WO1999011753A1 - SpoOJ2 OF STAPHYLOCOCCUS AUREUS - Google Patents

SpoOJ2 OF STAPHYLOCOCCUS AUREUS Download PDF

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Publication number
WO1999011753A1
WO1999011753A1 PCT/US1998/018365 US9818365W WO9911753A1 WO 1999011753 A1 WO1999011753 A1 WO 1999011753A1 US 9818365 W US9818365 W US 9818365W WO 9911753 A1 WO9911753 A1 WO 9911753A1
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WIPO (PCT)
Prior art keywords
polypeptide
glu
leu
lys
seq
Prior art date
Application number
PCT/US1998/018365
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English (en)
French (fr)
Inventor
Martin K. R. Burnham
Andrew Fosberry
John E. Hodgson
Elizabeth J. Lawlor
Martin Rosenberg
Judith Ward
Deborah D. Jaworski
Min Wang
Lisa K. Shilling
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Smithkline Beecham Corporation
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from US09/042,771 external-priority patent/US6080729A/en
Application filed by Smithkline Beecham Corporation filed Critical Smithkline Beecham Corporation
Priority to EP98943538A priority Critical patent/EP0970186A4/en
Priority to JP51703299A priority patent/JP2002511768A/ja
Publication of WO1999011753A1 publication Critical patent/WO1999011753A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/305—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F)
    • C07K14/31—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F) from Staphylococcus (G)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies

Definitions

  • This invention relates to newly identified polynucleotides and polypeptides, and their production and uses, as well as their vanants, agonists and antagonists, and their uses.
  • the invention relates to polynucleotides and polypeptides of the parB family, as well as their va ⁇ ants, hereinafter referred to as "spoOJ2,” “spoOJ2 polynucleot ⁇ de(s),” and “spoOJ2 polypept ⁇ de(s)” as the case may be.
  • Staphylococcal genes and gene products are particularly 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. They are known to produce two types of disease, invasive and toxigenic. Invasive infections are characte ⁇ zed generally by abscess formation effecting both skm surfaces and deep tissues. S. aureus is the second leading cause of bacteremia in cancer patients. Osteomyelitis, septic arth ⁇ tis, septic thrombophlebitis and acute bacte ⁇ al 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 exotoxms as opposed to tissue invasion and bacteremia. These conditions include: Staphylococcal food poisoning, scalded skin syndrome and toxic shock syndrome.
  • Staphylococcus aureus infections has ⁇ sen dramatically m the past few decades. This has been attributed 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 which 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.
  • polynucleotides and polypeptides such as the spoOJ2 embodiments of the invention, that have 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, dysfunction and disease.
  • identification and characte ⁇ zation of such factors and their antagonists and agonists to find ways to prevent, ameliorate or correct such infection, dysfunction and disease.
  • the present invention relates to spoOJ2, in particular spoOJ2 polypeptides and spoOJ2 polynucleotides, recombinant matenals and methods for their production.
  • the invention relates to methods for using such polypeptides and polynucleotides, including treatment of microbial diseases, amongst others.
  • the invention relates to methods for identifying agonists and antagonists using the materials provided 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 spoOJ2 expression or activity.
  • the invention relates to spoOJ2 polypeptides and polynucleotides as descnbed in greater detail below.
  • the invention relates to polypeptides and polynucleotides of a spoOJ2 of Staphylococcus aureus, which is related by amino acid sequence homology to YYAA_BACSU polypeptide.
  • the invention relates especially to spoOJ2 having the nucleotide and amino acid sequences set out in Table 1 as SEQ ID NO: 1 or 3 and SEQ ED NO:2 or 4 respectively.
  • 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 usefully employed in polynucleotides in general, including ⁇ bopolynucleotides.
  • a deposit containing a Staphylococcus aureus WCUH 29 strain has been deposited with the National Collections of Indust ⁇ al and Manne Bactena Ltd. (herein "NCIMB"), 23 St. Machar Dnve, Aberdeen AB2 IRY, Scotland on 11 September 1995 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 "the deposited strain” or as "the DNA of the deposited strain.”
  • the deposited strain contains a full length spoOJ2 gene.
  • the sequence of the polynucleotides contained 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 descnption of sequences herein.
  • the deposit of the deposited strain has been made under the terms of the Budapest Treaty on the International Recognition of the Deposit of Micro-organisms for Purposes of Patent Procedure.
  • the deposited strain will be irrevocably and without restnction or condition released to the public upon the issuance of a patent.
  • the deposited strain is provided merely as convenience to those of skill in the art and is not an admission that a deposit is required for enablement, such as that required under 35 U.S.C. ⁇ 112.
  • a license may be required to make, use or sell the deposited strain, and compounds de ⁇ ved therefrom, and no such license is hereby granted.
  • an isolated nucleic acid molecule encoding a mature polypeptide expressible by the Staphylococcus aureus WCUH 29 strain, which polypeptide is contained in the deposited strain.
  • spoOJ2 polynucleotide sequences in the deposited strain such as DNA and RNA, and amino acid sequences encoded thereby.
  • spoOJ2 polypeptide and polynucleotide sequences isolated from the deposited strain are substantially phylogenetically related to other proteins of the parB family.
  • spoOJ2 polypeptides of Staphylococcus aureus referred to herein as "spoOJ2" and “spoOJ2 polypeptides” as well as biologically, diagnostically, prophylactically, clinically or therapeutically useful vanants thereof, and compositions comp ⁇ sing the same.
  • the present invention further provides for an isolated polypeptide which: (a) compnses or consists of an amino acid sequence which has at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, yet more preferably at least 95% identity, most preferably at least 97-99% or exact identity, to that of SEQ ID NO:2 over the entire length of SEQ ID NO:2; (b) a polypeptide encoded by an isolated polynucleotide comprising or
  • (g) comprises or consists of an amino acid sequence which has at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, yet more preferably at least 95% identity, most preferably at least 97-99% or exact identity, to the ammo acid sequence of SEQ ID NO:2 over the entire length of SEQ ID NO:4.
  • polypeptides of the invention include a polypeptide of Table 1 [SEQ ID NO:2 or 4] (in particular the mature polypeptide) as well as polypeptides and fragments, particularly those which have the biological activity of spoOJ2, and also those which have at least 70% identity to a polypeptide of Table 1 [SEQ ID NO:l or 3] or the relevant portion, preferably at least 80% identity to a polypeptide of Table 1 [SEQ ID NO:2 or 4] and more preferably at least 90% identity to a polypeptide of Table 1 [SEQ ID NO:2 or 4] and still more preferably at least 95% identity to a polypeptide of Table 1 [SEQ ID NO:2 or 4] and also include portions of such polypeptides with such portion of the polypeptide generally containing at least 30 ammo acids and more preferably at least 50 amino acids.
  • the invention also includes a polypeptide consisting of or compnsmg a polypeptide of the formula: X-(R 1 ) m -(R 2 )-(R 3 ) n -Y wherein, at the amino terminus, X is hydrogen, a metal or any other moiety descnbed herein for modified polypeptides, and at the carboxyl terminus, Y is hydrogen, a metal or any other moiety descnbed herein for modified polypeptides, R j 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 invention, particularly an amino acid sequence selected from Table 1 or modified forms thereof.
  • R 2 is onented so that its ammo terminal amino acid residue is at the left, covalently bound to R and its carboxy terminal ammo acid residue is at the ⁇ ght, covalently bound to R3.
  • Any stretch of amino acid residues denoted by either R ⁇ or R3, where m and/or n is greater than 1, may be either a heteropolymer or a homopolymer, preferably a heteropolymer.
  • Other preferred embodiments of the invention are provided where m is an integer between 1 and 50, 100 or 500, and n is an integer between 1 and 50, 100, or 500.
  • a polypeptide of the invention is denved from Staphylococcus aureus, however, it may preferably be obtained from other organisms of the same taxonomic genus.
  • a polypeptide of the invention may also be obtained, for example, from organisms of the 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 any ammo acid sequence of any polypeptide of the invention.
  • fragments may be "free-standing,” or comp ⁇ sed withm a larger polypeptide of which they form a part or region, most preferably as a single continuous region in a single larger polypeptide.
  • Preferred fragments include, for example, truncation polypeptides having a portion of an ammo acid sequence of Table 1 [SEQ ID NO:2 or 4], or of va ⁇ ants thereof, such as a continuous se ⁇ es of residues that includes an ammo- and/or carboxyl-termmal ammo acid sequence.
  • Degradation forms of the polypeptides of the invention produced by or in a host cell, particularly a Staphylococcus aureus are also preferred.
  • fragments charactenzed by structural or functional attributes such as fragments that comp ⁇ se alpha-helix and alpha-helix forming regions, beta-sheet and beta-sheet-formmg regions, turn and turn-forming regions, coil and coil-form g regions, hydrophihc 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, 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.
  • biologically active fragments which are those fragments that mediate activities of spoOJ2, including those with a similar activity or an improved activity, or with a decreased undesirable activity
  • Fragments of the polypeptides of the invention may be employed for producing the corresponding full-length polypeptide by peptide synthesis; therefore, these vanants may be employed as intermediates for producing the full-length polypeptides of the invention.
  • the term "X” or "Xaa” may also be used in describing certain polypeptides of the invention. "X” and "Xaa” mean that any of the twenty naturally occurring amino acids may appear at such a designated position in the polypeptide sequence.
  • Polynucleotides It is an object of the invention to provide polynucleotides that encode spoOJ2 polypeptides, particularly polynucleotides that encode the polypeptide herein designated spoOJ2.
  • the polynucleotide compnses a region encoding spoOJ2 polypeptides comp ⁇ smg a sequence set out in Table 1 [SEQ ID NO:l or 3] which includes a full length gene, or a variant thereof.
  • SEQ ID NO:l or 3 a sequence set out in Table 1 [SEQ ID NO:l or 3] which includes a full length gene, or a variant thereof.
  • this full length gene is essential to the growth and/or survival of an organism which possesses it, such as Staphylococcus aureus.
  • isolated nucleic acid molecules encoding and/or expressing spoOJ2 polypeptides and polynucleotides, particularly Staphylococcus aureus spoOJ2 polypeptides and polynucleotides, including, for example, unprocessed RNAs, ⁇ bozyme RNAs, mRNAs, cDNAs, genomic DNAs, B- and Z-DNAs.
  • Further embodiments of the invention include biologically, diagnostically, prophylactically, clinically or therapeutically useful polynucleotides and polypeptides, and vanants thereof, and compositions comp ⁇ smg the same.
  • Another aspect of the invention relates to isolated polynucleotides, including at least one full length gene, that encodes a spoOJ2 polypeptide having a deduced ammo acid sequence of Table 1 [SEQ ID NO:2 or 4] and polynucleotides closely related thereto and vanants thereof.
  • spoOJ2 polypeptide from Staphylococcus aureus comprising or consisting of an ammo acid sequence of Table 1 [SEQ ID NO:2 or 4], or a vanant thereof.
  • a polynucleotide of the invention encoding spoOJ2 polypeptide may be obtained using standard cloning and screening methods, such as those for cloning and sequencing chromosomal DNA fragments from bactena using Staphylococcus aureus WCUH 29 cells as starting mate ⁇ al, followed by obtaining a full length clone.
  • a polynucleotide sequence of the invention such as a polynucleotide sequence given in Table 1 [SEQ ID NO:l or 3]
  • a library of clones of chromosomal DNA of Staphylococcus aureus WCUH 29 in E.coh or some other suitable host is probed with a radiolabeled oligonucleotide, preferably a 17-mer or longer, de ⁇ ved from a partial sequence.
  • Clones carrying DNA identical to that of the probe can then be distinguished using stringent hybridization conditions.
