA. accB (fabE).
  • accC. accD polypeptide self-association as well as an association of accA. accB (fabE).
  • accC. accD polypeptide can be coupled to a sensor chip at low site density such that covalently bound molecules will be monomenc Solution protein can then passed over the accA. accB (fabE).
  • accD polypeptide -coated surface and specific binding can be detected in real -time by monitoring the change in resonance angle caused by a change m local refractive index
  • This technique can be used to characterize the effect of small molecules on kinetic rates and equilibrium binding constants for accA, accB (fabE).
  • accC. accD polypeptide self-association as well as an association of accA. accB (fabE).
  • a scintillation proximity assay may be used to characterize the interaction between an association of accA. accB (fabE).
  • accC. accD polypeptide can be coupled to a scintillation-filled bead Addition of radio-labeled accA. accB (fabE).
  • accC. accD polypeptide results in binding where the radioactive source molecule is in close proximity to the scintillation fluid Thus, signal is emitted upon accA. accB (fabE).
  • accC. accD polypeptide bmdmg and compounds that prevent accA. accB (fabE).
  • accC. accD polypeptide self-association or an association of accA. accB (fabE).
  • accC. accD polypeptide and another polypeptide or small molecule will dimmish signal
  • a polypeptide and/or polynucleotide of d e mvention compnsmg contacting a polypeptide and/or polynucleotide of the mvention with a compound to be screened under conditions to permit bmdmg to or other mteraction between the compound and the polypeptide and/or polynucleotide to assess the bmdmg to or other mteraction with the compound, such bmdmg or mteraction preferably being associated with a second component capable of providing a detectable signal m response to the bmdmg or mteraction of the polypeptide and/or polynucleotide with the compound, and determining whether the compound bmds to or otherwise interacts with and activates or inhibits an activity or expression of the polypeptide and/or polynucle
  • accC. accD agomsts is a competitive assay that combmes accA. accB (fabE). accC. accD and a potential agomst with accA. accB (fabE). accC. accD- binding molecules, recombmant accA. accB (fabE). accC. accD bmdmg molecules, natural substrates or gands or substrate or ligand mimetics. under appropnate conditions for a competitive inhibition assay AccA. accB (fabE). accC.
  • accD can be labeled, such as by radioactivity or a colonmetnc compound, such that the number of accA. accB (fabE). accC. accD molecules bound to a bmdmg molecule or converted to product can be determined accurately to assess the effectiveness of the potential antagonist
  • a polypeptide and/or polynucleotide of the present invention may also be used in a method for the structure-based design of an agonist or antagonist of the polypeptide and/or polynucleotide. by (a) determining in the first instance the three- dimensional structure of the polypeptide and/or polynucleotide. or complexes thereof, (b) deducing the three-dimensional structure for the likely reactive s ⁇ te(s). binding s ⁇ te(s) or mot ⁇ f(s) of an agonist or antagonist, (c) synthesizing candidate compounds that are predicted to bind to or react ith the deduced bindmg s ⁇ te(s). reactive s ⁇ te(s). and/or motifts). and (d) testing whether the candidate compounds are indeed agonists or antagonists
  • the present mvention 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 accA. accB (fabE). accC. accD polypeptide and/or polynucleotide
  • the expression and/or activity of the polypeptide and/or polynucleotide is m excess, several approaches are available One approach compnses administering to an individual m need thereof an inhibitor compound (antagonist) as herem desc ⁇ bed. optionally m combmation with a pharmaceutically acceptable earner, m an amount effective to inhibit the function and/or expression of the polypeptide and/or polynucleotide. such as. for example, by blocking the bmdmg of hgands. substrates, receptors, enzymes, etc .
  • soluble forms of the polypeptides still capable of binding the ligand, substrate, enzymes, receptors, etc in competition w ith endogenous polypeptide and/or polynucleotide may be administered Ty pical examples of such competitors include fragments of the accA. accB (fabE). accC. accD polypeptide and/or polypeptide
  • accA. accB (fabE).
  • accC. accD polypeptide can be inhibited using expression blocking techniques This blocking may be targeted against any step in gene expression, but is preferably targeted against transcription and/or translation
  • An examples of a known technique of this sort involve the use of antisense sequences, either internally generated or separately administered (see. for example, O ' Connor. J Neurochem (1991) 56 560 in Ohgodeoxy nucleotides as Antisense Inhibitors of Gene Expression. CRC Press. Boca Raton. FL (1988)) Alternatively . ohgonucleotides that form triple helices with the gene can be supplied (see. for example.
  • 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 drugs
  • the polynucleotide sequences encoding the ammo terminal regions of the encoded protein or Shine-Delgarno or other translation facilitating sequences of the respective mRNA can be used to construct antisense sequences to control the expression of the coding sequence of interest
  • the mvention 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.
  • the molecules of the invention may be used in the prevention of adhesion of bactena. in particular gram positive and/or gram negative bacteria, to eukaryotic, preferably mammalian, extracellular matrix proteins on m-dwelling devices or to extracellular matnx protems in wounds, to block bactenal adhesion between eukaryotic, preferably mammalian, extracellular matrix proteins and bacterial accA. accB (fabE). accC. accD proteins that mediate tissue damage and/or.
  • accA. accB fabE
  • accC. accD agomsts and antagonists preferably bacte ⁇ static or bactencidal agomsts and antagonists
  • the antagonists and agomsts of the mvention may be employed, for instance, to prevent, inhibit and/or treat diseases Hehcobacter pylori (herein "H pylori”) bacteria infect the stomachs of ov er one-third of the world's population causing stomach cancer, ulcers, and gastritis (International Agency for Research on Cancer (1994) Schi t ⁇ s omes Liver Flukes and Hehcobacter Pylori (International Agency for Research on Cancer. L on.
  • Bodily mate ⁇ al(s) means any matenal denved from an individual or from an orgamsm infecting. infesting or inhabiting an individual, mcludmg but not limited to. cells, tissues and waste, such as. bone. blood, serum, cerebrospmal fluid, semen, saliva, muscle, cartilage, organ tissue, skin, urine, stool or autopsy matenals
  • D ⁇ sease(s) means any disease caused by or related to infection by a bactena. mcludmg , for example, disease, such as. infections of the upper respiratory tract (e g , otitis media, bacterial tracheitis. acute epiglottitis. thy roiditis). lower respiratory (e g . empyema. lung abscess), cardiac (e g . infective endocarditis). gastrointestinal (e g . secretory diarrhoea, splemc absces. retrope ⁇ toneal abscess). CNS (e g . cerebral abscess), e e (e g . blephantis.
  • infections of the upper respiratory tract e g , otitis media, bacterial tracheitis. acute epiglottitis. thy roiditis
  • lower respiratory e g . empyema. lung abscess
  • cardiac e g
  • Hett cell(s) is a cell that has been introduced (e g . transformed or transfected) or is capable of introduction (e g . transformation or transfection) by an exogenous polynucleotide sequence
  • Identity as known m the art. is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as the case may be. as determined by comparing the sequences In the art. "identity " also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be. as determined by the match between strings of such sequences "Identity " can be readilv calculated by known methods, including but not limited to those described m (Computational Molecular Biology. Lesk. A M . ed . Oxford Umversitv Press. New York. 1988. Biocomputwg Informatics and Genome Projects. Smith. D W . ed . Academic Press. New York. 1993.
  • Poly nucleotide embodiments further include an isolated poly nucleotide compnsmg a polynucleotide sequence having at least a 95. 97 or 100% identity to the reference sequence of SEQ ID NO 1. 3. 5. 7. wherein said polynucleotide sequence may be identical to the reference sequence of SEQ ID NO 1. 3. 5. 7 or may include up to a certain integer number of nucleotide alterations as compared to the reference sequence, wherein said alterations are selected from the group consisting of at least one nucleotide deletion, substitution, including transition and transversion.
  • alterations may occur at the 5' or 3' terminal positions of the reference nucleotide sequence or anywhere between those tenninal positions, interspersed either individuallv among the nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence, and wherein said number of nucleotide alterations is determined by multiplying the total number of nucleotides m SEQ ID NO 1. 3. 5. 7 by the integer defining the percent identity divided by 100 and then subtracting that product from said total number of nucleotides m SEQ ID NO 1. 3, 5, 7. or
  • n n is the number of nucleotide alterations.
  • x n is the total number of nucleotides in SEQ ID NO 1. 3. 5.
  • y is 0 95 for 95%, 0 97 for 97% or 1 00 for 100%. and • is the symbol for the multiplication operator, and wherein any non-mteger product of x n and y is rounded down to the nearest integer prior to subtracting it from x n
  • Polypeptide embodiments further include an isolated polypeptide compnsmg a polypeptide having at least a 95, 97 or 100% identity to a polypeptide reference sequence of SEQ ID NO 2, 4. 6. 8. wherein said polypeptide sequence may be identical to the reference sequence of SEQ ID NO 2. 4. 6.
  • alterations 8 may include up to a certain integer number of ammo acid alterations as compared to the reference sequence, wherein said alterations are selected from the group consisting of at least one amino acid deletion, substitution, including conservative and non-conservati e substitution, or insertion, and wherein said alterations may occur at the amino- or carboxy -terminal positions of the reference polypeptide sequence or anywhere between those terminal positions, interspersed either individually among the amino acids in the reference sequence or in one or more contiguous groups w ithin the reference sequence, and wherein said number of amino acid alterations is determined by multiplying the total number of amino acids in SEQ ID NO 2. 4. 6. 8 by the integer defining the percent identity divided by 100 and then subtracting that product from said total number of ammo acids in SEQ ID NO 2. 4. 6. 8. or
  • n a is the number of amino acid alterations
  • x a is the total number of amino acids m SEQ ID NO 2. 4.
  • y is 0 95 for 95%. 0 97 for 97% or 1 00 for 100%. and • is the symbol for the multiplication operator, and wherein any non-integer product of x a and y is rounded down to the nearest integer prior to subtracting it from x a
  • Ind ⁇ v ⁇ dual(s) means a multicellular eukaryote. mcludmg. but not limited to a metazoan. a mammal, an ovid. a bovid. a simian, a primate, and a human
  • Isolated means altered “by the hand of man” from its natural state. i e . if it occurs m nature, it has been changed or removed from its ongmal environment, or bodi For example, a polynucleotide or a polypeptide naturally present in a living orgamsm is not ' 'isolated.
  • Streptococcus Staphylococcus Bordetella Corynebacterium Mycobactenum Neissena Haemophilus. Act nomycetes Streptomycetes, Nocardia. Enterohacter, Yersima Fancisella Pasture Ha, M ⁇ raxella Acinetobacter Erysipelothnx Branhamella Actinobacillus, Streptobacillus Listena Calymmatobactenum Brucella Bacillus Clostndium. Treponema. Eschenchia Salmonella.
  • Polynucleot ⁇ de(s) generally refers to any polynbonucleotide or poly deoxy ⁇ bonucleotide, that may be unmodified RNA or DNA or modified RNA or DNA
  • Polynucleot ⁇ de(s) mclude without limitation, smgle- and double-stranded DNA.
  • DNA that is a mixture of smgle- and double-stranded regions or smgle-, double- and tnple-stranded regions, smgle- and double-stranded RNA. and RNA that is mixture of smgle- and double-stranded regions
  • hybnd molecules compnsmg DNA and RNA that mav be single-stranded or, more typically, double-stranded, or tnple-stranded regions or a mixture of smgle- and double-stranded regions
  • polynucleotide refers to tnple-stranded regions compnsmg RNA or DNA or both RNA and DNA
  • the strands m such regions mav be from the same molecule or from different molecules
  • the regions mav 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 t ⁇ ple-hehcal region often is an oligonucleotide As used here
  • polynucleot ⁇ de(s) also mcludes DNAs or RNAs as desc ⁇ bed above that comp ⁇ se one or more modified bases
  • DNAs or RNAs with backbones modified for stability or for other reasons are "polynucleot ⁇ de(s)” as that term is mtended herem
  • DNAs or RNAs compnsmg unusual bases, such as lnosine.
  • polynucleot ⁇ de(s) as it is employed herem embraces such chemically , enzvmatically or metabolicallv modified forms of polynucleotides, as well as the chemical forms of DNA and RNA charactenstic of viruses and cells, mcludmg. for example, simple and complex cells "Polynucleot ⁇ de(s)” also embraces short polynucleotides often refened to as ohgonucleot ⁇ de(s)
  • Polypept ⁇ de(s) refers to any peptide or protem compnsmg two or more ammo acids jomed to each other by peptide bonds or modified peptide bonds "Polypept ⁇ de(s)” refers to both short chains, commonly refened to as peptides.
  • Polypeptides may compnse ammo acids other than the 20 gene encoded ammo acids "Polypept ⁇ de(s)" mclude tiiose modified either by natural processes, such as processing and other post-translational modifications, but also by chemical modification techniques Such modifications are well desc ⁇ bed m basic texts and more detailed monographs, as well as a voluminous research literature, and they are well known to those of skill in the art It will be appreciated that the same type of modification may be present the same or v ary mg degree at several sites m a given polypeptide Also, a given polypeptide may compnse many types of modifications Modifications can occur anywhere m a polypeptide.
  • Modifications mclude. for example, acetylation. acv lation. ADP- ⁇ bosy lation. amidation. covalent attachment of flavin, covalent attachment of a heme moiety , covalent attachment of a nucleotide or nucleotide denvative. covalent attachment of a lipid or lipid denvative. covalent attachment of phosphotidylinositol. cross-linking, cy clization. disulfide bond formation, demetiiy lation. formation of covalent cross-links, formation of cysteine.
  • Cyclic, branched and branched circular polypeptides may result from posttranslational natural processes and may be made by entirely synthetic methods, as well "Recombmant expression system(s)” refers to expression sv stems or portions thereof or polynucleotides of the invention mtroduced or transformed mto a host cell or host cell ly sate for the production of the polynucleotides and polypeptides of the mvention ⁇ 'Va ⁇ ant(s) ' as the term is used herein, is a polynucleotide or polypeptide that differs from a reference poly nucleotide or polypeptide respectively, but retains essential properties
  • a typical v anant of a polynucleotide differs in nucleotide sequence from another, reference polynucleotide Changes in the nucleotide sequence of the variant may or may not alter the amino acid sequence of a polypeptide encoded by the reference polynucleot
  • a vanant and reference polypeptide may differ in amino acid sequence by one or more substitutions, additions, deletions in any combination
  • a substituted or inserted amino acid residue may or may not be one encoded by the genetic code
  • the present mvention also mcludes mclude vanants of each of the polypeptides of the mvention. that is polypeptides that vary from the referents by conserv ative ammo acid substitutions, whereby a residue is substituted by another with like charactenstics Typical such substitutions are among Ala. Val. Leu and He. among Ser and Thr, among the acidic residues Asp and Glu. among Asn and Gin.
  • a variant of a polynucleotide or polypeptide may be a naturally occurring such as an allehc variant, or it may be a variant that is not known to occur naturally
  • Non-naturally occurring variants of polynucleotides and polypeptides may be made by mutagenesis techniques, by direct synthesis, and by other recombinant methods known to skilled artisans
  • 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 m size are rendered blunt by treatment with exonuclease and DNA polymerase. and EcoRI linkers added Fragments are hgated mto the vector Lambda ZapII that has been cut with EcoRI. the hbrarv packaged bv standard procedures and E cob infected with the packaged library
  • the library is amplified by standard procedures Method 2
  • Total cellular DNA is partially hy droly zed w ith a one or a combination of restriction enzymes appropriate to generate a series of fragments for cloning into library vectors (e g . Rsal. Pall. Alul. Bshl235I). and such fragments are size-fractionated according to standard procedures EcoRI linkers are hgated to the DNA and the fragments then hgated into the vector Lambda ZapII that have been cut with EcoRI. the library packaged by standard procedures, and E cob infected with the packaged library The library is amplified by standard procedures Example 2 accB (fabE) Characterization
  • bacterial biotin ligase (BirA) is assayed using the biotm carboxylase carrier protein (BCCP/AccB/FabE) as a substrate for the reaction
  • BCCP is either radiolabelled or labelled with fluorescent tag
  • Biotinylated product of the BirA reaction is captured v ia streptavidin / avidin interaction
  • Conversion of substrate to product is followed by either fluorescence polarization, homogeneous time resolved fluorescence or scintillation proximitv assay Standard procedures are used.
