WO1999010012A1 - NOVEL priA - Google Patents

NOVEL priA Download PDF

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Publication number
WO1999010012A1
WO1999010012A1 PCT/US1998/017324 US9817324W WO9910012A1 WO 1999010012 A1 WO1999010012 A1 WO 1999010012A1 US 9817324 W US9817324 W US 9817324W WO 9910012 A1 WO9910012 A1 WO 9910012A1
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WO
WIPO (PCT)
Prior art keywords
polypeptide
polynucleotide
pria
seq
sequence
Prior art date
Application number
PCT/US1998/017324
Other languages
French (fr)
Inventor
Richard L. Warren
Original Assignee
Smithkline Beecham Corporation
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Smithkline Beecham Corporation filed Critical Smithkline Beecham Corporation
Priority to EP98942175A priority Critical patent/EP1009443A4/en
Priority to JP2000507401A priority patent/JP2001513337A/en
Publication of WO1999010012A1 publication Critical patent/WO1999010012A1/en

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/305—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F)
    • C07K14/31—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F) from Staphylococcus (G)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies

Definitions

  • This invention relates to newly identified polynucleotides and polypeptides, and their production and uses, as well as their va ⁇ ants, agonists and antagonists, and their uses.
  • the invention relates to polynucleotides and polypeptides of the p ⁇ mosome family, hereinafter referred to as "priA"
  • Staphylococcal genes and gene products are particularly preferred to employ Staphylococcal genes and gene products as targets for the development of antibiotics.
  • the Staphylococci make up a medically important genera of microbes They are known to produce two types of disease, invasive and toxigenic. Invasive infections are characte ⁇ zed generally by abscess formation effecting both skm surfaces and deep tissues S aureus is the second leading cause of bacteremia in cancer patients. Osteomyelitis, septic arthritis, septic thrombophlebitis and acute bacterial endocarditis are also relatively common. There are at least three clinical conditions resulting from the toxigenic properties of Staphylococci.
  • polypeptides of the invention have ammo acid sequence homology to a known priA in B. subhlis (p94461), H. influenzae (P44647), E. coh (P17888), R. rubrum (P05445), and a low sequence homology to S. cerevisiae (p20449) protein.
  • polynucleotides that encode priA polypeptides particularly polynucleotides that encode the polypeptide herein designated priA
  • the polynucleotide comprises a region encoding priA polypeptides comprising the sequence set out m Table 1 [SEQ ID NO:l], or a variant thereof.
  • a priA protein from Staphylococcus aureus comprising the ammo acid sequence of Table 1 [SEQ ID NO.2], or a variant thereof.
  • an isolated nucleic acid molecule encoding a mature polypeptide expressible by the Staphylococcus aureus WCUH 29 strain contained in the deposited strain.
  • a further aspect of the invention there are provided isolated nucleic acid molecules encoding priA, particularly Staphylococcus aureus priA, including mRNAs, cDNAs, genomic
  • DNAs are provided.
  • Further embodiments of the invention include biologically, diagnostically, prophylactically, clinically or therapeutically useful variants thereof, and compositions comprising the same.
  • a polynucleotide of the invention for therapeutic or prophylactic purposes, in particular genetic immunization.
  • particularly preferred embodiments of the invention are naturally occurring allehc va ⁇ ants of p ⁇ A and polypeptides encoded thereby.
  • polypeptides of Staphylococcus aureus referred to herein as priA as well as biologically, diagnostically, prophylactically, clinically or therapeutically useful variants thereof, and compositions comp ⁇ smg the same.
  • va ⁇ ants of priA polypeptide encoded by naturally occurring alleles of the p ⁇ A gene are va ⁇ ants of priA polypeptide encoded by naturally occurring alleles of the p ⁇ A gene.
  • priA polypeptides In a preferred embodiment of the invention there are provided methods for producing the aforementioned priA polypeptides. In accordance with yet another aspect of the invention, there are provided inhibitors to such polypeptides, useful as antibacterial agents, including, for example, antibodies.
  • a disease for example, 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, retrope ⁇ toneal abscess), CNS (e g , cerebral abscess), eye (e.g., blepharitis, conjunctivitis, keratitis, endophthalmitis, preseptal and orbital celluhtis, darcryocystitis), kidney and urinary tract (e.g., epididymitis, mtrarenal and pe ⁇ neph ⁇ c absces, toxic shock syndrome
  • infections of the upper respiratory tract e.g , otitis media, bacterial tracheitis, acute
  • antibodies against priA polypeptides there are provided antibodies against priA polypeptides.
  • methods for identifying compounds which bind to or otherwise interact with and inhibit or activate an activity of a polypeptide or polynucleotide of the invention comprising: contacting a polypeptide 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 or polynucleotide to assess the binding to or other interaction with the compound, such binding or interaction being associated with a second component capable of providing a detectable signal in response to the binding or interaction of the polypeptide or polynucleotide with the compound; and determining whether the compound binds to or otherwise interacts with and activates or inhibits an activity of the polypeptide or polynucleotide by detecting the presence or absence of a signal generated from the binding or interaction of the compound with the polypeptide or polynucleotide.
  • compositions comprising a priA polynucleotide or a priA polypeptide for administration to a cell or to a multicellular organism.
  • “Host cell” is a cell which has been transformed or transfected, or is capable of transformation or transfection by an exogenous polynucleotide sequence.
  • Identity is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as the case may be, as determined by 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; Biocoinputing: 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, J., et al., Nucleic Acids Research 12(1): 387 (1984)), BLASTP, BLASTN, and FASTA (Altschul, S.F. et al., J. Molec. Biol. 215: 403-410 (1990). The BLAST X program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S., et al, NCBI NLM NIH Bethesda, MD 20894;
  • Waterman algorithm may also be used to determine identity.
  • Gap Length Penalty 4 A program useful with these parameters is publicly available as the "gap" program from
  • 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:
  • Polynucleotide embodiments further include an isolated polynucleotide comprising a polynucleotide sequence having at least a 50, 60, 70, 80, 85, 90, 95, 97 or 100% identity to the reference sequence of SEQ ID NO.
  • polynucleotide sequence may be identical to the reference sequence of SEQ ID NO: 1 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 NO: l by the integer defining the percent identity divided by 100 and then subtracting that product from said total number of nucleotides in SEQ ID NO: 1, or:
  • n n is the number of nucleotide alterations
  • x n is the total number of nucleotides in SEQ ID NO: l
  • y is 0.50 for 50%, 0.60 for 60%, 0.70 for 70%, 0.80 for 80%, 0.85 for 85%, 0.90 for 90%, 0.95 for 95%, 0.97 for 97% or 1.00 for 100%
  • • is the symbol for the multiplication operator, and wherein any non-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 the polypeptide of SEQ ID NO:2 may create nonsense, missense or frameshift mutations in this coding sequence and thereby alter the polypeptide encoded by the polynucleotide following such alterations.
  • a polynucleotide sequence of the present invention may be identical to the reference sequence of SEQ ID NO:l, that is it may be 100% identical, or it may include up to a certain integer number of nucleic acid alterations as compared to the reference sequence such that the percent identity is less than 100% identity.
  • Such alterations are selected from the group consisting of at least one nucleic acid deletion, substitution, including transition and transversion, or insertion, and wherein said alterations may occur at the 5' or 3' terminal positions of the reference polynucleotide sequence or anywhere between those terminal positions, interspersed either individually among the nucleic acids in the reference sequence or in one or more contiguous groups within the reference sequence.