  • sequencing is then possible to extend the polynucleotide sequence m both directions to determine a full length gene sequence.
  • sequencing is performed, for example, using denatured double stranded DNA prepared from a plasmid clone. Suitable techniques are described by Mamatis, T., Fntsch, E.F. and Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, 2nd Ed.; Cold Sp ⁇ ng Harbor Laboratory Press, Cold Sp ⁇ ng Harbor, New York (1989). (see in particular Screening By Hybndization 1.90 and Sequencing Denatured Double-Stranded DNA Templates 13.70).
  • Direct genomic DNA sequencing may be performed, for example, using denatured double stranded DNA prepared from a plasmid clone. Suitable techniques are described by Mamatis, T., Fntsch, E.F. and Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, 2nd Ed.; Cold
  • SUBST ⁇ UTE SHEET RULE 26 also be performed to obtain a full length gene sequence.
  • each polynucleotide set out in Table 1 [SEQ ID NO:l or 3] was discovered in a DNA library de ⁇ ved from Staphylococcus aureus WCUH 29.
  • each DNA sequence set out in Table 1 [SEQ ID NO: 1 or 3] contains an open reading frame encoding a protein having about the number of amino acid residues set forth in Table 1 [SEQ ID NO:2 or 4] with a deduced molecular weight that can be calculated using amino acid residue molecular weight values well known to those skilled m the art.
  • the polynucleotide of SEQ ID NO:l, between nucleotide number 1 and the stop codon which begins at nucleotide number 838 of SEQ ID NO: 1 encodes the polypeptide of SEQ ID NO:2.
  • the present invention provides for an isolated polynucleotide compnsing or consisting of: (a) a polynucleotide sequence which has at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, yet more preferably at least 95% identity, even more preferably at least 97-99% or exact identity to SEQ ID NO:l over the entire length of SEQ ID NO:l; (b) a polynucleotide sequence encoding a polypeptide which has at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, yet more preferably at least 95% identity, even more preferably at least 97-99% or 100% exact, to the ammo acid sequence of SEQ ID NO:2, over the entire length of SEQ ID NO:2; or (c) a nucleotide sequence which has at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, yet more preferably at least 95% identity, even more preferably at least 97-99% or 100% exact,
  • a polynucleotide encoding a polypeptide of the present invention may be obtained by a process which comp ⁇ ses the steps of screening an appropnate library under st ⁇ ngent hybndization conditions with a labeled or detectable probe consisting of or compnsing the sequence of SEQ ID NO:l or 3 or a fragment thereof; and isolating a full-length gene and or genomic clones containing said polynucleotide sequence.
  • the invention provides a polynucleotide sequence identical over its entire length to a coding sequence (open reading frame) in Table 1 [SEQ ID NO:l or 3]. Also provided by the invention 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 in 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 invention may also contain at least one non-coding sequence, including for example, but not limited to at least one non-codmg 5' and 3' sequence, such as the transcnbed but non-translated sequences, termination signals (such as rho-dependent and rho-independent termination signals), ⁇ bosome binding sites, Kozak sequences, sequences that stabilize mRNA, lntrons, and polyadenylation signals.
  • the polynucleotide sequence may also compnse additional coding sequence encoding additional ammo acids. Tor example, a marker sequence that facilitates pu ⁇ fication of the fused polypeptide can be encoded.
  • the marker sequence is a hexa-histidine peptide, as provided m the pQE vector (Qiagen, Inc.) and descnbed in Gentz et al, Proc Natl. Acad. Set , USA 86- 821-824 (1989), or an HA peptide tag (Wilson et al , Cell 37 767 (1984), both of which may be useful in punfying polypeptide sequence fused to them.
  • Polynucleotides of the invention also include, but are not limited to, polynucleotides compnsing a structural gene and its naturally associated sequences that control gene expression.
  • a preferred embodiment of the invention is a polynucleotide of consisting of or compnsing nucleotide 1 to the nucleotide immediately upstream of or including nucleotide 838 set forth in SEQ ID NO:l of Table 1, both of which encode the spoOJ2 polypeptide.
  • the invention also includes a polynucleotide consisting of or compnsing a polynucleotide of the formula:
  • R 1 X-(R 1 ) m -(R 2 )-(R 3 ) n -Y
  • X is hydrogen, a metal or a modified nucleotide residue, or together with Y defines a covalent bond
  • Y is hydrogen, a metal, or a modified nucleotide residue, or together with X defines the covalent bond
  • each occ rence of R ⁇ and R3 is independently 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.
  • R 2 is oriented so that its 5' end nucleic acid residue is at the left, bound to R ⁇ and its 3' end nucleic acid residue is at the nght, bound to R3.
  • Any stretch of nucleic acid residues denoted by either R j and or R 2 , where m and/or n is greater than 1, may be either a heteropolymer or a homopolymer, preferably a heteropolymer.
  • the polynucleotide of the above formula is a closed, circular polynucleotide, which 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 preferred embodiments of the invention are provided where m is an integer between 1 and 50, 100 or 500, and n is an integer between 1 and 50, 100, or 500.
  • a polynucleotide of the invention is de ⁇ ved from Staphylococcus aureus, however, it may preferably be obtained from other organisms of the same taxonomic genus.
  • a polynucleotide of the invention may also be obtained, for example, from organisms of the same taxonomic family or order.
  • polynucleotide encoding a polypeptide encompasses polynucleotides that include a sequence encoding a polypeptide of the invention, particularly a bactenal polypeptide and more particularly a polypeptide of the Staphylococcus aureus spoOJ2 having an ammo acid sequence set out in Table 1 [SEQ ID NO:2 or 4].
  • the term also encompasses polynucleotides that include a single continuous region or discontinuous regions encoding the polypeptide (for example, polynucleotides interrupted by integrated phage, an integrated insertion sequence, an integrated vector sequence, an integrated transposon sequence, or due to RNA editing or genomic DNA reorganization) together with additional regions, that also may contain coding and/or non-coding sequences.
  • the invention further relates to vanants of the polynucleotides descnbed herein that encode va ⁇ ants of a polypeptide having a deduced amino acid sequence of Table 1 [SEQ ID NO:2 or 4]. Fragments of a polynucleotides of the invention may be used, for example, to synthesize full-length polynucleotides of the invention.
  • spoOJ2 va ⁇ ants that have the amino acid sequence of spoOJ2 polypeptide of Table 1 [SEQ ID NO:2 or 4] 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 combination.
  • spoOJ2 polypeptide of Table 1 [SEQ ID NO:2 or 4] 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 combination.
  • silent substitutions, additions and deletions that do not alter the properties and activities of spoOJ2 polypeptide.
  • polynucleotides that are at least 70% identical over their entire length to a polynucleotide encoding spoOJ2 polypeptide having an ammo acid sequence set out m Table 1 [SEQ ID NO:2 or 4], and polynucleotides that are complementary to such polynucleotides.
  • most highly preferred are polynucleotides that compnse a region that is at least 80% identical over its entire length to a polynucleotide encoding spoOJ2 polypeptide and polynucleotides complementary thereto.
  • polynucleotides at least 90% identical over their entire length to the same are particularly preferred, and among these particularly preferred polynucleotides, those with at least 95% are especially preferred. Furthermore, those with at least 97% are highly preferred among those with at least 95%, and among these those with at least 98% and at least 99% are particularly highly preferred, with at least 99% being the more preferred.
  • Preferred embodiments are polynucleotides encoding polypeptides that retain substantially the same biological function or activity as the mature polypeptide encoded by a DNA of Table 1 [SEQ ID NO:l or 3].
  • polynucleotides that hyb ⁇ dize, particularly under st ⁇ ngent conditions, to spoOJ2 polynucleotide sequences such as those polynucleotides in Table 1.
  • the invention further relates to polynucleotides that hybndize to the polynucleotide sequences provided herein.
  • the invention especially relates to polynucleotides that hybndize under st ⁇ ngent conditions to the polynucleotides descnbed herein.
  • st ⁇ ngent conditions and “st ⁇ ngent hybndization conditions” mean hybndization occurnng only if there is at least 95% and preferably at least 97% identity between the sequences.
  • a specific example of st ⁇ ngent hybndization conditions is overnight incubation at 42°C in a solution comprising: 50% formamide, 5x SSC (150mM NaCl, 15mM tnsodium 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 washing the hybndization support in O.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 Spnng Harbor, N.Y., (1989), particularly Chapter 11 therein. Solution hybndization may also be used with the polynucleotide sequences provided by the invention.
  • the invention also provides a polynucleotide consisting of or compnsing a polynucleotide sequence obtained by screening an appropnate library containing the complete gene for a polynucleotide sequence set forth m SEQ ID NO:l or 3 under stringent hybndization conditions with a probe having the sequence of said polynucleotide sequence set forth m SEQ ID NO: l or 3 or a fragment thereof; and isolating said polynucleotide sequence.
  • Fragments useful for obtaining such a polynucleotide include, for example, probes and primers fully described elsewhere herein.
  • the polynucleotides of the invention may be used as a hybndization probe for RNA, cDNA and genomic DNA to isolate full-length cDNAs and genomic clones encoding spoOJ2 and to isolate cDNA and genomic clones of other genes that have a high identity, particularly high sequence identity, to the spoOJ2 gene.
  • Such probes generally will compnse at least 15 nucleotide residues or base pairs.
  • 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 spoOJ2 gene may be isolated by screening using a DNA sequence provided in Table 1 [SEQ ID NO:l or 3] to synthesize an oligonucleotide probe.
  • a labeled oligonucleotide having a sequence complementary to that of a gene of the invention is then used to screen a library of cDNA, genomic DNA or mRNA to determine which members of the library the probe hyb ⁇ dizes to.
  • Nucleic acid amplification is then carried out to amplify the "missing" 5' end of the DNA using a combination of gene specific and adaptor specific oligonucleotide p ⁇ mers.
  • the PCR reaction is then repeated using "nested" pnmers, 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 pnmer 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 give a complete sequence, or carrying out a separate full-length PCR using the new sequence information for the design of the 5' primer.
  • polynucleotides and polypeptides of the invention may be employed, for example, as research reagents and mate ⁇ als for discovery of treatments of and diagnostics for diseases, particularly human diseases, as further discussed herein relating to polynucleotide assays.
  • polynucleotides of the invention that are o gonucleotides derived from a sequence of Table 1 [SEQ ID NOS:l or 2 or 3 or 4] 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 m bacteria in infected tissue. It is recognized that such sequences will also have utility diagnosis of the stage of infection and type of infection the pathogen has attained.
  • the invention also provides polynucleotides that encode a polypeptide that is the mature protein plus additional amino or carboxyl-termmal ammo acids, or ammo acids mtenor to the mature polypeptide (when the mature form has more than one polypeptide chain, for instance).
  • Such sequences may play a role in processing of a protein from precursor to a mature form, may allow protein transport, may lengthen or shorten protein half-life or may facilitate manipulation of a protein for assay or production, among other things.
  • the additional amino acids may be processed away from the mature protein by cellular enzymes.
  • a precursor protein, having a mature form of the polypeptide fused to one or more prosequences may be an inactive form of the polypeptide. When prosequences are removed such inactive precursors generally are activated. Some or all of the prosequences may be removed before activation. Generally, such precursors are called proproteins.
  • N may also be used in describing certain polynucleotides of the invention. “N” means that any of the four DNA or RNA nucleotides may appear at such a designated position in the DNA or RNA sequence, except it is preferred that N is not a nucleic acid that when taken in combination with adjacent nucleotide positions, when read in the correct reading frame, would have the effect of generating a premature termination codon in such reading frame.