  • Acety 1-CoA carboxylase is responsible for the ATP-dependent carboxy lation of acetyl-CoA to form malonyl-CoA. the first commited and the rate limiting step in fatty acid biosynthesis This activity is comprised of three separate components, biotin carboxylase. a homodimer of AccC. carboxyltransferase. a heterotetramer of AccA and AccD and biotm carboxy 1 earner protein.
  • AccB(FabE) Detailed descnptions of the components and assays for the Escherichia coh enzymes comprising acety 1-CoA carboxylase have been descnbed (Ras B Guchhait. S Efthimios Polakis.
  • the determination of expression during infection of a gene from Staphylococcus aureus Necrotic fatty tissue from a 72hour groin infection of Staphylococcus aureus WCUH29 m the mouse is eff ⁇ cienth disrupted and processed m the presence of chaotropic agents and RNAase inhibitor to provide a mixture of animal and bacterial RNA
  • the optimal conditions for disruption and processing to give stable preparations and high yields of bacterial RNA are follow ed by the use of hybridisation to a radiolabelled oligonucleotide specific to Staphy lococcus aureus 16S RNA on Northern blots The RNAase free. DNAase free.
  • RNA obtained are suitable for Reverse Transcription PCR (RT-PCR) using unique primer pairs designed from the sequence of each gene of Staphy lococcus aureus WCUH29 a) Isolation of tissue infected with Staphylococcus aureus WCUH29 from a mouse animal model of infection (groin) 10 ml volumes of sterile nutrient broth (No 2 Oxoid) are seeded with isolated, individual colonies of Staphylococcus aureus WCUH29 from an agar culture plate The cultures are incubated aerobicallv (static culture) at 37°C for 16-20 hours Four week old mice (female.18g-22g.
  • RT-PCR Reverse Transcription PCR
  • mice are each infected by subcutaneous injection of 0 5ml of this broth culture of Staphylococcus aureus WCUH29 (diluted in broth to approximately 10 8 cfu/ml ) into the anterior, right lower quadrant (groin area) Mice should be monitored regularly during the first 24 hours after infection, then daily until termination of study Animals with signs of systemic infection, I e lethargy, ruffled appearance, isolation from group, should be monitored closely and if signs progress to mo ⁇ bundancy. the animal should be culled immediately
  • mice are killed using carbon dioxide asphyxiation
  • mice should be killed individuallv rather than m groups
  • the dead animal is placed onto its back and the fur swabbed liberally with 70% alcohol
  • An initial incision using scissors is made through the skin of the abdominal left lower quadrant, travelling superiorly up to. then across the thorax
  • the incision is completed bv cutting inferiorly to the abdominal low er right quadrant
  • Care should be taken not to penetrate the abdominal w ll Holding the skm flap with forceps, the skin is gently pulled way from the abdomen
  • the exposed abscess which covers the peritoneal w all but generallv does not penetrate the muscle sheet completel . is excised, taking care not to puncture the viscera
  • the abscess/muscle sheet and other infected tissue may require cutting in sections, prior to flash-freezing in liquid nitrogen, thereby allowing easier storage in plastic collecting vials b) Isolation of Staphylococcus aureus WCUH29 RNA from infected tissue samples 4-6 infected tissue samples(each approx 0 5-0 7g) in 2ml screw -cap tubes are removed from -
  • RNA extraction is then continued according to the method given by the manufacturers of TRIzol Reagent I e -
  • the aqueous phase approx 0 6 ml. is transferred to a stenle eppendorf tube and 0 5 ml of isopropanol is added After 10 minutes at room temperature the samples are spun at 12.000 x g.
  • RNA pellet is washed ith 1 ml 75% ethanol A brief vortex is used to mix the sample before cent ⁇ fuging at 7.500 x g 4 °C for 5 minutes The ethanol is removed and the RNA pellet dried under vacuum for no more than 5 minutes Samples are then resuspended by repeated pipetting in 100 microhtres of DEPC treated water, followed by 5-10 minutes at 55 °C Finally . after at least 1 minute on ice.
  • RNA preparations are stored at -80 °C for up to one month
  • RNA precipitate can be stored at the wash stage of the protocol in 75% ethanol for at least one year at -20 °C
  • RNA isolation Quality of the RNA isolated is assessed by running samples on 1% agarose gels 1 x TBE gels stained with ethidium bromide are used to visualise total RNA y lelds
  • 2 2M fonnaldeh de gels are run and v acuum blotted to Hy bond-N (Amersham) The blot is then hy b ⁇ dised w ith a ' " P labelled ohgonucletide probe specific to 16s rRNA of Staphylococcus aureus ( K Greisen. M Loeffelholz.
  • RNA samples derived from infected tissue were inactivated and removed by treatment with TRIzol LS Reagent (Gibco BRL. Life Technologies) according to the manufacturers protocol DNAase treated RNA was resuspended in 73 microhtres of DEPC treated water with the addition of Rnasin as described in Method 1 d) The preparation of cDNA from RNA samples derived from infected tissue
  • PCR reactions are run on a Perkin Elmer GeneAmp PCR Sy stem 9600 as follows 5 minutes at 95 °C. then 50 cvcles of 30 seconds each at 94 °C. 42 °C and 72 °C followed by 3 minutes at 72 °C and then a hold temperature of 4 °C (the number of cycles is optimally 30-50 to determine the appearance or lack of a PCR product and optimally 8-30 cycles if an estimation of the starting quantitv of cDNA from the RT reaction is to be made).
  • RT/PCR controls may include +/- reverse transcnptase reactions. 16s rRNA primers or DNA specific pnmer pairs designed to produce PCR products from non-transcribed Staphylococcus aureus WCUH29 genomic sequences To test the efficiency of the primer pairs they are used in DNA PCR with Staphylococcus aureus WCUH29 total DNA PCR reactions are set up and run as described above using approx 1 nucrogram of DNA in place of the cDNA and 35 cycles of PCR
  • Primer pairs which fail to give the predicted sized product in either DNA PCR or RT/PCR are PCR failures and as such are unmformative Of those which give the correct size product with DNA PCR
  • two classes are distinguished m RT/PCR 1 Genes which are not transcribed in v vo reproducibly fail to give a product in RT/PCR.
  • 2 Genes which are transcribed in vivo reproducibly giv e the correct size product in RT/PCR and show a stronger signal m the +RT samples than the signal (if at all present) in -RT controls as did accD gene when examined in this way

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Abstract

The invention provides accA; accB (fabE); accC; accD polypeptides and polynucleotides encoding accA; accB (fabE); accC; accD polypeptides and methods for producing such polypeptides by recombinant techniques. Also provided are methods for utilizing accA; accB (fabE); accC; accD polypeptides to screen for antibacterial compounds.

Description

Acetyl Coenzyme A Carboxylase from Staphylococcus aureus
HELD OF THE INVENTION
This relates to newly identified polynucleotides and polypeptides. and their production and uses, as well as their
Figure imgf000002_0001
agonists and antagonists, and their uses In particular, the invention relates to polynucleotides and pohpeptides of the acetyl coenzyme A carboxylase farniK. as well as their \aπants. herein referred to as "accA. accB (fabE). accC. accD." "accA. accB (fabE). accC. accD polynucleotιde(s)." and "accA. accB (fabE). accC. accD polypeptιde(s)" as the case may be
BACKGROUND OF THE INVENTION
It is particularh preferred to employ Staphylococcal genes and gene products as targets for the development of antibiotics The Staphylococci make up a medically important genera of microbes The\ are known to produce tv\ o types of disease, invasive and toxigenic Invasive infections are characterized generalh by abscess formation effecting both skin surfaces and deep tissues S aureus is the second leading cause of bacteremia in cancer patients Osteomyelitis, septic arthritis, septic thrombophlebitis and acute bacterial endocarditis are also relatively common There are at least three clinical conditions resulting from the toxigenic properties of Staphylococci The manifestation of these diseases result from the actions of exotoxins as opposed to tissue invasion and bacteremia These conditions include Staphylococcal food poisoning, scalded skin syndrome and toxic shock syndrome The frequency of Staphylococcus aureus infections has nsen dramatically in the past few decades
This has been attnbuted to the emergence of multiply antibiotic resistant strains and an increasing population of people with weakened immune systems It is no longer uncommon to isolate Staphylococcus aureus strains that are resistant to some or all of the standard antibiotics This phenomenon has created an unmet medical need and demand for new anti-microbial agents, vaccines, drug screening methods, and diagnostic tests for this organism
Moreover, the drug discovery process is currently undergoing a fundamental revolution as it embraces "functional genomics," that is, high throughput genome- or gene-based biology This approach is rapidly superseding earlier approaches based on "positional cloning" and other methods Functional genomics relies heavily on the various tools of bioinformatics to identify gene sequences of potential interest from the many molecular biology databases now available as well as from other sources There is a continuing and significant need to identify and characterize further genes and other polynucleotides sequences and their related polypeptides. as targets for drug discovery Clearly, there exists a need for polynucleotides and pohpeptides. such as the accA. accB (fabE). accC. accD embodiments of the invention, that ha\e a present benefit of. among other things, being useful to screen compounds for antimicrobial activity Such factors are also useful to determine their role in pathogenesis of infection. dy sfunction and disease There is also a need for identification and charactenzation of such factors and dieir antagonists and agonists to find way s to prevent, ameliorate or correct such infection, dy sfunction and disease
SUMMARY OF THE INVENTION
The present invention relates to accA. accB (fabE). accC. accD. in particular accA. accB (fabE). accC. accD pohpeptides and accA. accB (fabE). accC. accD poKnucleotides. recombmant matenals and methods for their production In another aspect, the invention relates to mediods for usmg such pohpeptides and poKnucleotides. including treatment of microbial diseases, amongst others In a further aspect, the invention relates to methods for identifying agonists and antagonists using the materials pro \ ided by the invention, and for treating microbial infections and conditions associated with such infections with the identified agonist or antagonist compounds In a still further aspect, the invention relates to diagnostic assays for detecting diseases associated with microbial infections and conditions associated with such infections, such as assays for detecting accA. accB (fabE). accC. accD expression or activity
Vaπous changes and modifications within the spiπt and scope of the disclosed invention will become readily apparent to those skilled in the art from reading the following descriptions and from reading the other parts of the present disclosure
DESCRIPTION OF THE INVENTION
The invention relates to accA. accB (fabE). accC. accD polypeptides and poKnucleotides as descnbed m greater detail below In particular, the invention relates to pohpeptides and polynucleotides of a accA. accB (fabE). accC. accD of Staphylococcus aureus. ti at is related by amino acid sequence homology to yqhW of Bacillus subtihs and accA. accB (fabE), accC. accD of E cob polypeptide The invention relates especially to accA. accB (fabΕ). accC. accD having a nucleotide and ainino acid sequences set out in Table 1 as SΕQ ID NO 1, 3. 5. 7 and SΕQ ID NO 2. 4. 6. 8 respectively Note that sequences recited in the Sequence Listing below as "DNA" represent an exemplification of the invention, since those of ordinary skill will recognize that such sequences can be usefulK employed in polynucleotides in general including πbopolynucleotides
TABLE 1 Staphylococcus aureus accA; accB (fabE); accC; accD Polynucleotide and Polypeptide Sequences
(A) Staphylococcus aureus accA polynucleotide sequence [SEQ ID NO: 1].
