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WO2001023403A1 - ypga CLADE GENES - Google Patents

ypga CLADE GENES Download PDF

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Publication number
WO2001023403A1
WO2001023403A1 PCT/US2000/026450 US0026450W WO0123403A1 WO 2001023403 A1 WO2001023403 A1 WO 2001023403A1 US 0026450 W US0026450 W US 0026450W WO 0123403 A1 WO0123403 A1 WO 0123403A1
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WO
WIPO (PCT)
Prior art keywords
polypeptide
polynucleotide
ypga
group
isolated
Prior art date
Application number
PCT/US2000/026450
Other languages
French (fr)
Inventor
James R. Brown
Alison F. Chalker
David J. Holmes
Edwina Imogen Wilding
Original Assignee
Smithkline Beecham Corporation
Smithkline Beecham P.L.C.
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Application filed by Smithkline Beecham Corporation, Smithkline Beecham P.L.C. filed Critical Smithkline Beecham Corporation
Publication of WO2001023403A1 publication Critical patent/WO2001023403A1/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)
    • 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
    • C07K14/315—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Streptococcus (G), e.g. Enterococci
    • 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
    • C07K14/32—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Bacillus (G)

Definitions

  • This invention relates to newly identified polynucleotides and polypeptides, and their production and uses, as well as their variants, agonists and antagonists, and their uses.
  • the invention relates to polynucleotides and polypeptides of the ypgA clade family, as well as their variants, herein referred to as "ypgA clade genes," “ ypgA clade gene polynucleotide(s),” and “ypgA clade gene polypeptide(s),” as the case may be.
  • ypgA is found downstream of the gene encoding phosphomevalonate kinase and is part of the mevalonate kinase/decarboxylase operon. It is not found at this position in species of staphylococci, although a homologue is present in the genomes of the species examined. A ypgA homologue is also present in the ypgA clade in Borrelia b rgdoferi. No homologues were identified in eukaroytic organisms. Analysis of the amino-acid sequence for conserved motifs suggests that ypgA shows some distant homology to inosine 5'-monophosphate
  • ypgA is a member of the ypgA clade gene family.
  • the present invention relates to ypgA clade pathway genes, in particular ypgA polypeptides and 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 m
  • the invention relates to ypgA clade gene polypeptides and polynucleotides as desc ⁇ bed in greater detail below
  • the invention relates to polypeptides and polynucleotides of ypgA clade genes that are related by amino acid sequence homology to ypgA clade polypeptides from other species
  • the invention relates especially to ypgA clade genes having a high degree of homology to the nucleotide and amino acid sequences set out in Table 1 as SEQ ID NOs 1-16
  • the "ypgA clade gene family" of the invention means a set of genes encoding a set of polypeptides from Gram-positive bacteria falling within nodes A, B, C, D, E, F, and G of the phylogenetic tree depicted in Figure 1 comprising the genera Staphvlococcus, Streptococcus, and Enterococcus
  • ypgA clade gene(s) refers to a gene of the "ypgA clade pathway gene family", defined above "YpgA clade pathway gene polynucleot ⁇ de(s)” and “ypgA clade gene polypept ⁇ de(s)” means, respectively, a polynucleotide of the invention or polypeptide of the invention, as more particularly set forth elsewhere herein
  • the invention provides a set of genes encoding a set of polypeptides involved from Gram-positive bacteria falling within the clade defined by: node A of Figure 1 ; node B of Figure 1 ; node C of Figure 1 ; node D of Figure 1 ; node E of Figure 1 ; node F of Figure 1 ; and node G of the phylogenetic tree depicted in Figure 1.
  • the invention provides a set of genes encoding ypgA isolated from bacteria falling within the clade of Gram-positive bacteria of the phylogenetic tree depicted in Figure 1 comprising the species Staphylococcus aureus, Staphylococcus haemolyticus, Staphylococcus epidermidis, Streptococcus pneumoniae, Streptococcus pyogenes, Enterococcus faecium, Enter ococcus faecalis, and Bacillus subtilis.
  • 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 ribopolynucleotides.
  • Staphylococcus epidermidis ypgA polynucleotide sequence [SEQ ID NO: l].
  • Steptococcus pyogenes ypgA polynucleotide sequence [SEQ ID NO: 13].
  • Bacillus subtilis ypgA polynucleotide sequence [SEQ ID NO: 15]. 5 ' - GTGACTCGAGCAGAACGAAAAAGACAACACATCAATCATGCCTTGTCCAT CGGCCAGAAGCGGGAAACAGGTCTTGATGATATTACGTTTGTTCACGTCA GTCTGCCCGATCTTGCATTAGAACAAGTAGATATTTCCACAAAAATCGGC GAACTTTCAAGCAGTTCGCCGATTTTTATCAATGCAATGACTGGCGGCGG CGGAAAACTTACATATGAGATTAATAAATCGCTTGCGCGAGCGGCTTC AGGCTGGAATTCCCCTTGCTGTGGGATCGCAAATGTCAGCATTAAAAGAT CCATCAGAGCGTCTTTCCTATGAAATTGTTCGAAAGGAAAACCCAAACGG GCTGATTTTTGCCAACCTGGGAAGCGAGGCAACGGCTGCTCAGGCAAAGG AAGCCGTTGAGATGATTGGAGCAACGGCTGCTCAGGCAAAGG AAGCCGTT
  • a deposit comprising a Streptococcus pneumoniae 0100993 strain has been deposited with the National Collections of Industrial and Marine Bacteria Ltd. (herein "NCIMB"), 23 St Machar Drive, Aberdeen AB2 1RY, Scotland on 1 1 April 1996 and assigned deposit number 40794 The deposit was desc ⁇ bed as Streptococcus pneumoniae 0100993 on deposit
  • Streptococcus pneumoniae 0100993 DNA library in E coll was similarly deposited with the NCIMB and assigned deposit number 40800
  • the Streptococcus pneumoniae strain deposit is referred to herein as "the deposited strain” or as "the DNA of the deposited strain"
  • the deposited strain comp ⁇ ses a full-length ypgA gene of the ypgA clade gene family
  • the sequence of the polynucleotides comp ⁇ sed in the deposited strain, as well as the amino acid sequence of any polypeptide encoded thereby, are controlling in the event of any conflict with any desc ⁇ ption of sequences herein
  • Staphylococcus aureus WCUH 29 strain A deposit comp ⁇ smg a Staphylococcus aureus WCUH 29 strain has been deposited with the NCIMB), 23 St Machar Drive, Aberdeen AB2 1 RY, Scotland on 1 1 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 comp ⁇ ses a full-length ypgA gene of the ypgA clade gene family
  • the sequence of the polynucleotides comp ⁇ sed in the deposited strain, as well as the amino acid sequence of any polypeptide encoded thereby, are controlling in the event of any conflict with any desc ⁇ ption 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 rest ⁇ ction 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 ⁇ 1 12
  • 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 Streptococcus pneumoniae 0100993 strain, which polypeptide is comp ⁇ sed in the deposited strain
  • ypgA clade gene polynucleotide sequences in the deposited strain such as DNA and RNA, and amino acid sequences encoded thereby.
  • mevalonate pathway gene polypeptide and polynucleotide sequences isolated from the deposited strain are also provided by the invention.
  • an isolated nucleic acid molecule encoding a mature polypeptide expressible by the Staphylococcus aureus WCUH 29 strain, which polypeptide is comprised in the deposited strain.
  • ypgA clade gene polynucleotide sequences in the deposited strain such as DNA and RNA, and amino acid sequences encoded thereby.
  • Polypeptides Yp A clade gene polypeptides of the invention are substantially phylogenetically related to other proteins of the ypgA clade gene family.
  • Figure 1 shows the phylogenetic analysis of ypgA, a member of the ypgA clade gene family.
  • Phylogenetic trees are based on the neighbor-joining (NJ) method as implemented by the program NEIGHBOR of the PHYLIP 3.57c package (Felsenstein, J. 1993. Distributed by the author: http://evolution.genetics.washington. edu/phylip.html, Department of Genetics, University of Washington, Seattle.). The method used to create this phylogenetic tree is described in detail in Example 1.
  • polypeptides of ypgA clade genes referred to herein as "ypgA clade genes" and “ypgA clade gene polypeptides” as well as biologically, diagnostically, prophylactically, clinically or therapeutically useful variants thereof, and compositions comprising the same.
  • ypgA clade gene polypeptides encoded by naturally occurring alleles of a ypgA clade gene.
  • the present invention further provides for an isolated polypeptide that: (a) comprises or consists of an amino acid sequence that has at least 95% identity, most preferably at least 97- 99% or exact identity, to that of SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, and 16 over the entire length of said amino acid sequence; (b) a polypeptide encoded by an isolated polynucleotide comprising or consisting of a polynucleotide sequence that has at least 95% identity, even more preferably at least 97-99% or exact identity to SEQ ID NOs: l, 3, 5, 7, 9, 1 1, 13, and 15 over the entire length of said polynucleotide sequence; (c) a polypeptide encoded by an isolated polynucleotide comprising or consisting of a polynucleotide sequence encoding a polypeptide that has at least 95% identity, even more preferably at least 97-99% or exact identity, to the amino acid sequence of SEQ ID NOs 2, 4, 6, 8, 10, 12, 14, and 16 over the
  • polypeptides of the invention include the polypeptides of Table 1 [SEQ ID NOs 2, 4, 6, 8, 10, 12, 14, and 16] (in particular a mature polypeptide) as well as polypeptides and fragments, particularly those that has a biological activity of a ypgA clade gene, and also those that have at least 95% identity to a polypeptide of Table 1 [SEQ ID NOs 2, 4, 6, 8, 10, 12, 14, and 16] and also include portions of such polypeptides with such portion of the polypeptide generally comp ⁇ sing at least 30 amino acids and more preferably at least 50 amino acids
  • the invention also includes a polypeptide consisting of or comp ⁇ sing a polypeptide of the formula
  • a polypeptide of the invention is de ⁇ ved from a bacte ⁇ um of the ypgA clade gene family, 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 amino 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 comprised within 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 amino acid sequence of Table 1 [SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, and 16], or of variants thereof, such as a continuous series of residues that includes an amino- and/or carboxyl-terminal amino acid sequence.
  • Degradation forms of the polypeptides of the invention produced by or in a host cell, particularly a bacterium of the ypgA clade gene family, are also preferred.
  • fragments characterized by structural or functional attributes such as fragments that comprise alpha-helix and alpha-helix forming regions, beta-sheet and beta-sheet-forming regions, turn and turn-forming regions, coil and coil-forming regions, hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions, substrate binding region, and high antigenic index regions.
  • fragments include an isolated polypeptide comprising an amino acid sequence having at least 15, 20, 30, 40, 50 or 100 contiguous amino acids from the amino acid sequence of SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, and 16, 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 such amino acid sequence.
  • Fragments of the polypeptides of the invention may be employed for producing the corresponding full-length polypeptide by peptide synthesis; therefore, these variants may be employed as intermediates for producing the full-length polypeptides of the invention.
  • the polynucleotide comprises a region encoding ypgA clade gene polypeptides comprising a sequence set out in Table 1 [SEQ ID NOs: l, 3, 5, 7, 9, 1 1, 13, and 15] that includes a full length gene, or a variant thereof.
  • a full-length gene from the ypgA clade gene family is essential to the growth and/or survival of an organism that possesses it, such as a bacterium from the ypgA clade gene family.
  • isolated nucleic acid molecules encoding and/or expressing ypgA clade gene polypeptides and polynucleotides, particularly ypgA clade gene polypeptides and polynucleotides, including, for example, unprocessed RNAs, ribozyme RNAs, mRNAs, cDNAs, genomic DNAs, B- and Z-DNAs.
  • Further embodiments of the invention include biologically, diagnosttcally, prophylactically, clinically or therapeutically useful polynucleotides and polypeptides, and va ⁇ ants thereof, and compositions comp ⁇ sing the same
  • Another aspect of the invention relates to isolated polynucleotides, including at least one full length gene, that encodes a ypgA clade gene polypeptide having a deduced amino acid sequence of Table 1 [SEQ ID NOs 2, 4, 6, 8, 10, 12, 14, and 16] and polynucleotides closely related thereto and va ⁇ ants thereof
  • a ypgA clade gene polypeptide from a bacterium of the ypgA clade gene family comprising or consisting of an amino acid sequence of Table 1 [SEQ ID NOs 2, 4, 6, 8, 10, 12, 14, and 16], or a variant thereof
  • a polynucleotide of the invention encoding ypgA clade gene polypeptide may be obtained using standard cloning and screening methods, such as those for cloning and sequencing chromosomal DNA fragments from bacte ⁇ a using cells from a bacte ⁇ um of the ypgA clade gene family as starting matenal, followed by obtaining a full length clone
  • a polynucleotide sequence of the invention such as a polynucleotide sequence given in Table 1 [SEQ ID NOs 1, 3, 5, 7, 9, 1 1, 13, and 15]
  • a library of clones of chromosomal DNA from a bacteria of the ypgA clade gene family in E coh or some other suitable host is probed with a radiolabeled o
  • each DNA sequence set out in Table 1 contains an open reading frame encoding a protein having about the number of ammo acid residues set forth m Table 1 [SEQ ID NOs 2, 4, 6, 8, 10, 12, 14, and 16] with a deduced molecular weight that can be calculated using amino acid residue molecular weight values well known to those skilled in the art
  • the present invention provides for an isolated polynucleotide comprising or consisting of (a) a polynucleotide sequence that has at least 95% identity, even more preferably at least 97-99% or exact identity to SEQ ID NOs 1, 3, 5, 7, 9, 1 1, 13, and 15 over the entire length of SEQ ID NOs 1 , 3, 5, 7, 9, 1 1 , 13, and 15, (b) a polynucleotide sequence encoding a polypeptide that has at least 95% identity, even more preferably at least 97-99% or 100% exact, to the ammo acid sequence of SEQ ID NOs 2, 4, 6, 8, 10, 12, 14, and 16 over the entire length of said amino acid sequence
  • a polynucleotide encoding a polypeptide of the present invention, including homologs and orthologs from species other than a bacte ⁇ um of the ypgA clade gene family, may be obtained by a process that comp ⁇ ses the steps of screening an approp ⁇ ate library under st ⁇
  • the invention provides a polynucleotide sequence identical over its entire length to a coding sequence (open reading frame) in Table 1 [SEQ ID NOs 1 , 3, 5, 7, 9, 1 1 , 13, and 15] 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-protein sequence
  • the polynucleotide of the invention may also comp ⁇ se at least one non-coding sequence, including for example, but not limited to, at least one non-coding 5' and 3' sequence, such as the transc ⁇ bed but non- translated sequences, termination signals (such as rho-dependent and rho-independent termination signals), ⁇ bosome binding sites, Kozak sequences, sequences that stabilize mRNA, introns, and polyadeny
  • the invention also includes a polynucleotide consisting of or comp ⁇ sing a polynucleotide of the formula X-(R ] ) m -(R 2 )-(R 3 ) n -Y wherein, at the 5' end of the molecule, X is hydrogen, a metal or a modified nucleotide residue, or together with Y defines a covalent bond, and at the 3' end of the molecule, Y is hydrogen, a metal, or a modified nucleotide residue, or together with X defines the covalent bond, each occurrence 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, and R 2 is a nucleic acid sequence or modified nucleic acid sequence of the invention, particularly a nucleic acid sequence selected from Table 1 or a modified nucleic acid sequence thereof In the polynucleotide
  • a polynucleotide of the invention is de ⁇ ved from a bacte ⁇ um of the ypgA clade gene family, 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
  • the term "polynucleotide encoding a polypeptide" as used herein encompasses polynucleotides that include a sequence encoding a polypeptide of the invention, particularly a bacterial polypeptide and more particularly a polypeptide of a ypgA clade gene having an amino acid sequence set out in Table 1 [SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, and 16].
  • 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 comprise coding and/or non-coding sequences.
  • the invention further relates to variants of the polynucleotides described herein that encode variants of a polypeptide having a deduced amino acid sequence of Table 1 [SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, and 16]. Fragments of polynucleotides of the invention may be used, for example, to synthesize full-length polynucleotides of the invention.
  • polynucleotides encoding ypgA clade gene variants that have the amino acid sequence of one of the ypgA clade gene polypeptides of Table 1 [SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, and 16 in which several, a few, 5 to 10, 1 to 5, 1 to 3, 2, 1 or no amino acid residues are substituted, modified, deleted and/or added, in any combination.
  • ypgA clade gene polypeptide of Table 1 [SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, and 16 in which several, a few, 5 to 10, 1 to 5, 1 to 3, 2, 1 or no amino acid residues are substituted, modified, deleted and/or added, in any combination.
  • silent substitutions, additions and deletions that do not alter the properties and activities of a ypgA clade gene polypeptide.