  • a polynucleotide of the invention may encode a mature protein, a mature protein plus a leader sequence (which may be referred to as a preprotein), a precursor of a mature protein having one or more prosequences that are not the leader sequences of a preprotein, or a preproprotem, which is a precursor to a proprotein, having a leader sequence and one or more prosequences, which generally are removed dunng processing steps that produce active and mature forms of the polypeptide.
  • a leader sequence which may be referred to as a preprotein
  • a precursor of a mature protein having one or more prosequences that are not the leader sequences of a preprotein or a preproprotem, which is a precursor to a proprotein, having a leader sequence and one or more prosequences, which generally are removed dunng processing steps that produce active and mature forms of the polypeptide.
  • the invention also relates to vectors that comp ⁇ se a polynucleotide or polynucleotides of the invention, host cells that are genetically engineered with vectors of the invention and the production of polypeptides of the invention by recombinant techniques.
  • Cell-free translation systems can also be employed to produce such proteins using RNAs denved from the DNA constructs of the invention
  • Recombinant polypeptides of the present invention may be prepared by processes well known in those skilled in the art from genetically engineered host cells compnsing expression systems.
  • the present invention relates to expression systems which comp ⁇ se a polynucleotide or polynucleotides of the present invention, to host cells which are genetically engineered with such expression systems, and to the production of polypeptides of the invention by recombinant techniques.
  • host cells can be genetically engineered to incorporate expression systems or portions thereof or polynucleotides of the invention.
  • Introduction of a polynucleotide into the host cell can be effected by methods descnbed in 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 Spnng Harbor Laboratory Press, Cold Sp ⁇ ng Harbor, N.Y.
  • bacte ⁇ al cells such as cells of streptococci, staphylococci, enterococci E coli, streptomyces, cyanobacte ⁇ a, Bacillus subtilis, 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
  • plant cells such as cells of a gymnosperm or angiosper
  • vectors include, among others, chromosomal-, episomal- and virus-de ⁇ ved vectors, for example, vectors denved from bacte ⁇ al 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 combinations thereof, such as those denved from plasmid and bactenophage genetic elements, such as cosmids and phagemids.
  • vectors denved from bacte ⁇ al plasmids for example, vectors denved from bacte ⁇ al plasmids, from bactenophage, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements
  • the expression system constructs may contain control regions that regulate as well as engender expression.
  • any system or vector suitable to maintain, propagate or express polynucleotides and/or to express a polypeptide m a host may be used for expression in this regard.
  • the appropnate DNA sequence may be inserted into the expression system by any of a va ⁇ ety of well-known and routine techniques, such as, for example, those set forth in Sambrook et al, MOLECULAR CLONING, A LABORATORY MANUAL, (supra).
  • appropnate secretion signals may be incorporated into the expressed polypeptide. These signals may be endogenous to the polypeptide or they may be heterologous signals.
  • Polypeptides of the invention can be recovered and pu ⁇ fied from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography, and lectm chromatography. Most preferably, high performance liquid chromatography is employed for punf ⁇ cation. Well known techniques for refolding protein may be employed to regenerate active conformation when the polypeptide is denatured du ⁇ ng isolation and or pu ⁇ fication. Diagnostic, Prognostic, Serotyping and Mutation Assays
  • This invention is also related to the use of spoOJ2 polynucleotides and polypeptides of the invention for use as diagnostic reagents.
  • Detection of spoOJ2 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 organism to drugs.
  • Eukaryotes, particularly mammals, and especially humans, particularly those infected or suspected to be infected with an organism compnsing the spoOJ2 gene or protein may be detected at the nucleic acid or amino acid level by a vanety of well known techniques as well as by methods provided herein.
  • Polypeptides and polynucleotides for prognosis, diagnosis or other analysis may be obtained from a putatively infected and or infected individual's bodily matenals.
  • Polynucleotides from any of these sources may be used directly for detection or may be amplified enzymatically by using PCR or any other amplification technique pnor to analysis.
  • RNA, particularly mRNA, cDNA and genomic DNA may also be used m the same ways. Using amplification, characte ⁇ zation of the species and strain of infectious or resident organism present in an individual, may be made by an analysis of the genotype of a selected polynucleotide of the organism.
  • Deletions and insertions can be detected by a change in size of the amplified product in compa ⁇ son to a genotype of a reference sequence selected from a related organism, preferably a different species of the same genus or a different strain of the same species.
  • Point mutations can be identified by hyb ⁇ dizing amplified DNA to labeled spoOJ2 polynucleotide sequences. Perfectly or significantly matched sequences can be distinguished from imperfectly or more significantly mismatched duplexes by DNase or RNase digestion, for DNA or RNA respectively, or by detecting differences in melting temperatures or renaturation kinetics.
  • Polynucleotide sequence differences may also be detected by alterations in the electrophoretic mobility of polynucleotide fragments in gels as compared to a reference sequence. This may be earned out with or without denatu ⁇ ng agents. Polynucleotide differences may also be detected by direct DNA or RNA sequencing. See, for example, Myers et al , Science, 230: 1242 (1985). Sequence changes at specific locations also may be revealed by nuclease protection assays, such as RNase, VI and SI protection assay or a chemical cleavage method. See, for example, Cotton et al , Proc Natl Acad. Scl, USA, 85. 4397-4401 (1985).
  • an array of ohgonucleotides probes compnsing spoOJ2 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 well known and have general applicability and can be used to address a
  • the present invention relates to a diagnostic kit which compnses: (a) a polynucleotide of the present invention, preferably the nucleotide sequence of SEQ ID NO:l or 3, 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 or 4 or a fragment thereof; or (d) an antibody to a polypeptide of the present invention, preferably to the polypeptide of SEQ ID NO.2 or 4. It will be appreciated that m any such kit, (a), (b), (c) or (d) may comprise a substantial component. Such a kit will be of use in diagnosing a disease or susceptibility to a Disease, among others.
  • This invention also relates to the use of polynucleotides of the present invention as diagnostic reagents.
  • Detection of a mutated form of a polynucleotide of the invention, preferable, SEQ ID NO: 1 or 3, which 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, which results from under- expression, over-expression or altered expression of the polynucleotide.
  • Organisms, particularly infectious organisms, carrying mutations in such polynucleotide may be detected at the polynucleotide level by a vanety of techniques, such as those descnbed elsewhere herein.
  • the nucleotide sequences of the present invention are also valuable for organism chromosome identification.
  • the sequence is specifically targeted to, and can hybndize with, a particular location on an organism's chromosome, particularly to a Staphylococcus aureus chromosome.
  • the mapping of relevant sequences to chromosomes according to the present invention may be an important step in correlating those sequences with pathogenic potential and/or an ecological niche of an organism and/or drug resistance of an organism, as well as the essentiality of the gene to the organism.
  • the physical position of the sequence on the chromosome can be correlated with genetic map data. Such data may be found on-line in a sequence database.
  • the relationship between genes and diseases that have been mapped to the same chromosomal region are then identified through known genetic methods, for example, through linkage analysis (comhentance of physically adjacent genes) or mating studies, such as by conjugation.
  • the differences in 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 some or all organisms possessing the first phenotype but not in any organisms possessing the second phenotype, then the mutation is likely to be the causative agent of the first phenotype.
  • RNA from an organism carrying mutations or polymorphisms (allehc va ⁇ ations) in a polynucleotide and or polypeptide of the invention may also be detected at the polynucleotide or polypeptide level by a va ⁇ ety of techniques, to allow for serotyping, for example.
  • RT-PCR can be used to detect mutations in the RNA. It is particularly preferred to use RT-PCR in conjunction with automated detection systems, such as, for example, GeneScan.
  • RNA, cDNA or genomic DNA may also be used for the same purpose, PCR.
  • PCR pnmers complementary to a polynucleotide encoding spoOJ2 polypeptide can be used to identify and analyze mutations Examples of representative pnmers are shown below in Table 2.
  • the invention also includes pnmers 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, and at the 3' end of the molecule, Y is hydrogen, a metal or a modified nucleotide residue, R and R3 are any nucleic acid residue or modified nucleotide residue, m is an integer between 1 and 20 or zero , n is an integer between 1 and 20 or zero, and R is a pnmer sequence of the invention, particularly a pnmer sequence selected from Table 2.
  • R 2 is o ⁇ ented so that its 5' end nucleotide residue is at the left, bound to R ⁇ and its 3' end nucleotide residue is at the nght, bound to R3.
  • Any stretch of nucleic acid residues denoted by either R group, where m and or n is greater than 1, may be either a heteropolymer or a homopolymer, preferably a heteropolymer being complementary to a region of a polynucleotide of Table 1.
  • m and or n is an integer between 1 and 10.
  • the invention further provides these pnmers with 1, 2, 3 or 4 nucleotides removed from the 5' and/or the 3' end.
  • These pnmers may be used for, among other things, amplifying spoOJ2 DNA and/or RNA isolated from a sample denved from an individual, such as a bodily mate ⁇ al.
  • the pnmers may be used to amplify a polynucleotide isolated from an infected individual, such that the polynucleotide may then be subject to vanous techniques for elucidation of the polynucleotide sequence. In this way, mutations in the polynucleotide 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 invention 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:l or 3].
  • Increased or decreased expression of a spoOJ2 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.
  • a diagnostic assay in accordance with the invention for detecting over- expression of spoOJ2 polypeptide compared to normal control tissue samples may be used to detect the presence of an infection, for example.
  • Assay techniques that can be used to determine levels of a spoOJ2 polypeptide, in a sample denved from a host, such as a bodily matenal, are well-known to those of skill in the art.
  • Such assay methods include radioimmunoassays, competitive-binding assays, Western Blot analysis, antibody sandwich assays, antibody detection and ELISA assays.
  • the polynucleotides and polynucleotides of the invention may be used as reagents for differential screening methods.
  • differential screening and differential display methods known in the art m which the polynucleotides and polypeptides of the invention may be used.
  • the differential display technique is descnbed by Chuang et ah, J. Bacteriol 775.2026-2036 (1993). This method identifies those genes which are expressed in an organism by identifying mRNA present using randomly-p ⁇ med RT-PCR. By compa ⁇ ng pre-mfection and post infection profiles, genes up and down regulated dunng infection can be identified and the RT-PCR product sequenced and matched to ORF "unknowns.”
  • IVET In Vivo Expression Technology (IVET) is described by Camilh et al, Proc. Nat'l Acad. Set USA. 97.2634-2638 (1994). IVET identifies genes up-regulated during infection when compared to laboratory cultivation, implying an important role in infection. ORFs identified by this technique are implied to have a significant role in infection establishment and/or maintenance. In this technique random chromosomal fragments of target organism are cloned upstream of a promoter-less recombmase gene in a plasmid vector. This construct is introduced into the target organism which carries an antibiotic resistance gene flanked by resolvase sites.
  • the resistant pool is introduced into a host and at various times after infection bacteria may be recovered and assessed for the presence of antibiotic resistance.
  • the chromosomal fragment carried by each antibiotic sensitive bacte ⁇ um should cany a promoter or portion of a gene normally upregulated during infection. Sequencing upstream of the recombmase gene allows identification of the up regulated gene.
  • RT-PCR may also be used to analyze gene expression patterns.
  • messenger RNA is isolated from bacte ⁇ al infected tissue, e.g., 48 hour murme lung infections, and the amount of each mRNA species assessed by reverse transcription of the RNA sample primed with random hexanucleotides followed by PCR with gene specific primer pairs.
  • the determination of the presence and amount of a particular mRNA species by quantification of the resultant PCR product provides information on the bacterial genes which are franscnbed m the infected tissue.