5 ' - ATGTTAGATTTTGAAAAACCACTTTTTGAAATTCGAAATAAAATTGAATCTTTAAAAGAATCTCAAGATAAAAAT GATGTGGATTTACAAGAAGAAATTGACATGCTTGAAGCGTCATTGGAACGAGAAACTAAAAAAATATATACAAAT CTAAAACCATGGGATCGTGTGCAAATTGCGCGTTTGCAAGAAAGACCTACGACCCTAGATTATATTCCATATATC TTTGATTCGTTTATGGAACTACATGGTGATCGTAATTTTAGAGATGATCCAGCAATGATTGGTGGTATTGGCTTT TTAAATGGTCGTGCTGTTACAGTTATTGGACAACAACGTGGAAAAGATACAAAAGATAATATTTATCGAAATTTT GGTATGGCGCATCCAGAAGGTTATCGAAAAGCATTACGTTTAATGAAACAAGCTGAAAAATTCAATCGTCCTATC TTTACATTTATAGATACAAAAGGTGCATATCCTGGTAAAGCTGCTGAAGAACGTGGACAAAGTGAATCTATCGCA ACAAATTTGATTGAGATGGCTTCATTAAAAGTACCAGTTATTGCGATAGTCATTGGTGAAGGTGGCAGTGGAGGT GCTCTAGGTATTGGTATTGCCAATAAAGTATTGATGTTAGAGAATAGTACTTACTCTGTTATATCTCCTGAAGGT GCAGCGGCATTATTATGGAAAGACAGTAATTTGGCTAAAATTGCAGCTGAAACAATGAAAATTACTGCCCATGAT ATTAAGCAATTAGGTATTATAGATGATGTCATTTCTGAACCACTTGGCGGTGCACATAAAGATGTTGAACAGCAA GCTTTAGCTATTAAATCAGCGTTTGTTGCACAGTTAGATTCACTTGAGTCATTATCACGTGATGAAATTGCTAAT GATCGCTTTGAAAAATTCAGAAATATCGGTTCTTATATAGAATAA-3 '
Staphylococcus aureus accB (fabE) polynucleotide sequence [SEQ ID NO:3J. 5 ' -
ATGAACTTTAAAGAAATCAAAGAATTAATTGAAATTCTGGATAAATCAACTTTAACGGAAATCAATATTGAAGAT ACTAAAGGCAAAGTGACGCTTAAGAAAGAAAAAGAAACTGAGATTATCACGCCACAAATCTCACAAATGCCAGTT GAAGCTGCGGCAATGCCTATGCCTCAAGCACAATCAACTGATAGCAATAAAACTGAAGCTCCAAAGCCAACTTCA GATAATCACAAAACAATTAATGCACCTATGGTAGGTACATTTTACAAATCGCCATCTCCAGACGAAGAAGCATAT GTGCAAGTTGGGGACACTGTTTCAAATGAAACAACAGTGTGTATTTTAGAAGCAATGAAACTATTTAATGAAATT CAAGCAGAAATTTCAGGTGAAATTGTTGAAATCTTAGTAGAAGACGGACAAATGGTAGAGTATGGCCAACCGTTA TTTAAGGTGAAATAA-3 '
Staphylococcus aureus accC polynucleotide sequence [SEQ ID NO:5]. 5 ' -
ATGCTTCGTTGTTTAATTGCGAACAGAGGTGAAATTGCTGTAAGGATTATAAGAGCTTGCAGAGAATATGGGATT GAAACTGTAGCAGTTTATGCAAAAGGGGACGAGCAAAGCTTGCATGTACATTTAGCAGATCAAGCTATATGTATT GGGGAAGCTAATGCTTTAGATAGTTATTTAAATATTGACCGCATCATATCTGCTGCACAAATCACTGGTGCTAAT GCAATTCACCCAGGATATGGCTTTTTATCAGAATCAACAAAATTTGCTCAAACCGTTGAGGAACAAGGCATAGCG TTTATAGGGCCTACTAAAAAAACGATGGAAATGATGGGGGATAAAATAACAGCAAGACAAACAGTTCATCATGCA GGTGTTCCTGTGATTCCAGGATCAAATGGTGCTGTGAATCATGTGTCTGAAATTGAGAATCTTGCCAAAGACATC GGTTATCCAGTTGTTATCAAAGCTGCCAGTGGCGGTGGTGGTAAAGGTATACGTATTGTAAAGAAAGCTGAAGAT TTAGAAAAGGCATTTAAAGAAGCTAAAAGTGAAGGAAAAAAATACTTTGATGATGATCGTGTTTATGTTGAGGCT TTTATACCTGTTGCAAAACATGTTGAAGTTCAAGTTATGGGAGATGGGCAGGATAATTATGTACATTTAGGGGAA CGTGATTGTTCTGTACAACGTAAAAATCAAAAATTAATCGAAGAATCTCCTTGTGCAGCATTAACTGAAGAACGA CGACAACAAATATGTAACGATGCTGTTAAAGTTGCACGTGCAGCAAATTATAGAAGTGCAGGTACGATTGAATTT TTAGTAACTGATACCGCACATTACTTTATTGAAATGAATGCTCGAATACAAGTTGAACATACTGTTACAGAGATG CGAGCAGAACGTGATTTGGTTGCAGCACAACTGTATTTATTGGAACATAATCATTTACCTTTTTCACAAAGCGAT 5 ATTCAATTTAATGGTCATGTGATAGAAGCGAGAATAAATGCCGAAAATCCTGAAAAGAAATTTCAACCAACACCA GGTAAAGTGACAGCGTTACATTTACCTCAAGGCTTTAATGTGCGAGTCGATTCTTTACTATATCACGGTTATCAA GTTTCACCATATTATGATTCTTTAGTAGCTAAAGTAATTGTAAAATCTCATGATAGAGCTTCAGCAATTGATAAG TTGAAAGTCACCTTAGATGAAATGGTGATAGATGGATTTTCTACAACTGCCGATTTTTTATATGCAGTTTTAAAT TATCCACTATATCGTGATGGAGATGCAAAAGATGTTGATATTAAATTTTTAGAAAAACATCAAATTGTTAAAGGG ]() GTGGAATTATAA -3"
Staphylococcus aureus accD polynucleotide sequence [SEQ ID NO 7]
5 ' -
1 ATGTTTAAAGATTTTTTTAATCGAACAAAGAAAAAGAAATATCTTACAGTACAAGACTCTAAAAATAATGATGTG CCTGCAGGTATTATGACTAAGTGTCCAAAGTGTAAGAAAATTATGTACACAAAAGAATTAGCTGAAAATTTAAAT GTGTGCTTTAATTGTGATCATCATATTGCTTTAACTGCGTATAAACGTATAGAAGCAATTTCTGATGAAGGATCA TTTACAGAATTCGATAAGGGAATGACCTCTGCGAATCCATTAGATTTTCCAAGTTATTTAGAAAAAATTGAAAAG GACCAACAAAAGACAGGTCTTAAAGAAGCAGTTGTGACTGGTACAGCGCAACTAGATGGTATGAAATTTGGCGTT 0 GCTGTCATGGATTCACGTTTTAGAATGGGAAGTATGGGATCGGTTATCGGTGAAAAGATATGTCGCATCATTGAT TACTGCACTGAGAACCGTTTACCATTTATTCTTTTCTCTGCAAGTGGTGGTGCACGTATGCAAGAAGGTATTATT TCCTTGATGCAAATGGGTAAAACCAGTGTATCTTTAAAACGTCATTCTGACGCTGGACTATTATATATATCATAT TTAACACATCCAACTACTGGTGGTGTATCTGCAAGTTTTGCATCAGTTGGTGATATAAATTTAAGTGAGCCAAAA GCGTTGATAGGTTTTGCAGGTCGTCGAGTTATTGAACAGACAATAAACGAAAAATTGCCAGATGATTTCCAAACT
25 GCAGAATTTTTATTAGAGCATGGACAATTGGATAAAGTTGTACATCGTAATGATATGCGTCAAACATTGTCTGAA ATTCTAAAAATCCATCAAGAGGTGACTAAATAA-3 '
(B) Staphylococcus aureus accA. accB (fabE). accC. accD polypeptide sequence deduced from a 30 polynucleotide sequence in this table [SEQ ID NO 2] accA:
NH_-
MLDFEKPLFEI RNKI ESLKESQDKNDVDLQEEI DML--AS ERETKKI YTNLKP DRVQIARLQERPTTLDYI PYI FDS FMELHGDRNFRDDPAMI GGI GFLNGRAVTVI GQQRGKDTKDNIYRJIFGMAHPEGYRKALRLMKQAEKFNRPI 35 FTFIDTKGAYPGKAAEERGQSESIATNLIEMASLKVPVIAIVIGEGGSGGALGI GIANKVLMLENSTYSVI SPEG AAALL KDSNI-AKIAAETMKITAHDI KQLGI IDDVI SEPLGGAHKDVEQQALAI KSAFVAQLDSLESLSRDEIAN DRFEKFRNI GSYI E- COOH
accB (fabE): [SEQ ID NO 4] NH:- - KEIKELIEILDKSTLTEINIEDTKGIλ/T KKEKETEIITPQISQMPVEAA MPMPQAQS DSNKT--APKPTS DNHKTINAPMVGTFYKSPSPDEEAYVQVGDTVSNETTVCILEAMKLFNEIQAEISGEIVEI VEDGQMVEYGQPL FKVK -COOH
accC: [SEQ ID NO 6]
NH2-
MLRCLIANRGEIAVRI I RACREYGI ETVAVYAKGDEQS LHλΛILADQAI CI GEAl.ALDSYLNI DRI I SAAQITGAN
AIHPGYGFLSESTKFAQTVEEQGIAFI GPTKKTMEMMGDKITARQTλ HHAGVPVI PGSNGAVNHVSEI ENI-AKDI GYPWI KAASGGGGKGI RIVKKAEDLEKAFKEAKS EGKKYFDDDRVYVEAFI PVAKHVEVQVMGDGQDNYVHLGE RDCSVQRKNQKLI EES PCAALTEERRQQI CNDAVKVARAANYRSAGTI EFLVTDTAHYFI
Figure imgf000006_0001
RAERDLVAAQLYLLEHNHLPFSQSDIQFNGHVI EARINAENPEKKFQPTPGKVTALHLPQGFNVRVDSLLYHGYQ VS PYYDS LVAP VIVKSHDRAΞAI DKLKVTLDEMVI DGFSTTADFLYAVLNYPLYRDGDAKDVDIKFLEKHQIVKG VEL -COOH
accD: [SEQ ID NO 8]
NH2-
MFKDFFNRTKKKKYLTVQDSKNNDVPAGIMTKCPKCKKIMYTKE AEN NVCFNCDHHIALTAYKRIEAISDEGS FTEFDKGMTSANPLDFPSYLEKIEKDQQKTGLKEAWTGTAQLDGMKFGVAVMDSRFRMGSMGSVIGEKICRIID YCTENRLPFILFSASGGARMQEGIISLMQMGKTSVSLKRHSDAGLLYISYLTHPTTGGVSASFASVGDINLSEPK ALIGFAGRRVIEQTINEK PDDFQTAEF EHGQLDKWHRNDMRQTLSEILKIHQEλ TK -COOH
Deposited materials
A deposit compnsing a Staphylococcus aureus WCUH 29 strain has been deposited with the National Collections of Industπal and Marine Bactena Ltd (herein "NCIMB"), 23 St Machar Dπve, Aberdeen AB2 1RY. Scotland on 11 September 1 95 and assigned NCIMB Deposit No 40771. and referred to as Staphylococcus aureus WCUH29 on deposit The Staphylococcus aureus strain deposit is referred to herein as "d e deposited stram" or as "the DNA of the deposited strain "
The deposited stram compπses a full length accA. accB (fabE). accC. accD gene The sequence of the polynucleotides compπsed in the deposited strain, as well as the amino acid sequence of any polypeptide encoded thereby, are controlling m the event of any conflict with any descπption of sequences herem The deposit of the deposited stram has been made under the teπns of die Budapest Treaty on the
International Recognition of the Deposit of Micro-organisms for Purposes of Patent Procedure The deposited stram will be irrevocably and without restnction or condition released to the public upon the issuance of a patent The deposited stram is provided merely as convenience to those of skill in the art and is not an admission tliat a deposit is required for enablement. such as that required under 35 U S C §1 12 A license may be required to make, use or sell the deposited stram. and compounds den\ ed therefrom, and no such license is hereby granted In one aspect of the mvention there is provided an isolated nucleic acid molecule encoding a mature poKpeptide expressible by the Staphylococcus aureus WCUH 29 stram. which poKpeptide is compπsed m the deposited strain Further provided by the mvention are accA. accB (fabE). accC. accD polynucleotide sequences m the deposited stram. such as DNA and RNA. and ammo acid sequences encoded thereby Also provided by the invention are accA. accB (fabE). accC. accD poKpeptide and poKnucleotide sequences isolated from the deposited stram Polypeptides
AccA. accB (fabE). accC. accD poKpeptide of the mvention is substantially phylogenetically related to other proteins of the acety 1 coenzyme A carboxylase family
In one aspect of the mvention there are provided polypeptides of Staphylococcus aureus referred to herem as "accA. accB (fabE), accC. accD" and "accA. accB (fabE). accC. accD polypeptides" as well as biologically, diagnostically . prophylactically . clinically or therapeutically useful \anants thereof, and compositions compnsing the same
Among the particularly preferred embodiments of the mvention are vaπants of accA. accB (fabE). accC. accD poKpeptide encoded by naturally occurring alleles of a accA. accB (fabE). accC. accD gene The present mvention further provides for an isolated polypeptide that (a) comprises or consists of an ammo acid sequence that has at least 95% identity, most preferably at least 97-99% or exact identity , to that of SEQ ID NO 2. 4. 6. 8 over the entire length of SEQ ID NO 2. 4. 6. 8. (b) a polypeptide encoded by an isolated polynucleotide comprising or consisting of a polynucleotide sequence that has at least 95% identity, even more preferably at least 97-99% or exact identity to SEQ ID NO 1. 3. 5. 7 o\er the entire length of SEQ ID NO 1, 3, 5. 7, (c) a polypeptide encoded by an isolated polynucleotide comprising or consisting of a polynucleotide sequence encoding a polypeptide that has at least 95% identity, even more preferably at least 97-99% or exact identity . to the ammo acid sequence of SEQ ID NO 2. 4. 6. 8. over the entire length of SEQ ID NO 2. 4, 6, 8
The polypeptides of the mvention mclude a polypeptide of Table 1 [SEQ ID NO 2. 4 6. 8] (in particular a mature polypeptide) as well as polypeptides and fragments, particularly those that has a biological activity of accA. accB (fabE). accC. accD. and also those that ha\e at least 95% identity to a polypeptide of Table 1 [SEQ ID NO 2. 4. 6. 8] and also mclude portions of such polypeptides with such portion of the polypeptide generally compπsmg at least 30 ammo acids and more preferably at least 50 ammo acids The invention also cludes a polypeptide consisting of or compπsmg a poKpeptide of the fomiula
X-(Rl)m-(R2)-(R3)n-Y w herem at d e amino terminus. X is hydrogen, a metal or any other moietλ descπbed herem for modified polypeptides and at die carboxyl terminus. Y is hydrogen, a metal or any other moiety descπbed herem for modified polypeptides. Rj and R3 are any ammo acid residue or modified ammo acid residue, m is an integer between 1 and 1000 or zero, n is an integer between 1 and 1000 or zero, and R2 is an ammo acid sequence of the mvention. particularly an ammo acid sequence selected from Table 1 or modified forms thereof In the formula abo\ e. R2 is oπented so that its ammo terminal ammo acid residue is at the left, covalently bound to Ri and its terminal ammo acid residue is at the πght. covalently bound to R3 Any stretch of ammo acid residues denoted by either R] or R3. where m and/or n is greater than 1. may be either a heteropolymer or a homopohmer. preferably a heteropolymer Other preferred embodiments of the mvention are provided where m is an mteger between 1 and 50. 100 or 500. and n is an mteger between 1 and 50. 100, or 500
It is most preferred that a polypeptide of die mvention is demed from Staphylococcus aureus however it may preferably be obtained from other organisms of die same taxonomic genus A poKpeptide of the mvention also be obtained, for example, from organisms of die same taxonomic family or order
A fragment is a vaπant polypeptide having an ammo acid sequence that is entirely the same as part but not all of an\ ammo acid sequence of any poKpeptide of the mvention As with accA. accB (fabE). accC. accD polypeptides. fragments may be "free-standing." or compπsed within a larger polypeptide of which they form a part or region, most preferably as a smgle cont uous region m a smgle larger polypeptide Preferred fragments mclude, for example, truncation polypeptides havmg a portion of an ammo acid sequence of Table 1 [SEQ ID NO 2. 4. 6, 8], or of vaπants thereof, such as a contmuous seπes of residues that mcludes an ammo- and/or carboxyl-termmal amino acid sequence Degradation forms of the polypeptides of the mvention produced by or in a host cell, particularly a Staphylococcus aureus. are also preferred Further preferred are fragments characterized by structural or functional attributes such as fragments that compπse alpha-helix and alpha-helix formmg regions, beta-sheet and beta-sheet-formmg regions, turn and turn-formmg regions, coil and coil-forming regions, hydrophilic regions, hydrophobic regions alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions, substrate binding region, and high antigenic index regions
Further preferred fragments mclude an isolated polypeptide comprising an amino acid sequence having at least 15, 20, 30, 40, 50 or 100 contiguous amino acids from the amino acid sequence of SEQ ID NO 2, 4, 6, 8, or an isolated polypeptide comprising an amino acid sequence having at least 15, 20, 30, 40, 50 or 100 contiguous amino acids truncated or deleted from the amino acid sequence of SEQ ID NO.2, 4, 6, 8. Fragments of die polypeptides of the may be employ ed for producmg the corresponding full-length polypeptide by peptide synthesis, therefore, these \aπants may be emplo\ed as intermediates for producmg d e full-lengdi polypeptides of the mvention Polynucleotides It is an object of the mvention to provide polynucleotides that encode accA. accB (fabE). accC. accD polypeptides. particularly polynucleotides that encode a polypeptide herem designated accA. accB (fabE). accC. accD
In a particularly preferred embodiment of the mvention the polvnucleotide compπses a region encoding accA. accB (fabE). accC. accD polypeptides compnsmg a sequence set out m Table 1 [SEQ ID NO 1. 3. 5. 7] that mcludes a full length gene, or a variant thereof The Applicants believe that this fiiU length gene is essential to the growth and/or survival of an organism that possesses it. such as Staphylococcus aureus
As a further aspect of the mvention there are provided isolated nucleic acid molecules encoding and/or expressmg accA. accB (fabE). accC. accD polypeptides and polynucleotides. particularly Staphylococcus aureus accA. accB (fabE). accC. accD polypeptides and polynucleotides. mcludmg. for example, unprocessed RNAs. πbozyme RNAs. mRNAs. cDNAs. genomic DNAs. B- and Z-DNAs Further embodiments of the mvention mclude biologically, diagnostically. prophylactically. clinically or therapeutically useful polynucleotides and polypeptides. and vaπants thereof and compositions compnsmg die same Another aspect of the mvention relates to isolated polynucleotides. mcludmg at least one full length gene, that encodes a accA. accB (fabE). accC. accD poKpeptide havmg a deduced ammo acid sequence of Table 1 [SEQ ID NO 2. 4. 6. 8] and polynucleotides closely related thereto and vaπants thereof
In another particularly preferred embodiment of the invention there is a accA. accB (fabE). accC. accD polypeptide from Staphylococcus aureus comprising or consisting of an ammo acid sequence of Table 1 [SEQ ID NO 2. 4, 6. 8]. or a vaπant thereof
Usmg die mformation provided herem. such as a polynucleotide sequence set out m Table 1 [SEQ ID NO 1. 3. 5. 7J. a polynucleotide of die mvention encoding accA. accB (fabE). accC. accD polypeptide may be obtained usmg standard cloning and screening methods, such as those for cloning and sequencmg chromosomal DNA fragments from bacteπa using Staphylococcus aureus WCUH 29 cells as starting mateπal. followed bv obtainmg a full length clone For example, to obtain a polynucleotide sequence of the invention, such as a polynucleotide sequence given m Table 1 [SEQ ID NO 1. 3. 5. 7], typically a library of clones of chromosomal DNA of Staphylococcus aureus WCUH 29 in E cob or some other suitable host is probed with a radiolabeled ohgonucleotide. preferably a 17-mer or longer, derived from a partial sequence Clones carry ing DNA identical to that of the probe can then be distinguished usmg stringent hy bπdization conditions By sequencing the indiv ldual clones thus identified by hy bπdization with sequencing primers designed from the original polypeptide or poly nucleotide sequence it is then possible to extend the polynucleotide sequence in both directions to determine a full length gene sequence Conveniently , such sequencmg is performed, for example, using denatured double stranded DNA prepared from a plasmid clone Suitable techniques are described by Mamatis. T . Fπtsch. E F and Sambrook et al . MOLECULAR CLONING A LABORATORY MANUAL. 2nd Ed . Cold Spring Harbor Laboratory Press. Cold Spring Harbor. New York (1989) (see in particular Screening By Hybridization 1 90 and Sequencing Denatured Double-Stranded DNA Templates 13 70) Direct genomic DNA sequencing may also be performed to obtain a full length gene sequence Illustrative of the invention each polynucleotide set out m Table 1 [SEQ ID NO 1. 3. 5. 7] was discovered m a DNA library deπved from Staphylococcus aureus WCUH 29