  • Preferred isolated polynucleotide embodiments also include polynucleotide fragments, such as a polynucleotide comprising a nucleic acid sequence having at least 15, 20, 30, 40, 50 or 100 contiguous nucleic acids from the polynucleotide sequence of SEQ ID NOs: 1 , 3, 5, 7, 9, 1 1, 13, and 15, or an polynucleotide comprising a nucleic acid sequence having at least 15, 20, 30, 40, 50 or 100 contiguous nucleic acids truncated or deleted from the 5' and/or 3' end of the polynucleotide sequence of SEQ ID NOs: 1 , 3, 5, 7, 9, 11 , 13, and 15.
  • polynucleotide fragments such as a polynucleotide comprising a nucleic acid sequence having at least 15, 20, 30, 40, 50 or 100 contiguous nucleic acids from the polynucleotide sequence of SEQ ID NOs: 1 , 3, 5, 7, 9, 11 , 13, and 15.
  • polynucleotides that are at least 95% or 97% identical over their entire length to a polynucleotide encoding ypgA clade gene polypeptide having an amino acid sequence set out in Table 1 [SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, and 16], and polynucleotides that are complementary to such polynucleotides.
  • polynucleotides that comprise a region that is at least 95% are especially preferred.
  • 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 a mature polypeptide encoded by a DNA of Table 1 [SEQ ID NOs 1, 3, 5, 7, 9, 11, 13, and 15]
  • polynucleotides that hyb ⁇ dize, particularly under st ⁇ ngent conditions, to ypgA clade gene polynucleotide sequences such as those polynucleotides in Table 1
  • the invention further relates to polynucleotides that hyb ⁇ dize to the polynucleotide sequences provided herein
  • the invention especially relates to polynucleotides that hyb ⁇ dize under stringent conditions to the polynucleotides desc ⁇ bed herein
  • the terms "st ⁇ ngent conditions” and “stnngent hyb ⁇ dization conditions” mean hyb ⁇ dization occur ⁇ ng only if there is at least 95% and preferably at least 97% identity between the sequences
  • a specific example of stringent hybridization conditions is overnight incubation at 42°C in a solution comprising 50% formamide, 5x SSC (150mM NaCl, 15mM t ⁇ sodium citrate), 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 hybridization
  • the invention also provides a polynucleotide consisting of or comprising a polynucleotide sequence obtained by screening an appropriate library comprising a complete gene for a polynucleotide sequence set forth in SEQ ID NOs 1, 3, 5, 7, 9, 1 1 , 13, and 15 under stringent hybridization conditions with a probe having the sequence of said polynucleotide sequence set forth in SEQ ID NOs 1, 3, 5, 7, 9, 1 1, 13, and 15, or a fragment thereof, and isolating said polynucleotide sequence Fragments useful for obtaining such a polynucleotide include, for example, probes and primers fully desc ⁇ bed elsewhere herein It is preferred that polynucleotides of the invention encoding ypgA be isolated from
  • Gram-positive bacteria of the phylogenetic tree depicted in Figure 1 It is more particularly preferred that such bacteria of the invention are bacteria of the genera Staphylococcus, Streptococcus, Enterococcus, or Bacillus It is most particularly preferred that such bacteria of the invention are bacteria of the species Staphylococcus aureus, Staphylococcus haemolyticus, Staphylococcus epidermidis, Streptococcus pneumoniae, Streptococcus pyogenes, Enterococcus faecium, Enterococcus faecalis, or Bacillus subtilis
  • FIG. 1 For example, polynucleotides of the invention encoding ypgA falling within the clade defined by node A of Figure 1 , node B of Figure 1 , node C of Figure 1 , node C of Figure 1 , node D of Figure 1 , node E of Figure 1 , node F of Figure 1 , and node G of the phylogenetic tree of Figure 1
  • each is determined using the cladistical analyses disclosed herein, in Example 1
  • the polynucleotides of the invention may be used as a hybridization probe for RNA, cDNA and genomic DNA to isolate full-length cDNAs and genomic clones encoding a ypgA clade gene and to isolate cDNA and genomic clones of other genes that have a high identity, particularly high sequence identity, to a ypgA clade gene
  • Such probes generally will comp ⁇ se 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 ypgA clade gene may be isolated by screening using a DNA sequence provided in Table 1 [SEQ ID NOs 1, 3, 5, 7, 9, 1 1, 13, and 15] 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
  • 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
  • the polynucleotides of the invention that are oligonucleotides derived from any polynucleotide or polypeptide sequence of Table 1 may be used in the processes herein as described, but preferably for PCR, to determine whether or not the polynucleotides identified herein in whole or in part are transcribed in bacteria in infected tissue It is recognized that such sequences will also have utility in 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 a mature protein plus additional amino or carboxyl-terminal amino acids, or amino acids inte ⁇ or to a mature polypeptide (when a mature form has more than one polypeptide chain, for instance) Such sequences may play a role in processing of a protein from precursor to a mature form, may allow protein transport, may lengthen or shorten protein half-life or may facilitate manipulation of a protein for assay or production, among other things As generally is the case in vivo, the additional amino acids may be processed away from a 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
  • the entire polypeptide encoded by an open reading frame is often not required for activity Accordingly, it has become routine in molecular biology to map the boundanes of the p ⁇ mary structure required for activity with N-terminal and C- terminal deletion expenments
  • expe ⁇ ments utilize exonuclease digestion or convenient rest ⁇ ction sites to cleave coding nucleic acid sequence
  • Promega (Madison, WI) sell an Erase-a-baseTM system that uses Exonuclease III designed to facilitate analysis of the deletion products (protocol available at www promega com)
  • the digested endpoints can be repaired (e g , by ligation to synthetic linkers) to the extent necessary to preserve an open reading frame
  • the nucleic acid of SEQ ID NO 1 readily provides contiguous fragments of SEQ ID NO 2 sufficient to provide an activity, such as an enzymatic, binding or antibody-inducing activity
  • 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, that is a precursor to a proprotein, having a leader sequence and one or more prosequences, that 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 de ⁇ ved 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 comp ⁇ sing expression systems Accordingly, in a further aspect, the present invention relates to expression systems that comp ⁇ se a polynucleotide or polynucleotides of the present invention, to host cells that are genetically engineered with such expression systems, and to the production of polypeptides of the invention by recombinant techniques For recombinant production of the polypeptides of the invention, 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 described 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 Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989), such as, calcium phosphate transfection, DEAE-dextran mediated transfection, transvection, microinjection, cationic lipid-mediated transfection, electroporation, transduction, scrape loading, ballistic introduction and infection.
  • Suitable hosts include, but are not limited to a (i) 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, Borrelia, Leptospira, Spirillum, Campy lobacter, Shigella, Legionella, Pseudomonas, Aeromona
  • vectors include, among others, chromosomal-, episomal- and virus-de ⁇ ved vectors, for example, vectors de ⁇ ved from bacte ⁇ al plasmids, from bacte ⁇ ophage, 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 de ⁇ ved from combinations thereof, such as those denved from plasmid and bacte ⁇ ophage genetic elements, such as cosmids and phagemids
  • the expression system constructs may comp ⁇ se 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 in a host may be used for expression in this regard
  • the approp ⁇ ate DNA sequence may be inserted into the expression system by any of a vanety of well-known and routine techniques, such as, for example, those set forth in Sambrook, et al , MOLECULAR CLONING, A LABORA TOR Y MANUAL, supra
  • approp ⁇ ate 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 lectin chromatography Most preferably, high performance liquid chromatography is employed for pu ⁇ fication Well known techniques for refolding protein may be employed to regenerate active conformation when the polypeptide is denatured during isolation and or pu ⁇ fication Diagnostic, Prognostic, Serotyping and Mutation Assays
  • This invention is also related to the use of ypgA clade gene polynucleotides and polypeptides of the invention for use as diagnostic reagents Detection of ypgA clade gene polynucleotides and/or polypeptides in 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 comp ⁇ sing the ypgA clade gene or protein, may be detected at the nucleic acid or ammo acid level by a va ⁇ ety 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 materials Polynucleotides from any of these sources,
  • an array of oligonucleotides probes comprising ypgA clade gene 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 vanety of questions in molecular genetics including gene expression, genetic linkage, and genetic va ⁇ ability (see, for example, Chee, et al , Science, 274 610 (1996))
  • the present invention relates to a diagnostic kit that comprises
  • kits (a) a polynucleotide of the present invention, preferably the nucleotide sequence of SEQ ID NOs 1, 3, 5, 7, 9, 1 1, 13, and 15, 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 NOs 2, 4, 6, 8, 10, 12, 14, and 16, or a fragment thereof, or (d) an antibody to a polypeptide of the present invention, preferably to the polypeptide of SEQ ID NOs 2, 4, 6, 8, 10, 12, 14, and 16 It will be appreciated that in 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, preferably, SEQ ID NOs 1, 3, 5, 7, 9, 11, 13, and 15, that is associated with a disease or pathogenicity will provide a diagnostic tool that can add to, or define, a diagnosis of a disease, a prognosis of a course of disease, a determination of a stage of disease, or a susceptibility to a disease, that results from under-expression, over-expression or altered expression of the polynucleotide
  • Organisms, particularly infectious organisms, carrying mutations in such polynucleotide may be detected at the polynucleotide level by a vanety of techniques, such as those descnbed elsewhere herein
  • the invention further provides a process for diagnosing, disease, preferably bacterial infections, more preferably infections caused by a bacte ⁇ um of the ypgA clade gene family, 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 NOs 1, 3, 5, 7, 9, 1 1 , 13, and 15] Increased or decreased expression of a ypgA clade gene 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 a ypgA clade gene polypeptide compared to normal control tissue samples may be used to detect the presence of an infection, for example Assay techniques
  • Polypeptides and polynucleotides of the invention may also be used to assess the binding of small molecule substrates and ligands in, for example, cells, cell-free preparations, chemical bra ⁇ es, and natural product mixtures
  • substrates and ligands may be natural substrates and ligands 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, in particular the Diseases herein mentioned It is therefore desirable to devise screening methods to identify compounds that agonize (e g , stimulate) or that antagonize (e g , inhibit) the function of the polypeptide or polynucleotide
  • the present invention provides for a method of screening compounds to identify those that agonize or that antagonize the function of a polypeptide or polynucleotide of the
  • the screening methods may simply measure the binding of a candidate compound to the polypeptide or polynucleotide, or to cells or membranes bearing the polypeptide or polynucleotide, or a fusion protein of the polypeptide by means of a label directly or indirectly associated with the candidate compound Alternatively, the screening method may involve competition with a labeled competitor Further, these screening methods may test whether the candidate compound results in a signal generated by activation or inhibition of the polypeptide or polynucleotide, using detection systems appropriate to the cells comp ⁇ sing 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, in the absence of an agonist or antagonist, by testing whether the candidate compound results in inhibition of activation of the polypeptide or
  • polypeptides and antibodies that bind to and/or interact with a polypeptide of the present invention may also be used to configure screening methods for detecting the effect of added compounds on the production of mRNA and/or polypeptide in cells
  • an ELISA assay may be constructed for measuring secreted or cell associated levels of polypeptide using monoclonal and polyclonal antibodies by standard methods known in the art This can be used to discover agents that 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 that enhance (agonist) or block (antagonist) the action of a ypgA clade gene polypeptides or polynucleotides, particularly those compounds that are bactenstatic and/or bactencidal
  • the method of screening may involve high-throughput techniques
  • a synthetic reaction mix for agonists or antagonists, a synthetic reaction mix, a cellular compartment, such as a membrane, cell envelope or cell wall, or a preparation of any thereof, comp ⁇ sing a ypgA clade gene polypeptide and a labeled substrate or gand of such polypeptide is incubated in the absence or the presence of a candidate molecule that may be a ypgA clade gene agonist or antagonist
  • the ability of the candidate molecule to agonize or antagonize the ypgA clade gene polypeptide is reflected in decreased binding of the labeled hgand or decreased production of product from such substrate Molecule
  • ligand binding and crosslinking assays in which the polypeptide is labeled with a radioactive isotope (for instance, ⁇ * I), chemically modified (for instance, biotinylated), or fused to a peptide sequence suitable for detection or purification, and incubated with a source of the putative receptor (e.g., cells, cell membranes, cell supematants, tissue extracts, bodily materials).
  • a source of the putative receptor e.g., cells, cell membranes, cell supematants, tissue extracts, bodily materials.
  • Other methods include biophysical techniques such as surface plasmon resonance and spectroscopy. These screening methods may also be used to identify agonists and antagonists of the polypeptide that compete with the binding of the polypeptide to its receptor(s), if any. Standard methods for conducting such assays are well understood in the art.
  • the fluorescence polarization value for a fluorescently-tagged molecule depends on the rotational correlation time or tumbling rate.
  • Protein complexes such as formed by a ypgA clade gene polypeptide associating with another ypgA clade gene polypeptide or other polypeptide, labeled to comprise a fluorescently-labeled molecule will have higher polarization values than a fluorescently labeled monomeric protein. It is preferred that this method be used to characterize small molecules that disrupt polypeptide complexes.
  • Fluorescence energy transfer may also be used characterize small molecules that interfere with the formation of ypgA clade gene polypeptide dimers, trimers, tetramers or higher order structures, or structures formed by a ypgA clade gene polypeptide bound to another polypeptide.
  • a ypgA clade gene 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 dimerization will inhibit fluorescence energy transfer.
  • Surface plasmon resonance can be used to monitor the effect of small molecules on a ypgA clade gene polypeptide self-association as well as an association of a ypgA clade gene polypeptide and another polypeptide or small molecule.
  • a ypgA clade gene polypeptide can be coupled to a sensor chip at low site density such that covalently bound molecules will be monomeric.
  • Solution protein can then passed over the ypgA clade gene polypeptide -coated surface and specific binding can be detected in real-time by monitoring the change in resonance angle caused by a change in local refractive index.
  • This technique can be used to characterize the effect of small molecules on kinetic rates and equilibrium binding constants for ypgA clade gene polypeptide self-association as well as an association of a ypgA clade gene polypeptide and another polypeptide or small molecule
  • a scintillation proximity assay may be used to characterize the interaction between an association of a ypgA clade gene polypeptide with another ypgA clade gene polypeptide or a different polypeptide
  • a ypgA clade gene polypeptide can be coupled to a scintillation- filled bead Addition of radiolabeled ypgA clade gene polypeptide results in binding where the radioactive source molecule is in close proximity to the scintillation fluid
  • signal is emitted upon a ypgA clade gene polypeptide binding and compounds that prevent a ypgA clade gene polypeptide self-association or an association of a ypgA clade gene polypeptide and another polypeptide or small molecule will diminish signal
  • inventions provide methods for identifying compounds that 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 ypgA clade gene agonists is a competitive assay that combines a ypgA clade gene and a potential agonist with ypgA clade gene-binding molecules, recombinant ypgA clade gene binding molecules, natural substrates or ligands, or substrate or hgand mimetics, under appropnate conditions for a competitive inhibition assay
  • a ypgA clade gene can be labeled, such as by radioactivity or a colonmetnc compound, such that the number of ypgA clade gene molecules bound to a binding molecule or converted to product can be determined accurately to assess the effectiveness of the potential antagonist
  • a polypeptide and/or polynucleotide of the present invention may also be used in a method for the structure- based design of an agonist or antagonist of the polypeptide and/or polynucleotide, by (a) determining in the first
  • the present invention provides methods of treating abnormal conditions such as, for instance, a Disease, related to an excess of, an under-expression of, an elevated activity of, or a decreased activity of a ypgA clade gene polypeptide and/or polynucleotide
  • Typical examples of such competitors include fragments of a ypgA clade gene polypeptide and/or polypeptide
  • expression of a gene encoding an endogenous ypgA clade gene polypeptide can be inhibited using expression-blocking techniques This blocking may be targeted against any step in gene expression, but is preferably targeted against transcription and/or translation
  • An example of a known technique of this sort involves the use of antisense sequences, either internally generated or separately administered (see, for example, O'Connor, J Neurochem (1991) 56 560 in OLIGODEOXYNUCLEOTIDES AS ANTISENSE INHIBITORS OF GENE EXPRESSION, CRC Press, Boca Raton, FL ( 1988))
  • oligonucleotides that form triple helices with the gene can be supplied (see, for example, Lee, et al , Nucleic Acids Res ( 1979) 6 3073, Cooney, et al , Science (1988) 241 456, Dervan, et al , Science (1991) 251 1360)
  • These ohgomers can be administered per se or the relevant o
  • the encoded protein upon expression, can be used as a target for the screening of antibacterial drugs. Additionally, the polynucleotide sequences encoding the amino terminal regions of the encoded protein or Shine-Delgarno or other translation facilitating sequences of the respective mRNA can be used to 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 bacteria, in particular gram positive and/or gram negative bacteria, to eukaryotic, preferably mammalian, extracellular matrix proteins on in-dwelling devices or to extracellular matrix proteins in wounds; to block bacterial adhesion between eukaryotic, preferably mammalian, extracellular matrix proteins and bacterial ypgA clade gene proteins that mediate tissue damage and/or; to block the normal progression of pathogenesis in infections initiated other than by the implantation of in-dwelling devices or by other surgical techniques.