  • bactenal mRNA preparation need not be free of mammalian RNA. This allows the investigator to carry out a simple and quick RNA preparation from infected tissue to obtain bacterial mRNA species which are very short lived in the bacterium (m the order of 2 minute halfhves).
  • the bacterial mRNA is prepared from infected mu ⁇ ne lung tissue by mechanical disruption in the presence of TRIzole (GIBCO-BRL) for very short penods of time, subsequent processing according to the manufacturers of TRIzole reagent and DNAase treatment to remove contaminating DNA.
  • the process is optimized by finding those conditions which give a maximum amount of Staphylococcus aureus 16S nbosomal RNA as detected by probing Northerns with a suitably labeled sequence specific oligonucleotide probe.
  • a 5' dye labeled primer is used in each PCR primer pair in a PCR reaction which is terminated optimally between 8 and 25 cycles.
  • the PCR products are separated on 6% polyacrylamide gels with detection and quantification using GeneScanner (manufactured by ABI). Gridding and Polynucleotide Subtraction
  • the polynucleotides of the invention may be used as components of polynucleotide arrays, preferably high density arrays or grids. These high density arrays are particularly useful for diagnostic and prognostic purposes.
  • a set of spots each comprising a different gene, and further comprising a polynucleotide or polynucleotides of the invention may be used for probing, such as using hybridization or nucleic acid amplification, using a probes obtained or derived from a bodily sample, to determine the presence of a particular polynucleotide sequence or related sequence in an individual.
  • Such a presence may indicate the presence of a pathogen, particularly Staphylococcus aureus, and may be useful in diagnosing and/or prognosing disease or a course of disease.
  • a grid compnsing a number of variants of the polynucleotide sequence of SEQ ID NO: 1 or 3 are preferred. Also preferred is a compnsing a number of variants of a polynucleotide sequence encoding the polypeptide sequence of SEQ ID NO:2 or 4.
  • polypeptides and polynucleotides of the invention or vanants thereof, or cells expressing the same can be used as immunogens to produce antibodies lmmunospecific for such polypeptides or polynucleotides respectively.
  • antibodies against spoOJ2 polypeptides or polynucleotides there are provided antibodies against spoOJ2 polypeptides or polynucleotides.
  • Antibodies generated against the polypeptides or polynucleotides of the invention can be obtained by adm iste ⁇ ng the polypeptides and/or polynucleotides of the invention, or epitope- bea ⁇ ng fragments of either or both, analogues of either or both, or cells expressing either or both, to an animal, preferably a nonhuman, using routine protocols.
  • any technique known m the art that provides antibodies produced by continuous cell line cultures can be used. Examples include vanous techniques, such as those in Kohler, G. and Milstem, C , Nature 256 495-497 (1975), Kozbor et al , Immunology Today 4 72 (1983), Cole et al , pg. 77-96 in MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc. (1985).
  • phage display technology may be utilized to select antibody genes with binding activities towards a polypeptide of the invention either from repertoires of PCR amplified v-genes of lymphocytes from humans screened for possessing ant ⁇ -spoOJ2 or from naive libraries (McCafferty, et al , (1990), Nature 348, 552-554; Marks, et al, (1992) Biotechnology 10, 779-783).
  • the affinity of these antibodies can also be improved by, for example, chain shuffling (Clackson et ⁇ /. , ( 1991) Nature 352 628).
  • the above-desc ⁇ bed antibodies may be employed to isolate or to identify clones expressing the polypeptides or polynucleotides of the invention to punfy the polypeptides or polynucleotides by, for example, affinity chromatography
  • antibodies against spoOJ2-polypept ⁇ de or spoOJ2-polynucleot ⁇ de may be employed to treat infections, particularly bactenal infections.
  • Polypeptide vanants include antigemcally, epitopically or immunologically equivalent variants form a particular aspect of this invention.
  • a polypeptide or polynucleotide of the invention such as an antigemcally or immunologically equivalent denvative or a fusion protein of the polypeptide is used as an antigen to immunize a mouse or other animal such as a rat or chicken.
  • the fusion protein may provide stability to the polypeptide.
  • the antigen may be associated, for example by conjugation, with an immunogenic carrier protein for example bovine serum albumin, keyhole limpet haemocyanm or tetanus toxoid.
  • a multiple antigenic polypeptide compnsing multiple copies of the polypeptide, or an antigemcally or immunologically equivalent polypeptide thereof may be sufficiently antigenic to improve immunogenicity so as to obviate the use of a earner.
  • the antibody or variant thereof is modified to make it less immunogenic in the individual.
  • the individual is human the antibody may most preferably be "humanized," where the complimenta ⁇ ty determining region or regions of the hyb ⁇ doma- de ⁇ ved antibody has been transplanted into a human monoclonal antibody, for example as described in Jones et al. (1986), Nature 321, 522-525 or Tempest et al, (1991) Biotechnology 9, 266-273.
  • a polynucleotide of the invention for therapeutic or prophylactic purposes, in particular genetic immunization.
  • particularly preferred embodiments of the invention are naturally occurnng allelic va ⁇ ants of spoOJ2 polynucleotides and polypeptides encoded thereby.
  • the use of a polynucleotide of the invention in genetic immunization will preferably employ a suitable delivery method such as direct injection of plasmid DNA into muscles (Wolff et al, Hum Mol Genet (1992) 1: 363, Manthorpe et al, Hum. Gene Ther.
  • Polypeptides and polynucleotides of the invention may also be used to assess the binding of small molecule substrates and gands in, for example, cells, cell-free preparations, chemical branes, and natural product mixtures. These substrates and hgands may be natural substrates and hgands or may be structural or functional mimetics. See, e.g., Coligan et al, Current Protocols in Immunology 1(2). Chapter 5 (1991).
  • Polypeptides and polynucleotides of the present invention are responsible for many biological functions, including many disease states, m particular the Diseases hereinbefore mentioned. It is therefore desirable to devise screening methods to identify compounds which stimulate or which inhibit the function of the polypeptide or polynucleotide. Accordingly, in a further aspect, the present invention provides for a method of screening compounds to identify those which stimulate or which inhibit the function of a polypeptide or polynucleotide of the invention, as well as related polypeptides and polynucleotides. In general, agonists or antagonists may be employed for therapeutic and prophylactic purposes for such Diseases as hereinbefore mentioned.
  • Compounds may be identified from a vanety of sources, for example, cells, cell-free preparations, chemical hbra ⁇ es, and natural product mixtures.
  • Such agonists, antagonists or inhibitors so-identified may be natural or modified substrates, hgands, receptors, enzymes, etc., as the case may be, of spoOJ2 polypeptides and polynucleotides; or may be structural or functional mimetics thereof (see Coligan et al , Current Protocols in Immunology l(2):Chapter 5 (1991)).
  • the screening methods may simply measure the binding of a candidate compound to the polypeptide or polynucleotide, or to cells or membranes beanng the polypeptide or polynucleotide, or a fusion protein of the polypeptide by means of a label directly or indirectly associated with the candidate compound.
  • the screening method may involve competition with a labeled competitor.
  • these screening methods may test whether the candidate compound results in a signal generated by activation or inhibition of the polypeptide or polynucleotide, using detection systems appropriate to the cells comprising the polypeptide or polynucleotide. Inhibitors of activation are generally assayed in the presence of a known agonist and the effect on activation by the agonist by the presence of the candidate compound is observed.
  • Constitutively active polypeptide and/or constitutively expressed polypeptides and polynucleotides may be employed in screening methods for inverse agonists or inhibitors, in the absence of an agonist or inhibitor, by testing whether the candidate compound results in inhibition of activation of the polypeptide or polynucleotide, as the case may be.
  • the screening methods may simply comprise the steps of mixing a candidate compound with a solution containing a polypeptide or polynucleotide of the present invention, to form a mixture, measuring spoOJ2 polypeptide and/or polynucleotide activity in the mixture, and comparing the spoOJ2 polypeptide and/or polynucleotide activity of the mixture to a standard.
  • Fusion proteins such as those made from Fc portion and spoOJ2 polypeptide, as hereinbefore described, can also be used for high-throughput screening assays to identify antagonists of the polypeptide of the present invention, as well as of phylogenetically and and/or functionally related polypeptides (see D. Bennett et al., J Mol Recognition, 8:52-58 (1995); and K. Johanson et al, J Biol Chem, 270(16): 9459-9471 (1995)).
  • 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 measunng secreted or cell associated levels of polypeptide using monoclonal and polyclonal antibodies by standard methods known in the art. This can be used to discover agents which may inhibit or enhance the production of polypeptide (also called antagonist or agonist, respectively) from suitably manipulated cells or tissues.
  • the invention also provides a method of screening compounds to identify those which enhance (agonist) or block (antagonist) the action of spoOJ2 polypeptides or polynucleotides, particularly those compounds that are bacte ⁇ static and or bactencidal.
  • the method of screening may involve high-throughput techniques. For example, to screen for agonists or antagonists, a synthetic reaction mix, a cellular compartment, such as a membrane, cell envelope or cell wall, or a preparation of any thereof, compnsing spoOJ2 polypeptide and a labeled substrate or hgand of such polypeptide is incubated in the absence or the presence of a candidate molecule that may be a spoOJ2 agonist or antagonist.
  • the ability of the candidate molecule to agonize or antagonize the spoOJ2 polypeptide is reflected in decreased binding of the labeled hgand or decreased production of product from such substrate.
  • Molecules that bind gratuitously, i.e., without inducing the effects of spoOJ2 polypeptide are most likely to be good antagonists.
  • Molecules that bind well and, as the case may be, increase the rate of product production from substrate, increase signal transduction, or increase chemical channel activity are agonists. Detection of the rate or level of, as the case may be, production of product from substrate, signal transduction, or chemical channel activity may be enhanced by using a reporter system.
  • Reporter systems that may be useful m this regard include but are not limited to colo ⁇ met ⁇ c, labeled substrate converted into product, a reporter gene that is responsive to changes m spoOJ2 polynucleotide or polypeptide activity, and binding assays known m the art.
  • Polypeptides of the invention may be used to identify membrane bound or soluble receptors, if any, for such polypeptide, through standard receptor binding techniques known in the art.
  • hgand binding and crosshnking assays which the polypeptide is labeled with a radioactive isotope (for instance, 1 ⁇ 1), chemically modified (for instance, biotmylated), or fused to a peptide sequence suitable for detection or purification, and incubated with a source of the putative receptor (e.g., cells, cell membranes, cell supernatants, tissue extracts, bodily matenals).
  • a source of the putative receptor e.g., cells, cell membranes, cell supernatants, tissue extracts, bodily matenals.
  • Other methods include biophysical techniques such as surface plasmon resonance and spectroscopy. These screening methods may also be used to identify agonists and antagonists of the polypeptide which compete with the binding of the polypeptide to its receptor(s), if any. Standard methods for conducting such assays are well understood in the art.
  • the fluorescence polarization value for a fluorescently-tagged molecule depends on the rotational correlation time or tumbling rate.
  • Protein complexes such as formed by spoOJ2 polypeptide associating with another spoOJ2 polypeptide or other polypeptide, labeled to comprise a fluorescently-labeled molecule will have higher pola ⁇ zation values than a fluorescently labeled monome ⁇ c protein. It is preferred that this method be used to characterize small molecules that disrupt polypeptide complexes.
  • Fluorescence energy transfer may also be used charactenze small molecules that interfere with the formation of spoOJ2 polypeptide dimers, tnmers, tetramers or higher order structures, or structures formed by spoOJ2 polypeptide bound to another polypeptide.
  • SpoOJ2 polypeptide can be labeled with both a donor and acceptor fluorophore. Upon mixing of the two labeled species and excitation of the donor fluorophore, fluorescence energy transfer can be detected by observing fluorescence of the acceptor. Compounds that block dime ⁇ zation will inhibit fluorescence energy transfer.