Moreover, each DNA sequence set out in Table 1 [SEQ ID NO 1. 3. 5. 7] contains an open reading frame encoding a protem having about the number of ammo acid residues set forth in Table 1 [SEQ ID NO 2. 4, 6, 8] witii a deduced molecular weight that can be calculated usmg ammo acid residue molecular weight values well known to those skilled m the art The polynucleotide of SEQ ID NO 1. 3, 5. 7. between nucleotide number 1 (accA), 1 (accB/fabE). 1 (accC). 1 (accD) and the stop codon that begins at nucleotide number 943 (accA). 463 (accB/fabE), 1360 (accC). 856 (accD) of SEQ ID NO 1. 3. 5. 7, encodes the polypeptide of SEQ ID NO 2. 4, 6. 8 hi a further aspect, the present mvention provides for an isolated polynucleotide comprising or consisting of (a) a polynucleotide sequence that has at least 95% identity , even more preferably at least 97-99% or exact identity to SEQ ID NO 1. 3. 5. 7 over the entire length of SEQ ID NO 1. 3. 5. 7. or the entire length of that portion of SEQ ID NO 1. 3. 5. 7 which encodes SEQ ID NO 2. 4. 6. 8. (b) a polynucleotide sequence encoding a polypeptide that has at least 95% identity . even more preferably at least 97-99% or 100% exact, to the ammo acid sequence of SEQ ID NO 2. 4. 6. 8. over the entire length of SEQ ID NO 2. 4. 6, 8
A polynucleotide encoding a polypeptide of the present invention, mcludmg homologs and orthologs from species odier than Staphylococcus aureus. may be obtained by a process tiiat compπses the steps of screening an appropnate library under stringent hybπdization conditions w th a labeled or detectable probe consisting of or compnsmg the sequence of SEQ ID NO 1. 3. 5. 7 or a fragment thereof, and isolating a full- length gene and/or genomic clones compnsmg said polynucleotide sequence
The mvention provides a polynucleotide sequence identical over its entire length to a coding sequence (open reading frame) in Table 1 [SEQ ID NO 1. 3, 5. 7] Also provided by the mvention is a coding sequence for a mature polypeptide or a fragment thereof, by itself as well as a coding sequence for a mature polypeptide or a fragment m reading frame with another coding sequence, such as a sequence encoding a leader or secretory sequence, a pre-, or pro- or prepro-protem sequence The polynucleotide of the mvention may also compπse at least one non-coding sequence, mcludmg for example, but not limited to at least one non-coding 5 ' and 3' sequence, such as the transcπbed but non-translated sequences, termination signals (such as rho- dependent and rho-independent termination signals), πbosome binding sites. Kozak sequences, sequences that stabilize niRNA. mtrons. and poly adenylation signals The polynucleotide sequence may also compπse additional coding sequence encoding additional ammo acids For example, a marker sequence that facilitates punfication of a fused poKpeptide can be encoded In certain embodiments of the mvention. the marker sequence is a hexa-histidine peptide. as provided m the pQE vector (Qiagen. Inc ) and descπbed m Gentz et al . Proc Natl Acad Sa USA 86 821-824 (1989). or an HA peptide tag (Wilson et al . Cell 37 767 (1984). bodi of that may be useful m punfying polypeptide sequence fused to them Polynucleotides of the mvention also mclude. but are not limited to. polynucleotides compnsmg a structural gene and its naturally associated sequences diat control gene expression A preferred embodiment of the mvention is a polynucleotide of consistmg of or compnsmg nucleotide
1 (accA). 1 (accB/fabE). 1 (accC). 1 (accD) to the nucleotide immediately upstream of or mcludmg nucleotide 943 (accA). 463 (accB/fabE). 1360 (accC). 856 (accD) set forth m SEQ ID NO 1. 3. 5. 7 of Table 1. both of that encode a accA. accB (fabE). accC. accD polypeptide
The mvention also mcludes a polynucleotide consistmg of or compnsmg a polynucleotide of the formula
X-(Rι )m-(R2)-(R3)n-Y wherein, at the 5' end of the molecule. X is hydrogen, a metal or a modified nucleotide residue, or together with Y defines a covalent bond, and at the 3' end of the molecule. Y is hy drogen, a metal, or a modified nucleotide residue, or together with X defines the covalent bond, each occurrence of Rj and R3 is mdependently any nucleic acid residue or modified nucleic acid residue, m is an integer between 1 and 3000 or zero . n is an integer between 1 and 3000 or zero, and R2 is a nucleic acid sequence or modified nucleic acid sequence of the invention, particularly a nucleic acid sequence selected from Table 1 or a modified nucleic acid sequence thereof In the polynucleotide formula above. R? is oriented so that its 5' end nucleic acid residue is at the left, bound to Rj and its 3' end nucleic acid residue is at the right, bound to R3 Any stretch of nucleic acid residues denoted by either Rj and/or R2. where m and/or n is greater than 1. may be either a heteropolymer or a homopolymer. preferably a heteropolymer Where, in a preferred embodiment. X and Y together define a cov alent bond, the polynucleotide of the above formula is a closed, circular polynucleotide. that can be a double-stranded polynucleotide wherein the formula shows a first strand to which the second strand is complementary In another preferred embodiment m and/or n is an integer between 1 and 1000. Other prefened embodiments of the mvention are provided where m is an mteger between 1 and 50. 100 or 500. and n is an mteger between 1 and 50. 100. or 500 It is most prefened that a polynucleotide of the mvention is deπved from Staphylococcus aureus. however, it may preferably be obtamed from other organisms of the same taxonomic genus A polynucleotide of the invention may also be obtamed. for example, from organisms of the same taxonomic family or order
The tenn "polynucleotide encoding a polypeptide" as used herem encompasses polynucleotides that mclude a sequence encoding a polypeptide of the mvention. particularly a bactenal polypeptide and more particularly a polypeptide of the Staphvlococais aureus accA. accB (fabE). accC. accD having an ammo acid sequence set out in Table 1 [SEQ ID NO 2. 4. 6. 8] The term also encompasses polynucleotides that mclude a smgle contmuous region or discontinuous regions encoding the polypeptide (for example, polynucleotides interrupted by integrated phage. an mtegrated insertion sequence, an mtegrated vector sequence, an mtegrated transposon sequence, or due to RNA editing or genomic DNA reorganization) together witii additional regions, that also may compnse coding and/or non-coding sequences
The mvention further relates to vaπants of the polynucleotides descnbed herem that encode vaπants of a polypeptide havmg a deduced am o acid sequence of Table 1 [SEQ ID NO 2. 4, 6. 8] Fragments of polynucleotides of the mvention may be used, for example, to synthesize full-lengui polynucleotides of the mvention Further particularly preferred embodiments are polynucleotides encoding accA. accB (fabE). accC. accD vaπants. that have the ammo acid sequence of accA. accB (fabE). accC. accD polypeptide of Table 1 [SEQ ID NO 2. 4. 6. 8] m which several, a few. 5 to 10. 1 to 5. 1 to 3. 2. 1 or no ammo acid residues are substituted, modified, deleted and/or added, m any combmation Especially prefened among these are silent substitutions, additions and deletions, that do not alter the properties and activities of accA. accB (fabE). accC. accD polypeptide
Prefened isolated polynucleotide embodiments also mclude polynucleotide fragments, such as a polynucleotide comprising a nuclic acid sequence having at least 15, 20, 30, 40, 50 or 100 contiguous nucleic acids from the polynucleotide sequence of SEQ ID NO: l , 3, 5, 7, or an polynucleotide comprising a nucleic acid sequence having at least 15, 20, 30, 40, 50 or 100 contiguous nucleic acids truncated or deleted from the 5' and/or 3' end of the polynucleotide sequence of SEQ ID NO: l, 3, 5, 7.
Further prefened embodiments of the mvention are polynucleotides that are at least 95% or 97% identical over their entire length to a polynucleotide encoding accA. accB (fabE). accC. accD polypeptide having an ammo acid sequence set out m Table 1 [SEQ ID NO 2. 4. 6. 8]. and polynucleotides that are complementary to such polynucleotides Most highly prefened are polynucleotides that compπse a region that is at least 95% are especially prefened 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 prefened with at least 99% being the more prefened
Prefened embodiments are polynucleotides encoding polypeptides that retain substantially the same biological function or activity as a mature polypeptide encoded by a DNA of Table 1 [SEQ ID NO 1. 3. 5. 7] hi accordance with certain prefened embodiments of this mvention there are provided polynucleotides that hybndize. particularly under stringent conditions, to accA. accB (fabE). accC. accD polynucleotide sequences, such as diose polynucleotides m Table 1
The mvention further relates to polynucleotides that hybndize to die polynucleotide sequences provided herem In diis regard, the mvention especially relates to polynucleotides that hybndize under stringent conditions to the polvnucleotides descπbed herem As herem used, the terms "stringent conditions" and "stringent hybndization conditions" mean hvbndization occurring only if there is at least 95% and preferably at least 97% identity between the sequences A specific example of stringent hybridization conditions is overnight incubation at 42°C in a solution comprising 50% formamide. 5x SSC (150mM NaCl. 15mM tπsodium citrate). 50 mM sodium phosphate (pH7 6). 5x Denhardt's solution, 10% dextran sulfate. and 20 micrograms/ml of denatured, sheared salmon sperm DNA, followed by vvashmg the hybridization support in 0 lx SSC at about 65°C Hybridization and wash conditions are well known and exemplified in Sambrook. et al . Molecular Cloning A Laboratory Manual. Second Edition. Cold Spring Harbor. N Y . (1989). particularly Chapter 1 1 therein Solution hybridization may also be used with the polynucleotide sequences provided by the invention
The invention also provides a polynucleotide consisting of or comprising a polynucleotide sequence obtained by screening an appropriate library compnsmg a complete gene for a polynucleotide sequence set forth in SEQ ID NO 1. 3, 5. 7 under stringent hybridization conditions with a probe having the sequence of said polynucleotide sequence set forth in SEQ ID NO 1. 3. 5. 7 or a fragment thereof, and isolating said polynucleotide sequence Fragments useful for obtaining such a polynucleotide include, for example, probes and primers fully descnbed elsewhere herein
As discussed elsewhere herem regarding polynucleotide assays of die mvention. for instance, the polynucleotides of the mvention. may be used as a hvbndization probe for RNA. cDNA and genomic DNA to isolate full-length cDNAs and genomic clones encoding accA. accB (fabE). accC. accD and to isolate cDNA and genomic clones of other genes that have a high identity . particularly high sequence identity, to a accA. accB (fabE). accC. accD gene Such probes generally will compnse at least 15 nucleotide residues or base pairs Preferably such probes will have at least 30 nucleotide residues or base pairs and may have at least 50 nucleotide residues or base pairs Particularly preferred probes will have at least 20 nucleotide residues or base pairs and will have lee than 30 nucleotide residues or base pairs
A coding region of a accA. accB (fabE). accC. accD gene mav be isolated by screening usmg a DNA sequence provided m Table 1 [SEQ ID NO 1. 3. 5. 7] to svnthesize an oligonucleotide probe A labeled oligonucleotide havmg a sequence complementary to that of a gene of the v ention is then used to screen a library of cDNA genomic DNA or rnRNA to determine which members of the librarv the probe h bndizes to
There are sev eral methods available and well known to those skilled m the art to obtain full- length DNAs. or extend short DNAs, for example those based on the method of Rapid Amplification of cDNA ends (RACE) (see. for example. Frohman. et al . PNAS USA 85 8998-9002. 1988) Recent modifications of the technique, exemplified by the Marathon™ technologv (Clontech Laboratories Inc ) for example, hav e significantly simplified the search for longer cDNAs In the Marathon™ technology . cDNAs have been prepared from mRNA extracted from a chosen tissue and an 'adaptor' sequence hgated onto each end Nucleic acid amplification (PCR) is then carried out to amplify the "missing" 5' end of the DNA using a combination of gene specific and adaptor specific oligonucleotide primers The PCR reaction is then repeated using "nested" primers, that is, primers designed to anneal within the amplified product (typically an adaptor specific pnmer that anneals further 3' in the adaptor sequence and a gene specific primer that anneals further 5' in the selected gene sequence) The products of this reaction can then be analyzed by DNA sequencing and a full-length DNA constructed either by joining the product directly to the existing DNA to giv e a complete sequence, or carry ing out a separate full- length PCR using the new sequence information for the design of the 5' primer
The polynucleotides and polypeptides of the mvention mav be employ ed. for example, as research reagents and matenals for discovery of treatments of and diagnostics for diseases, particularly human diseases, as further discussed herem relating to polynucleotide assays
The polvnucleotides of the invention that are ohgonucleotides deπv ed from a sequence of Table 1 [SEQ ID NOS 1 or 2] may be used in the processes herein as described, but preferably for PCR. to determine whether or not the polynucleotides identified herein in whole or in part are transcribed in bacteria in infected tissue It is recognized that such sequences w ill also hav e utility in diagnosis of the stage of infection and type of infection the pathogen has attained
The mvention also provides polynucleotides that encode a polypeptide that is a mature protein plus additional ammo or carboxyl-terrninal ammo acids, or ammo acids uitenor to a mature polypeptide (when a mature form has more than one polypeptide chain, for instance) Such sequences may play a role m processing of a protem from precursor to a mature form may allow protein transport, mav lengthen or shorten protem half-life or may facilitate manipulation of a protem for assav or production, among other things As generally is the case in vivo, the additional ammo acids may be processed away from a mature protem b\ cellular enzymes For each and every polynucleotide of the mvention there is provided a polynucleotide complementary to it It is prefened that these complementary polynucleotides are fully complementary to each polynucleotide with which uiey are complementary
A precursor protem. having a mature form of the polypeptide fused to one or more prosequences may be an mactiv e form of the polypeptide When prosequences are removed such mactiv e precursors generally are activated Some or all of the prosequences may be removed before activation Generally . such precursors are called proproteins
As will be recognized, the entire polypeptide encoded by an open reading frame is often not required for activity Accordingly . it has become routme m molecular biology to map the boundanes of the primary structure required for activity with N-termmal and C-terminal deletion experiments These experiments utilize exonuclease digestion or convenient restnction sites to cleave coding nucleic acid sequence For example. Promega (Madison, WI) sell an Erase-a-base™ system that uses Exonuclease III designed to facilitate analysis of the deletion products (protocol available at www promega com) The digested endpoints can be repaired (e g . by hgation to synthetic linkers) to the extent necessary to preserve an open reading frame In this way. the nucleic acid of SEQ ID NO 1, 3. 5. 7 readily provides contiguous fragments of SEQ ID NO 2, 4. 6, 8 sufficient to provide an activity, such as an enzymatic, bmdmg or antibody -mducmg activity Nucleic acid sequences encoding such fragments of SEQ ID NO 2. 4. 6, 8 and vanants thereof as descπbed herem are within the mvention. as are polypeptides so encoded
In sum. a polynucleotide of the mvention may encode a mature protem. a mature protem plus a leader sequence (which may be refened to as a preprotein), a precursor of a mature protem having one or more prosequences that are not the leader sequences of a preprotein. or a preproprotein. that is a precursor to a proprotem. havmg a leader sequence and one or more prosequences. that generally are removed during process g steps that produce active and mature forms of the polypeptide Vectors, Host Cells, Expression Systems The mvention also relates to vectors that compnse a polynucleotide or polynucleotides of the mvention. host cells that are genetically engmeered with vectors of the mvention and the production of polypeptides of the mvention by recombinant techniques Cell-free translation systems can also be employed to produce such protems usmg RNAs deπved from the DNA constructs of the mvention Recombmant polypeptides of the present mvention may be prepared by processes well known m those skilled m the art from genetically engmeered host cells compnsmg expression systems Accordingl . m a further aspect, the present mvention relates to expression systems that compnse a polynucleotide or polynucleotides of the present mvention. to host cells that are genetically engmeered with such expression systems, and to the production of polypeptides of the mvention by recombmant techniques