  • the number of nucleic acid alterations for a given percent identity is determined by multiplying the total number of nucleic acids in SEQ ID NO: l by the integer defining the percent identity divided by 100 and then subtracting that product from said total number of nucleic acids in SEQ ID NO: 1, or:
  • Polypeptide embodiments further include an isolated polypeptide comprising a polypeptide having at least a 50,60, 70, 80, 85, 90, 95, 97 or 100% identity to a polypeptide reference sequence of SEQ ID NO 2, wherein said polypeptide sequence may be identical to the reference sequence of SEQ ID NO 2 or may include up to a certain integer number of ammo acid alterations as compared to the reference sequence, wherein said alterations are selected from the group consisting of at least one ammo acid deletion, substitution
  • n a is the number of ammo acid alterations
  • x a is the total number of ammo acids in SEQ ID NO 2
  • y is 0 50 for 50%, 0 60 for 60%, 0 70 for 70%, 0 80 for 80%, 0 85 for 85%, 0 90 for 90%, 0 95 for 95%, 0 97 for 97% or 1 00 for 100%
  • • is the symbol for the multiplication operator, and wherein any non-mteger product of x a and y is rounded down to the nearest integer prior to subtracting it from x a
  • a polypeptide sequence of the present invention may be identical to the reference sequence of SEQ ID NO 2, that is it may be 100% identical, or it may include up to a certain integer number of ammo acid alterations as compared to the reference sequence such that the percent identity is less than 100% identity
  • Such alterations are selected from the group consisting of at least one ammo acid deletion, substitution, including conservative and non-conservative substitution, or insertion, and wherein said alterations may occur at the ammo- or carboxy-termmal 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.
  • the number of ammo acid alterations for a given % identity is determined by multiplying the total number of ammo acids in SEQ ID NO: 2 by the integer defining the percent identity divided by 100 and then subtracting that product from said total number of ammo acids in SEQ ID NO:2, or:
  • n a is the number of ammo acid alterations
  • x a is the total number of amino acids in SEQ ID NO:2
  • y is, for instance 0 70 for 70%, 0.80 for 80%, 0.85 for 85% etc.
  • • is the symbol for the multiplication operator, and wherein any non-mteger product of x a and y is rounded down to the nearest integer prior to subtracting it from x a .
  • Isolated means altered “by the hand of man” from its natural state, i e , if it occurs in nature, it has been changed or removed from its original environment, or both
  • a polynucleotide or a polypeptide naturally present in a living organism is not “isolated,” but the same polynucleotide or polypeptide separated from the coexisting mate ⁇ als of its natural state is “isolated”, as the term is employed herein.
  • Polynucleot ⁇ de(s) generally refers to any poly ⁇ bonucleotide or polydeox ⁇ bonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA.
  • Polynucleot ⁇ de(s) include, without limitation, single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions or single-, double- and t ⁇ ple-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double- stranded regions, hyb ⁇ d molecules comp ⁇ smg DNA and RNA that may be single-stranded or, more typically, double-stranded, or t ⁇ ple-stranded regions, or a mixture of single- and double- stranded regions
  • polynucleotide as used herein refers to t ⁇ ple-stranded regions comprising RNA or DNA or both RNA and DNA.
  • the strands m 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 t ⁇ ple-hehcal region often is an ohgonucleotide.
  • polynucleot ⁇ de(s) also includes DNAs or RNAs as described above that contain one or more modified bases. Thus, DNAs or RNAs with backbones modified for stability or for other reasons are "polynucleot ⁇ de(s)" as that term is intended herein.
  • DNAs or RNAs comprising unusual bases, such as mosme, or modified bases, such as t ⁇ tylated 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 "polynucleot ⁇ de(s)" as it is employed herein embraces such chemically, enzymatically or metabohcally modified forms of polynucleotides, as well as the chemical forms of DNA and RNA characte ⁇ stic of viruses and cells, including, for example, simple and complex cells. "Polynucleot ⁇ de(s)” also embraces short polynucleotides often referred to as ohgonucleot ⁇ de(s)
  • Polypept ⁇ de(s) refers to any peptide or protein comprising two or more ammo acids joined to each other by peptide bonds or modified peptide bonds
  • Polypept ⁇ de(s) refers to both short chains, commonly referred to as peptides, ohgopeptides and ohgomers and to longer chains generally referred to as proteins. Polypeptides may contain ammo acids other than the 20 gene encoded ammo acids.
  • Polypept ⁇ de(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- ⁇ bosylation, amidahon, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a hpid or lipid de ⁇ vative, covalent attachment of phosphotidyhnositol, cross-linking, cychzation, disulfide bond formation, demethylation, formation of covalent cross-lmks, formation of cysteme, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, lodination, methylation, my ⁇ stoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, glycosylation, hpid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation
  • 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.
  • 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 m amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence, as discussed below.
  • a typical variant of a polypeptide differs m amino acid sequence from another, reference polypeptide. Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, in many regions, identical.
  • a variant and reference polypeptide may differ in ammo acid sequence by one or more substitutions, additions, deletions m any combination.
  • a substituted or inserted ammo acid residue may or may not be one encoded by the genetic code.
  • a variant of a polynucleotide or polypeptide may be a naturally occurring such as an allehc variant, or it may be a variant that is not known to occur naturally.
  • Non-naturally occu ⁇ ing variants of polynucleotides and polypeptides may be made by mutagenesis techniques, by direct synthesis, and by other recombinant methods known to skilled artisans.
  • the invention relates to priA polypeptides and polynucleotides as desc ⁇ bed in greater detail below.
  • the invention relates to polypeptides and polynucleotides of a p ⁇ A of Staphylococcus aureus, which is related by ammo acid sequence homology to p ⁇ A in B subtihs (p94461), H influenzae (P44647), E. coh (P17888), R rubrum (P05445), and a low sequence homology to S cerevisiae (p20449) polypeptide.
  • the invention relates especially to priA having the nucleotide and amino acid sequences set out in Table 1 [SEQ ID NO: 1] and Table 1 [SEQ ID NO: 2] respectively, and to the priA nucleotide sequences of the DNA in the deposited strain and amino acid sequences encoded thereby.
  • NCIMB National Collections of Industrial and Marine Bacteria Ltd
  • the Staphylococcus aureus strain deposit is referred to herein as "the deposited strain” or as "the DNA of the deposited strain.”
  • the deposited strain contains the full length priA gene.
  • the sequence of the polynucleotides contained in the deposited strain, as well as the ammo acid sequence of the 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
  • a license may be required to make, use or sell the deposited strain, and compounds derived therefrom, and no such license is hereby granted.
  • polypeptides of the invention include the polypeptide of Table 1 [SEQ ID NO:2] (in particular the mature polypeptide) as well as polypeptides and fragments, particularly those which have the biological activity of priA, and also those which have at least 70% identity to the polypeptide of Table 1 [SEQ ID NO:2] or the relevant portion, preferably at least 80% identity to the polypeptide of Table 1 [SEQ ID NO:2], and more preferably at least 90% similarity (more preferably at least 90% identity) to the polypeptide of Table 1 [SEQ ID NO:2] and still more preferably at least 95% simila ⁇ ty (still more preferably at least 95% identity) to the polypeptide of Table 1 [SEQ ID NO: 2] and also include portions of such polypeptides with such portion of the polypeptide generally containing at least 30 ammo acids and more preferably at least 50 ammo acids.
  • the invention also includes polypeptides of the formula set forth in Table 1 (D) wherein, at the amino terminus, X is hydrogen, and at the carboxyl terminus, Y is hydrogen or a metal, Ri and R2 is any amino acid residue, and n is an integer between 1 and 1000.
  • Any stretch of ammo acid residues denoted by either R group, where R is greater than 1, may be either a heteropolymer or a homopolymer, preferably a heteropolymer.