  • spoOJ2 polypeptide can be coupled to a sensor chip at low site density such that covalently bound molecules will be monome ⁇ c Solution protein can then passed over the spoOJ2 polypeptide -coated surface and specific binding can be detected in real-time by monitonng the change in resonance angle caused by a change in local refractive index.
  • This technique can be used to charactenze the effect of small molecules on kinetic rates and equihbnum binding constants for spoOJ2 polypeptide self- association as well as an association of spoOJ2 polypeptide and another polypeptide or small molecule.
  • a scintillation proximity assay may be used to charactenze the interaction between an association of spoOJ2 polypeptide with another spoOJ2 polypeptide or a different polypeptide .
  • spoOJ2 polypeptide can be coupled to a scintillation-filled bead. Addition of radio-labeled spoOJ2 polypeptide results in binding where the radioactive source molecule is in close proximity to the scintillation fluid. Thus, signal is emitted upon spoOJ2 polypeptide binding and compounds that prevent spoOJ2 polypeptide self-association or an association of spoOJ2 polypeptide and another polypeptide or small molecule will dimmish signal.
  • ICS biosensors have been described by AMBRI (Australian Membrane
  • methods for identifying compounds which bind to or otherwise interact with and inhibit or activate an activity or expression of a polypeptide and or polynucleotide of the invention compnsing: contacting a polypeptide and or polynucleotide of the invention with a compound to be screened under conditions to permit binding to or other interaction between the compound and the polypeptide and/or polynucleotide to assess the binding to or other interaction with the compound, such binding or interaction preferably being associated with a second component capable of providing a detectable signal in response to the binding or interaction of the polypeptide and/or polynucleotide with the compound; and determining whether the compound binds to or otherwise interacts with and activates or inhibits an activity or expression of the polypeptide and/or polynucleotide by detecting the presence or absence of a signal generated from the binding or interaction of the compound with the polypeptide and/or polynucleotide.
  • an assay for spoOJ2 agonists is a competitive assay that combines spoOJ2 and a potential agonist with spoOJ2-b ⁇ ndmg molecules, recombinant spoOJ2 binding molecules, natural substrates or hgands, or substrate or hgand mimetics, under appropnate conditions for a competitive inhibition assay.
  • spoOJ2 can be labeled, such as by radioactivity or a colonmetnc compound, such that the number of spoOJ2 molecules bound to a binding molecule or converted to product can be determined accurately to assess the effectiveness of the potential antagonist.
  • Potential antagonists include, among others, small organic molecules, peptides, polypeptides and antibodies that bind to a polynucleotide and/or polypeptide of the invention and thereby inhibit or extinguish its activity or expression.
  • Potential antagonists also may be small organic molecules, a peptide, a polypeptide such as a closely related protein or antibody that binds the same sites on a binding molecule, such as a binding molecule, without inducing spoOJ2- mduced activities, thereby preventing the action or expression of spoOJ2 polypeptides and/or polynucleotides by excluding spoOJ2 polypeptides and/or polynucleotides from binding.
  • Potential antagonists include a small molecule that binds to and occupies the binding site of the polypeptide thereby preventing binding to cellular binding molecules, such that normal biological activity is prevented.
  • small molecules include but are not limited to small organic molecules, peptides or peptide-hke molecules.
  • Other potential antagonists include antisense molecules (see Okano, J Neurochem. 56- 560 (1991); OLIGODEOXYNUCLEOTIDES AS ANTISENSE INHIBITORS OF GENE EXPRESSION, CRC Press, Boca Raton, FL ( 1988), for a descnption of these molecules).
  • Preferred potential antagonists include compounds related to and vanants of spoOJ2.
  • polypeptide antagonists include antibodies or, in some cases, ohgonucleotides or proteins which are closely related to the hgands, substrates, receptors, enzymes, etc., as the case may be, of the polypeptide, e.g., a fragment of the hgands, substrates, receptors, enzymes, etc.; or small molecules which bind to the polypeptide of the present invention but do not elicit a response, so that the activity of the polypeptide is prevented.
  • polypeptides of the invention are biomimetics, functional mimetics of the natural spoOJ2 polypeptide. These functional mimetics may be used for, among other things, antagonizing the activity of spoOJ2 polypeptide or as a antigen or immunogen m a manner descnbed elsewhere herein.
  • Functional mimetics of the polypeptides of the invention include but are not limited to truncated polypeptides.
  • preferred functional mimetics include, a polypeptide compnsing the polypeptide sequence set forth in SEQ ID NO:2 lacking 20, 30, 40, 50, 60, 70 or 80 ammo- or carboxy-termmal amino acid residues, including fusion proteins compnsing one or more of these truncated sequences.
  • Polynucleotides encoding each of these functional mimetics may be used as expression cassettes to express each mimetic polypeptide. It is preferred that these cassettes compnse 5' and 3' restnction sites to allow for a convenient means to hgate the cassettes together when desired. It is further preferred that these cassettes comp ⁇ se gene expression signals known m the art or descnbed elsewhere herein.
  • the present invention relates to a screening kit for identifying agonists, antagonists, hgands, receptors, substrates, enzymes, etc. for a polypeptide and/or polynucleotide of the present invention; or compounds which decrease or enhance the production of such polypeptides and/or polynucleotides , which compnses: (a) a polypeptide and/or a polynucleotide of the present invention; (b) a recombinant cell expressing a polypeptide and/or polynucleotide of the present invention; (c) a cell membrane expressing a polypeptide and/or polynucleotide of the present invention; or (d) antibody to a polypeptide and/or polynucleotide of the present invention; which polypeptide is preferably that of SEQ ID NO.2, and which polynucleotide is preferably that of SEQ ID NO:l.
  • a polypeptide and/or polynucleotide of the present invention may also be used in a method for the structure-based design of an agonist, antagonist or inhibitor of the polypeptide and/or polynucleotide, by: (a) determining m 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, antagonist or inhibitor; (c) synthesizing candidate compounds that are predicted to bind to or react with the deduced binding s ⁇ te(s), reactive s ⁇ te(s), and/or mot ⁇ f(s); and
  • the present invention provides methods of treating abnormal conditions such as, for instance, a Disease, related to either an excess of, an under-expression of, an elevated activity of, or a decreased activity of spoOJ2 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 comp ⁇ ses administe ⁇ ng to an individual m need thereof an inhibitor compound (antagonist) as herein descnbed, optionally m combination with a pharmaceutically acceptable earner, in an amount effective to inhibit the function and/or
  • SUBST ⁇ UTE SHEET RULE 26 expression of the polypeptide and/or polynucleotide such as, for example, by blocking the binding of hgands, substrates, receptors, enzymes, etc., or by inhibiting a second signal, and thereby alleviating the abnormal condition.
  • soluble forms of the polypeptides still capable of binding the hgand, substrate, enzymes, receptors, etc.
  • competition with endogenous polypeptide and/or polynucleotide may be administered. Typical examples of such competitors include fragments of the spoOJ2 polypeptide and/or polypeptide.
  • the present invention relates to genetically engineered soluble fusion proteins compnsing a polypeptide of the present invention, or a fragment thereof, and vanous portions of the constant regions of heavy or light chains of immunoglobulins of vanous subclasses (IgG, IgM, IgA, IgE).
  • an immunoglobulin is the constant part of the heavy chain of human IgG, particularly IgGl, where fusion takes place at the hinge region.
  • the Fc part can be removed simply by incorporation of a cleavage sequence which can be cleaved with blood clotting factor Xa.
  • this invention relates to processes for the preparation of these fusion proteins by genetic enginee ⁇ ng, and to the use thereof for drug screening, diagnosis and therapy.
  • a further aspect of the invention also relates to polynucleotides encoding such fusion proteins. Examples of fusion protein technology can be found m International Patent Application Nos. W094/29458 and W094/22914.
  • expression of the gene encoding endogenous spoOJ2 polypeptide can be inhibited using expression blocking techniques.
  • This blocking may be targeted against any step in gene expression, but is preferably targeted against transc ⁇ ption 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 Ohgodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, FL (1988)).
  • oligonucleotides which form triple helices with the gene can be supplied (see, for example, Lee et al, Nucleic Acids Res (1979) 3:173; 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 antibactenal compounds.
  • the encoded protein, upon expression, can be used as a target for the screening of antibacterial drugs.
  • polynucleotide sequences encoding the amino terminal regions of the encoded protein or Shine-Delgarno or other translation facilitating sequences of the respective mRNA can be used to construct antisense sequences to control the expression of the coding sequence of interest.
  • the invention also provides the use of the polypeptide, polynucleotide, agonist or antagonist of the invention to interfere with the initial physical interaction between a pathogen or pathogens and a eukaryotic, preferably mammalian, host responsible for sequelae of infection
  • the molecules of the invention may be used, in the prevention of adhesion of bactena, m particular gram positive and/or gram negative bactena, to eukaryotic, preferably mammalian, extracellular matnx proteins on in-dwelling devices or to extracellular matnx proteins in wounds; to block bactenal adhesion between eukaryotic, preferably mammalian, extracellular matnx proteins and bactenal spoOJ2 proteins that mediate tissue damage and or, to block the normal progression of pathogenesis in infections initiated other than by the implantation of m-dwelling devices or by other surgical techniques.
  • spoOJ2 agonists and antagonists preferably bactenstatic or bacte ⁇ cidal agonists and antagonists.
  • the antagonists and agonists of the invention may be employed, for instance, to prevent, inhibit and/or treat diseases.
  • H pylori Helicobacter pylori
  • bactena infect the stomachs of over one-third of the world's population causing stomach cancer, ulcers, and gastntis
  • International Agency for Research on Cancer (1994) Schistosomes, Liver Flukes and Helicobacter Pylori International Agency for Research on Cancer, Lyon, France, http.//www u ⁇ cc.ch/ecp/ecp2904 htm
  • the International Agency for Research on Cancer recently recognized a cause-and-effect relationship between H pylori and gast ⁇ c adenocarcinoma, classifying the bacte ⁇ um as a Group I (definite) carcinogen
  • Preferred antimicrobial compounds of the invention agonists and antagonists of spoOJ2 polypeptides and/or polynucleotides found using screens provided by the invention, or known in the art, particularly narrow-spectrum antibiotics, should be useful in the treatment of H pylori infection Such treatment should be useful in
  • Another aspect of the invention relates to a method for inducing an lmmunological response m an individual, particularly a mammal which compnses inoculating the individual with spoOJ2 polynucleotide and/or polypeptide, or a fragment or vanant thereof, adequate to produce antibody and/ or T cell immune response to protect said individual from infection, particularly bacterial infection and most particularly Staphylococcus aureus infection. Also provided are methods whereby such lmmunological response slows bactenal replication.
  • Yet another aspect of the invention relates to a method of inducing lmmunological response m an individual which comprises dehve ⁇ ng to such individual a nucleic acid vector, sequence or ⁇ bozyme to direct expression of spoOJ2 polynucleotide and/or polypeptide, or a fragment or a vanant thereof, for expressing spoOJ2 polynucleotide and or polypeptide, or a fragment or a vanant thereof in vivo in order to induce an lmmunological response, such as, to produce antibody and/ or T cell immune response, including, for example, cytokine-producing T cells or cytotoxic T cells, to protect said individual, preferably a human, from disease, whether that disease is already established withm the individual or not.
  • an lmmunological response such as, to produce antibody and/ or T cell immune response, including, for example, cytokine-producing T cells or cytotoxic T cells, to protect said individual, preferably a human, from disease, whether that disease
  • nucleic acid vector may compnse DNA, RNA, a nbozyme, a modified nucleic acid, a DNA RNA hybnd, a DNA-protein complex or an RNA-prote complex.