For recombmant production of the polypeptides of the mvention. host cells can be genetically engmeered to incorporate expression systems or portions thereof or polynucleotides of the invention Introduction of a polynucleotide mto the host cell can be effected by methods descπbed m many standard laboratory manuals, such as Davis, et al . BASIC METHODS IN MOLECULAR BIOLOGY. (1986) and Sambrook. et al . MOLECULAR CLONING A LABORATORY MANUAL. 2nd Ed . Cold Spπng Harbor Laboratory Press. Cold Spring Harbor, N Y (1989). such as. calcium phosphate transfection. DEAE-dextran mediated transfection. transvection. microinjection. cationic hpid-mediated transfection. electroporation. transduction. scrape loading, ballistic introduction and infection
Representative examples of appropπate hosts mclude bacteπal cells, such as cells of streptococci. staphylococci. enterococci E cob. streptomyces. cyanobactena. Bacillus subttbs, and Staphylococcus aureus. fungal cells, such as cells of a yeast. Kluveromyces . Saccharomyces . a basidiomycete. Candida albicans and Aspergillus. insect cells such as cells of Drosophila S2 and Spodoptera Sf9. animal cells such as CHO. COS. HeLa, C127, 3T3. BHK. 293, CV-1 and Bowes melanoma cells, and plant cells, such as cells of a gymnosperm or angiosperm A great vanety of expression systems can be used to produce the polypeptides of the mvention Such vectors mclude. among others, chromosomal-, episomal- and virus-deπved vectors, for example, vectors denved from bactenal plasmids. from bactenophage. from transposons. from yeast episomes. from insertion elements, from yeast chromosomal elements, from viruses such as baculoviruses. papova viruses, such as SV40. vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses, picornaviruses and retroviruses. and vectors denved from combmations thereof, such as those denved from plasmid and bactenophage genetic elements, such as cosmids and phagemids The expression system constructs may compπse control regions diat regulate as well as engender expression Generally, any system or vector suitable to maintain, propagate or express polynucleotides and/or to express a polypeptide in a host may be used for expression m diis regard The appropnate DNA sequence may be inserted mto the expression system by any of a vanety of well-known and routme techniques, such as. for example, those set forth in Sambrook et al . MOLECULAR CLONING, A LABORATORY MANUAL, (supra)
In recombmant expression systems in eukaryotes. for secretion of a translated protem mto the lumen of the endoplasmic reticulum. mto the penplasmic space or mto the extracellular environment, appropnate secretion signals mav be incorporated mto the expressed polypeptide These signals may be endogenous to the polypeptide or ti ey mav be heterologous signals
Polypeptides of the mvention can be recovered and punfied from recombmant cell cultures by well- known methods mcludmg ammomum sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatograph . phosphocellulose chromatography . hydrophobic mteraction chromatography. affinity' chromatography . hydroxylapatite chromatography . and lectm chromatography Most preferably . high performance liquid chromatography is employed for puπfication Well known techniques for refolding protem may be emplov ed to regenerate active conformation when the polypeptide is denatured during isolation and or punfication Diagnostic, Prognostic, Serotyping and Mutation Assays
This mvention is also related to the use of accA. accB (fabE). accC. accD polynucleotides and polypeptides of die mvention for use as diagnostic reagents Detection of ace A. accB (fabE). accC. accD polynucleotides and/or polypeptides m a eukaryote. particularly a mammal, and especially a human, will provide a diagnostic method for diagnosis of disease, staging of disease or response of an infectious orgamsm to drugs Eukarvotes, particularly mammals, and especially humans, particularly those infected or suspected to be infected with an orgamsm compnsmg the accA. accB (fabE). accC. accD gene or protein, may be detected at the nucleic acid or ammo acid level by a vanety of well known techniques as well as by methods provided herem
Polypeptides and polynucleotides for prognosis, diagnosis or other analy sis may be obtamed from a putatively infected and/or infected mdividual's bodily matenals Polynucleotides from any of these sources, particularly DNA or RNA. may be used directly for detection or may be amplified enzymatically by usmg PCR or any other amplification technique pnor to analysis RNA. particularly rnRNA. cDNA and genomic DNA may also be used m die same way s Usmg amplification, characteπzation of the species and stram of infectious or resident organism present m an individual, may be made by an analysis of the genotype of a selected polynucleotide of the orgamsm Deletions and insertions can be detected by a change m size of the amplified product m companson to a genotype of a reference sequence selected from a related organism, preferably a different species of the same genus or a different stram of the same species Pomt mutations can be identified by hybndizing amplified DNA to labeled accA. accB (fabE). accC. accD polynucleotide sequences Perfectly or significantly matched sequences can be distinguished from imperfectly or more significantlv mismatched duplexes by DNase or RNase digestion, for DNA or RNA respectively, or by detectmg differences m melting temperatures or renaturation kinetics Polynucleotide sequence differences may also be detected by alterations the electrophoretic mobility of polynucleotide fragments m gels as compared to a reference sequence This may be earned out with or without denaturing agents Polynucleotide differences may also be detected by direct DNA or RNA sequencing See. for example. My ers et al . Science 230 1242 (1985) Sequence changes at specific locations also may be revealed by nuclease protection assay s, such as RNase. VI and SI protection assay or a chemical cleavage method See. for example. Cotton et al . Proc Natl Acad Sci USA. 85 4397-4401 (1985) In another embodiment, an array of oligonucleotides probes compnsmg accA. accB (fabE). accC. accD nucleotide sequence or fragments thereof can be constructed to conduct efficient screening of. for example, genetic mutations, serotype. taxonomic classification or identification Array technology methods are vv ell known and hav e general applicability and can be used to address a v anety of questions m molecular genetics mcludmg gene expression, genetic linkage, and genetic vanabihty (see. for example. Chee et al . Science. 274 670 (1996))
Thus in another aspect, the present invention relates to a diagnostic kit that comprises (a) a polynucleotide of the present invention, preferably the nucleotide sequence of SEQ ID NO 1. 3. 5, 7. or a fragment thereof, (b) a nucleotide sequence complementary to that of (a), (c) a polypeptide of the present invention, preferably the polypeptide of SEQ ID NO 2. 4. 6. 8 or a fragment thereof, or (d) an antibody to a polypeptide of the present mvention. preferably to the polypeptide of SEQ ID NO 2. 4. 6, 8 It will be appreciated that in any such kit, (a), (b). (c) or (d) may comprise a substantial component Such a kit ill be of use in diagnosing a disease or susceptibility to a Disease, among others
This mvention also relates to the use of polynucleotides of the present mvention as diagnostic reagents Detection of a mutated form of a polynucleotide of the mvention. preferable. SEQ ID NO 1. 3. 5. 7. that is associated with a disease or pathogenicity will provide a diagnostic tool that can add to. or define, a diagnosis of a disease, a prognosis of a course of disease, a determination of a stage of disease, or a susceptibility to a disease, that results from under-expression. over-expression or altered expression of the polynucleotide Organisms, particularly infectious organisms, carrying mutations m such polynucleotide may be detected at the polynucleotide level by a vanety of techniques, such as those descπbed elsewhere herem The differences m a polynucleotide and/or polypeptide sequence between organisms possessing a first phenotype and organisms possessing a different, second different phenotype can also be determined If a mutation is observed in some or all organisms possessing the first phenotype but not in any organisms possessing the second phenotype. then the mutation is hkelv to be the causative agent of the first phenotype Cells from an organism carrying mutations or polymorphisms (allelic vanations) m a polynucleotide and/or polypeptide of the invention may also be detected at the polynucleotide or polypeptide level by a vanetv of techniques, to allow for serotyping. for example For example. RT-PCR can be used to detect mutations in the RNA It is particularly prefened to use RT-PCR m conjunction with automated detection systems, such as. for example. GeneScan RNA- cDNA or genomic DNA may also be used for the same purpose. PCR As an example. PCR primers complementary to a polynucleotide encoding accA. accB (fabE). accC. accD polypeptide can be used to identify and analy ze mutations The mvention further prov ides these primers with 1. 2. 3 or 4 nucleotides removed from the 5' and/or the 3' end These pπmers may be used for. among other thmgs. amplify mg accA. accB (fabE). accC. accD DNA and/or RNA isolated from a sample denved from an individual, such as a bodily matenal The pπmers may be used to amplify a polynucleotide isolated from an infected indi idual, such diat die polynucleotide mav then be subject to vanous techniques for elucidation of the polynucleotide sequence In this way. mutations m die polvnucleotide sequence may be detected and used to diagnose and/or prognose the infection or its stage or course, or to serotype and/or classify the infectious agent
The mvention further provides a process for diagnosing, disease, preferably bacterial infections, more preferably infections caused by Staphylococcus aureus. comprising determining from a sample derived from an individual, such as a bodily material, an increased level of expression of polynucleotide having a sequence of Table 1 [SEQ ID NO 1. 3. 5. 7] Increased or decreased expression of a accA. accB (fabE). accC. accD polynucleotide can be measured using any on of the methods well known in the art for the quantitation of polynucleotides, such as. for example, amplification. PCR. RT-PCR. RNase protection. Northern blotting, spectrometry and other hybridization methods
In addition, a diagnostic assay in accordance with the mvention for detectmg over-expression of accA. accB (fabE). accC. accD polypeptide compared to normal control tissue samples may be used to detect die presence of an infection, for example Assay techniques that can be used to determine levels of a accA. accB (fabE). accC. accD polypeptide. in a sample denved from a host, such as a bodily mateπal. are well- known to those of skill m the art Such assav methods mclude radioimmunoassav s. competitive-binding assays. Western Blot analysis, antibody sandwich assays, antibody detection and ELISA assays Antagonists and Agonists - Assays and Molecules Polypeptides and polynucleotides of the mvention may also be used to assess the bmdmg of small molecule substrates and hgands m, for example, cells, cell-free preparations, chemical hbranes. and natural product mixtures These substrates and hgands may be natural substrates and ligands or may be structural or functional mimetics See. e g , Cohgan et al . Current Protocols in Immunology 1(2) Chapter 5 (1991) Polypeptides and polynucleotides of the present mvention are responsible for many biological functions, mcludmg many disease states, m particular the Diseases herem mentioned It is therefore desirable to devise screening methods to identify compounds that agomze (e g . stimulate) or that antagonize (e g .inhibit) the function of the polypeptide or polynucleotide Accordingly, m a further aspect, the present mvention provides for a method of screening compounds to identify those that agonize or that antagonize the function of a polypeptide or polynucleotide of the mvention. as well as related polypeptides and polynucleotides In general, agonists or antagonists (e g . inhibitors) may be employ ed for therapeutic and prophy lactic purposes for such Diseases as herem mentioned Compounds may be identified from a v anety of sources, for example, cells, cell-free preparations, chemical hbranes. and natural product mixtures Such agonists and antagonists so-identified may be natural or modified substrates, hgands. receptors, enzymes, etc , as the case may be. of accA. accB (fabE). accC. accD polypeptides and polynucleotides. or may be structural or functional mimetics thereof (see Coligan et al . Current Protocols in Immunology 1(2) Chapter 5 (1991))
The screening methods may simply measure the bmdmg of a candidate compound to the polypeptide or poly nucleotide. or to cells or membranes bearing the poly peptide 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 w ith 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 sy stems appropriate to the cells comprising the poly peptide or polynucleotide Inhibitors of activ ation 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, in the absence of an agonist or antagomst, by testing whether the candidate compound results m inhibition of activation of the polypeptide or polynucleotide. as the case may be Further, the screenmg methods may simply comprise the steps of mixing a candidate compound with a solution comprising a polypeptide or polynucleotide of the present invention, to form a mixture, measuring accA. accB (fabE). accC. accD polypeptide and/or polynucleotide activity in the mixture, and comparing the accA. accB (fabE). accC, accD polypeptide and/or polynucleotide activity of the mixture to a standard Fusion proteins, such as those made from Fc portion and accA. accB (fabE). accC, accD polypeptide. as herein described, can also be used for high-throughput screening assays to identify antagonists of the polypeptide of the present invention, as well as of phylogenetically and and/or functionally related polypeptides (see D Bennett et al X Mol Recognition. 8 52-58 (1995). and K Johanson et a! . ) Biol Chem. 270(16) 9459-9471 (1995))
The polynucleotides, polypeptides and antibodies that bind to and/or interact with a polypeptide of the present invention may also be used to configure screening methods for detecting the effect of added compounds on the production of mRNA and/or polypeptide in cells For example, an ELISA assay may be constructed for measuring secreted or cell associated levels of poKpeptide usmg monoclonal and polyclonal antibodies by standard methods known in the art This can be used to discov er agents that may inhibit or enhance the production of poKpeptide (also called antagonist or agonist, respectiv ely ) from suitablv manipulated cells or tissues
The mv ention also provides a method of screening compounds to identifv those that enhance (agomst) or block (antagonist) the action of accA. accB (fabE). accC. accD polypeptides or polynucleotides. particularly those compounds that are bacteπstatic and/or bacteπcidal The mediod of screening may involve higli-throughput techniques For example, to screen for agomsts or antagonists, a synthetic reaction mix. a cellular compartment, such as a membrane, cell envelope or cell wall, or a preparation of any thereof, compnsmg accA. accB (fabE). accC. accD poKpeptide and a labeled substrate or ligand of such polypeptide is mcubated m die absence or the presence of a candidate molecule that may be a accA. accB (fabE). accC. accD agomst or antagonist The ability of the candidate molecule to agomze or antagomze the accA. accB (fabE). accC. accD polypeptide is reflected m decreased bmdmg of die labeled ligand or decreased production of product from such substrate Molecules that bmd gratuitously . i e . without mducmg the effects of accA. accB (fabE). accC. accD polypeptide are most likely to be good antagonists Molecules that bmd well and. as the case may be. mcrease die rate of product production from substrate, mcrease signal transduction. or mcrease chemical channel activity are agomsts Detection of the rate or lev el of. as the case may be, production of product from substrate, signal transduction. or chemical channel activity may be enhanced by usmg a reporter sy stem Reporter systems that may be useful in this regard mclude but are not limited to colonmetnc. labeled substrate converted mto product, a reporter gene that is responsive to changes m accA. accB (fabE). accC. accD polynucleotide or polypeptide activity, and bmdmg assay s known m the art Poly peptides 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 crosshnkmg assays in which the poKpeptide is labeled with a radioactive isotope (for instance. l^I). chemically modified (for instance, biotinylated). 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 supematants, tissue extracts, bodily materials) Other methods include biophysical techniques such as surface plasmon resonance and spectroscopv These screening methods may also be used to identify agonists and antagonists of the polypeptide that 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 correlation time or tumbling rate Protein complexes, such as formed by accA. accB (fabE). accC. accD polypeptide associating with another accA. accB (fabE). accC. accD polypeptide or other polypeptide. labeled to compπse a fluorescently-labeled molecule will have higher polarization values than a fluorescently labeled monomenc protein It is preferred that this method be used to characterize small molecules that disrupt poly peptide complexes