  • ypgA clade gene agonists and antagonists preferably bacteristatic or bactericidal 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 bacteria infect the stomachs of over one- third of the world's population causing stomach cancer, ulcers, and gastritis (International Agency for Research on Cancer (1994) Schistosomes, Liver Flukes and Helicobacter Pylori (International Agency for Research on Cancer, Lyon, France, http://www.uicc.ch/ecp/ecp 2904.htm). Moreover, the International Agency for Research on Cancer recently recognized a cause-and-effect relationship between H. pylori and gastric adenocarcinoma, classifying the bacterium as a Group I (definite) carcinogen.
  • Prefened antimicrobial compounds of the invention should be useful in the treatment of H. pylori infection. Such treatment should decrease the advent of H. /ry/o ⁇ -induced cancers, such as gastrointestinal carcinoma. Such treatment should also prevent, inhibit and/or cure gastric ulcers and gastritis.
  • Bodily material(s) means any material derived 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, cerebrospinal fluid, semen, saliva, muscle, cartilage, organ tissue, skin, urine, stool or autopsy materials.
  • Disease(s) means any disease caused by or related to infection by a bacteria, including, for example, otitis media, conjunctivitis, pneumonia, bacteremia, meningitis, sinusitis, pleural empyema and endocarditis, and most particularly meningitis, such as, for example, infection of cerebrospinal fluid, disease, such as, infections of the upper respiratory tract (e.g., otitis media, bacterial tracheitis, acute epiglottitis, thyroiditis), lower respiratory (e.g., empyema, lung abscess), cardiac (e.g., infective endocarditis), gastrointestinal (e.g., secretory diarrhoea, splenic absces, retroperitoneal abscess), CNS (e.g., cerebral abscess), eye (e.g., blepharitis, conjunctivitis, keratitis, endophthalmitis, preseptal
  • “Host cell(s)” is a cell that has been introduced (e.g., transformed or transfected) or is capable of introduction (e.g., transformation or transfection) by an exogenous polynucleotide sequence.
  • Identity is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as the case may be, as determined by comparing the sequences.
  • identity also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences.
  • Identity can be readily calculated by known methods, including but not limited to those described 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 Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, 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, et al , Nucleic Acids Research 12(1) 387 (1984)), BLASTP, BLASTN, and FASTA (Altschul, 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, et al , J Mol Biol 215 403-410 (1990)
  • Polynucleotide embodiments further include an isolated polynucleotide comprising a polynucleotide sequence having at least a 95, 97 or 100% identity to the reference sequence of SEQ ID NOs 1 , 3, 5, 7, 9, 11 , 13, and 15, wherein said polynucleotide sequence may be identical to the reference sequence of SEQ ID NOs 1, 3, 5, 7, 9, 1 1 , 13, and 15, 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 in the reference sequence or in one or more contiguous groups within the reference sequence, and wherein said number of nucleotide alterations is determined by multiplying the total number of nucleotides
  • n n is the number of nucleotide alterations
  • x n is the total number of nucleotides in SEQ ID NOs 1 , 3, 5, 7, 9, 1 1 , 13, and 15, y is 0 95 for 95%, 0 97 for 97% or 1 00 for 100%
  • • 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
  • Alterations of a polynucleotide sequence encoding a polypeptide of SEQ ID NOs 2, 4, 6, 8, 10, 12, 14, and 16, may create nonsense, missense or frameshift mutations in this coding sequence and thereby alter the polypeptide encoded by the polynucleotide following such alterations
  • Polypeptide embodiments further include an isolated polypeptide comprising a polypeptide having at least a 95, 97 or 100% identity to a polypeptide reference sequence of SEQ ID NOs 2, 4, 6, 8, 10, 12, 14, and
  • n a is the number of amino acid alterations
  • x a is the total number of amino acids in SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, and 16
  • y is 0.95 for 95%, 0.97 for 97% or 1.00 for 100%
  • • 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 .
  • “Individual(s)” means a multicellular eukaryote, including, but not limited to, a metazoan, a mammal, an ovid, a bovid, a simian, a primate, and a human.
  • “Isolated” means altered “by the hand of man” from its natural state, i.e., if it occurs in nature, it has been changed or removed from its original environment, or both.
  • a polynucleotide or a polypeptide naturally present in a living organism is not “isolated," but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is “isolated", as the term is employed herein.
  • a polynucleotide or polypeptide that is introduced into an organism by transformation, genetic manipulation or by any other recombinant method is "isolated” even if it is still present in said organism, which organism may be living or non-living.
  • Organism(s) means a (i) 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, Borrelia, Leptospira, Spirillum, Campy lobacter, Shigella, Legionella, Pseudomonas, Aeromonas,
  • Polynucleotide(s) generally refers to any polyribonucleotide or polydeoxyribonucleotide, that may be unmodified RNA or DNA or modified RNA or DNA.
  • Polynucleotide(s) include, without limitation, single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions or single-, double- and triple-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double- stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double- stranded, or triple-stranded regions, or a mixture of single- and double- stranded regions.
  • polynucleotide refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA.
  • the strands in such regions may be from the same molecule or from different molecules.
  • the regions may include all of one or more of the molecules, but more typically involve only a region of some of the molecules.
  • One of the molecules of a triple-helical region often is an oligonucleotide.
  • the term "polynucleotide(s)” also includes DNAs or RNAs as described above that comprise one or more modified bases. Thus, DNAs or RNAs with backbones modified for stability or for other reasons are "polynucleotide(s)" as that term is intended herein.
  • DNAs or RNAs comprising unusual bases, such as inosine, or modified bases, such as tritylated bases, to name just two examples are polynucleotides as the term is used herein. It will be appreciated that a great variety of modifications have been made to DNA and RNA that serve many useful purposes known to those of skill in the art.
  • polynucleotide(s) as it is employed herein embraces such chemically, enzymatically or metabolically modified forms of polynucleotides, as well as the chemical forms of DNA and RNA characteristic of viruses and cells, including, for example, simple and complex cells.
  • Polynucleotide(s) also embraces short polynucleotides often referred to as oligonucleotide(s).
  • Polypeptide(s) refers to any peptide or protein comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds.
  • Polypeptide(s) refers to both short chains, commonly referred to as peptides, oligopeptides and ohgomers and to longer chains generally referred to as proteins. Polypeptides may comprise amino acids other than the 20 gene encoded amino acids.
  • Polypeptide(s) include those modified either by natural processes, such as processing and other post-translational modifications, but also by chemical modification techniques.
  • Modifications include, for example, acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP-ribosylation, selenoylation
  • 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
  • Va ⁇ ant(s) is a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide respectively, but retains essential properties
  • a typical variant of a polynucleotide differs in nucleotide sequence from another, reference polynucleotide Changes in the nucleotide sequence of the variant may or may not alter the ammo acid sequence of a polypeptide encoded by the reference polynucleotide Nucleotide changes may result in amino acid substitutions, additions, deletions, fusion proteins and truncations in the polypeptide encoded by the reference sequence, as discussed below
  • a typical variant of a polypeptide differs in amino acid sequence from another, reference polypeptide Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, in many regions, identical
  • a variant and reference polypeptide may differ in amino acid sequence by one or more substitutions, additions, deletions in any
  • Phylogenetic trees were constructed by neighbor-joining (N-J) and maximum parsimony (MP) methods for each set of alignments. N-J trees were based on pairwise distances between amino acid sequences using the programs NEIGHBOR and PROTDIST of the PHYLIP 3.57c package (Felsenstein, J. 1993. Distributed by the author: http devolution . genetics.washington.edu/phylip.html, Department of Genetics, University of Washington, Seattle.) The "Dayhoff ' program option was invoked in the latter program which estimates the expected amino acid replacements per position (EAARP) using a replacement model based on the Dayhoff 120 matrix.
  • EAARP expected amino acid replacements per position

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Abstract

The invention provides ypgA clade polypeptides and polynucleotides encoding ypgA clade polypeptides and methods for producing such polypeptides by recombinant techniques. Also provided are methods for utilizing ypgA clade polypeptides to screen for antibacterial compounds.

Description

ypgA CLADE GENES
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims priority to the earlier provisional U.S. application, Serial No. 60/156,176, which was filed on September 27, 1999, the contents of which are herein incorporated by reference in their entirety. FIELD OF THE INVENTION
This invention relates to newly identified polynucleotides and polypeptides, and their production and uses, as well as their variants, agonists and antagonists, and their uses. In particular, the invention relates to polynucleotides and polypeptides of the ypgA clade family, as well as their variants, herein referred to as "ypgA clade genes," " ypgA clade gene polynucleotide(s)," and "ypgA clade gene polypeptide(s)," as the case may be. BACKGROUND OF THE INVENTION
In the species of streptococci and enterococci, examined, ypgA is found downstream of the gene encoding phosphomevalonate kinase and is part of the mevalonate kinase/decarboxylase operon. It is not found at this position in species of staphylococci, although a homologue is present in the genomes of the species examined. A ypgA homologue is also present in the ypgA clade in Borrelia b rgdoferi. No homologues were identified in eukaroytic organisms. Analysis of the amino-acid sequence for conserved motifs suggests that ypgA shows some distant homology to inosine 5'-monophosphate
(IMP) dehyrogenase and other NAD+ dependent enzymes. Accordingly, ypgA is a member of the ypgA clade gene family.
Moreover, the drug discovery process is currently undergoing a fundamental revolution as it embraces "functional genomics," that is, high throughput genome- or gene-based biology. This approach is rapidly superseding earlier approaches based on "positional cloning" and other methods. Functional genomics relies heavily on the various tools of bioinformatics to identify gene sequences of potential interest from the many molecular biology databases now available as well as from other sources. There is a continuing and significant need to identify and characterize further genes and other polynucleotides sequences and their related polypeptides, as targets for drug discovery.
Clearly, there exists a need for polynucleotides and polypeptides, such as the ypgA clade gene 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 There is also a need for identification and charactenzation of such factors and their antagonists and agonists to find ways to prevent, ameliorate or correct such infection, dysfunction and disease SUMMARY OF THE INVENTION The present invention relates to ypgA clade pathway genes, in particular ypgA polypeptides and polynucleotides, recombinant matenals and methods for their production In another aspect, the invention relates to methods for using such polypeptides and polynucleotides, including treatment of microbial diseases, amongst others In a further aspect, 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 In a still further aspect, the invention relates to diagnostic assays for detecting diseases associated with microbial infections and conditions associated with such infections, such as assays for detecting ypgA clade gene expression or activity Vanous changes and modifications within the spint and scope of the disclosed invention will become readily apparent to those skilled in the art from reading the following descπptions and from reading the other parts of the present disclosure DESCRIPTION OF THE INVENTION
The invention relates to ypgA clade gene polypeptides and polynucleotides as descπbed in greater detail below In particular, the invention relates to polypeptides and polynucleotides of ypgA clade genes that are related by amino acid sequence homology to ypgA clade polypeptides from other species The invention relates especially to ypgA clade genes having a high degree of homology to the nucleotide and amino acid sequences set out in Table 1 as SEQ ID NOs 1-16 As used herein, the "ypgA clade gene family" of the invention means a set of genes encoding a set of polypeptides from Gram-positive bacteria falling within nodes A, B, C, D, E, F, and G of the phylogenetic tree depicted in Figure 1 comprising the genera Staphvlococcus, Streptococcus, and Enterococcus
As used herein, "ypgA clade gene(s)" refers to a gene of the "ypgA clade pathway gene family", defined above "YpgA clade pathway gene polynucleotιde(s)" and "ypgA clade gene polypeptιde(s)" means, respectively, a polynucleotide of the invention or polypeptide of the invention, as more particularly set forth elsewhere herein Preferably, the invention provides a set of genes encoding a set of polypeptides involved from Gram-positive bacteria falling within the clade defined by: node A of Figure 1 ; node B of Figure 1 ; node C of Figure 1 ; node D of Figure 1 ; node E of Figure 1 ; node F of Figure 1 ; and node G of the phylogenetic tree depicted in Figure 1. Still more preferably, the invention provides a set of genes encoding ypgA isolated from bacteria falling within the clade of Gram-positive bacteria of the phylogenetic tree depicted in Figure 1 comprising the species Staphylococcus aureus, Staphylococcus haemolyticus, Staphylococcus epidermidis, Streptococcus pneumoniae, Streptococcus pyogenes, Enterococcus faecium, Enter ococcus faecalis, and Bacillus subtilis. The 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 ribopolynucleotides.
TABLE 1
YpgA clade Genes — Polynucleotide and Polypeptide Sequences
(1) Staphylococcus epidermidis ypgA polynucleotide sequence [SEQ ID NO: l].
5 ' -ATGAGTGACTCACAAAGAGAACAGAGGAAAAATGAACATGTAGAAATCGC
AATGTCACAAAAAGATGCGCTGGTTTCAGATTTTGATAAAGTGAGATTTG TTCATCATTCCATCCCCAGTATTGATGTTAGTCAAGTCGATATGACAAGT
CATACTACGAAATTCGATTTGGCATATCCAATCTATATAAATGCAATGAC
TGGTGGAAGTGATTGGACAAAACAAATTAATGAAAAATTAGCAATTGTTG
CTAGAGAAACTGGAATTGCAATGGCGGTGGGATCAACACATGCAGCTTTG
CGCAATCCTAATATGATTGAAACATTTAGCATTGTGCGTAAAACAAATCC CAAAGGAACAATTTTCAGCAATGTGGGTGCCGATGTACCAGTGGATAAAG
CTCTACAAGCGGTTGAATTATTAGATGCTCAAGCGCTACAAATTCATGTG
AACTCACCTCAAGAATTAGTCATGCCTGAAGGGAACCGTGAATTTGCTTC
ATGGATGTCAAATATTGAATCTATTGTTAAACGCGTTGATGTTCCAGTTA
TTATTAAAGAAGTTGGTTTCGGAATGAGTAAAGAGACATTACAAGCGTTA TATGATATTGGTGTTAACTATGTTGATGTCAGTGGGCGCGGTGGAACTAA
TTTCGTTGATATTGAAAATGAAAGACGTTCGAATAAAGATATGAATTATT
TATCTCAGTGGGGACAATCTACCGTAGAATCCTTACTTGAGAGTACTGAA
TTTCAAGATCGATTAAATATTTTTGCTAGCGGTGGCTTACGTACACCACT
CGATGCTGTAAAATGTTTAGCATTAGGTGCAAAAGCAATAGGGATGTCTC GACCGTTTTTAAATCAAGTAGAACAATCAGGTATCACAAATACCGTAGAC
TATGTAGAGTCTTTTATTCAACATATGAAAAAAATTATGACGATGTTAGA
TGCGCCGAACATTGAGCGTTTACGACAAGCAGATATCGTAATGAGCCCGG
AGTTAATATCATGGATCAATCAACGTGGCCTTCATTTAAATAGAAAATAA-3 ' (2) Staphylococcus epidermidis ypgA polypeptide sequence deduced from a polynucleotide sequence in this table [SEQ ID NO:2].
NH2-MSDSQREQRKNEHVEIAMSQKDALVSDFDKVRFVHHSIPSIDVSQVDMTS HTTKFDLAYPIYINAMTGGSDWTKQINEK AIVARETGIAMAVGSTHAAL RNPNMIETFSIVRKTNPKGTIFSNVGADVPVDKALQAVE LDAQALQIHV NSPQELVMPEGNREFAS MSNIESIVKRVDVPVIIKEVGFGMSKETLQAL YDIGWYVDVSGRGGTNFVDIENERRSNKDMNYLSQWGQSTVES LESTE FQDRLNIFASGGLRTPLDAVKCLALGAKAIGMSRPFLNQVEQSGITNTVD YVESFIQHMKKIMTMLDAPNIER RQADIVMSPELIS INQRGLHLNRK-COOH
(3) Staphylococcus haemolyticus ypgA polynucleotide sequence [SEQ ID NO:3].