  • a further aspect of the invention relates to an lmmunological composition that when introduced into an individual, preferably a human, capable of having induced within it an lmmunological response, induces an lmmunological response in such individual to a spoOJ2 polynucleotide and/or polypeptide encoded therefrom, wherein the composition comprises a recombinant spoOJ2 polynucleotide and or polypeptide encoded therefrom and/or comp ⁇ ses DNA and/or RNA which encodes and expresses an antigen of said spoOJ2 polynucleotide, polypeptide encoded therefrom, or other polypeptide of the invention.
  • the lmmunological response may be used therapeutically or prophylactically and may take the form of antibody immunity and/or cellular immunity, such as cellular immunity ansing from CTL or CD4+ T cells.
  • a spoOJ2 polypeptide or a fragment thereof may be fused with co-protein or chemical moiety which may or may not by itself produce antibodies, but which is capable of stabilizing the first protein and producing a fused or modified protein which will have antigenic and/or immunogenic properties, and preferably protective properties.
  • fused recombinant protein preferably further comprises an antigenic co-protem, such as hpoprotein D from Hemoph us in ⁇ uenzae, Glutathione-S-transferase (GST) or beta-galactosidase, or any other relatively large co-protem which solubihzes the protein and facilitates production and punf ⁇ cation thereof.
  • an antigenic co-protem such as hpoprotein D from Hemoph us in ⁇ uenzae, Glutathione-S-transferase (GST) or beta-galactosidase, or any other relatively large co-protem which solubihzes the protein and facilitates production and punf ⁇ cation thereof.
  • the co-protein may act as an adjuvant m the sense of providing a generalized stimulation of the immune system of the organism receiving the protein.
  • the co- protein may be attached to either the amino- or carboxy-terminus of the first protein.
  • compositions particularly vaccine compositions, and methods compnsing the polypeptides and/or polynucleotides of the invention and immunostimulatory DNA sequences, such as those descnbed in Sato, Y. et al. Science 273: 352 (1996).
  • methods using the descnbed polynucleotide or particular fragments thereof, which have been shown to encode non-vanable regions of bacterial cell surface proteins m polynucleotide constructs used in such genetic immunization expenments m animal models of infection with Staphylococcus aureus. Such expe ⁇ ments will be particularly useful for identifying protein epitopes able to provoke a prophylactic or therapeutic immune response.
  • a polypeptide of the invention may be used as an antigen for vaccination of a host to produce specific antibodies which protect against invasion of bactena, for example by blocking adherence of bacteria to damaged tissue.
  • tissue damage examples include wounds in skin or connective tissue caused, for example, by mechanical, chemical, thermal or radiation damage or by implantation of indwelling devices, or wounds in the mucous membranes, such as the mouth, throat, mammary glands, urethra or vagina.
  • the invention also includes a vaccine formulation which compnses an immunogenic recombinant polypeptide and/or polynucleotide of the invention together with a suitable earner, such as a pharmaceutically acceptable earner. Since the polypeptides and polynucleotides may be broken down in the stomach, each is preferably administered parenterally, including, for example, administration that is subcutaneous, intramuscular, intravenous, or mtradermal.
  • Formulations suitable for parenteral administration include aqueous and non-aqueous stenle injection solutions which may contain anti-oxidants, buffers, bactenstatic compounds and solutes which render the formulation lsotomc with the bodily fluid, preferably the blood, of the individual; and aqueous and non-aqueous stenle suspensions which may include suspending agents or thickening agents.
  • the formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampoules and vials and may be stored m a freeze-d ⁇ ed condition requinng only the addition of the stenle liquid earner immediately prior to use.
  • the vaccine formulation may also include adjuvant systems for enhancing the immunogenicity of the formulation, such as oil-m water systems and other systems known in the art.
  • adjuvant systems for enhancing the immunogenicity of the formulation such as oil-m water systems and other systems known in the art.
  • the dosage will depend on the specific activity of the vaccine and can be readily determined by routine experimentation. While the invention has been described with reference to certain spoOJ2 polypeptides and polynucleotides, it is to be understood that this covers fragments of the naturally occurnng polypeptides and polynucleotides, and similar polypeptides and polynucleotides with additions, deletions or substitutions which do not substantially affect the immunogenic properties of the recombinant polypeptides or polynucleotides.
  • compositions compnsing a spoOJ2 polynucleotide and/or a spoOJ2 polypeptide for administration to a cell or to a multicellular organism are provided.
  • the invention also relates to compositions compnsing a polynucleotide and/or a polypeptides discussed herein or their agonists or antagonists.
  • the polypeptides and polynucleotides of the invention may be employed m combination with a non-stenle or stenle earner or earners for use with cells, tissues or organisms, such as a pharmaceutical earner suitable for administration to an individual.
  • Such earners may include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol and combinations thereof.
  • the formulation should suit the mode of administration.
  • the invention further relates to diagnostic and pharmaceutical packs and kits compnsing one or more containers filled with one or more of the ingredients of the aforementioned compositions of the invention.
  • Polypeptides, polynucleotides and other compounds of the invention may be employed alone or in conjunction with other compounds, such as therapeutic compounds.
  • the pharmaceutical compositions may be administered in any effective, convenient manner including, for instance, administration by topical, oral, anal, vaginal, intravenous, mtrapentoneal, intramuscular, subcutaneous, mtranasal or intradermal routes among others.
  • the active agent may be administered to an individual as an mjectable composition, for example as a stenle aqueous dispersion, preferably lsotomc.
  • the composition may be formulated for topical application for example in the form of ointments, creams, lotions, eye ointments, eye drops, ear drops, mouthwash, impregnated dressings and sutures and aerosols, and may contain appropnate conventional additives, including, for example, preservatives, solvents to assist drug penetration, and emollients m ointments and creams.
  • Such topical formulations may also contain compatible conventional earners, for example cream or ointment bases, and ethanol or oleyl alcohol for lotions.
  • Such earners may constitute from about 1% to about 98% by weight of the formulation; more usually they will constitute up to about 80% by weight of the formulation.
  • the present invention provides for pharmaceutical compositions compnsing a therapeutically effective amount of a polypeptide and/or polynucleotide, such as the soluble form of a polypeptide and/or polynucleotide of the present invention, agonist or antagonist peptide or small molecule compound, in combination with a pharmaceutically acceptable earner or excipient.
  • a pharmaceutically acceptable earner or excipient include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof.
  • the invention further relates to pharmaceutical packs and kits compnsing one or more containers filled with one or more of the ingredients of the aforementioned compositions of the invention.
  • Polypeptides, polynucleotides and other compounds of the present invention may be employed alone or in conjunction with other compounds, such as therapeutic compounds.
  • composition will be adapted to the route of administration, for instance by a systemic or an oral route.
  • Preferred forms of systemic administration include injection, typically by intravenous injection. Other injection routes, such as subcutaneous, intramuscular, or mtrapentoneal, can be used.
  • Alternative means for systemic administration include transmucosal and transdermal admimstratton using penetrants such as bile salts or fusidic acids or other detergents.
  • penetrants such as bile salts or fusidic acids or other detergents.
  • oral administration may also be possible.
  • Administration of these compounds may also be topical and/or localized, in the form of salves, pastes, gels, and the like.
  • the daily dosage level of the active agent will be from 0.01 mg/kg to 10 mg/kg, typically around 1 mg/kg.
  • the physician in any event will determine the actual dosage which will be most suitable for an individual and will vary with the age, weight and response of the particular individual.
  • the above dosages are exemplary of the average case. There can, of course, be individual instances where higher or lower dosage ranges are mented, and such are within the scope of this invention.
  • In-dwelling devices include surgical implants, prosthetic devices and catheters, i.e., devices that are introduced to the body of an individual and remain in position for an extended time.
  • Such devices include, for example, artificial joints, heart valves, pacemakers, vascular grafts, vascular catheters, cerebrospinal fluid shunts, unnary catheters, continuous ambulatory pentoneal dialysis (CAPD) catheters.
  • CAPD continuous ambulatory pentoneal dialysis
  • composition of the invention may be administered by injection to achieve a systemic effect against relevant bacteria shortly before insertion of an in-dwelling device. Treatment may be continued after surgery dunng the m-body time of the device.
  • the composition could also be used to broaden pe ⁇ operative cover for any surgical technique to prevent bacterial wound infections, especially Staphylococcus aureus wound infections.
  • compositions of this invention may be used generally as a wound treatment agent to prevent adhesion of bactena to matnx proteins exposed m wound tissue and for prophylactic use in dental treatment as an alternative to, or in conjunction with, antibiotic prophylaxis.
  • the composition of the invention may be used to bathe an indwelling device immediately before insertion.
  • the active agent will preferably be present at a concentration of l ⁇ g/ml to lOmg/ml for bathing of wounds or indwelling devices.
  • a vaccine composition is conveniently in mjectable form. Conventional adjuvants may be employed to enhance the immune response.
  • a suitable unit dose for vaccination is 0.5-5 microgram/kg of antigen, and such dose is preferably administered 1-3 times and with an interval of 1-3 weeks. With the indicated dose range, no adverse toxicological effects will be observed with the compounds of the invention which would preclude their administration to suitable individuals. Sequence Databases, Sequences in a Tangible Medium, and Algorithms
  • Polynucleotide and polypeptide sequences form a valuable information resource with which to determine their 2- and 3 -dimensional structures as well as to identify further sequences of similar homology. These approaches are most easily facilitated by sto ⁇ ng the sequence in a computer readable medium and then using the stored data in a known macromolecular structure program or to search a sequence database using well known searching tools, such as GCC.
  • polynucleotide and polypeptide sequences of the invention are particularly useful as components m databases useful for search analyses as well as in sequence analysis algonthms.
  • sequence databases Sequence Databases, Sequences in a Tangible Medium, and Algorithms
  • polynucleotide of the invention and “polynucleotide sequence of the invention” mean any detectable chemical or physical charactenstic of a polynucleotide of the invention that is or may be reduced to or stored m a tangible medium, preferably a computer readable form.
  • chromatographic scan data or peak data, photographic data or scan data therefrom, called bases, and mass spectrographic data for example, chromatographic scan data or peak data, photographic data or scan data therefrom, called bases, and mass spectrographic data.
  • polypeptide of the invention and “polypeptide sequence of the invention” mean any detectable chemical or physical characteristic of a polypeptide of the invention that is or may be reduced to or stored in a tangible medium, preferably a computer readable form. For example, chromatographic scan data or peak data, photographic data or scan data therefrom, and mass spectrographic data.
  • the invention provides a computer readable medium having stored thereon polypeptide sequences of the invention and/or polynucleotide sequences of the invention.
  • a computer readable medium compnsing and having stored thereon a member selected from the group consisting of: a polynucleotide compnsing the sequence of a polynucleotide of the invention; a polypeptide compnsing the sequence of a polypeptide sequence of the invention; a set of polynucleotide sequences wherein at least one of the sequences compnses the sequence of a polynucleotide sequence of the invention; a set of polypeptide sequences wherein at least one of the sequences comprises the sequence of a polypeptide sequence of the invention; a data set representing a polynucleotide sequence compnsing the sequence of polynucleotide sequence of the invention; a data set representing a polynucleotide sequence encoding a polypeptide sequence compnsing the sequence of a polypeptide sequence of the invention; a polynucleotide compnsing the sequence of a polynucleot
  • the computer readable medium can be any composition of matter used to store information or data, including, for example, commercially available floppy disks, tapes, chips, hard dnves, compact disks, and video disks.