Fluorescence energy transfer may also be used characterize small molecules that interfere with the formation of accA. accB (fabE). accC. accD polypeptide dimers. tnmers. tetramers or higher order structures, or structures formed by accA. accB (fabE). accC. accD polypeptide bound to another polypeptide AccA. accB (fabE). accC. accD 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 bv obser ing fluorescence of the acceptor Compounds that block dimeπzation will inhibit fluorescence energv transfer Surface plasmon resonance can be used to monitor the effect of small molecules on accA. accB (fabE). accC. accD polypeptide self-association as well as an association of accA. accB (fabE). accC. accD polypeptide and another polypeptide or small molecule AccA. accB (fabE). accC. accD polypeptide can be coupled to a sensor chip at low site density such that covalently bound molecules will be monomenc Solution protein can then passed over the accA. accB (fabE). accC. accD polypeptide -coated surface and specific binding can be detected in real -time by monitoring the change in resonance angle caused by a change m local refractive index This technique can be used to characterize the effect of small molecules on kinetic rates and equilibrium binding constants for accA, accB (fabE). accC. accD polypeptide self-association as well as an association of accA. accB (fabE). accC. accD polypeptide and another polypeptide or small molecule A scintillation proximity assay may be used to characterize the interaction between an association of accA. accB (fabE). accC. accD polypeptide with another accA. accB (fabE). accC. accD polypeptide or a different polypeptide AccA, accB (fabE). accC. accD polypeptide can be coupled to a scintillation-filled bead Addition of radio-labeled accA. accB (fabE). accC. accD polypeptide results in binding where the radioactive source molecule is in close proximity to the scintillation fluid Thus, signal is emitted upon accA. accB (fabE). accC. accD polypeptide bmdmg and compounds that prevent accA. accB (fabE). accC. accD polypeptide self-association or an association of accA. accB (fabE). accC. accD polypeptide and another polypeptide or small molecule will dimmish signal
In other embodiments of the mvention there are provided methods for identifying compounds that bmd to or otherwise mteract with and inhibit or activate an activity or expression of a polypeptide and/or polynucleotide of d e mvention compnsmg contacting a polypeptide and/or polynucleotide of the mvention with a compound to be screened under conditions to permit bmdmg to or other mteraction between the compound and the polypeptide and/or polynucleotide to assess the bmdmg to or other mteraction with the compound, such bmdmg or mteraction preferably being associated with a second component capable of providing a detectable signal m response to the bmdmg or mteraction of the polypeptide and/or polynucleotide with the compound, and determining whether the compound bmds 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 d e bmdmg or mteraction of the compound with the polypeptide and/or polynucleotide
Anod er example of an assay for accA. accB (fabE). accC. accD agomsts is a competitive assay that combmes accA. accB (fabE). accC. accD and a potential agomst with accA. accB (fabE). accC. accD- binding molecules, recombmant accA. accB (fabE). accC. accD bmdmg molecules, natural substrates or gands or substrate or ligand mimetics. under appropnate conditions for a competitive inhibition assay AccA. accB (fabE). accC. accD can be labeled, such as by radioactivity or a colonmetnc compound, such that the number of accA. accB (fabE). accC. accD molecules bound to a bmdmg molecule or converted to product can be determined accurately to assess the effectiveness of the potential antagonist
It will be readily appreciated by the skilled artisan that a polypeptide and/or polynucleotide of the present invention may also be used in a method for the structure-based design of an agonist or antagonist of the polypeptide and/or polynucleotide. by (a) determining in the first instance the three- dimensional structure of the polypeptide and/or polynucleotide. or complexes thereof, (b) deducing the three-dimensional structure for the likely reactive sιte(s). binding sιte(s) or motιf(s) of an agonist or antagonist, (c) synthesizing candidate compounds that are predicted to bind to or react ith the deduced bindmg sιte(s). reactive sιte(s). and/or motifts). and (d) testing whether the candidate compounds are indeed agonists or antagonists
It will be further appreciated that this will normally be an iterative process, and this iterative process may be performed using automated and computer-controlled steps
In a further aspect, the present mvention 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 accA. accB (fabE). accC. accD polypeptide and/or polynucleotide
If the expression and/or activity of the polypeptide and/or polynucleotide is m excess, several approaches are available One approach compnses administering to an individual m need thereof an inhibitor compound (antagonist) as herem descπbed. optionally m combmation with a pharmaceutically acceptable earner, m an amount effective to inhibit the function and/or expression of the polypeptide and/or polynucleotide. such as. for example, by blocking the bmdmg of hgands. substrates, receptors, enzymes, etc . or by inhibiting a second signal, and thereby alleviating the abnormal condition In another approach, soluble forms of the polypeptides still capable of binding the ligand, substrate, enzymes, receptors, etc in competition w ith endogenous polypeptide and/or polynucleotide may be administered Ty pical examples of such competitors include fragments of the accA. accB (fabE). accC. accD polypeptide and/or polypeptide
In still another approach, expression of the gene encoding endogenous accA. accB (fabE). accC. accD polypeptide can be inhibited using expression blocking techniques This blocking may be targeted against any step in gene expression, but is preferably targeted against transcription and/or translation An examples of a known technique of this sort involve the use of antisense sequences, either internally generated or separately administered (see. for example, O'Connor. J Neurochem (1991) 56 560 in Ohgodeoxy nucleotides as Antisense Inhibitors of Gene Expression. CRC Press. Boca Raton. FL (1988)) Alternatively . ohgonucleotides that form triple helices with the gene can be supplied (see. for example. Lee et al Nucleic Acids Res (1979) 6 3073. Cooney et al Science (1988) 241 456. Dervan et al . Science (1991) 251 1360) These ohgomers can be administered per se or the relevant ohgomers can be expressed in vivo
Each of the polynucleotide sequences provided herein may be used in the discovery and development of antibacterial compounds The encoded protein, upon expression, can be used as a target for the screening of antibacterial drugs Additionally, the polynucleotide sequences encoding the ammo terminal regions of the encoded protein or Shine-Delgarno or other translation facilitating sequences of the respective mRNA can be used to construct antisense sequences to control the expression of the coding sequence of interest The mvention also provides the use of the polypeptide. polynucleotide. agonist or antagonist of the invention to interfere with the initial physical interaction between a pathogen or pathogens and a eukaryotic. preferably mammalian, host responsible for sequelae of infection In particular, the molecules of the invention may be used in the prevention of adhesion of bactena. in particular gram positive and/or gram negative bacteria, to eukaryotic, preferably mammalian, extracellular matrix proteins on m-dwelling devices or to extracellular matnx protems in wounds, to block bactenal adhesion between eukaryotic, preferably mammalian, extracellular matrix proteins and bacterial accA. accB (fabE). accC. accD proteins that mediate tissue damage and/or. to block the normal progression of pathogenesis in infections initiated other than by the implantation of in-dwelling devices or by other surgical techniques In accordance with vet another aspect of the mvention. there are provided accA. accB (fabE). accC. accD agomsts and antagonists, preferably bacteπstatic or bactencidal agomsts and antagonists
The antagonists and agomsts of the mvention may be employed, for instance, to prevent, inhibit and/or treat diseases Hehcobacter pylori (herein "H pylori") bacteria infect the stomachs of ov er one-third of the world's population causing stomach cancer, ulcers, and gastritis (International Agency for Research on Cancer (1994) Schi tυs omes Liver Flukes and Hehcobacter Pylori (International Agency for Research on Cancer. L on. France, http //vvvwv uicc ch/ecp/ecp2904 htm) Moreover, the International Agency for Research on Cancer recently recognized a cause-and-effect relationship between H pylori and gastric adenocarcinoma. classifying the bactenum as a Group I (definite) carcinogen Preferred antimicrobial compounds of the invention (agonists and antagonists of accA. accB (fabE). accC. accD polypeptides and/or polynucleotides) found using screens provided bv the invention, or known in the art. particularly narrow -spectrum antibiotics, should be useful in the treatment of H pylori infection Such treatment should decrease the advent of H /.v/σr. -induced cancers, such as gastrointestinal carcinoma Such treatment should also prevent, inhibit and/or cure gastric ulcers and gastritis
All publications and references, including but not limited to patents and patent applications, cited in this specification are herein incorporated by reference in their entirety as if each individual publication or reference were specifically and individually indicated to be incorporated by reference herein as being fully set forth Any patent application to which this application claims priority is also incorporated by reference herein in its entirety in the manner described above for publications and references
GLOSSARY The following definitions are provided to facilitate understanding of certain terms used frequently herem
"Bodily mateπal(s) means any matenal denved from an individual or from an orgamsm infecting. infesting or inhabiting an individual, mcludmg but not limited to. cells, tissues and waste, such as. bone. blood, serum, cerebrospmal fluid, semen, saliva, muscle, cartilage, organ tissue, skin, urine, stool or autopsy matenals
"Dιsease(s)" means any disease caused by or related to infection by a bactena. mcludmg , for example, disease, such as. infections of the upper respiratory tract (e g , otitis media, bacterial tracheitis. acute epiglottitis. thy roiditis). lower respiratory (e g . empyema. lung abscess), cardiac (e g . infective endocarditis). gastrointestinal (e g . secretory diarrhoea, splemc absces. retropeπtoneal abscess). CNS (e g . cerebral abscess), e e (e g . blephantis. conjunctivitis, keratitis. endophthalmitis. preseptal and orbital celluhtis, darcry ocy stitis). kidney and urinary tract (e g . epididymrtis. intrarenal and peπnephπc absces, toxic shock syndrome), skm (e g . impetigo, folhculitis. cutaneous abscesses, celluhtis. wound infection, bactenal myositis) bone and jomt (e g . septic arthπtis. osteomyelitis) "Host cell(s)" is a cell that has been introduced (e g . transformed or transfected) or is capable of introduction (e g . transformation or transfection) by an exogenous polynucleotide sequence
"Identity ." as known m the art. is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as the case may be. as determined by comparing the sequences In the art. "identity " also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be. as determined by the match between strings of such sequences "Identity " can be readilv calculated by known methods, including but not limited to those described m (Computational Molecular Biology. Lesk. A M . ed . Oxford Umversitv Press. New York. 1988. Biocomputwg Informatics and Genome Projects. Smith. D W . ed . Academic Press. New York. 1993. Computer Analysis of Sequence Data. Part I. Griffm. A M . and Griffin. H G . eds . Humana Press. New Jersey . 1994. Sequence Analysis in Molecular Biology, von Heinje. G . Academic Press. 1987. and Sequence Analysis Primer. Gπbskov. M and Devereux. J . eds . M Stockton Press. New York. 1991. and Caπllo. H . and Lipman. D . SIAM ./ Applied Math 48 1073 (1988) Methods to determine identity are designed to give the largest match betw een the sequences tested Moreover. methods to determine identity are codified in publicly available computer programs Computer program methods to determine identity between two sequences include, but are not limited to. the GCG program package (Devereux. J . et al . Nucleic Acids Research 12(1) 387 (1984)). BLASTP, BLASTN. and FASTA (Altschul. S F et al , J Molec Biol 215 403-410 (1990) The BLAST X program is publicly available from NCBI and other sources (BLAST Manual. Altschul. S . et al . NCBI NLM NIH Bethesda. MD 20894. Altschul, S . et al . . I Mol Biol 215 403-410 (1990) The well known Smith Waterman algorithm may also be used to determine identity
Parameters for polypeptide sequence comparison include the follow mg Algorithm Needleman and Wunsch J Mol Biol 48 443-453 (1970) Comparison matrix BLOSSUM62 from Hentikoff and Hentikoff, Proc Natl Acad Sci USA 89 10915-10919 (1992) Gap Penalty 12 Gap Length Penalty 4
A program useful with these parameters is publicly available as the "gap" program from Genetics Computer Group. Madison WI The aforementioned parameters are the default parameters for peptide comparisons (along with no penalty for end gaps)
Parameters for polynucleotide comparison include the following Algonthm Needleman and Wunsch. J Mol Biol 48 443-453 (1970) Comparison matrix matches = +10. mismatch = 0 Gap Penalty 50 Gap Length Penalty 3
Available as The "gap" program from Genetics Computer Group. Madison WI These are the default parameters for nucleic acid comparisons A preferred meaning for "identity " for polynucleotides and polypeptides. as the case may be. are provided in (1 ) and (2) below
( 1 ) Poly nucleotide embodiments further include an isolated poly nucleotide compnsmg a polynucleotide sequence having at least a 95. 97 or 100% identity to the reference sequence of SEQ ID NO 1. 3. 5. 7. wherein said polynucleotide sequence may be identical to the reference sequence of SEQ ID NO 1. 3. 5. 7 or may include up to a certain integer number of nucleotide alterations as compared to the reference sequence, wherein said alterations are selected from the group consisting of at least one nucleotide deletion, substitution, including transition and transversion. or insertion, and wherein said alterations may occur at the 5' or 3' terminal positions of the reference nucleotide sequence or anywhere between those tenninal positions, interspersed either individuallv among the nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence, and wherein said number of nucleotide alterations is determined by multiplying the total number of nucleotides m SEQ ID NO 1. 3. 5. 7 by the integer defining the percent identity divided by 100 and then subtracting that product from said total number of nucleotides m SEQ ID NO 1. 3, 5, 7. or
nn < xn - (χn • y),
wherein nn is the number of nucleotide alterations. xn is the total number of nucleotides in SEQ ID NO 1. 3. 5. 7, y is 0 95 for 95%, 0 97 for 97% or 1 00 for 100%. and • is the symbol for the multiplication operator, and wherein any non-mteger product of xn and y is rounded down to the nearest integer prior to subtracting it from xn Alterations of a polynucleotide sequence encodmg the polypeptide of SEQ ID NO 2. 4, 6, 8 may create nonsense, missense or frameshift mutations in this coding sequence and thereby alter the polypeptide encoded by the polynucleotide following such alterations