  • a fragment is a va ⁇ ant polypeptide having an ammo acid sequence that entirely is the same as part but not all of the ammo acid sequence of the aforementioned 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, a single larger polypeptide
  • An example of such a fragment is the polypeptide of SEQ ID NO 6
  • Preferred fragments include, for example, truncation polypeptides having a portion of the ammo acid sequence of Table 1 [SEQ ID NO.2], or of variants thereof, such as a continuous series of residues that includes the ammo terminus, or a continuous series of residues that includes the carboxyl terminus
  • truncation polypeptides having a portion of the ammo acid sequence of Table 1 [SEQ ID NO.2] or of variants thereof, such as a continuous series of residues that includes the ammo terminus, or a continuous series of residues that includes the carboxyl terminus
  • Degradation forms of the polypeptides of the invention m a host cell, particularly a Staphylococcus aureus, 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-formmg regions, turn and turn-forming regions, coil and coil-forming regions, hydrophihc regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions, substrate binding region, and high antigenic index regions.
  • biologically active fragments which are those fragments that mediate activities of p ⁇ A, including those with a similar activity or an improved activity, or with a decreased undesirable activity
  • fragments that are antigenic or immunogenic in an animal especially in a human
  • Variants that are fragments of the polypeptides of the invention may be employed for producing the corresponding full-length polypeptide by peptide synthesis; therefore, these va ⁇ ants may be employed as intermediates for producing the full-length polypeptides of the invention.
  • An example of such a fragment is the polynucleotide of SEQ ID NO: 5.
  • Another aspect of the invention relates to isolated polynucleotides that encode the priA polypeptide having the deduced ammo acid sequence of Table 1 [SEQ ID NO.2] and polynucleotides closely related thereto and variants thereof
  • a polynucleotide of the invention encoding priA polypeptide may be obtained using standard cloning and screening methods, such as those for cloning and sequencing chromosomal DNA fragments from bacteria using Staphylococcus aureus WCUH 29 cells as starting material, followed by obtaining a full length clone.
  • a library of clones of chromosomal DNA of Staphylococcus aureus WCUH 29 in E coh or some other suitable host is probed with a radiolabeled ohgonucleotide, preferably a 17-mer or longer, derived from a partial sequence. Clones carrying DNA identical to that of the probe can then be distinguished using stringent conditions.
  • sequencing is performed using denatured double stranded DNA prepared from a plasmid clone Suitable techniques are described by Mamatis, T., F ⁇ tsch, E.F.
  • the DNA sequence set out in Table 1 [ SEQ ID NO 1] contains an open reading frame encoding a protein having about or exactly the number of ammo acid residues set forth in Table 1 [SEQ ID NO:2] with a deduced molecular weight that can be calculated using ammo acid residue molecular weight values well known in the art.
  • the open reading frame may be correlated with the polypeptide of Table 1 [SEQ ID NO: 2] by using a codon chart well known in the art or any algo ⁇ thm for detecting open reading frames, such as the known methods of Framesearch (Wisconsin Package of the Genetics Computer Group (GCG) software), TBLASTN (Blast software from NCBI), and FASTA (TFASTX of Pearson & Lipman; PNAS 85. 2444 (1988).
  • P ⁇ A of the invention is structurally related to other proteins of the p ⁇ mosome family, as shown by the results of sequencing the DNA encoding priA of the deposited strain
  • the protein exhibits greatest homology to priA in B subtihs (p94461) protein among known proteins.
  • P ⁇ A of Table 1 [SEQ ID NO 2] has about 61% identity (I e, 250/410) over its entire length and about 80.7% similarity (1 e , 331/410) over its entire length with the ammo acid sequence of the p ⁇ A in B subtihs (p94461) polypeptide.
  • the invention provides a polynucleotide sequence identical over its entire length to the coding sequence in Table 1 [SEQ ID NO.l]. Also provided by the invention is the coding sequence for the mature polypeptide or a fragment thereof, by itself as well as the coding sequence for the mature polypeptide or a fragment in reading frame with other coding sequence, such as those encoding a leader or secretory sequence, a pre-, or pro- or prepro- protem sequence.
  • the polynucleotide may also contain non-codmg sequences, including for example, but not limited to non-codmg 5' and 3' sequences, such as the transc ⁇ bed, non- translated sequences, termination signals, ⁇ bosome binding sites, sequences that stabilize mRNA, mtrons, polyadenylation signals, and additional coding sequence which encode additional ammo acids.
  • non-codmg sequences including for example, but not limited to non-codmg 5' and 3' sequences, such as the transc ⁇ bed, non- translated sequences, termination signals, ⁇ bosome binding sites, sequences that stabilize mRNA, mtrons, polyadenylation signals, and additional coding sequence which encode additional ammo acids.
  • a marker sequence that facilitates purification of the fused polypeptide can be encoded.
  • the marker sequence is a hexa-histidme peptide, as provided in the pQE vector (Qiagen, Inc.) and
  • Polynucleotides of the invention also include, but are not limited to, polynucleotides comprising a structural gene and its naturally associated sequences that control gene expression.
  • a preferred embodiment of the invention is the polynucleotide comprising nucleotide set forth in SEQ ID NO: 1 of Table 1 which encodes the p ⁇ A polypeptide.
  • the invention also includes polynucleotides of the formula set forth in Table 1 (C) wherein, at the 5' end of the molecule, X is hydrogen, and at the 3' end of the molecule, Y is hydrogen or a metal, Ri and R2 is any nucleic acid residue, and n is an integer between 1 and 1000. Any stretch of nucleic acid residues denoted by either R group, where R is greater than 1 , may be either a heteropolymer or a homopolymer, preferably a heteropolymer.
  • polynucleotide encoding a polypeptide encompasses polynucleotides that include a sequence encoding a polypeptide of the invention, particularly a bacte ⁇ al polypeptide and more particularly a polypeptide of the Staphylococcus aureus priA having the ammo acid sequence set out in Table 1 [SEQ ID NO:2].
  • the term also encompasses polynucleotides that include a single continuous region or discontinuous regions encoding the polypeptide (for example, interrupted by integrated phage or an insertion sequence or editing) together with additional regions, that also may contain coding and/or non-codmg sequences.
  • the invention further relates to va ⁇ ants of the polynucleotides described herein that encode for variants of the polypeptide having the deduced ammo acid sequence of Table 1 [SEQ ID NO.2].
  • Va ⁇ ants that are fragments of the polynucleotides of the invention may be used to synthesize full-length polynucleotides of the invention.
  • Further particularly preferred embodiments are polynucleotides encoding priA va ⁇ ants, that have the ammo acid sequence of priA polypeptide of Table 1 [SEQ ID NO:2] in which several, a few, 5 to 10, 1 to 5, 1 to 3, 2, 1 or no ammo acid residues are substituted, deleted or added, in any combination.
  • silent substitutions, additions and deletions that do not alter the properties and activities of p ⁇ A.
  • polynucleotides that are at least 70%) identical over their entire length to a polynucleotide encoding p ⁇ A polypeptide having the amino acid sequence set out in Table 1 [SEQ ID NO:2], and polynucleotides that are complementary to such polynucleotides.
  • polynucleotides that comprise a region that is at least 80% identical over its entire length to a polynucleotide encoding p ⁇ A polypeptide of the deposited strain and polynucleotides complementary thereto.
  • polynucleotides at least 90% identical over their entire length to the same are particularly preferred, and among these particularly preferred polynucleotides, those with at least 95% are especially preferred Furthermore, those with at least 97% are highly preferred among those with at least 95%, and among these those with at least 98%o and at least 99% are particularly highly preferred, with at least 99% being the more preferred.
  • Preferred embodiments are polynucleotides that encode polypeptides that retain substantially the same biological function or activity as the mature polypeptide encoded by the DNA of Table 1 [SEQ ID NO: 1].
  • the invention further relates to polynucleotides that hybridize to the herein above- described sequences.
  • the invention especially relates to polynucleotides that hyb ⁇ dize under st ⁇ ngent conditions to the herein above-described polynucleotides.
  • stringent conditions and “stringent hybridization conditions” mean hybridization will occur only if there is at least 95% and preferably at least 97% identity between the sequences.
  • An example of stringent hybridization conditions is overnight incubation at 42°C m 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 denatured, sheared salmon sperm DNA, followed by washing the hybridization support in O.lx SSC at about 65°C.