  • methods for the analysis of character sequences or strings include, for example, methods of sequence homology analysis, such as identity and similanty analysis, RNA structure analysis, sequence assembly, cladistic analysis, sequence motif analysis, open reading frame determination, nucleic acid base calling, nucleic acid base tnmmmg, and sequencing chromatogram peak analysis.
  • a computer based method for performing homology identification. This method comp ⁇ ses the steps of providing a first polynucleotide sequence compnsing the sequence a polynucleotide of the invention in a computer readable medium; and compa ⁇ ng said first polynucleotide sequence to at least one second polynucleotide or polypeptide sequence to identify homology.
  • a computer based method is also provided for performing homology identification, said method compnsing the steps of: providing a first polypeptide sequence compnsing the
  • SUBST ⁇ UTE SHEET (RULE 26) sequence of a polypeptide of the invention in a computer readable medium; and companng said first polypeptide sequence to at least one second polynucleotide or polypeptide sequence to identify homology.
  • a computer based method is still further provided for polynucleotide assembly, said method comprising the steps of: providing a first polynucleotide sequence compnsing the sequence of a polynucleotide of the invention in a computer readable medium; and screening for at least one overlapping region between said first polynucleotide sequence and at least one second polynucleotide or polypeptide sequence.
  • a computer based method is still further provided for polynucleotide assembly, said method compnsing the steps of: providing a first polypeptide sequence compnsing a polypeptide of the invention m a computer readable medium; and screening for at least one overlapping region between said first polypeptide sequence and at least one second polynucleotide or polypeptide sequence.
  • a computer readable medium having stored thereon a member selected from the group consisting of: a polynucleotide compnsing the sequence of SEQ ID NO:l or 3; a polypeptide compnsing the sequence of SEQ ID NO:2 or 4; a set of polynucleotide sequences wherein at least one of said sequences compnses the sequence of SEQ ID NO:l or 3; a set of polypeptide sequences wherein at least one of said sequences compnses the sequence of SEQ ID NO:2 or 4; a data set representing a polynucleotide sequence compnsing the sequence of SEQ ID NO:l or 3; a data set representing a polynucleotide sequence encoding a polypeptide sequence compnsing the sequence of SEQ ID NO:2 or 4; a polynucleotide compnsing the sequence of SEQ ID NO:l or 3; a polypeptide comprising the sequence of SEQ
  • a further preferred embodiment of the invention provides a computer based method for performing homology identification, said method compnsing the steps of providing a polynucleotide sequence compnsing the sequence of SEQ ID NO: 1 or 3 in a computer readable medium; and companng said polynucleotide sequence to at least one polynucleotide or polypeptide sequence to identify homology.
  • a still further preferred embodiment of the invention provides a computer based method for performing homology identification, said method compnsing the steps of: providing a polypeptide sequence comprising the sequence of SEQ ID NO:2 or 4 in a computer readable medium; and companng said polypeptide sequence to at least one polynucleotide or polypeptide sequence to identify homology.
  • a further embodiment of the invention provides a computer based method for polynucleotide assembly, said method comprising the steps of: providing a first polynucleotide sequence compnsing the sequence of SEQ ID NO:l or 3 in a computer readable medium; and screening for at least one overlapping region between said first polynucleotide sequence and a second polynucleotide sequence.
  • a further embodiment of the invention provides a computer based method for performing homology identification, said method compnsing the steps of: providing a polynucleotide sequence compnsing the sequence of SEQ ID NO: 1 or 3 in a computer readable medium; and comparing said polynucleotide sequence to at least one polynucleotide or polypeptide sequence to identify homology.
  • Antibody ( ⁇ es) as used herein includes polyclonal and monoclonal antibodies, chime ⁇ c, single chain, and humanized antibodies, as well as Fab fragments, including the products of an Fab or other immunoglobulin expression library.
  • Antigemcally equivalent de ⁇ vat ⁇ ve(s) encompasses a polypeptide, polynucleotide, or the equivalent of either which will be specifically recognized by certain
  • SUBST ⁇ UTE SHEET RULE 26 antibodies which, when raised to the protein, polypeptide or polynucleotide according to the invention, interferes with the immediate physical interaction between pathogen and mammalian host.
  • Bispecific ant ⁇ body( ⁇ es) means an antibody compnsing at least two antigen binding domains, each domain directed against a different epitope.
  • Bodily mate ⁇ al(s) means any mate ⁇ al denved from an individual or from an organism infecting, infesting or inhabiting an individual, including but not limited to, cells, tissues and waste, such as, bone, blood, serum, cerebrospmal fluid, semen, saliva, muscle, cartilage, organ tissue, skin, unne, stool or autopsy mate ⁇ als.
  • D ⁇ sease(s) means any disease caused by or related to infection by a bactena, including , for example, disease, such as, infections of the upper respiratory tract (e.g., otitis media, bactenal tracheitis, acute epiglottitis, thyroiditis), lower respiratory (e.g., empyema, lung abscess), cardiac (e.g., infective endocarditis), gastrointestinal (e.g., secretory diarrhoea, splenic absces, retropentoneal abscess), CNS (e.g., cerebral abscess), eye (e.g., blephantis, conjunctivitis, keratitis, endophthalmitis, preseptal and orbital cellulitts, darcryocystitis), kidney and unnary tract (e.g., epididymitis, intrarenal and pennephnc absces, toxic shock syndrome), skm
  • Fusion prote ⁇ n(s) refers to a protein encoded by two, often unrelated, fused genes or fragments thereof.
  • EP-A-0464 discloses fusion proteins comprising various portions of constant region of immunoglobulin molecules together with another human protein or part thereof.
  • employing an immunoglobulin Fc region as a part of a fusion protein is advantageous for use in therapy and diagnosis resulting m, for example, improved pharmacokmetic properties [see, e g , EP-A 0232262].
  • “Host cell(s)” is a cell which has been transformed or transfected, or is capable of transformation or transfection by an exogenous polynucleotide sequence.
  • Identity is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as the case may be, as determined by companng the sequences.
  • identity also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between st ⁇ ngs of such sequences.
  • Identity can be readily calculated by known methods, including but not limited to those descnbed in (Computational Molecular Biology, Lesk, A.M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D.W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A.M., and Griffin, H.G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Hemje, G., Academic Press, 1987; and Sequence Analysis Primer, G ⁇ bskov, M.
  • Methods to determine identity are designed to give the largest match between 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, J. Mol Biol 215: 403-410 (1990).
  • the well known Smith Waterman algorithm may also be used to determine identity.
  • Parameters for polypeptide sequence compa ⁇ son include the following: Algonthm: 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 compansons (along with no penalty for end gaps).
  • Gap Length Penalty 3 Available as: The "gap” program from Genetics Computer Group, Madison WI. These are the default parameters for nucleic acid compa ⁇ sons.
  • Polynucleotide embodiments further include an isolated polynucleotide comprising a polynucleotide sequence having at least a 50, 60, 70, 80, 85, 90, 95, 97 or 100% identity to the reference sequence of SEQ ID NO:l, wherein said polynucleotide sequence may be identical to the reference sequence of SEQ ID NO:l 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 terminal positions, interspersed either individually among the nucleotides m the reference sequence or in one or more contiguous groups within the reference sequence
  • n n is the number of nucleotide alterations
  • x n is the total number of nucleotides in SEQ ID NO: l
  • y is 0.50 for 50%, 0.60 for 60%, 0.70 for 70%, 0.80 for 80%, 0.85 for 85%, 0.90 for 90%, 0.95 for 95%, 0.97 for 97% or 1.00 for 100%
  • • 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 .
  • Alterations of a polynucleotide sequence encoding the polypeptide of SEQ ID NO:2 may create nonsense, missense or frameshift mutations in this coding sequence and thereby alter the polypeptide encoded by the polynucleotide following such alterations.
  • a polynucleotide sequence of the present invention may be identical to the reference sequence of SEQ ID NO: 1 , that is it may be 100% identical, or it may include up to a certain integer number of nucleic acid alterations as compared to the reference sequence such that the percent identity is less than 100% identity.
  • Such alterations are selected from the group consisting of at least one nucleic acid deletion, substitution, including transition and transversion, or insertion, and wherein said alterations may occur at the 5' or 3' terminal positions of the reference polynucleotide sequence or anywhere between those terminal positions, interspersed either individually among the nucleic acids m the reference sequence or in one or more contiguous groups withm the reference sequence.
  • the number of nucleic acid alterations for a given percent identity is determined by multiplying the total number of nucleic acids in SEQ ID NO:l by the integer defining the percent identity divided by 100 and then subtracting that product from said total number of nucleic acids in SEQ ID NO:l, or:
  • n n is the number of nucleic acid alterations
  • x n is the total number of nucleic acids in SEQ ID NO:l
  • y is, for instance 0.70 for 70%, 0.80 for 80%, 0.85 for 85% etc.
  • • is the symbol for the multiplication operator, and wherein any non-integer 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 compnsing a polypeptide having at least a 50,60, 70, 80, 85, 90, 95, 97 or 100% identity to a polypeptide reference sequence of SEQ ID NO:2, wherein said polypeptide sequence may be identical to the reference sequence of SEQ ID NO:2 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 ammo acid deletion, substitution, including conservative and non-conservative 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 ammo acids in the reference sequence or m one or more contiguous groups withm the reference sequence, and wherein said number of ammo acid alterations is determined by multiplying the total number of amino acids in SEQ ID NO:2 by the integer defining the percent identity divided by 100 and then subtracting that
  • n a is the number of ammo acid alterations
  • x a is the total number of ammo acids in SEQ ID NO:2
  • y is 0.50 for 50%, 0.60 for 60%, 0.70 for 70%, 0.80 for 80%, 0.85 for 85%, 0.90 for 90%, 0.95 for 95%, 0.97 for 97% or 1.00 for 100%
  • • is the symbol for the multiplication operator, and wherein any non-mteger product of x a and y is rounded down to the nearest integer prior to subtracting it from x a .
  • a polypeptide sequence of the present invention may be identical to the reference sequence of SEQ ID NO:2, that is it may be 100% identical, or it may include up to a certain integer number of ammo acid alterations as compared to the reference sequence such that the percent identity is less than 100% identity.
  • Such alterations are selected from the group consisting of at least one ammo acid deletion, substitution, including conservative and non-conservative substitution, or insertion, and wherein said alterations may occur at the ammo- or carboxy-terminal positions of the reference polypeptide sequence or anywhere between those terminal positions, interspersed either individually among the ammo acids in the reference sequence or in one or more contiguous groups withm the reference sequence.
  • the number of ammo acid alterations for a given % identity is determined by multiplying the total number of amino acids m SEQ ID NO:2 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, or:
  • n a is the number of ammo acid alterations
  • x a is the total number of ammo acids in SEQ ID NO:2
  • y is, for instance 0.70 for 70%, 0.80 for 80%, 0.85 for 85% etc.
  • • 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 .
  • “Immunologically equivalent de ⁇ vat ⁇ ve(s)” as used herein encompasses a polypeptide, polynucleotide, or the equivalent of either which when used in a suitable formulation to raise antibodies in a vertebrate, the antibodies act to interfere with the immediate physical interaction between pathogen and mammalian host.
  • " lmmunospecific ' ' means that characte ⁇ stic of an antibody whereby it possesses substantially greater affinity for the polypeptides of the invention or the polynucleotides of the
  • SUBSTTTUTE SHEET RULE 26 invention than its affinity for other related polypeptides or polynucleotides respectively, particularly those polypeptides and polynucleotides in the pnor art.