5 ' -ATGAGTGACTTTCAAAGAGAACAGAGGAAAAATGAACATGTTGAAATAGC AATGGCTCAAAGTGATGCACCTCAATCAGACTTTGATCGAGTAAGATTCG TGCATCATTCAATCCCTAACATAAATGTAGATGAAGTAGACTTAACAAGT CGAACAACAGATTTTGATATGACATATCCAATTTATATCAATGCGATGAC AGGCGGCAGTGAATGGACAAAACAAATTAATGCGAAACTTGCAGTTGTCG CTAGAGAAACTGGATTAGCAATGGCTGTAGGCTCTACACATGCCGCTTTG AGAAATCCTAAAATGGCGGAATCATTCAGTATTGCACGTCAAACCAACCC AGAAGGTATAATTTTTAGTAATGTTGGTGCCGATGTTCCTGTAGATAAAG CCGTAGAAGCGGTTAGTTTATTAGATGCACAGGCTTTACAAATTCATGTG AATGCACCACAAGAACTTGTGATGCCTGAAGGAAATCGTGAATTCTCGAC TTGGTTAGATAATGTAGCAGCTATTGTTCAACGTGTTGATGTTCCAGTTA TTATTAAAGAAGTTGGCTTCGGCATGAGCAAAGAACTATATAAAGATTTA ATTGATGTTGGTGTTACATACGTTGATGTAAGTGGTAAAGGTGGAACAAA CTTTGTCACAATTGAAAATGAGCGTCGTTCAAATAAAGATATGGATTATC TTGCAAATTGGGGACAGTCAACTGTAGAGTCATTGCTTGAAAGTTCTGCT TATCAAGATTCACTTAATGTCTTTGCTAGTGGTGGTGTACGTACTCCTCT AGATGTTGTTAAGAGCCTAGCATTAGGTGCTAAAGCTGTAGGTATGTCTC GACCATTTTTAAATCAAGTTGAAAATGGGGGCATTACGACAACGATTGAG TATGTTGAGTCATTTATTGAACATACGAAATCAATTATGACAATGTTAAA TGCACGAGATATTAGTGAATTAAAACAAAGTAAGTTCGTTTTCGACCATA AGTTAATGTCATGGATTGAACAACGTGGTTTAGACATTCATAGAGGGTAA-3 '
(4) Staphylococcus haemolyticus ypgA polypeptide sequence deduced from a polynucleotide sequence in this table [SEQ ID NO:4].
NH2-MSDFQREQRKNEHVEIAMAQSDAPQSDFDRVRFVHHSIPNINVDEVDLTS RTTDFDMTYPIYINAMTGGSEWTKQINAKLAWARETGLAMAVGSTHAAL RNPKMAESFSIARQTNPEGIIFSNVGADVPVDKAVEAVSLLDAQALQIHV NAPQELV PEGNREFSTWLDNVAAIVQRVDVPVIIKEVGFGMSKE YKDL IDVGVTYVDVSGKGGTNFVTIENERRSNKDMDYLANWGQSTVESLLESSA YQDSLNVFASGGVRTPLDWKSLALGAKAVGMSRPFLNQVENGGITTTIE YVESFI EHTKSIMTMLNARDI SELKQSKFVFDHKLMSWI EQRGLDIHRG - COOH
(5) Stapyhlococcus aureus ypgA polynucleotide sequence [SEQ ID NO:5]. 5 ' - ATGAGTGATTTTCAAAGAGAACAGAGAAAAAATGAACATGTTGAAATAGC AATGGCCCAATCTGACGCAATGCATTCAGATTTTGATAAGATGCGTTTTG TGCATCATTCTATCCCATCAATTAATGTAAATGATATCGATTTGACATCA CAGACACCTGACTTAACGATGGCATATCCGGTTTATATTAATGCAATGAC GGGTGGTAGCGAGTGGACGAAAAACATCAATGAAAAGCTAGCTGTAGTTG C AGAGAAACTGGCTTAGCGATGGCAGTTGGATCAACACATGCGGCATTG AGAAATCCACGCATGGCTGAGACGTTTACGATTGCGCGAAAAATGAATCC TGAAGGCATGATTTTTAGCAATGTTGGTGCGGACGTACCAGTAGAAAAGG CTTTGGAAGCAGTTGAATTACTTGAGGCACAAGCGTTACAAATCCATGTT AATTCTCCTCAAGAATTAGTTATGCCTGAAGGGAATCGTGAATTTGTGAC TTGGTTAGATAATATAGCGTCGATTGTATCACGAGTGTCTGTTCCAGTCA TTATAAAAGAAGTTGGATTTGGTATGAGCAAAGAATTAATGCATGACTTA CAACAAATAGGCGTTAAGTATGTCGATGTTAGTGGCAAAGGTGGTACTAA CTTTGTAGATATTGAAAATGAACGTCGTGCAAATAAAGATATGGATTACT TATCATCATGGGGACAGTCTACAGTTGAGTCATTACTTGAAACAACGGCT TATCAAAGCGAAATTTCAGTTTTCGCGAGTGGTGGTTTACGTACACCACT CGATGCAATTAAAAGTTTAGCACTTGGCGCAAAGGCAACTGGTATGTCAC GTCCGTTTTTAAATCAAGTTGAAAATAATGGCATTGCACATACAGTAGCT TATGTAGAATCATTTATTGAACACATGAAATCAATAATGACGATGTTAGA TGCTAAAAATATTGACGACTTAACACAAAAACAAATCGTATTTAGTCCGG AAATATTGTCATGGATAGAACAACGTAGCTTGAATA ACATCGAGGATAA-3 '
(6) Stapyhlococcus aureus ypgA polypeptide sequence deduced from a polynucleotide sequence in this table [SEQ ID NO:6].
NH2 -MSDFQREQRKNEHVEIAMAQSDAMHSDFDKMRFVHHSIPSINVNDIDLTS QTPDLTMAYPVYINAMTGGSEWTKNINEKLAWARETGLAMAVGSTHAAL RNPRMAETFTIARKMNPEGMIFSNVGADVPVEKALEAVELLEAQALQIHV NSPQELVMPEGNREFVTWLDNIASIVΞRVSVPVI IKEVGFGMSKELMHDL QQ I GVKYVDVSGKGGTNF VD I ENERRANKDMDYL S S GQ S TVE S L LETTA YQSEISVFASGGLRTPLDAIKSLALGAKATGMSRPFLNQVENNGIAHTVA YVESFIEHMKSIMTMLDAKNIDDLTQKQIVFSPEILSWIEQRSLNIHRG-COOH
(7) Enterococcus faecium ypgA polynucleotide sequence [SEQ ID NO:7].
5 ' -ATGAATCGAAAAGACGAACATGTTTCACTAGCTAAAGCCTTTCATGATAA ACAAAAAAACGAATTTGACTTCGTACGGATCATCCATAACCCTTTGCCGC AAATAGCAGTTTCTGATGTCGATCTAAGTACACAGGCAGTTGGGTTTACA CTAAGCAGCCCATTTTATATCAATGCGATGACTGGCGGAAGCGAAAAAAC AAAAAAAATCAACCAAGATCTTGCAATCGTCGCAAGAGAAGCAGATTTGA TGATTGCCACTGGTTCAGTCAGCGCTGCCTTGAAAGACCCTTCTTTAGCC GATACTTATACGATTATGCGACAAGAATATCCTCACGGGAAAATCATTGC CAACATCGGTGCAGGGACTTCTGTCGAAAGAGCCCAAGAAGCTATCCGAT TGTTTCACGCAGATGCCTTACAGATTCATTTGAATGCACCTCAAGAGTTG GTTATGCCTGAGGGAGACCGTGATTTTACCAATTGGAAAGTACTTATTCA AGAGACTCAAACAGCCATTGACGTCCCCCTCATCGTAAAAGAAGTCGGTT TTGGCATGACAAGAGAAACGCTCAATGATCTAGCTTCTTTAGGAGTTCAT ACAGTTGATATCAGCGGTCGAAGCGGAACAAGCTTCACCCAGATCGAAAA CGCCCGTCGCTCCAAGCGGGAACTGAGTTATTTAGCTGACTGGGGGCAAT CGACAGTCTCTTCATTGCTTGAAGCAAATGAAGCAGACACTTCTATGGAA ATCCTAGCCTCCGGAGGTATCCGCAATGCCTATGACATATTTAAAGCCCT TTGTCTCGGCGCAAATGCAGTGGGTACTTCAGGAACCGTACTGACGCATC TGATGAATCATGGCGTAGAAGAAACTATTATACTGATGAAGCAATGGCAA GAAGAGCTTCGTCTGCTTTATACTATGGTCGGCGCAACGAATACCGCGGC CCTACACCAACACTCTCTGATCTTTTCCGGACCAGTGAAGGATTGGTGCG AAGCAAGAGGGATCGACTTAGTGAAGTATGGAAGACGCACAGAAAAGAGC GTGGGACAAAAGTAA-3 '
(8) Enterococcus faecium ypgA polypeptide sequence deduced from a polynucleotide sequence in this table [SEQ ID NO: 8].
NH2-MNRKDEHVSLAKAFHDKQKNEFDFVRIIHNPLPQIAVSDVDLSTQAVGFT LSSPFYINAMTGGSEKTKKINQDLAIVAREADLMIATGSVSAALKDPSLA DTYTIMRQEYPHGKIIANIGAGTSVERAQEAIRLFHADALQIHLNAPQEL VMPEGDRDFTNWKVLIQETQTAIDVPLIVKEVGFGMTRETLNDLASLGVH TVDISGRSGTSFTQIENARRSKRELSYLADWGQSTVSSLLEANEADTSME ILASGGIRNAYDIFKALCLGANAVGTSGTVLTHLMNHGVEETIILMKQWQ EELRLLYTMVGATNTAALHQHSLIFSGPVKDWCEARGIDLVKYGRRTEKS VGQK-COOH
(9) Enterococcus faecalis ypgA polynucleotide sequence [SEQ ID NO:9].
5 ' -ATGAATCGAAAAGACGAACATGTTTCACTAGCTAAAGCCTTTCATGATAA
ACAAAAAAACGAATTTGACTTCGTACGGATCATCCATAACCCTTTGCCGC
AAATAGCAGTTTCTGATGTCGATCTAAGTACACAGGCAGTTGGGTTTACA CTAAGCAGCCCATTTTATATCAATGCGATGACTGGCGGAAGCGAAAAAAC AAAAAAAATCAACCAAGATCTTGCAATCGTCGCAAGAGAAGCAGATTTGA TGATTGCCACTGGTTCAGTCAGCGCTGCCTTGAAAGACCCTTCTTTAGCC GATACTTATACGATTATGCGACAAGAATATCCTCACGGGAAAATCATTGC CAACATCGGTGCAGGGACTTCTGTCGAAAGAGCCCAAGAAGCTATCCGAT TGTTTCACGCAGATGCCTTACAGATTCATTTGAATGCACCTCAAGAGTTG GTTATGCCTGAGGGAGACCGTGATTTTACCAATTGGAAAGTACTTATTCA AGAGACTCAAACAGCCATTGACGTCCCCCTCATCGTAAAAGAAGTCGGTT TTGGCATGACAAGAGAAACGCTCAATGATCTAGCTTCTTTAGGAGTTCAT ACAGTTGATATCAGCGGTCGAAGCGGAACAAGCTTCACCCAGATCGAAAA CGCCCGTCGCTCCAAGCGGGAACTGAGTTATTTAGCTGACTGGGGGCAAT CGACAGTCTCTTCATTGCTTGAAGCAAATGAAGCAGACACTTCTATGGAA ATCCTAGCCTCCGGAGGTATCCGCAATGCCTATGACATATTTAAAGCCCT TTGTCTCGGCGCAAATGCAGTGGGTACTTCAGGAACCGTACTGACGCATC TGATGAATCATGGCGTAGAAGAAACTATTATACTGATGAAGCAATGGCAA GAAGAGCTTCGTCTGCTTTATACTATGGTCGGCGCAACGAATACCGCGGC CCTACACCAACACTCTCTGATCTTTTCCGGACCAGTGAAGGATTGGTGCG AAGCAAGAGGGATCGACTTAGTGAAGTATGGAAGACGCACAGAAAAGAGC GTGGGACAAAAGTAA-3 '
(10) Enterococcus faecalis ypgA polypeptide sequence deduced from a polynucleotide sequence in this table [SEQ ID NO: 10]. NH2-MNRKDEHLSLAKAFHKEKSNDFDRVRFVHQSFAESAVNEVDISTSFLSFQ LPQPFYVNAMTGGSQRAKEINQQLGIIAKETGLLVATGSVSAALKDASLA DTYQIMRKENPDGLIFANIGAGLGVEEAKRALDLFQANALQIHVNVPQEL VMPEGDRDFTNWLTKIEAIVQAVEVPVIVKEVGFGMSQETLEKLTSIGVQ AVDVSGQGGTSFTQIENARRKKRELSFLDDWGQSTVISLLESQNWQKKLT ILGSGGVRNSLDIVKGLALGAKSMGVAGTILASLMSKNGLENTLALVQQW
QEEVKMLYTLLGKKTTEELTSTALILDPVLVNWCHNRGIDSTVFAKR -COOH
(1 1) Streptococcus pneumoniae ypgA polynucleotide sequence [SEQ ID NO: l 1].
5 ' -ATGACGACAAATCGTAAGGACGAGCATATCCTCTATGCCCTTGAGCAG AAAAGTTCCTATAATAGCTTTGATGAGGTGGAGCTGATTCATTCTTCCTTG CCTCTTTACAATCTGGATGAAATCGATCTTTCGACAGAGTTTGCTGGTCGA AAGTGGGACTTTCCTTTTTATATCAATGCCATGACTGGTGGAAGTAATAAG GGAAGAGAAATCAATCAAAAGCTGGCTCAGGTGGCGGAATCCTGTGGTATT TTATTTGTAACGGGTTCTTATAGCGCAGCCCTCAAAAATCCAACGGATGAT TCTTTTTCTGTCAAGTCTAGTCATCCCAATCTCCTCCTTGGAACCAATATT GGATTGGACAAGCCTGTCGAGTTAGGACTTCAGACTGTAGAAGAGATGAAT CCTGTTCTATTGCAAGTGCATGTCAATGTCATGCAGGAATTACTCATGCCC GAGGGAGAAAGGAAGTTTAGAAGCTGGCAATCGCATCTAGCAGATTATATC AAGCAAATTCCCGTTCCTATTGTCCTCAAGGAAGTGGGCTTTGGAATGGAT GCCAAGACAATCGAAAGAGCCTATGAATTCGGTGTTCGTACAGTGGACCTA TCGGGTCGTGGTGGCACCAGCTTTGCCTATATCGAAAACCGTCGTAGTGGC CAGCGTGATTACCTCAATCAATGGGGTCAGTCTACCATGCAGGCCCTTCTC AATGCCCAAGAATGGAAAGATAAGGTCGAACTCTTGGTTAGTGGAGGGGTT CGGAATCCGCTGGATATGATTAAGTGCTTGGTTTTTGGTGCTAAGGCTGTG GGATTGTCACGAACCGTTCTGGAATTGGTTGAAACCTACACAGTTGAAGAA GTGATTGGCATTGTCCAAGGCTGGAAAGCAGATCTACGCTTGATTATGTGT TCCCTTAACTGTGCCACCATAGCAGATCTACAAAAAGTAGACTATCTTCTT TATGGAAAATTAAAAGAAGCAAAGGATCAGATGAAAAAGGCGTAA -3 '
(12) Streptococcus pneumoniae ypgA polypeptide sequence deduced from a polynucleotide sequence in this table [SEQ ID NO: 12]. NH2-MTTNRKDEHILYALEQKSSYNSFDEVELIHSSLPLYNLDEIDLSTEFAGR KWDFPFYINAMTGGSNKGREINQKLAQVAESCGILFVTGSYSAALKNPTD DSFSVKSSHPNLLLGTNIGLDKPVELGLQTVEEMNPVLLQVHVNVMQELL MPEGERKFRSWQSHLADYIKQIPVPIVLKEVGFGMDAKTIERAYEFGVRT VDLSGRGGTSFAYIENRRSGQRDYLNQWGQSTMQALLNAQEWKDKVELLV SGGVRNPLDMIKCLVFGAKAVGLSRTVLELVETYTVEEVIGIVQGWKADL RLIMCSLNCATIADLQKVDYLLYGKLKEAKDQMKKA-COOH
(13) Steptococcus pyogenes ypgA polynucleotide sequence [SEQ ID NO: 13].
5 ' -ATGACTAACCGTAAAGATGATCACATCAAATATGCTCTCAAGTACCAATC GCCTTATAATGCTTTTGATGACATAGAACTCATACACCATTCCTTACCTA GCTATGATTTGTCTGATATTGATCTCAGTACTCATTTTGCTGGGCAAGAC TTCGACTTTCCCTTTTACATCAATGCCATGACAGGAGGAAGTCAAAAAGG CAAAGCTGTCAATGAAAAATTGGCCAAAGTAGCAGCAGCAACAGGGATTG TCATGGTGACAGGGTCTTATAGCGCTGCTTTAAAAAATCCTAACGACGAT TCCTATCGTTTACATGAGGTGGCAGATAACTTGAAACTAGCCACGAATAT TGGTCTAGATAAACCTGTGGCGCTAGGACAACAAACGGTTCAAGAAATGC AGCCCCTCTTTTTACAGGTTCATGTGAATGTGATGCAAGAGTTGCTGATG CCAGAGGGTGAGCGCGTCTTTCATACCTGGAAAAAACACCTCGCTGAATA CGCTAGTCAAATACCAGTTCCTGTCATTCTCAAAGAAGTTGGTTTTGGCA TGGATGTCAATAGTATCAAGCTAGCACATGACCTAGGCATTCAAACCTTT GATATTTCAGGTAGAGGAGGAACTTCATTTGCTTACATTGAAAATCAAAG AGGGGGAGACCGCTCTTACTTAAACGATTGGGGACAAACCACTGTTCAGT GCTTACTGAATGCACAAGGACTGATGGACCAAGTGGAAATCTTAGCTTCG GGTGGTGTCAGACACCCCTTGGACATGATTAAGTGTTTTGTCTTAGGAGC ACGTGCAGTGGGACTCTCACGCACCGTTTTAGAATTGGTCGAAAAATACC CAACCGAGCGTGTGATTGCTATCGTTAATGGCTGGAAAGAAGAATTAAAA ATCATTATGTGTGCTCTTGACTGTAAAACTATTAAAGAATTAAAGGGAGT CGACTACTTACTATATGGACGCTTGCAGCAGGTCAATTAG-3 '
(14) Steptococcus pyogenes ypgA polypeptide sequence deduced from a polynucleotide sequence in this table [SEQ ID NO: 14].