  • Hybridization and wash conditions are well known and exemplified in Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (1989), particularly Chapter 11 therein.
  • the invention also provides a polynucleotide consisting essentially of a polynucleotide sequence obtainable by screening an appropriate library containing the complete gene for a polynucleotide sequence set forth in SEQ ID NO: l under stringent hybridization conditions with a probe having the sequence of said polynucleotide sequence set forth m SEQ ID NO:l or a fragment thereof; and isolating said DNA sequence. Fragments useful for obtaining such a polynucleotide include, for example, probes and primers described elsewhere herein.
  • 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 priA and to isolate cDNA and genomic clones of other genes that have a high sequence similarity to the priA gene.
  • Such probes generally will comp ⁇ se at least 15 bases.
  • such probes will have at least 30 bases and may have at least 50 bases.
  • Particularly preferred probes will have at least 30 bases and will have 50 bases or less.
  • the coding region of the priA gene may be isolated by screening using the DNA sequence provided in SEQ ID NO.
  • a labeled ohgonucleotide 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.
  • the 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 disease, particularly human disease, as further discussed herein relating to polynucleotide assays.
  • Polynucleotides of the invention that are ohgonucleotides derived from the sequences of SEQ ID NOS.1 and/or 2 may be used in the processes herein as described, but preferably for PCR, to determine whether or not the polynucleotides identified herein in whole or in part are transcribed in bacteria in infected tissue. It is recognized that such sequences 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 may encode a polypeptide that is the mature protein plus additional ammo or carboxyl-termmal ammo acids, or ammo acids interior to the mature polypeptide (when the mature form has more than one polypeptide chain, for instance).
  • Such sequences may play a role in processing of a protein from precursor to a mature form, may allow protein transport, may lengthen or shorten protein half-life or may facilitate manipulation of a protein for assay or production, among other things.
  • the additional amino acids may be processed away from the mature protein by cellular enzymes.
  • a precursor protein, having the mature form of the polypeptide fused to one or more prosequences may be an inactive form of the polypeptide.
  • inactive precursors 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 proprotems.
  • a polynucleotide of the invention may encode a mature protein, a mature protein plus a leader sequence (which may be referred to as a preprotein), a precursor of a mature protein having one or more prosequences that are not the leader sequences of a preprotein, or a preproprotem, which is a precursor to a proprotem, having a leader sequence and one or more prosequences, which generally are removed during processing steps that produce active and mature forms of the polypeptide.
  • a leader sequence which may be referred to as a preprotein
  • a precursor of a mature protein having one or more prosequences that are not the leader sequences of a preprotein or a preproprotem, which is a precursor to a proprotem, having a leader sequence and one or more prosequences, which generally are removed during processing steps that produce active and mature forms of the polypeptide.
  • Vectors, host cells, expression The invention also relates to vectors that comprise 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 derived from the DNA constructs 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 desc ⁇ bed 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 Sp ⁇ ng Harbor Laboratory Press, Cold Sp ⁇ ng Harbor, N.Y. (1989), such as, calcium phosphate transfection, DEAE-dextran mediated transfection, transvection, micromjection, catiomc hpid-mediated transfection, electroporation, transduction, scrape loading, ballistic introduction and infection.
  • approp ⁇ ate hosts include bacterial cells, such as streptococci, staphylococci, enterococci E coli, streptomyces and Bacillus subtihs cells; fungal cells, such as yeast cells and Aspergillus cells; insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, 293 and Bowes melanoma cells; and plant cells.
  • bacterial cells such as streptococci, staphylococci, enterococci E coli, streptomyces and Bacillus subtihs cells
  • fungal cells such as yeast cells and Aspergillus cells
  • insect cells such as Drosophila S2 and Spodoptera Sf9 cells
  • animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, 293 and Bowes melanoma cells
  • Such vectors include, among others, chromosomal
  • the expression system constructs may contain control regions that regulate as well as engender expression.
  • any system or vector suitable to maintain, propagate or express polynucleotides and/or to express a polypeptide in a host may be used for expression m this regard
  • the appropriate DNA sequence may be inserted into the expression system by any of a variety of well-known and routine techniques, such as, for example, those set forth in Sambrook et al, MOLECULAR CLONING, A LABORATORY MANUAL, (supra).
  • appropriate 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 purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography, and lectin chromatography. Most preferably, high performance liquid chromatography is employed for purification. Well known techniques for refolding protein may be employed to regenerate active conformation when the polypeptide is denatured du ⁇ ng isolation and or purification. Diagnostic Assays
  • This invention is also related to the use of the priA polynucleotides of the invention for use as diagnostic reagents. Detection of priA in a eukaryote, particularly a mammal, and especially a human, will provide a diagnostic method for diagnosis of a disease. Eukaryotes (herein also "individuals)”), particularly mammals, and especially humans, particularly those infected or suspected to be infected with an organism comprising the p ⁇ A gene may be detected at the nucleic acid level by a va ⁇ ety of techniques.
  • Nucleic acids for diagnosis may be obtained from an infected individual's cells and tissues, such as bone, blood, muscle, cartilage, and skin Genomic DNA may be used directly for detection or may be amplified enzymatically by using PCR or other amplification technique prior to analysis RNA or cDNA may also be used in the same ways Using amplification, characterization of the species and strain of prokaryote present in an individual, may be made by an analysis of the genotype of the prokaryote gene Deletions and insertions can be detected by a change m size of the amplified product in comparison to the genotype of a reference sequence Point mutations can be identified by hybridizing amplified DNA to labeled priA polynucleotide sequences Perfectly matched sequences can be distinguished from mismatched duplexes by RNase digestion or by differences m melting temperatures DNA sequence differences may also be detected by alterations in the electrophoretic mobility of the DNA fragments in gels, with or without denaturing agents, or by direct DNA sequencing See
  • RT-PCR can be used to detect mutations It is particularly preferred to used RT- PCR in conjunction with automated detection systems, such as, for example, GeneScan RNA or cDNA may also be used for the same purpose, PCR or RT-PCR
  • PCR p ⁇ mers complementary to a nucleic acid encoding priA can be used to identify and analyze mutations Examples of representative primers are shown below in Table 2
  • the invention further provides these primers with 1 , 2, 3 or 4 nucleotides removed from the 5' and/or the 3' end.
  • These primers may be used for, among other things, amplifying priA DNA isolated from a sample derived from an individual.
  • the p ⁇ mers may be used to amplify the gene isolated from an infected individual such that the gene may then be subject to various techniques for elucidation of the DNA sequence. In this way, mutations in the DNA sequence may be detected and used to diagnose infection and to serotype and/or classify the infectious agent.
  • the invention further provides a process for diagnosing, disease, preferably bacterial infections, more preferably infections by Staphylococcus aureus, and most preferably disease, such as, infections of the upper respiratory tract (e.g., otitis media, bacte ⁇ al tracheitis, acute epiglottitis, thyroiditis), lower respiratory (e.g., empyema, lung abscess), cardiac (e.g , infective endocarditis), gastrointestinal (e.g., secretory diarrhoea, splenic absces, retrope ⁇ toneal abscess), CNS (e.g., cerebral abscess), eye (e.g., blepharitis, conjunctivitis, keratitis, endophthalmitis, preseptal and orbital celluhtis, darcryocystitis), kidney and u ⁇ nary tract (e.g., epididymitis, mtrarenal and pe ⁇ ne
  • Increased or decreased expression of p ⁇ A polynucleotide can be measured using any on of the methods well known m the art for the quantation of polynucleotides, such as, for example, amplification, PCR, RT-PCR, RNase protection, Northern blotting and other hybridization methods.
  • a diagnostic assay in accordance with the invention for detecting over- expression of priA protein 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 p ⁇ A protein, in a sample derived from a host are well-known to those of skill in the art. Such assay methods include radioimmunoassays, competitive-binding assays, Western Blot analysis and ELISA assays.