  • “Ind ⁇ v ⁇ dual(s)” means a multicellular eukaryote, including, but not limited to a metazoan, a mammal, an ovid, a bovid, a simian, a p ⁇ mate, and a human "Isolated” means altered “by the hand of man” from its natural state, i e , if it occurs in nature, it has been changed or removed from its o ⁇ gmal environment, or both.
  • a polynucleotide or a polypeptide naturally present in a living organism is not “isolated,” but the same polynucleotide or polypeptide separated from the coexisting mate ⁇ als of its natural state is “isolated”, as the term is employed herein Moreover, a polynucleotide or polypeptide that is introduced into an organism by transformation, genetic manipulation or by any other recombinant method is "isolated” even if it is still present in said organism, which organism may be living or non-living
  • Orgamsm(s) means a (l) prokaryote, including but not limited to, a member of the genus Streptococcus, Staphylococcus, Bordetella, Corynebacterium, Mycobacterium, Neisseria, Haemophilus, Actinomycetes, Streptomycetes, Nocardia, Enterobacter, Yersinia, Fancisella, Pasturella, Moraxella, Acinetobacter, Erysipelothrix, Branhamella, Actinobacillus, Streptobacillus, Listeria, Calymmatobacterium, Brucella, Bacillus, Clostridium, Treponema, Escherichia, Salmonella, Kleibsiella, Vibrio, Proteus, Erwinia, Borreha, Leptospira, Spirillum, Campy lobacter, Shigella, Legwnella, Pseudomonas, Aeromona
  • Polynucleot ⁇ de(s) generally refers to any polynbonucleotide or polydeoxynbonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA
  • Polynucleot ⁇ de(s) include, without limitation, single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions or single-, double- and tnple-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybnd molecules compnsing DNA and RNA that may be single-stranded or, more typically, double-stranded, or tnple-stranded regions, or a mixture of single- and double-stranded regions
  • polynucleotide as used herein refers to tnple-stranded regions compnsing RNA or DNA or both RNA and DNA The strands in such regions may be
  • polynucleot ⁇ de(s) also includes DNAs or RNAs as descnbed above that contain 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 intended herein
  • DNAs or RNAs compnsing unusual bases, such as mosine, or modified bases, such as tntylated bases, to name just two examples are polynucleotides as the term is used herein
  • polynucleot ⁇ de(s) as it is employed herein embraces such chemically, enzymatically or metabolically modified forms of polynucleotides, as well as the
  • Polypept ⁇ de(s) refers to any peptide or protein compnsing two or more ammo acids joined to each other by peptide bonds or modified peptide bonds. "Polypept ⁇ de(s)” refers to both short chains, commonly referred to as peptides, ohgopeptides and ohgomers and to longer chams generally referred to as proteins Polypeptides may contain ammo acids other than the 20 gene
  • Polypept ⁇ de(s) include those modified either by natural processes, such as processing and other post-translational modifications, but also by chemical modification techniques. Such modifications are well descnbed in basic texts and in more detailed monographs, as well as in a voluminous research literature, and they are well known to those of skill in the art. It will be appreciated that the same type of modification may be present in the same or varying degree at several sites in a given polypeptide. Also, a given polypeptide may contain many types of modifications. Modifications can occur anywhere in a polypeptide, including the peptide backbone, the ammo acid side-chains, and the amino or carboxyl termini.
  • Modifications include, for example, acetylation, acylation, ADP-nbosylation, 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 pid denvative, covalent attachment of phosphotidyhnositol, cross-linking, cychzation, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteme, formation of pyroglutamate, formylation, gamma-carboxylation, GPI anchor formation, hydroxylation, lodination, methylation, my ⁇ stoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, glycosylation, hpid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP-nbosy
  • Polypeptides may be branched or cyclic, with or without branching. Cyclic, branched and branched circular polypeptides may result from post-translational natural processes and may be made by entirely synthetic methods, as well.
  • Recombinant expression system(s) refers to expression systems or portions thereof or polynucleotides of the invention introduced or transformed into a host cell or host cell lysate for the production of the polynucleotides and polypeptides of the invention.
  • “Subtraction set” is one or more, but preferably less than 100, polynucleotides compnsing at least one polynucleotide of the invention
  • “Va ⁇ ant(s)” as the term is used herein is a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide respectively, but retains essential properties.
  • a typical vanant of a polynucleotide differs m nucleotide sequence from another, reference polynucleotide. Changes in the nucleotide sequence of the vanant may or may not alter the ammo 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 vanant of a polypeptide differs m amino acid sequence from another, reference polypeptide. Generally, differences are limited so that the sequences of the reference polypeptide and the vanant are closely similar overall and, in 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 invention also includes include vanants of each of the polypeptides of the invention, that is polypeptides that vary from the referents by conservative 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 Tyr. Particularly preferred are va ⁇ ants in which several, 5-10, 1-5, 1-3, 1-2 or 1 amino 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 earned out using standard techniques, which are well known and routine to those of skill m the art, except where otherwise descnbed in detail. The examples are illustrative, but do not limit the invention.
  • the polynucleotide having a DNA sequence given in Table 1 [SEQ ID NO: 1 or 3] was obtained from a library of clones of chromosomal DNA of Staphylococcus aureus in E. coli.
  • SUBSTITUTE SHEET RULE 25 DNAs was used to construct the contiguous DNA sequence in SEQ ID NO: 1.
  • Libranes may be prepared by routine methods, for example: Methods 1 and 2 below.
  • Total cellular DNA is mechanically sheared by passage through a needle in order to size-fractionate according to standard procedures.
  • DNA fragments of up to 1 lkbp in size are rendered blunt by treatment with exonuclease and DNA polymerase, and EcoRI linkers added. Fragments are ligated into the vector Lambda ZapII that has been cut with EcoRI, the library packaged by standard procedures and E.coh infected with the packaged library.
  • the library is amplified by standard procedures.
  • Total cellular DNA is partially hydrolyzed with a one or a combination of restriction enzymes appropnate to generate a senes 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 ligated to the DNA and the fragments then ligated into the vector Lambda ZapII that have been cut with EcoRI, the library packaged by standard procedures, and E.coh infected with the packaged library.
  • the library is amplified by standard procedures.
  • Necrotic fatty tissue from a four day groin infection of Staphylococcus aureus WCUH29 in the mouse is efficiently disrupted and processed m the presence of chaotropic agents and RNAase inhibitor to provide a mixture of animal and bactenal RNA.
  • the optimal conditions for disruption and processing to give stable preparations and high yields of bactenal RNA are followed by the use of hybndisation to a radiolabelled oligonucleotide specific to Staphylococcus aureus 16S RNA on Northern blots.
  • RNAase free, DNAase free, DNA and protein free preparations of RNA obtained are suitable for Reverse Transcnption PCR (RT-PCR) using unique pnmer pairs designed from the sequence of each gene of Staphylococcus aureus WCUH29.
  • RT-PCR Reverse Transcnption PCR
  • mice 10 ml. volumes of stenle nut ⁇ ent broth (No.2 Oxoid) are seeded with isolated, individual colonies of Staphylococcus aureus WCUH29 from an agar culture plate. The cultures are incubated aerobically (static culture) at 37°C for 16-20 hours . 4 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 (grom area). Mice should be monitored regularly dunng 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. To minimise delay between death and tissue processing /storage, mice should be killed individually rather than in 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 supe ⁇ orly up to, then across the thorax. The incision is completed by cutting mfe ⁇ orly to the abdominal lower nght quadrant. Care should be taken not to penetrate the abdominal wall. Holding the skin flap with forceps, the skm is gently pulled way from the abdomen.
  • the exposed abscess which covers the pentoneal wall but generally does not penetrate the muscle sheet completely, is excised, taking care not to puncture the viscera
  • the abscess/muscle sheet and other infected tissue may require cutting in sections, p ⁇ or to flash-freezing m liquid nitrogen, thereby allowing easier storage in plastic collecting vials.
  • tissue samples (each approx 0.5-0.7g) in 2ml screw-cap tubes are removed from -80oC. storage into a dry 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.
  • tissue sample 1ml of TRIzol Reagent (Gibco BRL, Life Technologies) is added followed by enough 0.1mm zirconia sihca beads to almost fill the tube, the lid is replaced taking care not to get any beads into 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).
  • In vivo grown bactena require longer treatment than in vitro grown Staphylococcus aureus Staphylococcus which are disrupted by a 30 second bead-beat.
  • the tubes After bead-beatmg the tubes are chilled on ice before opening in a fume-hood as heat generated during disruption may degrade the TRIzol and release cyanide.
  • 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 transfe ⁇ ed to a stenle eppendorf tube and 0.5 ml of isopropanol is added.
  • 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 with 1 ml 75% ethanol.
  • a b ⁇ ef vortex is used to mix the sample before centnfuging at 7,500 x g, 4 °C for 5 minutes.
  • the ethanol is removed and the RNA pellet dned 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.
  • the 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 yields.
  • 2.2M formaldehyde gels are run and vacuum blotted to Hybond-N (Amersham). The blot is then hybndised with a 32 P labelled o gonucletide probe specific to 16s rRNA of Staphylococcus aureus ( K.Greisen, M. Loeffelholz, A. Purohit and D. Leong. J.Clm. (1994) Microbiol. 32 335-351 ).
  • oligonucleotide of SEQ ID NO:5 is used as a probe.
  • the size of the hybndising band is compared to that of control RNA isolated from in vitro grown Staphylococcus aureus WCUH29 in the Northern blot. Correct sized bactenal 16s rRNA bands can be detected in total RNA samples which show extensive degradation of the mammalian RNA when visualised on TBE gels.
  • DNA is removed from 73 microhtre samples of RNA by a 15 mmute 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.
  • DNAase is inactivated and removed by treatment with TRIzol LS Reagent (Gibco BRL, Life Technologies) according to the manufacturers protocol. DNAase treated RNA is resuspended in 73 microhtres of DEPC treated water with the addition of Rnasin as described m Method 1.
  • PCR reactions are set up on ice in 0.2ml tubes by adding the following components: 45 microhtres PCR SUPERMIX (Gibco BRL, Life Technologies); 1 microhtre 50mM MgC12 , to adjust final concentration to 2.5mM; 1 microhtre PCR pnmers(opt ⁇ mally 18-25 basepairs in length and designed to possess similar annealing temperatures), each pnmer at lOmM initial concentration; and 2 microhtres cDNA.
  • PCR reactions are run on a Perkm Elmer GeneAmp PCR System 9600 as follows: 5 minutes at 95 °C, then 50 cycles 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-
  • PCR product if present, sizes estimated by compa ⁇ son to a 100 bp DNA Ladder (Gibco BRL, Life Technologies).
  • PCR products are conveniently labelled by the use of a labelled PCR pnmer (e.g. labelled at the 5'end with a dye) a suitable aliquot of the
  • PCR product is run out on a polyacrylamide sequencing gel and its presence and quantity detected using a suitable gel scanning system (e.g. ABI PnsmTM 377 Sequencer using
  • RT/PCR controls may include +/- reverse transc ⁇ ptase reactions, 16s rRNA primers or DNA specific pnmer pairs designed to produce PCR products from non-transcnbed
  • PCR reactions are set up and run as descnbed above using approx. 1 microgram of DNA m place of the cDNA and 35 cycles of PCR.
  • Pnmer pairs which fail to give the predicted sized product in either DNA PCR or RT/PCR are PCR failures and as such are uninformative. Of those which give the correct size product with DNA PCR two classes are distinguished in RT/PCR. 1. Genes which are not franscnbed in vivo reproducibly fail to give a product in RT/PCR; and 2. Genes which are franscnbed in vivo reproducibly give the correct size product in RT/PCR and show a stronger signal in the +RT samples than the signal (if at all present) in -RT controls.

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