NH2-MTNRKDDHIKYALKYQSPYNAFDDIELIHHSLPSYDLSDIDLSTHFAGQD FDFPFYINAMTGGSQKGKAVNEKLAKVAAATGIVMVTGSYSAALKNPNDD SYRLHEVADNLKLATNIGLDKPVALGQQTVQEMQPLFLQVHVNVMQELLM PEGERVFHTWKKHLAEYASQIPVPVILKEVGFGMDVNSIKLAHDLGIQTF DISGRGGTSFAYIENQRGGDRSYLNDWGQTTVQCLLNAQGLMDQVEILAS GGVRHPLDMIKCFVLGARAVGLSRTVLELVEKYPTERVIAIVNGWKEELK IIMCALDCKTIKELKGVDYLLYGRLQQVN-COOH
(15) Bacillus subtilis ypgA polynucleotide sequence [SEQ ID NO: 15]. 5 ' - GTGACTCGAGCAGAACGAAAAAGACAACACATCAATCATGCCTTGTCCAT CGGCCAGAAGCGGGAAACAGGTCTTGATGATATTACGTTTGTTCACGTCA GTCTGCCCGATCTTGCATTAGAACAAGTAGATATTTCCACAAAAATCGGC GAACTTTCAAGCAGTTCGCCGATTTTTATCAATGCAATGACTGGCGGCGG CGGAAAACTTACATATGAGATTAATAAATCGCTTGCGCGAGCGGCTTCTC AGGCTGGAATTCCCCTTGCTGTGGGATCGCAAATGTCAGCATTAAAAGAT CCATCAGAGCGTCTTTCCTATGAAATTGTTCGAAAGGAAAACCCAAACGG GCTGATTTTTGCCAACCTGGGAAGCGAGGCAACGGCTGCTCAGGCAAAGG AAGCCGTTGAGATGATTGGAGCAAACGCACTGCAGATCCACCTCAATGTG ATTCAGGAAATTGTGATGCCTGAAGGGGACAGAAGCTTTAGCGGCGCATT GAAACGCATTGAACAAATTTGCAGCCGGGTCAGTGTACCGGTCATTGTGA AAGAAGTCGGCTTCGGTATGAGCAAAGCATCAGCAGGAAAGCTGTATGAA GCTGGTGCTGCAGCTGTTGACATTGGCGGTTACGGGGGAACAAATTTCTC GAAAATCGAAAATCTCCGAAGACAGCGGCAAATCTCCTTTTTTAATTCGT GGGGCATTTCGACAGCTGCAAGTTTGGCGGAAATCCGCTCTGAGTTTCCT GCAAGCACCATGATCGCCTCTGGCGGTCTGCAAGATGCGCTTGACGTGGC AAAGGCAATTGCGCTGGGGGCCTCTTGCACCGGAATGGCAGGGCATTTTT TAAAAGCGCTGACTGACAGCGGTGAGGAAGGACTGCTTGAGGAGATTCAG CTGATCCTTGAGGAATTAAAGTTGATTATGACCGTGCTGGGTGCCAGAAC AATTGCCGATTTACAAAAGGCGCCCCTTGTGATCAAAGGTGAAACCCATC ATTGGCTCACAGAGAGAGGGGTCAATACATCAAGCTATAGTGTGCGATAA-3 '
( 16) Bacillus subtilis ypgA polypeptide sequence deduced from a polynucleotide sequence in this table [SEQ ID NO: 16].
NH2-MTRAERKRQHINHALSIGQKRETGLDDITFVHVSLPDLALEQVDISTKIG ELSSSSPIFINAMTGGGGKLTYEINKSLARAASQAGIPLAVGSQMSALKD PSERLSYEIVRKENPNGLIFANLGSEATAAQAKEAVEMIGANALQIHLNV IQEIVMPEGDRSFSGALKRIEQICSRVSVPVIVKEVGFGMSKASAGKLYE AGAAAVDIGGYGGTNFSKIENLRRQRQISFFNSWGISTAASLAEIRSEFP ASTMIASGGLQDALDVAKAIALGASCTGMAGHFLKALTDSGEEGLLEEIQ LILEELKLIMTVLGARTIADLQKAPLVIKGETHHWLTERGVNTSSYSVR-COOH
Deposited materials
A deposit comprising a Streptococcus pneumoniae 0100993 strain has been deposited with the National Collections of Industrial and Marine Bacteria Ltd. (herein "NCIMB"), 23 St Machar Drive, Aberdeen AB2 1RY, Scotland on 1 1 April 1996 and assigned deposit number 40794 The deposit was descπbed as Streptococcus pneumoniae 0100993 on deposit
On 17 April 1996, a Streptococcus pneumoniae 0100993 DNA library in E coll was similarly deposited with the NCIMB and assigned deposit number 40800 The Streptococcus pneumoniae strain deposit is referred to herein as "the deposited strain" or as "the DNA of the deposited strain "
The deposited strain compπses a full-length ypgA gene of the ypgA clade gene family The sequence of the polynucleotides compπsed in the deposited strain, as well as the amino acid sequence of any polypeptide encoded thereby, are controlling in the event of any conflict with any descπption of sequences herein
A deposit compπsmg a Staphylococcus aureus WCUH 29 strain has been deposited with the NCIMB), 23 St Machar Drive, Aberdeen AB2 1 RY, Scotland on 1 1 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 compπses a full-length ypgA gene of the ypgA clade gene family The sequence of the polynucleotides compπsed in the deposited strain, as well as the amino acid sequence of any polypeptide encoded thereby, are controlling in the event of any conflict with any descπption 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 restπction 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 § 1 12 A license may be required to make, use or sell the deposited strain, and compounds deπved therefrom, and no such license is hereby granted
In one aspect of the invention there is provided an isolated nucleic acid molecule encoding a mature polypeptide expressible by the Streptococcus pneumoniae 0100993 strain, which polypeptide is compπsed in the deposited strain Further provided by the invention are ypgA clade gene polynucleotide sequences in the deposited strain, such as DNA and RNA, and amino acid sequences encoded thereby. Also provided by the invention are mevalonate pathway gene polypeptide and polynucleotide sequences isolated from the deposited strain.
In another aspect of the invention there is provided an isolated nucleic acid molecule encoding a mature polypeptide expressible by the Staphylococcus aureus WCUH 29 strain, which polypeptide is comprised in the deposited strain. Further provided by the invention are ypgA clade gene polynucleotide sequences in the deposited strain, such as DNA and RNA, and amino acid sequences encoded thereby. Also provided by the invention are ypgA clade gene polypeptide and polynucleotide sequences isolated from the deposited strain. Polypeptides Yp A clade gene polypeptides of the invention are substantially phylogenetically related to other proteins of the ypgA clade gene family. Figure 1 shows the phylogenetic analysis of ypgA, a member of the ypgA clade gene family. Phylogenetic trees are based on the neighbor-joining (NJ) method as implemented by the program NEIGHBOR of the PHYLIP 3.57c package (Felsenstein, J. 1993. Distributed by the author: http://evolution.genetics.washington. edu/phylip.html, Department of Genetics, University of Washington, Seattle.). The method used to create this phylogenetic tree is described in detail in Example 1.
In one aspect of the invention, there are provided polypeptides of ypgA clade genes referred to herein as "ypgA clade genes" and "ypgA clade gene polypeptides" as well as biologically, diagnostically, prophylactically, clinically or therapeutically useful variants thereof, and compositions comprising the same.
Among the particularly preferred embodiments of the invention are variants of ypgA clade gene polypeptides encoded by naturally occurring alleles of a ypgA clade gene.
The present invention further provides for an isolated polypeptide that: (a) comprises or consists of an amino acid sequence that has at least 95% identity, most preferably at least 97- 99% or exact identity, to that of SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, and 16 over the entire length of said amino acid sequence; (b) a polypeptide encoded by an isolated polynucleotide comprising or consisting of a polynucleotide sequence that has at least 95% identity, even more preferably at least 97-99% or exact identity to SEQ ID NOs: l, 3, 5, 7, 9, 1 1, 13, and 15 over the entire length of said polynucleotide sequence; (c) a polypeptide encoded by an isolated polynucleotide comprising or consisting of a polynucleotide sequence encoding a polypeptide that has at least 95% identity, even more preferably at least 97-99% or exact identity, to the amino acid sequence of SEQ ID NOs 2, 4, 6, 8, 10, 12, 14, and 16 over the entire length of said amino acid sequence
The polypeptides of the invention include the polypeptides of Table 1 [SEQ ID NOs 2, 4, 6, 8, 10, 12, 14, and 16] (in particular a mature polypeptide) as well as polypeptides and fragments, particularly those that has a biological activity of a ypgA clade gene, and also those that have at least 95% identity to a polypeptide of Table 1 [SEQ ID NOs 2, 4, 6, 8, 10, 12, 14, and 16] and also include portions of such polypeptides with such portion of the polypeptide generally compπsing at least 30 amino acids and more preferably at least 50 amino acids
The invention also includes a polypeptide consisting of or compπsing a polypeptide of the formula
X-(R1)m-(R2)-(R3)n-Y wherein, at the amino terminus, X is hydrogen, a metal or any other moiety descπbed herein for modified polypeptides, and at the carboxyl terminus, Y is hydrogen, a metal or any other moiety descπbed herein for modified polypeptides, Rj and R3 are any ammo acid residue or modified amino acid residue, m is an integer between 1 and 1000 or zero, n is an integer between 1 and 1000 or zero, and R2 is an amino acid sequence of the invention, particularly an amino acid sequence selected from Table 1 or modified forms thereof In the formula above, R2 is oπented so that its amino terminal amino acid residue is at the left, covalently bound to Rj and its carboxy terminal amino acid residue is at the πght, covalently bound to R3 Any stretch of amino acid residues denoted by either Ri 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
It is most preferred that a polypeptide of the invention is deπved from a bacteπum of the ypgA clade gene family, 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 vaπant polypeptide having an amino acid sequence that is entirely the same as part but not all of any ammo acid sequence of any polypeptide of the invention As with ypgA clade gene polypeptides, fragments may be "free-standing," or comprised within 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 amino acid sequence of Table 1 [SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, and 16], or of variants thereof, such as a continuous series of residues that includes an amino- and/or carboxyl-terminal amino acid sequence. Degradation forms of the polypeptides of the invention produced by or in a host cell, particularly a bacterium of the ypgA clade gene family, are also preferred. Further preferred are fragments characterized by structural or functional attributes such as fragments that comprise alpha-helix and alpha-helix forming regions, beta-sheet and beta-sheet-forming regions, turn and turn-forming regions, coil and coil-forming regions, hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions, substrate binding region, and high antigenic index regions.
Further preferred fragments 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 NOs:2, 4, 6, 8, 10, 12, 14, and 16, 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 such amino acid sequence.
Fragments of the polypeptides of the invention may be employed for producing the corresponding full-length polypeptide by peptide synthesis; therefore, these variants may be employed as intermediates for producing the full-length polypeptides of the invention.
Polynucleotides
It is an object of the invention to provide polynucleotides that encode ypgA clade gene polypeptides, particularly polynucleotides that encode a polypeptide herein designated a ypgA clade gene.
In a particularly preferred embodiment of the invention the polynucleotide comprises a region encoding ypgA clade gene polypeptides comprising a sequence set out in Table 1 [SEQ ID NOs: l, 3, 5, 7, 9, 1 1, 13, and 15] that includes a full length gene, or a variant thereof. As provided by the invention, a full-length gene from the ypgA clade gene family is essential to the growth and/or survival of an organism that possesses it, such as a bacterium from the ypgA clade gene family. As a further aspect of the invention there are provided isolated nucleic acid molecules encoding and/or expressing ypgA clade gene polypeptides and polynucleotides, particularly ypgA clade gene polypeptides and polynucleotides, including, for example, unprocessed RNAs, ribozyme RNAs, mRNAs, cDNAs, genomic DNAs, B- and Z-DNAs. Further embodiments of the invention include biologically, diagnosttcally, prophylactically, clinically or therapeutically useful polynucleotides and polypeptides, and vaπants thereof, and compositions compπsing the same
Another aspect of the invention relates to isolated polynucleotides, including at least one full length gene, that encodes a ypgA clade gene polypeptide having a deduced amino acid sequence of Table 1 [SEQ ID NOs 2, 4, 6, 8, 10, 12, 14, and 16] and polynucleotides closely related thereto and vaπants thereof
In another particularly preferred embodiment of the invention there is a ypgA clade gene polypeptide from a bacterium of the ypgA clade gene family comprising or consisting of an amino acid sequence of Table 1 [SEQ ID NOs 2, 4, 6, 8, 10, 12, 14, and 16], or a variant thereof
Using the information provided herein, such as a polynucleotide sequence set out in Table 1 [SEQ ID NOs 1 , 3, 5, 7, 9, 1 1, 13, and 15], a polynucleotide of the invention encoding ypgA clade gene polypeptide may be obtained using standard cloning and screening methods, such as those for cloning and sequencing chromosomal DNA fragments from bacteπa using cells from a bacteπum of the ypgA clade gene family as starting matenal, followed by obtaining a full length clone For example, to obtain a polynucleotide sequence of the invention, such as a polynucleotide sequence given in Table 1 [SEQ ID NOs 1, 3, 5, 7, 9, 1 1, 13, and 15], typically a library of clones of chromosomal DNA from a bacteria of the ypgA clade gene family in E coh or some other suitable host, is probed with a radiolabeled ohgonucleotide, preferably a 17-mer or longer, deπved from a partial sequence Clones carrying DNA identical to that of the probe can then be distinguished using stringent hybridization conditions By sequencing the individual clones thus identified by hybridization with sequencing primers designed from the original polypeptide or polynucleotide sequence it is then possible to extend the polynucleotide sequence in both directions to determine a full length gene sequence Conveniently, such sequencing is performed, for example, using denatured double stranded DNA prepared from a plasmid clone Suitable techniques are described by Mamatis, T , Fπtsch, E F and Sambrook, et al , MOLECULAR CLONING, A LABORATORY MANUAL, 2nd Ed , Cold Spπng Harbor Laboratory Press, Cold Spnng Harbor, New York (1989) (see in particular Screening By Hybridization 1 90 and Sequencing Denatured Double-Stranded DNA Templates 13 70) Direct genomic DNA sequencing may also be performed to obtain a full length gene sequence Illustrative of the invention, each polynucleotide set out in Table 1 [SEQ ID NOs 1, 3, 5, 7, 9, 1 1 , 13, and 15] was discovered in a DNA library deπved from bacteπa of the ypgA clade gene family
Moreover, each DNA sequence set out in Table 1 [SEQ ID NOs 1, 3, 5, 7, 9, 1 1, 13, and 15] contains an open reading frame encoding a protein having about the number of ammo acid residues set forth m Table 1 [SEQ ID NOs 2, 4, 6, 8, 10, 12, 14, and 16] with a deduced molecular weight that can be calculated using amino acid residue molecular weight values well known to those skilled in the art
In a further aspect, the present invention provides for an isolated polynucleotide comprising or consisting of (a) a polynucleotide sequence that has at least 95% identity, even more preferably at least 97-99% or exact identity to SEQ ID NOs 1, 3, 5, 7, 9, 1 1, 13, and 15 over the entire length of SEQ ID NOs 1 , 3, 5, 7, 9, 1 1 , 13, and 15, (b) a polynucleotide sequence encoding a polypeptide that has at least 95% identity, even more preferably at least 97-99% or 100% exact, to the ammo acid sequence of SEQ ID NOs 2, 4, 6, 8, 10, 12, 14, and 16 over the entire length of said amino acid sequence A polynucleotide encoding a polypeptide of the present invention, including homologs and orthologs from species other than a bacteπum of the ypgA clade gene family, may be obtained by a process that compπses the steps of screening an appropπate library under stπngent hybπdization conditions with a labeled or detectable probe consisting of or compπsing the sequences of SEQ ID NOs 1, 3, 5, 7, 9, 1 1, 13, and 15, or a fragment thereof, and isolating a full-length gene and/or genomic clones compπsing 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 NOs 1 , 3, 5, 7, 9, 1 1 , 13, and 15] 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-protein sequence The polynucleotide of the invention may also compπse at least one non-coding sequence, including for example, but not limited to, at least one non-coding 5' and 3' sequence, such as the transcπbed but non- translated sequences, termination signals (such as rho-dependent and rho-independent termination signals), πbosome binding sites, Kozak sequences, sequences that stabilize mRNA, introns, and polyadenylation signals The polynucleotide sequence may also comprise additional coding sequence encoding additional amino acids For example, a marker sequence that facilitates puπfication of a fused polypeptide can be encoded In certain embodiments of the invention, the marker sequence is a hexa-histidme peptide, as provided in the pQE vector (Qiagen, Inc ) and descπbed in Gentz, et al , Proc Natl Acad Sci , USA 86 821-824 (1989), or an HA peptide tag (Wilson, et al , Cell 37 767 (1984)), both of that may be useful m purifying polypeptide sequence fused to them Polynucleotides of the invention also include, but are not limited to, polynucleotides compπsing a structural gene and its naturally associated sequences that control gene expression
The invention also includes a polynucleotide consisting of or compπsing a polynucleotide of the formula X-(R] )m-(R2)-(R3)n-Y wherein, at the 5' end of the molecule, X is hydrogen, a metal or a modified nucleotide residue, or together with Y defines a covalent bond, and at the 3' end of the molecule, Y is hydrogen, a metal, or a modified nucleotide residue, or together with X defines the covalent bond, each occurrence 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, and R2 is a nucleic acid sequence or modified nucleic acid sequence of the invention, particularly a nucleic acid sequence selected from Table 1 or a modified nucleic acid sequence thereof In the polynucleotide formula above, R is oriented so that its 5' end nucleic acid residue is at the left, bound to Ri and its 3' end nucleic acid residue is at the right, bound to R3 Any stretch of nucleic acid residues denoted by either Ri and/or R2, where m and/or n is greater than 1 , may be either a heteropolymer or a homopolymer, preferably a heteropolymer Where, in a preferred embodiment, X and Y together define a covalent bond, the polynucleotide of the above formula is a closed, circular polynucleotide, that can be a double-stranded polynucleotide wherein the formula shows a first strand to which the second strand is complementary In another preferred embodiment m and/or n is an integer between 1 and 1000. Other 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
It is most preferred that a polynucleotide of the invention is deπved from a bacteπum of the ypgA clade gene family, 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 The term "polynucleotide encoding a polypeptide" as used herein encompasses polynucleotides that include a sequence encoding a polypeptide of the invention, particularly a bacterial polypeptide and more particularly a polypeptide of a ypgA clade gene having an amino acid sequence set out in Table 1 [SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, and 16]. 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 comprise coding and/or non-coding sequences. The invention further relates to variants of the polynucleotides described herein that encode variants of a polypeptide having a deduced amino acid sequence of Table 1 [SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, and 16]. Fragments of polynucleotides of the invention may be used, for example, to synthesize full-length polynucleotides of the invention.