  • Antibodies The polypeptides of the invention or va ⁇ ants thereof, or cells expressing them can be used as an immunogen to produce antibodies lmmunospecific for such polypeptides.
  • Antibodies as used herein includes monoclonal and polyclonal antibodies, chime ⁇ c, single chain, simianized antibodies and humanized antibodies, as well as Fab fragments, including the products of an Fab lmmunolglobulm expression library.
  • Antibodies generated against the polypeptides of the invention can be obtained by administering the polypeptides or epitope-bearmg fragments, analogues or cells to an animal, preferably a nonhuman, using routine protocols.
  • any technique known in the art that provides antibodies produced by continuous cell line cultures can be used. Examples include various techniques, such as those in Kohler, G.
  • phage display technology may be utilized to select antibody genes with binding activities towards the polypeptide either from repertoires of PCR amplified v- genes of lymphocytes from humans screened for possessing anti-p ⁇ A or from naive libraries (McCafferty, J. et al, (1990), Nature 348, 552-554; Marks, J. et al, (1992) Biotechnology 10, 779-783).
  • the affinity of these antibodies can also be improved by chain shuffling (Clackson, T. et al., (1991) Nature 352, 624-628).
  • each domain may be directed against a different epitope - termed 'bispecific' antibodies.
  • the above-desc ⁇ bed antibodies may be employed to isolate or to identify clones expressing the polypeptides to purify the polypeptides by affinity chromatography.
  • antibodies against p ⁇ A- polypeptide may be employed to treat infections, particularly bacte ⁇ al infections and especially 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, retrope ⁇ toneal abscess), CNS (e.g., cerebral abscess), eye (e.g., blepharitis, conjunctivitis, keratitis, endophthalmitis, preseptal and orbital celluhtis, darcryocystitis), kidney and u ⁇ nary tract (e.g., epididymitis, mtrarenal and pe ⁇ neph ⁇ c absces, toxic shock syndrome
  • Polypeptide variants include antigemcally, epitopically or immunologically equivalent variants that form a particular aspect of this invention.
  • the term "antigemcally equivalent derivative” as used herein encompasses a polypeptide or its equivalent which will be specifically recognized by certain antibodies which, when raised to the protein or polypeptide according to the invention, interfere with the immediate physical interaction between pathogen and mammalian host.
  • the term “immunologically equivalent derivative” as used herein encompasses a peptide or its equivalent which when used in a suitable formulation to raise antibodies in a vertebrate, the antibodies act to interfere with the immediate physical interaction between pathogen and mammalian host.
  • the polypeptide such as an antigemcally or immunologically equivalent derivative or a fusion protein thereof is used as an antigen to immunize a mouse or other animal such as a rat or chicken.
  • the fusion protein may provide stability to the polypeptide.
  • the antigen may be associated, for example by conjugation, with an immunogenic carrier protein for example bovine serum albumin (BSA) or keyhole limpet haemocyanm (KLH).
  • BSA bovine serum albumin
  • KLH keyhole limpet haemocyanm
  • a multiple antigenic peptide comprising multiple copies of the protein or polypeptide, or an antigemcally or immunologically equivalent polypeptide thereof may be sufficiently antigenic to improve lmmunogenicity so as to obviate the use of a carrier.
  • the antibody or variant thereof is modified to make it less immunogenic in the individual.
  • the antibody may most preferably be "humanized"; where the comphmenta ⁇ ty determining reg ⁇ on(s) of the hyb ⁇ doma-de ⁇ ved antibody has been transplanted into a human monoclonal antibody , for example as described m Jones, P. et al. (1986), Nature 321, 522-525 or Tempest et al.,(1991) Biotechnology 9, 266-273.
  • a polynucleotide of the invention in genetic immunization will preferably employ a suitable delivery method such as direct injection of plasmid DNA into muscles (Wolff et al., Hum Mol Genet 1992, 1 :363, Manthorpe et al., Hum. Gene Ther.
  • Polypeptides of the invention may also be used to assess the binding of small molecule substrates and hgands in, for example, cells, cell-free preparations, chemical hbra ⁇ es, and natural product mixtures.
  • substrates and hgands may be natural substrates and hgands or may be structural or functional mimetics. See, e g , Cohgan et al , Current Protocols in Immunology 1(2) Chapter 5 (1991).
  • the invention also provides a method of screening compounds to identify those which enhance (agonist) or block (antagonist) the action of priA polypeptides or polynucleotides, particularly those compounds that are bacte ⁇ ostatic and/or bacteriocidal.
  • the method of screening may involve high-throughput techniques.
  • a synthetic reaction mix for example, a synthetic reaction mix, a cellular compartment, such as a membrane, cell envelope or cell wall, or a preparation of any thereof, comprising priA polypeptide and a labeled substrate or ligand of such polypeptide is incubated in the absence or the presence of a candidate molecule that may be a priA agonist or antagonist
  • a candidate molecule that may be a priA agonist or antagonist
  • the ability of the candidate molecule to agonize or antagonize the priA polypeptide is reflected in decreased binding of the labeled ligand or decreased production of product from such substrate.
  • Molecules that bind gratuitously, i e., without inducing the effects of priA polypeptide are most likely to be good antagonists
  • Molecules that bind well and increase the rate of product production from substrate are agonists. Detection of the rate or level of production of product from substrate may be enhanced by using a reporter system Reporter systems that may be useful in this regard include but are not limited to colo ⁇ met ⁇ c labeled substrate converted into product, a reporter gene that is responsive to changes in priA polynucleotide or polypeptide activity, and binding assays known in the art.
  • an assay for priA antagonists is a competitive assay that combines priA and a potential antagonist with p ⁇ A-bindmg molecules, recombinant priA binding molecules, natural substrates or hgands, or substrate or ligand mimetics, under appropriate conditions for a competitive inhibition assay P ⁇ A can be labeled, such as by radioactivity or a colo ⁇ met ⁇ c compound, such that the number of priA molecules bound to a binding molecule or converted to product can be determined accurately to assess the effectiveness of the potential antagonist.
  • Potential antagonists include small organic molecules, peptides, polypeptides and antibodies that bind to a polynucleotide or polypeptide of the invention and thereby inhibit or extinguish its activity
  • Potential antagonists also may be small organic molecules, a peptide, a polypeptide such as a closely related protein or antibody that binds the same sites on a binding molecule, such as a binding molecule, without inducing p ⁇ A-induced activities, thereby preventing the action of priA by excluding p ⁇ A from binding.
  • Potential antagonists include a small molecule that binds to and occupies the binding site of the polypeptide thereby preventing binding to cellular binding molecules, such that normal biological activity is prevented.
  • small molecules include but are not limited to small organic molecules, peptides or peptide-hke molecules
  • Other potential antagonists include antisense molecules (see Okano, J Neurochem 56 560 (1991); OLIGODEOXYNUCLEOTIDES AS ANTISENSE INHIBITORS OF GENE EXPRESSION, CRC Press, Boca Raton, FL (1988), for a description of these molecules).
  • Preferred potential antagonists include compounds related to and vanants of priA.
  • Each of the DNA sequences provided herein may be used in the discovery and development of antibacterial compounds.
  • the encoded protein upon expression, can be used as a target for the screening of antibacterial drugs Additionally, the DNA sequences encoding the ammo terminal regions of the encoded protein or Shine-Delgarno or other translation facilitating sequences of the respective mRNA can be used to construct antisense sequences to control the expression of the coding sequence of interest
  • the invention also provides the use of the polypeptide, polynucleotide or inhibitor of the invention to interfere with the initial physical interaction between a pathogen and 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 bacteria, to mammalian extracellular matrix proteins on m-dwellmg devices or to extracellular matrix proteins in wounds; to block priA protem-mediated mammalian cell invasion by, for example, initiating phosphorylation of mammalian tyrosme kmases (Rosenshme et al, Infect Immun (50:2211 (1992); to block bacterial adhesion between mammalian extracellular matrix proteins and bacte ⁇ al p ⁇ A proteins that mediate tissue damage and, to block the normal progression of pathogenesis m infections initiated other than by the implantation of m-dwelhng devices or by other surgical techniques.