(2) Polypeptide embodiments further include an isolated polypeptide compnsmg a polypeptide having at least a 95, 97 or 100% identity to a polypeptide reference sequence of SEQ ID NO 2, 4. 6. 8. wherein said polypeptide sequence may be identical to the reference sequence of SEQ ID NO 2. 4. 6. 8 or may include up to a certain integer number of ammo acid alterations as compared to the reference sequence, wherein said alterations are selected from the group consisting of at least one amino acid deletion, substitution, including conservative and non-conservati e substitution, or insertion, and wherein said alterations may occur at the amino- or carboxy -terminal positions of the reference polypeptide sequence or anywhere between those terminal positions, interspersed either individually among the amino acids in the reference sequence or in one or more contiguous groups w ithin the reference sequence, and wherein said number of amino acid alterations is determined by multiplying the total number of amino acids in SEQ ID NO 2. 4. 6. 8 by the integer defining the percent identity divided by 100 and then subtracting that product from said total number of ammo acids in SEQ ID NO 2. 4. 6. 8. or
na < xa - (xa • y),
wherein na is the number of amino acid alterations, xa is the total number of amino acids m SEQ ID NO 2. 4. 6. 8 y is 0 95 for 95%. 0 97 for 97% or 1 00 for 100%. and • is the symbol for the multiplication operator, and wherein any non-integer product of xa and y is rounded down to the nearest integer prior to subtracting it from xa
"Indιvιdual(s)" means a multicellular eukaryote. mcludmg. but not limited to a metazoan. a mammal, an ovid. a bovid. a simian, a primate, and a human
"Isolated" means altered "by the hand of man" from its natural state. i e . if it occurs m nature, it has been changed or removed from its ongmal environment, or bodi For example, a polynucleotide or a polypeptide naturally present in a living orgamsm is not ' 'isolated.'' but the same polynucleotide or polypeptide separated from the coexisting matenals of its natural state is "isolated", as the term is employed herem Moreover, a polynucleotide or poKpeptide that is mtroduced mto an orgamsm by transformation, genetic manipulation or by any other recombmant method is "isolated" even if it is still present in said organism, which orgamsm may be living or non-living "Organιsm(s)" means a (I) prokaryote, mcludmg but not limited to. a member of the genus
Streptococcus Staphylococcus, Bordetella Corynebacterium Mycobactenum Neissena Haemophilus. Act nomycetes Streptomycetes, Nocardia. Enterohacter, Yersima Fancisella Pasture Ha, Mυraxella Acinetobacter Erysipelothnx Branhamella Actinobacillus, Streptobacillus Listena Calymmatobactenum Brucella Bacillus Clostndium. Treponema. Eschenchia Salmonella. Kleibsiella Vibrio, Proteus, Erwima Borreba Leptospira, Spirillum, Campylobactcr Shigella, Legionella Pseudomonas, Aeromonas Rickettsia Chlamydia, Borreba and Mycoplasma. and further mcludmg. but not limited to. a member of the species or group. Group A Streptococcus Group B Streptococcus, Group C Streptococcus Group D Streptococcus Group G Streptococcus Streptococcus pneumoniae Streptococcus pyogenes Streptococcus agalactiac Streptococcus faecahs Streptococcus faecium Streptococcus durans Neissena gonorrheae Neissena meningitidis Staphylococcus aureus Staphylococcus epidermidis Cυrynebactenum diptheriae Gardnerella vaginabs Mycobacterium tuberculosis Mycobactenum bovis Mycobacterium ulcerans Mycobacterium leprae Achnomyctes israehi Listena monocytogenes Bordetella pertusis Bordatella parapertusis Bordetella bronchiseptica Eschenchia cob Shigella dysentenae Haemophilus influenzae Haemophήus aegyptius Haemophilus parainfluenzae. Haemophilus ducreyi Bordetella Salmonella typhi Citrobacter freundu Proteus mirabibs Proteus vulgaπs, Yersima pestis Kleibsiella pneumoniae Serratia marcessens Serratia hquefaciens Vibrio cholera Shigella dysenteru Shigella flexnen Pseudυmonas aerugmosa Franscisella tularensis Brucella abortis Bacillus anthracis Bacillus cereus Clostridium perfringens Clostridium tetani Clostridium botulmum Treponema palbdum Rickettsia nckettsii and Chlamydia trachomitis (n) an archaeon mcludmg but not limited to Archaebacter. and (in) a unicellular or filamentous eukaryote. mcludmg but not limited to. a protozoan, a fungus, a member of the genus Saccharomvces Kluveromyces or Candida and a member of the species Saccharomyces cenviseae Kluveromyces lactis or Candida albicans "Polynucleotιde(s)" generally refers to any polynbonucleotide or poly deoxyπbonucleotide, that may be unmodified RNA or DNA or modified RNA or DNA "Polynucleotιde(s)" mclude without limitation, smgle- and double-stranded DNA. DNA that is a mixture of smgle- and double-stranded regions or smgle-, double- and tnple-stranded regions, smgle- and double-stranded RNA. and RNA that is mixture of smgle- and double-stranded regions, hybnd molecules compnsmg DNA and RNA that mav be single-stranded or, more typically, double-stranded, or tnple-stranded regions or a mixture of smgle- and double-stranded regions In addition, "polynucleotide" as used herem refers to tnple-stranded regions compnsmg RNA or DNA or both RNA and DNA The strands m such regions mav be from the same molecule or from different molecules The regions mav 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 tπple-hehcal region often is an oligonucleotide As used herem. the term "polvnucleotιde(s)" also mcludes DNAs or RNAs as descπbed above that compπse one or more modified bases Thus DNAs or RNAs with backbones modified for stability or for other reasons are "polynucleotιde(s)" as that term is mtended herem Moreover. DNAs or RNAs compnsmg unusual bases, such as lnosine. or modified bases, such as tiit lated bases, to name just two examples, are polynucleotides as the term is used herem It will be appreciated that a great vanety of modifications have been made to DNA and RNA that serve many useful purposes known to those of skill in the art The term "polynucleotιde(s)" as it is employed herem embraces such chemically , enzvmatically or metabolicallv modified forms of polynucleotides, as well as the chemical forms of DNA and RNA charactenstic of viruses and cells, mcludmg. for example, simple and complex cells "Polynucleotιde(s)" also embraces short polynucleotides often refened to as ohgonucleotιde(s)
"Polypeptιde(s)" refers to any peptide or protem compnsmg two or more ammo acids jomed to each other by peptide bonds or modified peptide bonds "Polypeptιde(s)" refers to both short chains, commonly refened to as peptides. ohgopeptides and ohgomers and to longer chains generally refened to as proteins Polypeptides may compnse ammo acids other than the 20 gene encoded ammo acids "Polypeptιde(s)" mclude tiiose modified either by natural processes, such as processing and other post-translational modifications, but also by chemical modification techniques Such modifications are well descπbed m basic texts and more detailed monographs, as well as a voluminous research literature, and they are well known to those of skill in the art It will be appreciated that the same type of modification may be present the same or v ary mg degree at several sites m a given polypeptide Also, a given polypeptide may compnse many types of modifications Modifications can occur anywhere m a polypeptide. mcludmg the peptide backbone, the ammo acid side-chains, and the amino or carboxy 1 termini Modifications mclude. for example, acetylation. acv lation. ADP-πbosy lation. amidation. covalent attachment of flavin, covalent attachment of a heme moiety , covalent attachment of a nucleotide or nucleotide denvative. covalent attachment of a lipid or lipid denvative. covalent attachment of phosphotidylinositol. cross-linking, cy clization. disulfide bond formation, demetiiy lation. formation of covalent cross-links, formation of cysteine. formation of pyroglutamate. formvlation, gamma-carboxylation. GPI anchor formation, hydroxylation. lodination. methylation. mvnstoylation. oxidation, proteolytic processmg. phosphorylation. prenylation. racemization. glycosvlation. lipid attachment, sulfation. gamma-carboxylation of glutamic acid residues, hydroxylation and ADP-πbosylation. selenoylation. sulfation. transfer-RNA mediated addition of ammo acids to proteins, such as arginylation. and ubiquitination See. for instance. PROTEINS - STRUCTURE AND MOLECULAR PROPERTIES. 2nd Ed . T E Creighton. W H Freeman and Company . New York (1993) and Wold. F . Posttranslational Protem Modifications Perspectives and Prospects, pgs 1-12 m POSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS. B C Johnson. Ed . Academic Press. New York (1983). Seifter et al . Meth Enzymol 182 626-646 (1990) and Rattan et al . Protein Synthesis Posttranslational Modifications and Aging. Ann N Y Acad Sci 663 48-62 (1992) Polypeptides may be branched or cy clic. with or without branching Cyclic, branched and branched circular polypeptides may result from posttranslational natural processes and may be made by entirely synthetic methods, as well "Recombmant expression system(s)" refers to expression sv stems or portions thereof or polynucleotides of the invention mtroduced or transformed mto a host cell or host cell ly sate for the production of the polynucleotides and polypeptides of the mvention ■'Vaπant(s)' as the term is used herein, is a polynucleotide or polypeptide that differs from a reference poly nucleotide or polypeptide respectively, but retains essential properties A typical v anant of a polynucleotide differs in nucleotide sequence from another, reference polynucleotide Changes in the nucleotide sequence of the variant may or may not alter the amino acid sequence of a polypeptide encoded by the reference polynucleotide Nucleotide changes may result in ammo acid substitutions, additions, deletions, fusion proteins and truncations in the polypeptide encoded by the reference sequence, as discussed below A typical variant of a polypeptide differs in ammo acid sequence from another, reference poly peptide Generally . differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and. m many regions, identical A vanant and reference polypeptide may differ in amino acid sequence by one or more substitutions, additions, deletions in any combination A substituted or inserted amino acid residue may or may not be one encoded by the genetic code The present mvention also mcludes mclude vanants of each of the polypeptides of the mvention. that is polypeptides that vary from the referents by conserv ative ammo acid substitutions, whereby a residue is substituted by another with like charactenstics Typical such substitutions are among Ala. Val. Leu and He. among Ser and Thr, among the acidic residues Asp and Glu. among Asn and Gin. and among the basic residues Lys and Arg. or aromatic residues Phe and TVT Particularly prefened are vanants m which several. 5-10. 1-5. 1-3, 1-2 or 1 ammo acids are substituted, deleted, or added m any combination A variant of a polynucleotide or polypeptide may be a naturally occurring such as an allehc variant, or it may be a variant that is not known to occur naturally Non-naturally occurring variants of polynucleotides and polypeptides may be made by mutagenesis techniques, by direct synthesis, and by other recombinant methods known to skilled artisans
EXAMPLES
The examples below are carried out usmg standard techniques, that are well known and routme to those of skill m the art. except where otherwise descπbed m detail The examples are illustrative, but do not limit the mvention Example 1 Strain selection, Library Production and Sequencing
The polynucleotide havmg a DNA sequence given in Table 1 [SEQ ID NO 1. 3. 5. 7] was obtained from a library of clones of chromosomal DNA of Staphylococcus aureus in E cob The sequencing data from two or more clones compnsmg overlapping Staphylococcus aureus DNAs was used to construct the contiguous DNA sequence in SEQ ID NO 1. 3. 5. 7 Libraries may be prepared by routine methods, for example Methods 1 and 2 below Total cellular DNA is isolated from Staphylococcus aureus WCUH 29 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 m size are rendered blunt by treatment with exonuclease and DNA polymerase. and EcoRI linkers added Fragments are hgated mto the vector Lambda ZapII that has been cut with EcoRI. the hbrarv packaged bv standard procedures and E cob infected with the packaged library The library is amplified by standard procedures Method 2
Total cellular DNA is partially hy droly zed w ith a one or a combination of restriction enzymes appropriate to generate a series of fragments for cloning into library vectors (e g . Rsal. Pall. Alul. Bshl235I). and such fragments are size-fractionated according to standard procedures EcoRI linkers are hgated to the DNA and the fragments then hgated into the vector Lambda ZapII that have been cut with EcoRI. the library packaged by standard procedures, and E cob infected with the packaged library The library is amplified by standard procedures Example 2 accB (fabE) Characterization
Use of AccB (FabE) as substrate for assay of biotin ligase activity
Activity of bacterial biotin ligase (BirA) is assayed using the biotm carboxylase carrier protein (BCCP/AccB/FabE) as a substrate for the reaction BCCP is either radiolabelled or labelled with fluorescent tag Biotinylated product of the BirA reaction is captured v ia streptavidin / avidin interaction Conversion of substrate to product is followed by either fluorescence polarization, homogeneous time resolved fluorescence or scintillation proximitv assay Standard procedures are used Example 3 accA; accB (fabE); accC; accD Characterization Use of AccA; AccB (FabE); AccC; AccD to screen for inhibitors
Acety 1-CoA carboxylase is responsible for the ATP-dependent carboxy lation of acetyl-CoA to form malonyl-CoA. the first commited and the rate limiting step in fatty acid biosynthesis This activity is comprised of three separate components, biotin carboxylase. a homodimer of AccC. carboxyltransferase. a heterotetramer of AccA and AccD and biotm carboxy 1 earner protein. AccB(FabE) Detailed descnptions of the components and assays for the Escherichia coh enzymes comprising acety 1-CoA carboxylase have been descnbed (Ras B Guchhait. S Efthimios Polakis. et al. Aetyl-CoA Carboxylase System of Eschenchia coh, Purification and Properties of the Biotin Carboxylase. Carboxyltransferase and Carboxy 1 Carrier Protem Components The Journal of Biological Chemistry 249(20) 663-6645. 1974. and also Carol Z Blanchard and Grov er L Waldrop. Ov erexpression and Kinetic Characterization of the Carboxv Itranferase Component of Acety 1-CoA Carboxv lase The Journal of Biological Chemistry 273(30) 19140-19145. 1998) Included are spectrophotometπc and radiolabel assays to measure the biotm carboxylase and carboxyltransferase half reactions Also described are radiolabel assay s to measure the overall forward and overall reverse reactions In addition, one might be able to use luciferase to follow ATP production as an indirect meausure of the entire reverse reaction Example 4 accD Characterization
The determination of expression during infection of a gene from Staphylococcus aureus Necrotic fatty tissue from a 72hour groin infection of Staphylococcus aureus WCUH29 m the mouse is effϊcienth disrupted and processed m the presence of chaotropic agents and RNAase inhibitor to provide a mixture of animal and bacterial RNA The optimal conditions for disruption and processing to give stable preparations and high yields of bacterial RNA are follow ed by the use of hybridisation to a radiolabelled oligonucleotide specific to Staphy lococcus aureus 16S RNA on Northern blots The RNAase free. DNAase free. DNA and protein free preparations of RNA obtained are suitable for Reverse Transcription PCR (RT-PCR) using unique primer pairs designed from the sequence of each gene of Staphy lococcus aureus WCUH29 a) Isolation of tissue infected with Staphylococcus aureus WCUH29 from a mouse animal model of infection (groin) 10 ml volumes of sterile nutrient broth (No 2 Oxoid) are seeded with isolated, individual colonies of Staphylococcus aureus WCUH29 from an agar culture plate The cultures are incubated aerobicallv (static culture) at 37°C for 16-20 hours Four week old mice (female.18g-22g. strain MF1) are each infected by subcutaneous injection of 0 5ml of this broth culture of Staphylococcus aureus WCUH29 (diluted in broth to approximately 108 cfu/ml ) into the anterior, right lower quadrant (groin area) Mice should be monitored regularly during the first 24 hours after infection, then daily until termination of study Animals with signs of systemic infection, I e lethargy, ruffled appearance, isolation from group, should be monitored closely and if signs progress to moπbundancy. the animal should be culled immediately