Further particularly preferred embodiments are polynucleotides encoding ypgA clade gene variants, that have the amino acid sequence of one of the ypgA clade gene polypeptides of Table 1 [SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, and 16 in which several, a few, 5 to 10, 1 to 5, 1 to 3, 2, 1 or no amino acid residues are substituted, modified, deleted and/or added, in any combination. Especially preferred among these are silent substitutions, additions and deletions that do not alter the properties and activities of a ypgA clade gene polypeptide. Preferred isolated polynucleotide embodiments also include polynucleotide fragments, such as a polynucleotide comprising a nucleic acid sequence having at least 15, 20, 30, 40, 50 or 100 contiguous nucleic acids from the polynucleotide sequence of SEQ ID NOs: 1 , 3, 5, 7, 9, 1 1, 13, and 15, or an polynucleotide comprising a nucleic acid sequence having at least 15, 20, 30, 40, 50 or 100 contiguous nucleic acids truncated or deleted from the 5' and/or 3' end of the polynucleotide sequence of SEQ ID NOs: 1 , 3, 5, 7, 9, 11 , 13, and 15.
Further preferred embodiments of the invention are polynucleotides that are at least 95% or 97% identical over their entire length to a polynucleotide encoding ypgA clade gene polypeptide having an amino acid sequence set out in Table 1 [SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, and 16], and polynucleotides that are complementary to such polynucleotides. Most highly preferred are polynucleotides that comprise a region that is 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 a mature polypeptide encoded by a DNA of Table 1 [SEQ ID NOs 1, 3, 5, 7, 9, 11, 13, and 15]
In accordance with certain preferred embodiments of this invention there are provided polynucleotides that hybπdize, particularly under stπngent conditions, to ypgA clade gene polynucleotide sequences, such as those polynucleotides in Table 1
The invention further relates to polynucleotides that hybπdize to the polynucleotide sequences provided herein In this regard, the invention especially relates to polynucleotides that hybπdize under stringent conditions to the polynucleotides descπbed herein As herein used, the terms "stπngent conditions" and "stnngent hybπdization conditions" mean hybπdization occurπng only if there is at least 95% and preferably at least 97% identity between the sequences A specific example of stringent hybridization conditions is overnight incubation at 42°C in a solution comprising 50% formamide, 5x SSC (150mM NaCl, 15mM tπsodium citrate), 50 mM sodium phosphate (pH7 6), 5x Denhardt's solution, 10% dextran sulfate, and 20 micrograms/ml of denatured, sheared salmon sperm DNA, followed by washing the hybridization support in 0 lx SSC at about 65°C Hybridization and wash conditions are well known and exemplified in Sambrook, et al , MOLECULAR CLONING A LABORATORY MANUAL, Second Edition, Cold Spring Harbor, N Y , ( 1989), particularly Chapter 1 1 therein Solution hybridization may also be used with the polynucleotide sequences provided by the invention
The invention also provides a polynucleotide consisting of or comprising a polynucleotide sequence obtained by screening an appropriate library comprising a complete gene for a polynucleotide sequence set forth in SEQ ID NOs 1, 3, 5, 7, 9, 1 1 , 13, and 15 under stringent hybridization conditions with a probe having the sequence of said polynucleotide sequence set forth in SEQ ID NOs 1, 3, 5, 7, 9, 1 1, 13, and 15, or a fragment thereof, and isolating said polynucleotide sequence Fragments useful for obtaining such a polynucleotide include, for example, probes and primers fully descπbed elsewhere herein It is preferred that polynucleotides of the invention encoding ypgA be isolated from
Gram-positive bacteria of the phylogenetic tree depicted in Figure 1 It is more particularly preferred that such bacteria of the invention are bacteria of the genera Staphylococcus, Streptococcus, Enterococcus, or Bacillus It is most particularly preferred that such bacteria of the invention are bacteria of the species Staphylococcus aureus, Staphylococcus haemolyticus, Staphylococcus epidermidis, Streptococcus pneumoniae, Streptococcus pyogenes, Enterococcus faecium, Enterococcus faecalis, or Bacillus subtilis
Further embodiments of the invention include, for example, polynucleotides of the invention encoding ypgA falling within the clade defined by node A of Figure 1 , node B of Figure 1 , node C of Figure 1 , node C of Figure 1 , node D of Figure 1 , node E of Figure 1 , node F of Figure 1 , and node G of the phylogenetic tree of Figure 1
Polynucleotides encoding any polypeptide defined by a cladistical model set forth herein are also embodiments of the invention Polypeptides defined by a cladistical model set forth herein are also embodiments of the invention
For any clade provided in the invention, it is preferred that each is determined using the cladistical analyses disclosed herein, in Example 1
As discussed elsewhere herein regarding polynucleotide assays of the invention, for instance, the polynucleotides of the invention, may be used as a hybridization probe for RNA, cDNA and genomic DNA to isolate full-length cDNAs and genomic clones encoding a ypgA clade gene and to isolate cDNA and genomic clones of other genes that have a high identity, particularly high sequence identity, to a ypgA clade gene Such probes generally will compπse at least 15 nucleotide residues or base pairs Preferably, such probes will have at least 30 nucleotide residues or base pairs and may have at least 50 nucleotide residues or base pairs Particularly preferred probes will have at least 20 nucleotide residues or base pairs and will have lee than 30 nucleotide residues or base pairs
A coding region of a ypgA clade gene may be isolated by screening using a DNA sequence provided in Table 1 [SEQ ID NOs 1, 3, 5, 7, 9, 1 1, 13, and 15] 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 hybπdizes to
There are several methods available and well known to those skilled in the art to obtain full-length DNAs, or extend short DNAs, for example those based on the method of Rapid Amplification of cDNA ends (RACE) (see, for example, Frohman, et al , PNAS USA 85 8998-9002, 1988) Recent modifications of the technique, exemplified by the Marathon™ technology (Clontech Laboratories Inc ) for example, have significantly simplified the search for longer cDNAs In the Marathon™ technology, cDNAs have been prepared from mRNA extracted from a chosen tissue and an 'adaptor' sequence ligated onto each end Nucleic acid amplification (PCR) is then carried out to amplify the "missing" 5' end of the DNA using a combination of gene specific and adaptor specific oligonucleotide primers The PCR reaction is then repeated using "nested" primers, that is, primers designed to anneal within the amplified product (typically an adaptor specific primer that anneals further 3' in the adaptor sequence and a gene specific primer that anneals further 5' in the selected gene sequence) The products of this reaction can then be analyzed by DNA sequencing and a full-length DNA constructed either by joining the product directly to the existing DNA to give a complete sequence, or carrying out a separate full-length PCR using the new sequence information for the design of the 5' primer
The polynucleotides and polypeptides of the invention may be employed, for example, as research reagents and mateπals for discovery of treatments of and diagnostics for diseases, particularly human diseases, as further discussed herein relating to polynucleotide assays The polynucleotides of the invention that are oligonucleotides derived from any polynucleotide or polypeptide sequence of Table 1 may be used in the processes herein as described, but preferably for PCR, to determine whether or not the polynucleotides identified herein in whole or in part are transcribed in bacteria in infected tissue It is recognized that such sequences will also have utility in 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 a mature protein plus additional amino or carboxyl-terminal amino acids, or amino acids inteπor to a mature polypeptide (when a mature form has more than one polypeptide chain, for instance) Such sequences may play a role in processing of a protein from precursor to a mature form, may allow protein transport, may lengthen or shorten protein half-life or may facilitate manipulation of a protein for assay or production, among other things As generally is the case in vivo, the additional amino acids may be processed away from a mature protein by cellular enzymes
For each and every polynucleotide of the invention there is provided a polynucleotide complementary to it It is preferred that these complementary polynucleotides are fully complementary to each polynucleotide with which they are complementary
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
As will be recognized, the entire polypeptide encoded by an open reading frame is often not required for activity Accordingly, it has become routine in molecular biology to map the boundanes of the pπmary structure required for activity with N-terminal and C- terminal deletion expenments These expeπments utilize exonuclease digestion or convenient restπction sites to cleave coding nucleic acid sequence For example, Promega (Madison, WI) sell an Erase-a-base™ system that uses Exonuclease III designed to facilitate analysis of the deletion products (protocol available at www promega com) The digested endpoints can be repaired (e g , by ligation to synthetic linkers) to the extent necessary to preserve an open reading frame In this way, for example, the nucleic acid of SEQ ID NO 1 readily provides contiguous fragments of SEQ ID NO 2 sufficient to provide an activity, such as an enzymatic, binding or antibody-inducing activity Nucleic acid sequences encoding such fragments of the polypeptide sequences of Table 1 and vaπants thereof as descπbed herein are within the invention, as are polypeptides so encoded
In sum, 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, that is a precursor to a proprotein, having a leader sequence and one or more prosequences, that generally are removed dunng processing steps that produce active and mature forms of the polypeptide
Vectors, Host Cells, Expression Systems
The invention also relates to vectors that compπse 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 deπved 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 compπsing expression systems Accordingly, in a further aspect, the present invention relates to expression systems that compπse a polynucleotide or polynucleotides of the present invention, to host cells that are genetically engineered with such expression systems, and to the production of polypeptides of the invention by recombinant techniques For recombinant production of the polypeptides of the invention, 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 described 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 Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989), such as, calcium phosphate transfection, DEAE-dextran mediated transfection, transvection, microinjection, cationic lipid-mediated transfection, electroporation, transduction, scrape loading, ballistic introduction and infection. Representative examples of appropriate hosts include, but are not limited to a (i) 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, Borrelia, Leptospira, Spirillum, Campy lobacter, Shigella, Legionella, Pseudomonas, Aeromonas, Rickettsia, Chlamydia, Borrelia and Mycoplasma, and further including, but not limited to, a member of the species or group, Group A Streptococcus, Group B Streptococcus, Group C Streptococcus, Group D Streptococcus, Group G Streptococcus, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus faecalis, Streptococcus faecium, Streptococcus durans, Neisseria gonorrheae, Neisseria meningitidis, Staphylococcus aureus, Staphylococcus epidermidis, Corynebacterium diptheriae, Gardnerella vaginalis, Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium ulcerans, Mycobacterium leprae, Actinomyctes israelii, Listeria monocytogenes, Bordetella pertusis, Bordatella parapertusis, Bordetella bronchiseptica, Escherichia coli, Shigella dysenteriae, Haemophilus influenzae, Haemophilus aegyptius, Haemophilus parainfluenzae, Haemophilus ducreyi, Bordetella, Salmonella typhi, Citrobacter freundii, Proteus mirabilis, Proteus vulgaris, Yersinia pestis, Kleibsiella pneumoniae, Serratia marcessens, Serratia liquefaciens, Vibrio cholera, Shigella dysenterii, Shigella flexneri, Pseudomonas aeruginosa, Franscisella tularensis, Brucella abortis, Bacillus anthracis, Bacillus cereus, Clostridium perfringens, Clostridium tetani, Clostridium botulinum, Treponema pallidum, Rickettsia rickettsii and Chlamydia trachomitis, (ii) an archaeon, including but not limited to Archaebacter, and (iii) a unicellular or filamentous eukaryote, including but not limited to, a protozoan, a fungus, a member of the genus Saccharomyces, Kluveromyces, or Candida, and a member of the species Saccharomyces ceriviseae, Kluveromyces lactis or Candida albicans, insect cells such as cells of Drosophda S2 and Spodoptera Sf9, animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, 293, CV- 1 and Bowes melanoma cells, and plant cells, such as cells of a gymnosperm or angiosperm
A great vaπety of expression systems can be used to produce the polypeptides of the invention Such vectors include, among others, chromosomal-, episomal- and virus-deπved vectors, for example, vectors deπved from bacteπal plasmids, from bacteπophage, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses such as baculoviruses, papova viruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses, picornaviruses and retroviruses, and vectors deπved from combinations thereof, such as those denved from plasmid and bacteπophage genetic elements, such as cosmids and phagemids The expression system constructs may compπse control regions that regulate as well as engender expression
Generally, any system or vector suitable to maintain, propagate or express polynucleotides and/or to express a polypeptide in a host may be used for expression in this regard The appropπate DNA sequence may be inserted into the expression system by any of a vanety of well-known and routine techniques, such as, for example, those set forth in Sambrook, et al , MOLECULAR CLONING, A LABORA TOR Y MANUAL, supra
In recombinant expression systems in eukaryotes, for secretion of a translated protein into the lumen of the endoplasmic reticulum, into the peπplasmic space or into the extracellular environment, appropπate 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 puπfied from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography, and lectin chromatography Most preferably, high performance liquid chromatography is employed for puπfication Well known techniques for refolding protein may be employed to regenerate active conformation when the polypeptide is denatured during isolation and or puπfication Diagnostic, Prognostic, Serotyping and Mutation Assays
This invention is also related to the use of ypgA clade gene polynucleotides and polypeptides of the invention for use as diagnostic reagents Detection of ypgA clade gene polynucleotides and/or polypeptides in 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 compπsing the ypgA clade gene or protein, may be detected at the nucleic acid or ammo acid level by a vaπety 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 materials Polynucleotides from any of these sources, particularly DNA or RNA, may be used directly for detection or may be amplified enzymatically by using PCR or any other amplification technique pπor to analysis RNA, particularly mRNA, cDNA and genomic DNA may also be used in the same ways Using amplification, characteπzation 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 compaπson 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 hybπdizing amplified DNA to labeled ypgA clade gene 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 denatuπng agents Polynucleotide differences may also be detected by direct DNA or RNA sequencing See, e g , 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 S 1 protection assay or a chemical cleavage method See, e g , Cotton, et al , Proc Natl Acad Sci , USA, 85 4397-4401 (1985)
In another embodiment, an array of oligonucleotides probes comprising ypgA clade gene 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 vanety of questions in molecular genetics including gene expression, genetic linkage, and genetic vaπability (see, for example, Chee, et al , Science, 274 610 (1996))
Thus in another aspect, the present invention relates to a diagnostic kit that comprises
(a) a polynucleotide of the present invention, preferably the nucleotide sequence of SEQ ID NOs 1, 3, 5, 7, 9, 1 1, 13, and 15, 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 NOs 2, 4, 6, 8, 10, 12, 14, and 16, or a fragment thereof, or (d) an antibody to a polypeptide of the present invention, preferably to the polypeptide of SEQ ID NOs 2, 4, 6, 8, 10, 12, 14, and 16 It will be appreciated that in 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, preferably, SEQ ID NOs 1, 3, 5, 7, 9, 11, 13, and 15, that is associated with a disease or pathogenicity will provide a diagnostic tool that can add to, or define, a diagnosis of a disease, a prognosis of a course of disease, a determination of a stage of disease, or a susceptibility to a disease, that results from under-expression, over-expression or altered expression of the polynucleotide Organisms, particularly infectious organisms, carrying mutations in such polynucleotide may be detected at the polynucleotide level by a vanety of techniques, such as those descnbed elsewhere herein 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 in 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 Cells from an organism carrying mutations or polymorphisms (allelic vanations) in a polynucleotide and/or polypeptide of the invention may also be detected at the polynucleotide or polypeptide level by a vanety of techniques, to allow for serotyping, for example 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 As an example, PCR pπmers complementary to a polynucleotide encoding a ypgA clade gene polypeptide can be used to identify and analyze mutations The invention further provides these pnmers with 1, 2, 3 or 4 nucleotides removed from the 5' and/or the 3' end These pπmers may be used for, among other things, amplifying ypgA clade DNA and/or RNA isolated from a sample denved from an individual, such as a bodily mateπal 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 a bacteπum of the ypgA clade gene family, 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 NOs 1, 3, 5, 7, 9, 1 1 , 13, and 15] Increased or decreased expression of a ypgA clade gene polynucleotide can be measured using any on of the methods well known in the art for the quantitation of polynucleotides, such as, for example, amplification, PCR, RT- PCR, RNase protection, Northern blotting, spectrometry and other hybridization methods In addition, a diagnostic assay in accordance with the invention for detecting over- expression of a ypgA clade gene 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 ypgA clade gene polypeptide, in a sample derived 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