  • the antagonists and agonists of the invention may be employed, for instance, to inhibit and treat 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, retrope ⁇ toneal abscess), CNS (e.g., cerebral abscess), eye (e.g., blepha ⁇ tis, conjunctivitis, keratitis, endophthalmitis, preseptal and orbital celluhtis, darcryocystitis), kidney and u ⁇ nary tract (e.g., epididymitis, mtrarenal and pe ⁇ neph ⁇ c absces, toxic shock syndrome), skin (e.g., imp
  • H pylori Helicobacter pylori bacteria mfect 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/ecp2904.htm).
  • 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.
  • Preferred antimicrobial compounds of the invention (agonists and antagonists of priA) found using screens provided by the invention, particularly broad-spectrum antibiotics, should be useful in the treatment of H pylori infection. Such treatment should decrease the advent of H pylori- d ced cancers, such as gastrointestinal carcinoma Such treatment should also cure gastric ulcers and gastritis.
  • Another aspect of the invention relates to a method for inducing an immunological response in an individual, particularly a mammal which comprises inoculating the individual with priA, or a fragment or variant thereof, adequate to produce antibody and/ or T cell immune response to protect said individual from infection, particularly bacterial infection and most particularly Staphylococcus aureus infection Also provided are methods whereby such immunological response slows bacterial replication Yet another aspect of the invention relates to a method of inducing immunological response in an individual which comprises delivering to such individual a nucleic acid vector to direct expression of p ⁇ A, or a fragment or a variant thereof, for expressing priA, or a fragment or a variant thereof in vivo in order to induce an immunological response, such as, to produce antibody and/ or T cell immune response, including, for example, cytokine-producmg T cells or cytotoxic T cells, to protect said individual from disease, whether that disease is already established withm the individual or not
  • One way of administering the gene is by accelerating it
  • a further aspect of the invention relates to an immunological composition which, when introduced into an individual capable or having induced withm it an immunological response, induces an immunological response in such individual to a p ⁇ A or protein coded therefrom, wherein the composition comprises a recombinant priA or protein coded therefrom comprising DNA which codes for and expresses an antigen of said p ⁇ A or protein coded therefrom.
  • the immunological response may be used therapeutically or prophylactically and may take the form of antibody immunity or cellular immunity such as that arising from CTL or CD4+ T cells.
  • a p ⁇ A polypeptide or a fragment thereof may be fused with co-protein which may not by itself produce antibodies, but is capable of stabilizing the first protein and producing a fused protein which will have immunogenic and protective properties.
  • fused recombinant protein preferably further comprises an antigenic co-protem, such as hpoprotein D from Hemophilus influenzae, Glutathione-S-transferase (GST) or beta- galactosidase, relatively large co-protems which solubihze the protein and facilitate production and purification thereof.
  • the co-protem may act as an adjuvant in the sense of providing a generalized stimulation of the immune system.
  • compositions particularly vaccine compositions, and methods comprising the polypeptides or polynucleotides of the invention and immunostimulatory DNA sequences, such as those described in Sato, Y. et al. Science 273: 352 (1996).
  • polynucleotide or particular fragments thereof which have been shown to encode non-variable regions of bacterial cell surface proteins in DNA constructs used in such genetic immunization experiments in animal models of infection with Staphylococcus aureus will be particularly useful for identifying protein epitopes able to provoke a prophylactic or therapeutic immune response. It is believed that this approach will allow for the subsequent preparation of monoclonal antibodies of particular value from the requisite organ of the animal successfully resisting or clearing infection for the development of prophylactic agents or therapeutic treatments of bacte ⁇ al infection, particularly Staphylococcus aureus infection, m mammals, particularly humans
  • the polypeptide may be used as an antigen for vaccination of a host to produce specific antibodies which protect against invasion of bacteria, for example by blocking adherence of bacteria to damaged tissue.
  • tissue damage include wounds in skin or connective tissue caused, e.g., by mechanical, chemical or thermal damage or by implantation of indwelling devices, or wounds in the mucous membranes, such as the mouth, mammary glands, urethra or vagina.
  • the invention also includes a vaccine formulation which comprises an immunogenic recombinant protein of the invention together with a suitable carrier. Since the protein may be broken down in the stomach, it is preferably administered parenterally, including, for example, administration that is subcutaneous, intramuscular, intravenous, or mtradermal.
  • Formulations suitable for parenteral administration include aqueous and non- aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacte ⁇ ostats and solutes which render the formulation lnsoto c with the bodily fluid, preferably the blood, of the individual; and aqueous and non-aqueous sterile suspensions which may include suspending agents or thickening agents
  • the formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampules and vials and may be stored m a freeze-d ⁇ ed condition requiring only the addition of the sterile liquid carrier immediately prior to use
  • the vaccine formulation may also include adjuvant systems for enhancing the lmmunogenicity of the formulation, such as oil-m water systems and other systems known in the art. The dosage will depend on the specific activity of the vaccine and can be readily determined by routine experimentation.
  • the invention also relates to compositions comp ⁇ sing the polynucleotide or the polypeptides discussed above or their agonists or antagonists.
  • the polypeptides of the invention may be employed in combination with a non-sterile or sterile carrier or earners for use with cells, tissues or organisms, such as a pharmaceutical carrier suitable for administration to a subject.
  • Such compositions comprise, for instance, a media additive or a therapeutically effective amount of a polypeptide of the invention and a pharmaceutically acceptable earner or excipient.
  • earners may include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol and combinations thereof
  • the formulation should suit the mode of administration.
  • the invention further relates to diagnostic and pharmaceutical packs and kits comprising one or more containers filled with one or more of the ingredients of the aforementioned compositions of the invention.
  • Polypeptides and other compounds of the invention may be employed alone or in conjunction with other compounds, such as therapeutic compounds.
  • compositions may be administered m any effective, convenient manner including, for instance, administration by topical, oral, anal, vaginal, intravenous, mtrape ⁇ toneal, intramuscular, subcutaneous, mtranasal or mtradermal routes among others.
  • the active agent may be administered to an individual as an mjectable composition, for example as a sterile aqueous dispersion, preferably lsotomc.
  • the composition may be formulated for topical application for example in the form of ointments, creams, lotions, eye ointments, eye drops, ear drops, mouthwash, impregnated dressings and sutures and aerosols, and may contain appropriate conventional additives, including, for example, preservatives, solvents to assist drug penetration, and emollients in ointments and creams
  • Such topical formulations may also contain compatible conventional carriers, for example cream or ointment bases, and ethanol or oleyl alcohol for lotions.
  • Such ca ⁇ iers may constitute from about 1% to about 98% by weight of the formulation, more usually they will constitute up to about 80% by weight of the formulation.
  • the daily dosage level of the active agent will be from 0 01 mg/kg to 10 mg/kg, typically around 1 mg/kg
  • the physician m any event will determine the actual dosage which will be most suitable for an individual and will vary with the age, weight and response of the particular individual.
  • the above dosages are exemplary of the average case There can, of course, be individual instances where higher or lower dosage ranges are merited, and such are withm the scope of this invention.
  • In-dwelling devices include surgical implants, prosthetic devices and catheters, i.e., devices that are introduced to the body of an individual and remain in position for an extended time
  • Such devices include, for example, artificial joints, heart valves, pacemakers, vascular grafts, vascular catheters, cerebrospinal fluid shunts, urinary catheters, continuous ambulatory peritoneal dialysis (CAPD) catheters.
  • composition of the invention may be administered by injection to achieve a systemic effect against relevant bacteria shortly before insertion of an in-dwelling device Treatment may be continued after surgery during the m-body time of the device.