Visible external signs of lesion development will be seen 24-48h after infection Examination of the abdomen of the animal will show the raised outline of the abscess beneath the skin The localised lesion should remain in the right lower quadrant, but mav occasionally spread to the left lower quadrant, and superiorly to the thorax On occasions, the abscess may rupture through the overlying skin lay ers In such cases the affected animal should be culled immediately and the tissues sampled if possible Failure to cull the animal may result in the necrotic skin tissue overlying the abscess being sloughed off exposing the abdominal muscle wall
Approximatelv 96 hours after infection, animals are killed using carbon dioxide asphyxiation To minimise delay between death and tissue processing /storage, mice should be killed individuallv rather than m groups The dead animal is placed onto its back and the fur swabbed liberally with 70% alcohol An initial incision using scissors is made through the skin of the abdominal left lower quadrant, travelling superiorly up to. then across the thorax The incision is completed bv cutting inferiorly to the abdominal low er right quadrant Care should be taken not to penetrate the abdominal w ll Holding the skm flap with forceps, the skin is gently pulled way from the abdomen The exposed abscess, which covers the peritoneal w all but generallv does not penetrate the muscle sheet completel . is excised, taking care not to puncture the viscera
The abscess/muscle sheet and other infected tissue may require cutting in sections, prior to flash-freezing in liquid nitrogen, thereby allowing easier storage in plastic collecting vials b) Isolation of Staphylococcus aureus WCUH29 RNA from infected tissue samples 4-6 infected tissue samples(each approx 0 5-0 7g) in 2ml screw -cap tubes are removed from -
80°C storage into a drv ice ethanol bath In a microbiological safety cabinet the samples are disrupted individually whilst the remaining samples are kept cold in the dry ice ethanol bath To disrupt the bacteria within the tissue sample 1ml of TRIzol Reagent (Gibco BRL. Life Technologies) is added followed by enough 0 1mm zircoma/sihca beads to almost fill the tube, the lid is replaced taking care not to get any beads mto the screw thread so as to ensure a good seal and eliminate aerosol generation The sample is then homogenised m a Mmi-BeadBeater Type BX-4 (Biospec Products) Necrotic fatty tissue isstrain treated for 100 seconds at 5000 rpm in order to achieve bacterial lysis In vivo grown bacteria require longer treatment than in vitro grown Staphylococcus aureus Staphylococcus which are disrupted by a 30 second bead-beat After bead-beating the tubes are chilled on ice before opening in a fume-hood as heat generated during disruption may degrade the TRIzol and release cyanide
200 microhtres of chloroform is then added and the tubes shaken by hand for 15 seconds to ensure complete mixing After 2-3 minutes at room temperature the tubes are spun down at 12.000 x g, 4°C for 15mmutes and RNA extraction is then continued according to the method given by the manufacturers of TRIzol Reagent I e - The aqueous phase, approx 0 6 ml. is transferred to a stenle eppendorf tube and 0 5 ml of isopropanol is added After 10 minutes at room temperature the samples are spun at 12.000 x g. 4 °C for 10 minutes The supernatant is removed and discarded then the RNA pellet is washed ith 1 ml 75% ethanol A brief vortex is used to mix the sample before centπfuging at 7.500 x g 4 °C for 5 minutes The ethanol is removed and the RNA pellet dried under vacuum for no more than 5 minutes Samples are then resuspended by repeated pipetting in 100 microhtres of DEPC treated water, followed by 5-10 minutes at 55 °C Finally . after at least 1 minute on ice. 200 units of Rnasm (Promega) is added RNA preparations are stored at -80 °C for up to one month For longer term storage the RNA precipitate can be stored at the wash stage of the protocol in 75% ethanol for at least one year at -20 °C
Quality of the RNA isolated is assessed by running samples on 1% agarose gels 1 x TBE gels stained with ethidium bromide are used to visualise total RNA y lelds To demonstrate the isolation of bacterial RNA from the infected tissue 1 x MOPS. 2 2M fonnaldeh de gels are run and v acuum blotted to Hy bond-N (Amersham) The blot is then hy bπdised w ith a '" P labelled ohgonucletide probe specific to 16s rRNA of Staphylococcus aureus ( K Greisen. M Loeffelholz. A Purohit and D Leong J Chn (1994) Microbiol 32 335-351 ) An oligonucleotide of the sequence 5 ' - gctcctaaaaggttactccaccggc -3 ' [SEQ ID NO 9] is used as a probe The Size of the hybridising band is compared to that of control RNA isolated from in vitro grown Staphylococcus aureus WCUH29 in the Northern blot Correct sized bacterial 16s rRNA bands can be detected m total RNA samples which show extensive degradation of the mammalian RNA when visualised on TBE gels c) The removal of DNA from Staphylococcus aureus WCUH29-derived RNA DNA was removed from 73 microhtre samples of RNA by a 15 minute treatment on ice with
3 units of DNAasel. amplification grade (Gibco BRL. Life Technologies) in the buffer supplied with the addition of 200 units of Rnasin (Promega) in a final volume of 90 microhtres
The DNAase was inactivated and removed by treatment with TRIzol LS Reagent (Gibco BRL. Life Technologies) according to the manufacturers protocol DNAase treated RNA was resuspended in 73 microhtres of DEPC treated water with the addition of Rnasin as described in Method 1 d) The preparation of cDNA from RNA samples derived from infected tissue
10 microhtre samples of DNAase treated RNA are reverse transcribed using a SuperScnpt Preamphfication System for First Strand cDNA Synthesis kit (Gibco BRL. Life Technologies) according to the manufacturers instructions 1 nanogram of random hexamers is used to prime each reaction Controls without the addition of SuperScnptll reverse transcπptase are also run Both +/- RT samples are treated ith RNaseH before proceeding to the PCR reaction e) The use of PCR to determine the presence of a bacterial cDNA species PCR reactions are set up on ice m 0 2ml tubes by adding the following components 45 microhtres PCR SUPERMIX (Gibco BRL. Life Technologies) 1 microhtre 50mM MgCl . to adjust final concentration to 2 5mM. 1 microhtre PCR pπmers(optιmally 18-25 basepairs in length and designed to possess similar annealing temperatures), each pnmer at 1 OmM initial concentration, and 2 microhtres cDNA
PCR reactions are run on a Perkin Elmer GeneAmp PCR Sy stem 9600 as follows 5 minutes at 95 °C. then 50 cvcles of 30 seconds each at 94 °C. 42 °C and 72 °C followed by 3 minutes at 72 °C and then a hold temperature of 4 °C (the number of cycles is optimally 30-50 to determine the appearance or lack of a PCR product and optimally 8-30 cycles if an estimation of the starting quantitv of cDNA from the RT reaction is to be made). 10 microhtre ahquots are then run out on 1% 1 x TBE gels stained with ethidium bromide with PCR product, if present, sizes estimated by comparison to a 100 bp DNA Ladder (Gibco BRL. Life Technologies) Alternativ ely if the PCR products are com eniently labelled by the use of a labelled PCR primer (e g labelled at the 5 'end with a dye) a suitable aliquot of the PCR product is run out on a poly aery lamide sequencing gel and its presence and quantity detected using a suitable gel scanning system (e g ABI Prism™ 377 Sequencer using GeneScan™ software as supplied by Perkin Elmer)
RT/PCR controls may include +/- reverse transcnptase reactions. 16s rRNA primers or DNA specific pnmer pairs designed to produce PCR products from non-transcribed Staphylococcus aureus WCUH29 genomic sequences To test the efficiency of the primer pairs they are used in DNA PCR with Staphylococcus aureus WCUH29 total DNA PCR reactions are set up and run as described above using approx 1 nucrogram of DNA in place of the cDNA and 35 cycles of PCR
Primer pairs which fail to give the predicted sized product in either DNA PCR or RT/PCR are PCR failures and as such are unmformative Of those which give the correct size product with DNA PCR two classes are distinguished m RT/PCR 1 Genes which are not transcribed in v vo reproducibly fail to give a product in RT/PCR. and 2 Genes which are transcribed in vivo reproducibly giv e the correct size product in RT/PCR and show a stronger signal m the +RT samples than the signal (if at all present) in -RT controls as did accD gene when examined in this way

Claims

What is claimed is:
1 An isolated poly peptide selected from the group consisting of
(1) an isolated polypeptide compnsmg an ammo acid having at least 95% identity to the ammo acid sequence of SEQ ID NO 2. 4. 6. 8 ov er the entire length of SEQ ID NO 2. 4. 6. 8.
(π) an isolated polypeptide comprising the amino acid sequence of SEQ ID NO 2. 4. 6. 8. (in) an isolated polypeptide that is the amino acid sequence of SEQ ID NO 2. 4. 6. 8. and (iv) a polypeptide that is encoded by a recombinant polynucleotide compnsmg the polyncleotide sequence of SEQ ID NO 1. 3. 5. 7
2 An isolated polynucleotide selected from the group consistmg of
(I) an isolated polynucleotide compnsmg a polynucleotide sequence encodmg a polypeptide that has at least 95% identit, to the ammo acid sequence of SEQ ID NO 2. 4. 6. 8. over the entire length of SEQ ID NO 2. 4. 6. 8.
(n) an isolated pol nucleotide compnsmg a polynucleotide sequence that has at least 95% identity over its entire length to a polynucleotide sequence encodmg the polypeptide of SEQ ID
NO 2. 4. 6. 8.
(in) an isolated polynucleotide compnsmg a nucleotide sequence that has at least 95% identity to that of SEQ ID NO 1, 3, 5, 7 over the entire length of SEQ ID NO 1. 3. 5. 7.
(iv) an isolated polynucleotide compnsmg a nucleotide sequence encodmg the polypeptide of SEQ ID
NO 2. 4. 6. 8.
(v) an isolated polynucleotide that is the polynucleotide of SEQ ID NO 1.
(vi) an isolated polynucleotide of at least 30 nucleotides m length obtainable by screening an appropnate library under stringent hybndization conditions with a probe having the sequence of SEQ
ID NO 1. 3. 5. 7 or a fragment thereof of of at least 30 nucleotides m length,
(vu) an isolated polynucleotide encodmg a mature polypeptide expressed by the accA. accB (fabE). accC. accD gene compnsed in the Staphylococcus aureus. and
(vm) a polynucleotide sequence complementary to said isolated polynucleotide of (i). (n), (in), (iv). (v). (vι) or (vn)
3 A method for the treatment of an individual (1) in need of enhanced activity or expression of or immunological response to the polypeptide of claim 1 comprising the step of administering to the individual a therapeuticallv effecti e amount of an antagonist to said polypeptide. or
(n) having need to inhibit activity or expression of the polypeptide of claim 1 comprising
(a) administering to the individual a therapeuticallv effective amount of an antagonist to said polypeptide. or
(b) administering to the individual a nucleic acid molecule that inhibits the expression of a polynucleotide sequence encoding said polypeptide.
(c) administering to the individual a therapeuticallv effectiv e amount of a polypeptide that competes with said polypeptide for its ligand. substrate, or receptor, or
(d) administering to the individual an amount of a polypeptide that induces an immunological response to said polypeptide in said individual
4 A process for diagnosing or prognosing a disease or a susceptibility to a disease in an individual related to expression or activity of the polypeptide of claim 1 m an mdividual comprising the step of
(a) determining the presence or absence of a mutation in the nucleotide sequence encoding said polypeptide in an organism in said individual, or
(b) analyzing for the presence or amount of said polypeptide expression in a sample derived from said individual
5 A process for producing a polypeptide selected from the group consistmg of
(I) an isolated polypeptide comprising an amino acid sequence selected from the group having at least 95% identity to the ammo acid sequence of SEQ ID NO 2. 4. 6. 8 over the entire length of SEQ ID NO 2. 4. 6. 8, (n) an isolated polypeptide compnsmg the amino acid sequence of SEQ ID NO 2, 4, 6. 8. (in) an isolated polypeptide that is the amino acid sequence of SEQ ID NO 2. 4, 6. 8. and (iv) a poly peptide that is encoded by a recombinant poly nucleotide comprising the polynucleotide sequence of SEQ ID NO 1, 3. 5, 7, comprising the step of cultuπng a host cell under conditions sufficient for the production of the poKpeptide
6 A process for producing a host cell compnsmg an expression sy stem or a membrane thereof expressing a polypeptide selected from the group consisting of
(1) an isolated polypeptide comprising an amino acid sequence selected from the group having at least 95% identity to the amino acid sequence of SEQ ID NO 2. 4. 6. 8 ov er the entire length of SEQ ID NO 2. 4. 6. 8.
(n) an isolated polypeptide comprising the ammo acid sequence of SEQ ID NO 2. 4. 6. 8.
(in) an isolated polypeptide that is the amino acid sequence of SEQ ID NO 2. 4. 6. 8. and
(iv) a polypeptide that is encoded by a recombinant polynucleotide comprising the polynucleotide sequence of SEQ ID NO 1. 3. 5, 7. said process comprising the step of transforming or transfecting a cell with an expression system compnsmg a polynucleotide capable of producing said polypeptide of (I). (ii). (in) or (iv) when said expression system is present in a compatible host cell such the host cell, under appropriate culture conditions, produces said polypeptide of (I). (n), (in) or (iv)
7 A host cell or a membrane expressing a polypeptide selected from the group consisting of (I) an isolated polypeptide comprising an ammo acid sequence selected from the group havmg at least 95% identity to the amino acid sequence of SEQ ID NO 2. 4. 6. 8 over the entire length of SEQ
ID NO 2, 4. 6. 8.
(n) an isolated polypeptide compnsmg the amino acid sequence of SEQ ID NO 2, 4, 6. 8. (in) an isolated polypeptide that is the amino acid sequence of SEQ ID NO 2, 4. 6. 8, and (iv) a polypeptide that is encoded by a recombinant polynucleotide comprising the polynucleotide sequence of SEQ ID NO 1. 3. 5, 7
8 An antibody lmmunospecific for the polypeptide of claim 1
9 A method for screening to identify' compounds that agomze or that inhibit the function of the polypeptide of clami 1 that compnses a method selected from the group consistmg of (a) measuring the bmdmg of a candidate compound to the poly peptide (or to the cells or membranes bearing the polypeptide) or a fusion protein thereof by means of a label directly or indirectly associated w ith the candidate compound.
(b) measuring the bmdmg of a candidate compound to the polypeptide (or to the cells or membranes bearing the poly peptide) or a fusion protein thereof in the presence of a labeled competitor.
(c) testing whether the candidate compound results in a signal generated by activation or inhibition of the polypeptide. using detection systems appropriate to the cells or cell membranes beanng the poKpeptide.
(d) mixing a candidate compound with a solution comprising a polypeptide of claim 1. to form a mixture, measuring activ ity of the polypeptide in the mixture, and comparing the activity of the mixture to a standard, or
(e) detecting the effect of a candidate compound on the production of mRNA encoding said polypeptide and said polypeptide in cells, using for instance, an ELISA assay
10 An agonist or antagonist to the polypeptide of claim 1
PCT/US2000/016726 1999-06-22 2000-06-16 Acetyl coenzyme a carboxylase from staphylococcus aureus WO2000078782A1 (en)

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DATABASE GENSEQ 16 March 1999 (1999-03-16), BARASH S. C. ET AL.: "Polynucleotide(s) and proteins derived from Staphylococcus aureus stored on computer readable medium and used in the production of anti-S. aureus vaccins", XP002933713, accession no. gencore *
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EP2840131A3 (en) * 2006-05-19 2015-04-08 LS9, Inc. Production of fatty acids and derivatives thereof
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US11434512B2 (en) 2006-05-19 2022-09-06 Genomatica, Inc. Production of fatty acid esters
EP2087096A2 (en) * 2006-10-20 2009-08-12 Arizona Board Of Regents For And On Behalf Arizona State University Modified cyanobacteria
EP2087096A4 (en) * 2006-10-20 2009-11-25 Univ Arizona Modified cyanobacteria
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