Antagonists and Agonists - Assays and Molecules
Polypeptides and polynucleotides of the invention may also be used to assess the binding of small molecule substrates and ligands in, for example, cells, cell-free preparations, chemical braπes, and natural product mixtures These substrates and ligands may be natural substrates and ligands 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, in particular the Diseases herein mentioned It is therefore desirable to devise screening methods to identify compounds that agonize (e g , stimulate) or that antagonize (e g , 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 that agonize or that antagonize the function of a polypeptide or polynucleotide of the invention, as well as related polypeptides and polynucleotides In general, agonists or antagonists (e g , inhibitors) may be employed for therapeutic and prophylactic purposes for such Diseases as herein mentioned Compounds may be identified from a vanety of sources, for example, cells, cell-free preparations, chemical hbraπes, and natural product mixtures Such agonists and antagonists so-identified may be natural or modified substrates, ligands, receptors, enzymes, etc , as the case may be, of ypgA clade gene polypeptides and polynucleotides, or may be structural or functional mimetics thereof (see Coligan, et al , Current Protocols in Immunology 1(2) Chapter 5 (1991))
The screening methods may simply measure the binding of a candidate compound to the polypeptide or polynucleotide, or to cells or membranes bearing the polypeptide or polynucleotide, or a fusion protein of the polypeptide by means of a label directly or indirectly associated with the candidate compound Alternatively, the screening method may involve competition with a labeled competitor Further, these screening methods may test whether the candidate compound results in a signal generated by activation or inhibition of the polypeptide or polynucleotide, using detection systems appropriate to the cells compπsing 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, in the absence of an agonist or antagonist, by testing whether the candidate compound results in inhibition of activation of the polypeptide or polynucleotide, as the case may be Further, the screening methods may simply comprise the steps of mixing a candidate compound with a solution comprising a polypeptide or polynucleotide of the present invention, to form a mixture, measuring ypgA clade gene polypeptide and/or polynucleotide activity in the mixture, and comparing the ypgA clade gene polypeptide and/or polynucleotide activity of the mixture to a standard Fusion proteins, such as those made from Fc portion and ypgA clade gene polypeptide, as herein described, can also be used for high-throughput screening assays to identify antagonists of the polypeptide of the present invention, as well as of phylogenetically and and/or functionally related polypeptides (see Bennett, et al , J Mol Recognition, 8 52-58 (1995), and Johanson, et al , J Biol Chem , 270( 16) 9459-9471 ( 1995))
The polynucleotides, polypeptides and antibodies that bind to and/or interact with a polypeptide of the present invention may also be used to configure screening methods for detecting the effect of added compounds on the production of mRNA and/or polypeptide in cells For example, an ELISA assay may be constructed for measuring secreted or cell associated levels of polypeptide using monoclonal and polyclonal antibodies by standard methods known in the art This can be used to discover agents that 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 that enhance (agonist) or block (antagonist) the action of a ypgA clade gene polypeptides or polynucleotides, particularly those compounds that are bactenstatic 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, compπsing a ypgA clade gene polypeptide and a labeled substrate or gand of such polypeptide is incubated in the absence or the presence of a candidate molecule that may be a ypgA clade gene agonist or antagonist The ability of the candidate molecule to agonize or antagonize the ypgA clade gene 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 a ypgA clade gene polypeptide are most likely to be good antagonists Molecules that bind well and, as the case may be, increase the rate of product production from substrate, increase signal transduction, or increase chemical channel activity are agonists Detection of the rate or level of, as the case may be, production of product from substrate, signal transduction, or chemical channel activity may be enhanced by using a reporter system Reporter systems that may be useful in this regard include but are not limited to colonmetnc, labeled substrate converted into product, a reporter gene that is responsive to changes in ypgA clade gene polynucleotide or polypeptide activity, and binding assays known in the art Polypeptides of the invention may be used to identify membrane bound or soluble receptors, if any, for such polypeptide, through standard receptor binding techniques known in the art. These techniques include, but are not limited to, ligand binding and crosslinking assays in which the polypeptide is labeled with a radioactive isotope (for instance, ^*I), chemically modified (for instance, biotinylated), or fused to a peptide sequence suitable for detection or purification, and incubated with a source of the putative receptor (e.g., cells, cell membranes, cell supematants, tissue extracts, bodily materials). Other methods include biophysical techniques such as surface plasmon resonance and spectroscopy. These screening methods may also be used to identify agonists and antagonists of the polypeptide that compete with the binding of the polypeptide to its receptor(s), if any. Standard methods for conducting such assays are well understood in the art.
The fluorescence polarization value for a fluorescently-tagged molecule depends on the rotational correlation time or tumbling rate. Protein complexes, such as formed by a ypgA clade gene polypeptide associating with another ypgA clade gene polypeptide or other polypeptide, labeled to comprise a fluorescently-labeled molecule will have higher polarization values than a fluorescently labeled monomeric protein. It is preferred that this method be used to characterize small molecules that disrupt polypeptide complexes.
Fluorescence energy transfer may also be used characterize small molecules that interfere with the formation of ypgA clade gene polypeptide dimers, trimers, tetramers or higher order structures, or structures formed by a ypgA clade gene polypeptide bound to another polypeptide. A ypgA clade gene 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 dimerization will inhibit fluorescence energy transfer. Surface plasmon resonance can be used to monitor the effect of small molecules on a ypgA clade gene polypeptide self-association as well as an association of a ypgA clade gene polypeptide and another polypeptide or small molecule. A ypgA clade gene polypeptide can be coupled to a sensor chip at low site density such that covalently bound molecules will be monomeric. Solution protein can then passed over the ypgA clade gene polypeptide -coated surface and specific binding can be detected in real-time by monitoring the change in resonance angle caused by a change in local refractive index. This technique can be used to characterize the effect of small molecules on kinetic rates and equilibrium binding constants for ypgA clade gene polypeptide self-association as well as an association of a ypgA clade gene polypeptide and another polypeptide or small molecule
A scintillation proximity assay may be used to characterize the interaction between an association of a ypgA clade gene polypeptide with another ypgA clade gene polypeptide or a different polypeptide A ypgA clade gene polypeptide can be coupled to a scintillation- filled bead Addition of radiolabeled ypgA clade gene polypeptide results in binding where the radioactive source molecule is in close proximity to the scintillation fluid Thus, signal is emitted upon a ypgA clade gene polypeptide binding and compounds that prevent a ypgA clade gene polypeptide self-association or an association of a ypgA clade gene polypeptide and another polypeptide or small molecule will diminish signal
Other embodiments of the invention provide methods for identifying compounds that bind to or otherwise interact with and inhibit or activate an activity or expression of a polypeptide and/or polynucleotide of the invention compnsing contacting a polypeptide and/or polynucleotide of the invention with a compound to be screened under conditions to permit binding to or other interaction between the compound and the polypeptide and/or polynucleotide to assess the binding to or other interaction with the compound, such binding or interaction preferably being associated with a second component capable of providing a detectable signal in response to the binding or interaction of the polypeptide and/or polynucleotide with the compound, and determining whether the compound binds to or otherwise interacts with and activates or inhibits an activity or expression of the polypeptide and/or polynucleotide by detecting the presence or absence of a signal generated from the binding or interaction of the compound with the polypeptide and/or polynucleotide
Another example of an assay for ypgA clade gene agonists is a competitive assay that combines a ypgA clade gene and a potential agonist with ypgA clade gene-binding molecules, recombinant ypgA clade gene binding molecules, natural substrates or ligands, or substrate or hgand mimetics, under appropnate conditions for a competitive inhibition assay A ypgA clade gene can be labeled, such as by radioactivity or a colonmetnc compound, such that the number of ypgA clade gene molecules bound to a binding molecule or converted to product can be determined accurately to assess the effectiveness of the potential antagonist It will be readily appreciated by the skilled artisan that a polypeptide and/or polynucleotide of the present invention may also be used in a method for the structure- based design of an agonist or antagonist of the polypeptide and/or polynucleotide, by (a) determining in the first instance the three-dimensional structure of the polypeptide and/or polynucleotide, or complexes thereof, (b) deducing the three-dimensional structure for the likely reactive sιte(s), binding sιte(s) or motιf(s) of an agonist or antagonist, (c) synthesizing candidate compounds that are predicted to bind to or react with the deduced binding sιte(s), reactive sιte(s), and/or motιf(s), and (d) testing whether the candidate compounds are indeed agonists or antagonists
It will be further appreciated that this will normally be an iterative process, and this iterative process may be performed using automated and computer-controlled steps
In a further aspect, the present invention provides methods of treating abnormal conditions such as, for instance, a Disease, related to an excess of, an under-expression of, an elevated activity of, or a decreased activity of a ypgA clade gene polypeptide and/or polynucleotide
If the expression and/or activity of the polypeptide and/or polynucleotide is in excess, several approaches are available One approach compπses administering to an individual in need thereof an inhibitor compound (antagonist) as herein described, optionally in combination with a pharmaceutically acceptable earner, in an amount effective to inhibit the function and/or expression of the polypeptide and/or polynucleotide, such as, for example, by blocking the binding of ligands, substrates, receptors, enzymes, etc , or by inhibiting a second signal, and thereby alleviating the abnormal condition In another approach, soluble forms of the polypeptides still capable of binding the hgand, substrate, enzymes, receptors, etc , in competition with endogenous polypeptide and/or polynucleotide may be administered
Typical examples of such competitors include fragments of a ypgA clade gene polypeptide and/or polypeptide
In still another approach, expression of a gene encoding an endogenous ypgA clade gene polypeptide can be inhibited using expression-blocking techniques This blocking may be targeted against any step in gene expression, but is preferably targeted against transcription and/or translation An example of a known technique of this sort involves the use of antisense sequences, either internally generated or separately administered (see, for example, O'Connor, J Neurochem (1991) 56 560 in OLIGODEOXYNUCLEOTIDES AS ANTISENSE INHIBITORS OF GENE EXPRESSION, CRC Press, Boca Raton, FL ( 1988)) Alternatively, oligonucleotides that form triple helices with the gene can be supplied (see, for example, Lee, et al , Nucleic Acids Res ( 1979) 6 3073, Cooney, et al , Science (1988) 241 456, Dervan, et al , Science (1991) 251 1360) These ohgomers can be administered per se or the relevant ohgomers can be expressed in vivo Each of the polynucleotide sequences provided herein may be used in the discovery and development of antibacterial compounds. The encoded protein, upon expression, can be used as a target for the screening of antibacterial drugs. Additionally, the polynucleotide sequences encoding the 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. In particular, the molecules of the invention may be used: in the prevention of adhesion of bacteria, in particular gram positive and/or gram negative bacteria, to eukaryotic, preferably mammalian, extracellular matrix proteins on in-dwelling devices or to extracellular matrix proteins in wounds; to block bacterial adhesion between eukaryotic, preferably mammalian, extracellular matrix proteins and bacterial ypgA clade gene proteins that mediate tissue damage and/or; to block the normal progression of pathogenesis in infections initiated other than by the implantation of in-dwelling devices or by other surgical techniques.
In accordance with yet another aspect of the invention, there are provided ypgA clade gene agonists and antagonists, preferably bacteristatic or bactericidal agonists and antagonists. The antagonists and agonists of the invention may be employed, for instance, to prevent, inhibit and/or treat diseases.
Helicobacter pylori (herein "H. pylori") bacteria infect the stomachs of over one- third of the world's population causing stomach cancer, ulcers, and gastritis (International Agency for Research on Cancer (1994) Schistosomes, Liver Flukes and Helicobacter Pylori (International Agency for Research on Cancer, Lyon, France, http://www.uicc.ch/ecp/ecp 2904.htm). Moreover, the International Agency for Research on Cancer recently recognized a cause-and-effect relationship between H. pylori and gastric adenocarcinoma, classifying the bacterium as a Group I (definite) carcinogen. Prefened antimicrobial compounds of the invention (agonists and antagonists of ypgA clade gene polypeptides and/or polynucleotides) found using screens provided by the invention, or known in the art, particularly nanow-spectrum antibiotics, should be useful in the treatment of H. pylori infection. Such treatment should decrease the advent of H. /ry/oπ-induced cancers, such as gastrointestinal carcinoma. Such treatment should also prevent, inhibit and/or cure gastric ulcers and gastritis.
GLOSSARY
The following definitions are provided to facilitate understanding of certain terms used frequently herein.
"Bodily material(s)" means any material derived 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, cerebrospinal fluid, semen, saliva, muscle, cartilage, organ tissue, skin, urine, stool or autopsy materials.
"Disease(s)" means any disease caused by or related to infection by a bacteria, including, for example, otitis media, conjunctivitis, pneumonia, bacteremia, meningitis, sinusitis, pleural empyema and endocarditis, and most particularly meningitis, such as, for example, infection of cerebrospinal fluid, disease, such as, infections of the upper respiratory tract (e.g., otitis media, bacterial tracheitis, acute epiglottitis, thyroiditis), lower respiratory (e.g., empyema, lung abscess), cardiac (e.g., infective endocarditis), gastrointestinal (e.g., secretory diarrhoea, splenic absces, retroperitoneal abscess), CNS (e.g., cerebral abscess), eye (e.g., blepharitis, conjunctivitis, keratitis, endophthalmitis, preseptal and orbital cellulitis, darcryocystitis), kidney and urinary tract (e.g., epididymitis, intrarenal and perinephric absces, toxic shock syndrome), skin (e.g., impetigo, folliculitis, cutaneous abscesses, cellulitis, wound infection, bacterial myositis) bone and joint (e.g., septic arthritis, osteomyelitis).
"Host cell(s)" is a cell that has been introduced (e.g., transformed or transfected) or is capable of introduction (e.g., transformation or transfection) by an exogenous polynucleotide sequence.