  • composition could also be used to broaden pe ⁇ operative cover for any surgical technique to prevent bacterial wound infections, especially Staphylococcus aureus wound infections.
  • compositions of this invention may be used generally as a wound treatment agent to prevent adhesion of bacteria to matrix proteins exposed in wound tissue and for prophylactic use in dental treatment as an alternative to, or m conjunction with, antibiotic prophylaxis.
  • composition of the invention may be used to bathe an indwelling device immediately before insertion
  • the active agent will preferably be present at a concentration of l ⁇ g/ml to lOmg/ml for bathing of wounds or indwelling devices.
  • a vaccine composition is conveniently in mjectable form. Conventional adjuvants may be employed to enhance the immune response.
  • a suitable unit dose for vaccination is 0.5-5 microgram/kg of antigen, and such dose is preferably administered 1-3 times and with an interval of 1-3 weeks. With the indicated dose range, no adverse toxicological effects will be observed with the compounds of the invention which would preclude their administration to suitable individuals
  • Example 1 Strain selection, Library Production and Sequencing
  • the polynucleotide having the DNA sequence given in SEQ ID NO:l was obtained from a library of clones of chromosomal DNA of Staphylococcus aureus in E. coli.
  • the sequencing data from two or more clones containing overlapping Staphylococcus aureus DNAs was used to construct the contiguous DNA sequence m SEQ ID NO: l .
  • Libraries may be prepared by routine methods, for example: Methods 1 and 2 below.
  • Total cellular DNA is mechanically sheared by passage through a needle in order to size-fractionate according to standard procedures.
  • DNA fragments of up to l lkbp in size are rendered blunt by treatment with exonuclease and DNA polymerase, and EcoRI linkers added. Fragments are hgated into the vector Lambda ZapII that has been cut with EcoRI, the library packaged by standard procedures and E coli infected with the packaged library.
  • the library is amplified by standard procedures
  • Method 2 Total cellular DNA is partially hydrolyzed with a one or a combination of restriction enzymes appropriate to generate a series of fragments for cloning into library vectors (e.g., Rsal, Pall, Alul, Bshl235I), and such fragments are size-fractionated according to standard procedures.
  • EcoRI linkers are hgated to the DNA and the fragments then hgated into the vector Lambda ZapII that have been cut with EcoRI, the library packaged by standard procedures, and E coli infected with the packaged library.
  • the library is amplified by standard procedures.
  • Example 2 PriA Characterization
  • Replication of the lagging strand of DNA is mediated by a multiprotein complex composed of proteins n , priA, n", dnaT, dnaB, dnaC, and dnaG.
  • This complex is referred to as a p ⁇ mosome.
  • Purified p ⁇ A has ATPase, hehcase, translocase, and p ⁇ mosome assembly activities (Lee EH, Masai H, Allen GC Jr, Kornberg A, Proc Natl Acad Sci USA 1990 Jun, 87 (12)- 4620-4624) (Sandier SJ, Samra HS, Clark AJ Genetics 1996 May;143(l):5-13).
  • This gene may be essential in recombination and DNA repair since it binds to D-loops, interacts with recG and has hehcase activity (McGlynn P, Al-Deib AA, Liu J, Marians KJ, Lloyd RG J Mol Biol 1997 Jul 11 ;270(2):212-221)
  • the 3'-5' DNAhehcase activity of priA inhibits recombination (Al-Deib AA, Mahdi AA, Lloyd RG Bacte ⁇ ol 1996 Dec;178(23):6782-6789).
  • Insertion mutants of priA are Rec- and UVS which is a function of its p ⁇ mosome assembly function (Sandier SJ, Samra HS, Clark AJ Genetics 1996 May;143(l):5-13). Null mutants of priA are also defective in transductional and conjugational recombination (Kogoma T, Cadwell GW, Barnard KG, Asai T J Bacte ⁇ ol 1996 Mar;178(5): 1258-1264). Null mutants can be suppressed by mutations mapping to dnaC.

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Abstract

The invention provides priA polypeptides and DNA (RNA) encoding priA polypeptides and methods for producing such polypeptides by recombinant techniques. Also provided are methods for utilizing priA polypeptides to screen for antibacterial compounds.

Description

NOVEL priA
FIELD OF THE INVENTION
This invention relates to newly identified polynucleotides and polypeptides, and their production and uses, as well as their vaπants, agonists and antagonists, and their uses. In particular, m these and m other regards, the invention relates to polynucleotides and polypeptides of the pπmosome family, hereinafter referred to as "priA"
BACKGROUND OF THE INVENTION It is particularly preferred to employ Staphylococcal genes and gene products as targets for the development of antibiotics. The Staphylococci make up a medically important genera of microbes They are known to produce two types of disease, invasive and toxigenic. Invasive infections are characteπzed generally by abscess formation effecting both skm surfaces and deep tissues S aureus is the second leading cause of bacteremia in cancer patients. Osteomyelitis, septic arthritis, septic thrombophlebitis and acute bacterial endocarditis are also relatively common. There are at least three clinical conditions resulting from the toxigenic properties of Staphylococci. The manifestation of these diseases result from the actions of exotoxins as opposed to tissue invasion and bacteremia These conditions include: Staphylococcal food poisoning, scalded skin syndrome and toxic shock syndrome. The frequency of Staphylococcus aureus infections has πsen dramatically in the past
20 years. This has been attributed to the emergence of multiply antibiotic resistant strains and an increasing population of people with weakened immune systems. It is no longer uncommon to isolate Staphylococcus aureus strains which are resistant to some or all of the standard antibiotics. This has created a demand for both new anti-microbial agents and diagnostic tests for this organism.
Clearly, there is a need for factors, such as the compounds of the invention, that have a present benefit of being useful to screen compounds for antibiotic 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 characterization of such factors and their antagonists and agonists which can play a role in preventing, ameliorating or correcting infections, dysfunctions or diseases.
The polypeptides of the invention have ammo acid sequence homology to a known priA in B. subhlis (p94461), H. influenzae (P44647), E. coh (P17888), R. rubrum (P05445), and a low sequence homology to S. cerevisiae (p20449) protein. SUMMARY OF THE INVENTION
It is an object of the invention to provide polypeptides that have been identified as priA polypeptides of the invention by homology between the ammo acid sequence set out in Table 1 [SEQ ID NO: 2] and a known ammo acid sequence or sequences of other proteins such as pπA in B subtihs (p94461), H influenzae (P44647), E coh (P17888), R rubrum
(P05445), and a low sequence homology to S cerevisiae (p20449) protein.
It is a further object of the invention to provide polynucleotides that encode priA polypeptides, particularly polynucleotides that encode the polypeptide herein designated priA In a particularly preferred embodiment of the invention the polynucleotide comprises a region encoding priA polypeptides comprising the sequence set out m Table 1 [SEQ ID NO:l], or a variant thereof.
In another particularly preferred embodiment of the invention there is a priA protein from Staphylococcus aureus comprising the ammo acid sequence of Table 1 [SEQ ID NO.2], or a variant thereof.
In accordance with 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 contained in the deposited strain.
A further aspect of the invention there are provided isolated nucleic acid molecules encoding priA, particularly Staphylococcus aureus priA, including mRNAs, cDNAs, genomic
DNAs. Further embodiments of the invention include biologically, diagnostically, prophylactically, clinically or therapeutically useful variants thereof, and compositions comprising the same.
In accordance with another aspect of the invention, there is provided the use of a polynucleotide of the invention for therapeutic or prophylactic purposes, in particular genetic immunization. Among the particularly preferred embodiments of the invention are naturally occurring allehc vaπants of pπA and polypeptides encoded thereby.
Another aspect of the invention there are provided polypeptides of Staphylococcus aureus referred to herein as priA as well as biologically, diagnostically, prophylactically, clinically or therapeutically useful variants thereof, and compositions compπsmg the same.