"Identity," as known in the art, is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as the case may be, as determined by comparing the sequences. In the art, "identity" also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences. "Identity" can be readily calculated by known methods, including but not limited to those described 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 Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991 ; and Carillo, H„ and Lipman, D., SIAM J. Applied Math., 48: 1073 (1988). 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, et al , Nucleic Acids Research 12(1) 387 (1984)), BLASTP, BLASTN, and FASTA (Altschul, 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, 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 comparison include the following Algorithm
Needleman and Wunsch, J Mol Biol 48 443-453 (1970)
Comparison matrix BLOSSUM62 from Hentikoff and Hentikoff, Proc Natl Acad Sci USA, %9 10915-10919 (1992) Gap Penalty 12 Gap Length Penalty 4
A program useful with these parameters is publicly available as the "gap" program from Genetics Computer Group, Madison WI The aforementioned parameters are the default parameters for peptide comparisons (along with no penalty for end gaps)
Parameters for polynucleotide comparison include the following Algorithm Needleman and Wunsch, J Mol Biol 48 443-453 (1970) Comparison matrix matches = +10, mismatch = 0 Gap Penalty 50 Gap Length Penalty 3 Available as The ' gap' program from Genetics Computer Group, Madison WI These are the default parameters for nucleic acid comparisons
A prefened meaning for "identity" for polynucleotides and polypeptides, as the case may be, are provided in ( 1 ) and (2) below
(1) Polynucleotide embodiments further include an isolated polynucleotide comprising a polynucleotide sequence having at least a 95, 97 or 100% identity to the reference sequence of SEQ ID NOs 1 , 3, 5, 7, 9, 11 , 13, and 15, wherein said polynucleotide sequence may be identical to the reference sequence of SEQ ID NOs 1, 3, 5, 7, 9, 1 1 , 13, and 15, 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 in the reference sequence or in one or more contiguous groups within the reference sequence, and wherein said number of nucleotide alterations is determined by multiplying the total number of nucleotides in SEQ ID NOs 1 , 3, 5, 7, 9, 1 1 , 13, and 15, by the integer defining the percent identity divided by 100 and then subtracting that product from said total number of nucleotides in SEQ ID NOs 1, 3, 5, 7, 9, 1 1 , 13, and 15, or
nn < xn - (xn • y),
wherein nn is the number of nucleotide alterations, xn is the total number of nucleotides in SEQ ID NOs 1 , 3, 5, 7, 9, 1 1 , 13, and 15, y is 0 95 for 95%, 0 97 for 97% or 1 00 for 100%, and • is the symbol for the multiplication operator, and wherein any non-integer product of xn and y is rounded down to the nearest integer prior to subtracting it from xn Alterations of a polynucleotide sequence encoding a polypeptide of SEQ ID NOs 2, 4, 6, 8, 10, 12, 14, and 16, may create nonsense, missense or frameshift mutations in this coding sequence and thereby alter the polypeptide encoded by the polynucleotide following such alterations (2) Polypeptide embodiments further include an isolated polypeptide comprising a polypeptide having at least a 95, 97 or 100% identity to a polypeptide reference sequence of SEQ ID NOs 2, 4, 6, 8, 10, 12, 14, and 16, wherein said polypeptide sequence may be identical to a reference sequence of SEQ ID NOs 2, 4, 6, 8, 10, 12, 14, and 16, or may include up to a certain integer number of amino acid alterations as compared to the reference sequence, wherein said alterations are selected from the group consisting of at least one amino acid deletion, substitution, including conservative and non-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 in one or more contiguous groups within the reference sequence, and wherein said number of amino acid alterations is determined by multiplying the total number of amino acids in SEQ ID NOs 2, 4, 6, 8, 10, 12, 14, and 16, by the integer defining the percent identity divided by 100 and then subtracting that product from said total number of am o acids in SEQ ID NOs 2, 4, 6, 8, 10, 12, 14, and 16, or na < xa - (xa • y),
wherein na is the number of amino acid alterations, xa is the total number of amino acids in SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, and 16, y is 0.95 for 95%, 0.97 for 97% or 1.00 for 100%, and • is the symbol for the multiplication operator, and wherein any non-integer product of xa and y is rounded down to the nearest integer prior to subtracting it from xa.
"Individual(s)" means a multicellular eukaryote, including, but not limited to, a metazoan, a mammal, an ovid, a bovid, a simian, a primate, and a human. "Isolated" means altered "by the hand of man" from its natural state, i.e., if it occurs in nature, it has been changed or removed from its original environment, or both. For example, a polynucleotide or a polypeptide naturally present in a living organism is not "isolated," but the same polynucleotide or polypeptide separated from the coexisting materials 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.
"Organism(s)" means a (i) 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, Borrelia, Leptospira, Spirillum, Campy lobacter, Shigella, Legionella, Pseudomonas, Aeromonas, Rickettsia, Chlamydia, Borrelia and Mycoplasma, and further including, but not limited to, a member of the species or group, Group A Streptococcus, Group B Streptococcus, Group C Streptococcus, Group D Streptococcus, Group G Streptococcus, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus faecalis, Streptococcus faecium, Streptococcus durans, Neisseria gonorrheae, Neisseria meningitidis, Staphylococcus aureus, Staphylococcus epidermidis, Corynebacterium diptheriae, Gardnerella vaginalis, Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium ulcerans, Mycobacterium leprae, Actinomyctes israelii, Listeria monocytogenes, Bordetella pertusis, Bordatella parapertusis, Bordetella bronchiseptica, Escherichia coli, Shigella dysenteriae, Haemophilus influenzae, Haemophilus aegyptius, Haemophilus parainfluenzae, Haemophilus ducreyi, Bordetella, Salmonella typhi, Citrobacter freundii, Proteus mirabilis, Proteus vulgaris, Yersinia pestis, Kleibsiella pneumoniae, Serratia marcessens, Serratia liquefaciens, Vibrio cholera, Shigella dysenterii, Shigella flexneri, Pseudomonas aeruginosa, Franscisella tularensis, Brucella abortis, Bacillus anthracis, Bacillus cereus, Clostridium perfringens, Clostridium tetani, Clostridium botulinum, Treponema pallidum, Rickettsia rickettsii and Chlamydia trachomitis, (ii) an archaeon, including but not limited to Archaebacter, and (iii) a unicellular or filamentous eukaryote, including but not limited to, a protozoan, a fungus, a member of the genus Saccharomyces, Kluveromyces, or Candida, and a member of the species Saccharomyces ceriviseae, Kluveromyces lactis, or Candida albicans.
"Polynucleotide(s)" generally refers to any polyribonucleotide or polydeoxyribonucleotide, that may be unmodified RNA or DNA or modified RNA or DNA. "Polynucleotide(s)" include, without limitation, single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions or single-, double- and triple-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double- stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double- stranded, or triple-stranded regions, or a mixture of single- and double- stranded regions. In addition, "polynucleotide" as used herein refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA. The strands in such regions may be from the same molecule or from different molecules. The regions may include all of one or more of the molecules, but more typically involve only a region of some of the molecules. One of the molecules of a triple-helical region often is an oligonucleotide. As used herein, the term "polynucleotide(s)" also includes DNAs or RNAs as described above that comprise one or more modified bases. Thus, DNAs or RNAs with backbones modified for stability or for other reasons are "polynucleotide(s)" as that term is intended herein. Moreover, DNAs or RNAs comprising unusual bases, such as inosine, or modified bases, such as tritylated bases, to name just two examples, are polynucleotides as the term is used herein. It will be appreciated that a great variety of modifications have been made to DNA and RNA that serve many useful purposes known to those of skill in the art. The term "polynucleotide(s)" as it is employed herein embraces such chemically, enzymatically or metabolically modified forms of polynucleotides, as well as the chemical forms of DNA and RNA characteristic of viruses and cells, including, for example, simple and complex cells. "Polynucleotide(s)" also embraces short polynucleotides often referred to as oligonucleotide(s). "Polypeptide(s)" refers to any peptide or protein comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds. "Polypeptide(s)" refers to both short chains, commonly referred to as peptides, oligopeptides and ohgomers and to longer chains generally referred to as proteins. Polypeptides may comprise amino acids other than the 20 gene encoded amino acids. "Polypeptide(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 described 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 comprise many types of modifications. Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains, and the amino or carboxyl termini. Modifications include, for example, acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP-ribosylation, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins, such as arginylation, and ubiquitination. See, for instance, PROTEINS - STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed., T. E. Creighton, W. H. Freeman and Company, New York (1993) and Wold, F., Posttranslational Protein Modifications: Perspectives and Prospects, pgs. 1-12 in POSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C. Johnson, Ed., Academic Press, New York (1983); Seifter, et al., Meth. Enzymol. 182:626-646 (1990) and Rattan, et al., Ann. NY. Acad. Sci. 663: 48-62 (1992). 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
"Vaπant(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 variant of a polynucleotide differs in nucleotide sequence from another, reference polynucleotide Changes in the nucleotide sequence of the variant may or may not alter the ammo acid sequence of a polypeptide encoded by the reference polynucleotide Nucleotide changes may result in amino acid substitutions, additions, deletions, fusion proteins and truncations in the polypeptide encoded by the reference sequence, as discussed below A typical variant of a polypeptide differs in amino acid sequence from another, reference polypeptide Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, in many regions, identical A variant 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 vaπants of each of the polypeptides of the invention, that is polypeptides that vary from the referents by conservative amino acid substitutions, whereby a residue is substituted by another with like characteπstics 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 vanants in which several, 5-10, 1-5, 1-3, 1-2 or 1 amino acids are substituted, deleted, or added in any combination A vanant of a polynucleotide or polypeptide may be a naturally occurring such as an allelic variant, or it may be a variant that is not known to occur naturally Non-naturally occumng 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, that are well known and routine to those of skill in the art, except where otherwise descπbed in detail The examples are illustrative, but do not limit the invention Example 1 Phylogenetic Analysis of YpgA clade Genes
Homologous ypgA protein sequences retrieved from public and proprietary genomic sequence databases using the software BLASTP and TBLASTN (Altschul, et al, Nucleic Acids Res. 25:3389-3402 (1997)). The proteins were initially aligned using the program CLUSTALW vl .7
(Thompson, et al, Nucleic Acids Research 22: 4673-4680 (1994)) with the BLOSUM62 (Henikoff, et al, Genomics 19:97-107 (1994) (http://blocks.fhcrc.org/blocks/)) similarity matrix, and gap opening and extension penalties of 10.0 and 0.05, respectively. The multiple sequence alignments were further refined manually using the program SEQLAB of the GCG v9.0 software package (Genetics Computer Group, Madison WI, USA).
Phylogenetic trees were constructed by neighbor-joining (N-J) and maximum parsimony (MP) methods for each set of alignments. N-J trees were based on pairwise distances between amino acid sequences using the programs NEIGHBOR and PROTDIST of the PHYLIP 3.57c package (Felsenstein, J. 1993. Distributed by the author: http devolution . genetics.washington.edu/phylip.html, Department of Genetics, University of Washington, Seattle.) The "Dayhoff ' program option was invoked in the latter program which estimates the expected amino acid replacements per position (EAARP) using a replacement model based on the Dayhoff 120 matrix. The programs SEQBOOT and CONSENSE were used to estimate the confidence limits of branching points from 1000 bootstrap replications. MP analysis was done using the software package PAUP* (Swofford, D.L. 1999. PAUP*. Phylogenetic Analysis Using Parsimony (*and Other Methods). Version 4. Sinauer Associates, Sunderland, Massachusetts.) Given the large size of the dataset, it was not possible to exhaustively search for the total number of minimal length trees. Instead, the numbers and lengths of minimal trees were estimated from 100 replicate random heuristic searches, while confidence limits of branch points were estimated by 1000 bootstrap replications.
Both MP and N-J methods showed strong statistical support for clustering together ypgA proteins from Gram-positive cocci (node F) and all Gram-positive bacteria (node G). The clusters of Gram-positive bacterial enzymes showed high levels of sequence identity, 41 -80% (based on the length of the shorter sequence without gaps from the edited alignment). Thus, Gram-positive ypgA proteins show higher levels of overall sequence similarity and cluster together as a single group or clade according to various phylogenetic methodologies. All publications and references, including but not limited to patents and patent applications, cited in this specification are herein incorporated by reference in their entirety as if each individual publication or reference were specifically and individually indicated to be incorporated by reference herein as being fully set forth. Any patent application to which this application claims priority is also incorporated by reference herein in its entirety in the manner described above for publications and references.

Claims

What is claimed is:
1. An isolated ypgA clade gene polynucleotide, wherein said polynucleotide is derived from a Gram-positive bacterium comprised within the phylogenetic tree of Figure 1.
2. An isolated ypgA clade gene polynucleotide as claimed in Claim 1, wherein said bacterium is a genus selected from the group consisting of: Staphylococcus, Streptococcus, and Enterococcus, and Bacillus.
3. An isolated ypgA clade gene polynucleotide as claimed in Claim 2, wherein the bacterium is a species selected from the group consisting of: Staphylococcus aureus,
Staphylococcus haemolyticus, Staphylococcus epidermidis, Streptococcus pneumoniae, Streptococcus pyogenes, Enterococcus faecium, Enterococcus faecalis, and Bacillus subtilis
4. An isolated ypgA clade gene polypeptide encoded by the polynucleotide of Claim 1.
5. An isolated ypgA clade gene polypeptide encoded by the polynucleotide of Claim 2.
6. An isolated ypgA clade gene polypeptide encoded by the polynucleotide of Claim 3.
7. An isolated ypgA clade gene family polynucleotide.
8. The polynucleotide of Claim 7, wherein said polynucleotide is isolated from a genus of a Gram-positive bacterium selected from the group consisting of: Staphylococcus, Streptococcus, Enterococcus, and Bacillus.
9. An isolated polynucleotide encoding ypgA isolated from a bacterium selected from the group consisting of: Staphylococcus, Streptococcus, Enterococcus, Bacillus, Staphylococcus aureus, Staphylococcus haemolyticus, Staphylococcus epidermidis, Streptococcus pneumoniae, Streptococcus pyogenes, Enterococcus faecium, Enterococcus faecalis, and Bacillus subtilis.
10. An isolated polynucleotide encoding a ypgA clade family polypeptide falling within the clade defined by a node selected from the group consisting of: node A of Figure 1 , node B of Figure 1 , node C of Figure 1 , node C of Figure 1 , node D of Figure 1 , node E of Figure 1 , node F of Figure 1 , and node G of Figure 1
1 1 A process for producing a polypeptide selected from the group consisting of (l) an isolated polypeptide comprising an amino acid sequence selected from the group having at least 95% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs 2, 4, 6, 8, 10, 12, 14, and 16,
(n) an isolated polypeptide comprising an ammo acid sequence selected from the group consisting of SEQ ID NOs 2, 4, 6, 8, 10, 12, 14, and 16, (in) an isolated polypeptide that is an amino acid sequence selected from the group consisting of SEQ ID NOs 2, 4, 6, 8, 10, 12, 14, and 16, and
(iv) a polypeptide that is encoded by a recombinant polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NOs 1 , 3, 5, 7, 9, 1 1, 13, and 15, comprising the step of culturing a host cell under conditions sufficient for the production of the polypeptide
12 A process for producing a host cell comprising an expression system or a membrane thereof expressing a polypeptide selected from the group consisting of (l) an isolated polypeptide comprising an amino acid sequence selected from the group having at least 95% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs 2, 4, 6, 8, 10, 12, 14, and 16,
(n) an isolated polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs 2, 4, 6, 8, 10, 12, 14, and 16, (in) an isolated polypeptide that is an amino acid sequence selected from the group consisting of SEQ ID NOs 2, 4, 6, 8, 10, 12, 14, and 16, and
(iv) a polypeptide that is encoded by a recombinant polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NOs 1, 3, 5, 7, 9, 1 1 , 13, and 15, said process comprising the step of transforming or transfecting a cell with an expression system compπsing a polynucleotide capable of producing said polypeptide of (l), (n), (in) or (iv) when said expression system is present in a compatible host cell such the host cell, under appropriate culture conditions, produces said polypeptide of (I), (n), (in) or (iv)
13. A host cell or a membrane expressing a polypeptide selected from the group consisting of:
(i) an isolated polypeptide comprising an amino acid sequence selected from the group having at least 95% identity to an amino acid sequence selected from the group consisting of over the entire length of: SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, and 16;
(ii) an isolated polypeptide comprising an amino acid sequence selected from the group consisting of: SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, and 16;
(iii) an isolated polypeptide that is an amino acid sequence selected from the group consisting of: SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, and 16; and
(iv) a polypeptide that is encoded by a recombinant polynucleotide comprising a polynucleotide sequence selected from the group consisting of: SEQ ID NOs: l, 3, 5, 7, 9, 1 1 , 13, and 15.
14. An antibody immunospecific for the polypeptide of Claim 1.
15. A method for screening to identify compounds that agonize or that inhibit the function of the polypeptide of Claim 1 that comprises a method selected from the group consisting of:
(a) measuring the binding of a candidate compound to the polypeptide (or to the cells or membranes bearing the polypeptide) or a fusion protein thereof by means of a label directly or indirectly associated with the candidate compound;
(b) measuring the binding of a candidate compound to the polypeptide (or to the cells or membranes bearing the polypeptide) or a fusion protein thereof in the presence of a labeled competitor; (c) testing whether the candidate compound results in a signal generated by activation or inhibition of the polypeptide, using detection systems appropriate to the cells or cell membranes bearing the polypeptide;
(d) mixing a candidate compound with a solution comprising a polypeptide of claim 1 , to form a mixture, measuring activity of the polypeptide in the mixture, and comparing the activity of the mixture to a standard; or
(e) detecting the effect of a candidate compound on the production of mRNA encoding said polypeptide and said polypeptide in cells, using for instance, an ELISA assay.
PCT/US2000/026450 1999-09-27 2000-09-27 ypga CLADE GENES WO2001023403A1 (en)

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