Among the particularly preferred embodiments of the invention are vaπants of priA polypeptide encoded by naturally occurring alleles of the pπA gene.
In a preferred embodiment of the invention there are provided methods for producing the aforementioned priA polypeptides. In accordance with yet another aspect of the invention, there are provided inhibitors to such polypeptides, useful as antibacterial agents, including, for example, antibodies.
In accordance with certain preferred embodiments of the invention, there are provided products, compositions and methods for assessing priA expression, treating disease, for example, 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, retropeπtoneal abscess), CNS (e g , cerebral abscess), eye (e.g., blepharitis, conjunctivitis, keratitis, endophthalmitis, preseptal and orbital celluhtis, darcryocystitis), kidney and urinary tract (e.g., epididymitis, mtrarenal and peπnephπc absces, toxic shock syndrome), skm (e.g., impetigo, follicuhtis, cutaneous abscesses, celluhtis, wound infection, bacteπal myositis) bone and joint (e g., septic arthritis, osteomyelitis), assaying genetic variation, and administering a pπA polypeptide or polynucleotide to an organism to raise an immunological response against a bacteria, especially a Staphylococcus aureus bacteria In accordance with certain preferred embodiments of this and other aspects of the invention there are provided polynucleotides that hybridize to priA polynucleotide sequences, particularly under stringent conditions.
In certain preferred embodiments of the invention there are provided antibodies against priA polypeptides. In other embodiments of the invention there are provided methods for identifying compounds which bind to or otherwise interact with and inhibit or activate an activity of a polypeptide or polynucleotide of the invention comprising: contacting a polypeptide 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 or polynucleotide to assess the binding to or other interaction with the compound, such binding or interaction being associated with a second component capable of providing a detectable signal in response to the binding or interaction of the polypeptide or polynucleotide with the compound; and determining whether the compound binds to or otherwise interacts with and activates or inhibits an activity of the polypeptide or polynucleotide by detecting the presence or absence of a signal generated from the binding or interaction of the compound with the polypeptide or polynucleotide.
In accordance with yet another aspect of the invention, there are provided pnA agonists and antagonists, preferably bacteπostatic or bacteriocidal agonists and antagonists. In a further aspect of the invention there are provided compositions comprising a priA polynucleotide or a priA polypeptide for administration to a cell or to a multicellular organism.
Various changes and modifications within the spirit and scope of the disclosed invention will become readily apparent to those skilled in the art from reading the following descriptions and from reading the other parts of the present disclosure.
GLOSSARY
The following definitions are provided to facilitate understanding of certain terms used frequently herein.
"Host cell" is a cell which has been transformed or transfected, or is capable of 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; Biocoinputing: 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, J., et al., Nucleic Acids Research 12(1): 387 (1984)), BLASTP, BLASTN, and FASTA (Altschul, S.F. et al., J. Molec. Biol. 215: 403-410 (1990). The BLAST X program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S., et al, NCBI NLM NIH Bethesda, MD 20894;
Altschul, S., et al, J. Mol. Biol. 215: 403-410 (1990). The well known Smith
Waterman algorithm may also be used to determine identity.
Parameters for polypeptide sequence 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. 89: 10915-10919 (1992)
Gap Penalty: 12
Gap Length Penalty: 4 A program useful with these parameters is publicly available as the "gap" program from
Genetics Computer Group, Madison WI. The aforementioned parameters are the default parameters for peptide comparisons (along with no penalty for end gaps).
Parameters for polynucleotide comparison include the following' 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 preferred meaning for "identity" for polynucleotides and polypeptides, as the case may be, are provided m (1) and (2) below.
(1) Polynucleotide embodiments further include an isolated polynucleotide comprising a polynucleotide sequence having at least a 50, 60, 70, 80, 85, 90, 95, 97 or 100% identity to the reference sequence of SEQ ID NO. l, wherein said polynucleotide sequence may be identical to the reference sequence of SEQ ID NO: 1 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 NO: l by the integer defining the percent identity divided by 100 and then subtracting that product from said total number of nucleotides in SEQ ID NO: 1, or:
nn < xn - (xn • y),
wherein nn is the number of nucleotide alterations, xn is the total number of nucleotides in SEQ ID NO: l, y is 0.50 for 50%, 0.60 for 60%, 0.70 for 70%, 0.80 for 80%, 0.85 for 85%, 0.90 for 90%, 0.95 for 95%, 0.97 for 97% or 1.00 for 100%, 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 the polypeptide of SEQ ID NO:2 may create nonsense, missense or frameshift mutations in this coding sequence and thereby alter the polypeptide encoded by the polynucleotide following such alterations.
By way of example, a polynucleotide sequence of the present invention may be identical to the reference sequence of SEQ ID NO:l, that is it may be 100% identical, or it may include up to a certain integer number of nucleic acid alterations as compared to the reference sequence such that the percent identity is less than 100% identity. Such alterations are selected from the group consisting of at least one nucleic acid deletion, substitution, including transition and transversion, or insertion, and wherein said alterations may occur at the 5' or 3' terminal positions of the reference polynucleotide sequence or anywhere between those terminal positions, interspersed either individually among the nucleic acids in the reference sequence or in one or more contiguous groups within the reference sequence. The number of nucleic acid alterations for a given percent identity is determined by multiplying the total number of nucleic acids in SEQ ID NO: l by the integer defining the percent identity divided by 100 and then subtracting that product from said total number of nucleic acids in SEQ ID NO: 1, or:
nn ≤ xn (χ n • y)> wherein nn is the number of nucleic acid alterations, xn is the total number of nucleic acids in SEQ ID NO 1, y is, for instance 0 70 for 70%, 0 80 for 80%, 0 85 for 85% etc , • is the symbol for the multiplication operator, and wherein any non-integer product of xn and y is rounded down to the nearest integer prior to subtracting it from xn (2) Polypeptide embodiments further include an isolated polypeptide comprising a polypeptide having at least a 50,60, 70, 80, 85, 90, 95, 97 or 100% identity to a polypeptide reference sequence of SEQ ID NO 2, wherein said polypeptide sequence may be identical to the reference sequence of SEQ ID NO 2 or may include up to a certain integer number of ammo acid alterations as compared to the reference sequence, wherein said alterations are selected from the group consisting of at least one ammo acid deletion, substitution, including conservative and non-conservative substitution, or insertion, and wherein said alterations may occur at the ammo- or carboxy-termmal 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 ammo acid alterations is determined by multiplying the total number of ammo acids in SEQ ID NO 2 by the integer defining the percent identity divided by 100 and then subtracting that product from said total number of ammo acids in SEQ ID NO 2, or
na < xa - (xa • y),
wherein na is the number of ammo acid alterations, xa is the total number of ammo acids in SEQ ID NO 2, y is 0 50 for 50%, 0 60 for 60%, 0 70 for 70%, 0 80 for 80%, 0 85 for 85%, 0 90 for 90%, 0 95 for 95%, 0 97 for 97% or 1 00 for 100%, and • is the symbol for the multiplication operator, and wherein any non-mteger product of xa and y is rounded down to the nearest integer prior to subtracting it from xa
By way of example, a polypeptide sequence of the present invention may be identical to the reference sequence of SEQ ID NO 2, that is it may be 100% identical, or it may include up to a certain integer number of ammo acid alterations as compared to the reference sequence such that the percent identity is less than 100% identity Such alterations are selected from the group consisting of at least one ammo acid deletion, substitution, including conservative and non-conservative substitution, or insertion, and wherein said alterations may occur at the ammo- or carboxy-termmal 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. The number of ammo acid alterations for a given % identity is determined by multiplying the total number of ammo acids in SEQ ID NO: 2 by the integer defining the percent identity divided by 100 and then subtracting that product from said total number of ammo acids in SEQ ID NO:2, or:
na < xa - (xa • y),
wherein na is the number of ammo acid alterations, xa is the total number of amino acids in SEQ ID NO:2, y is, for instance 0 70 for 70%, 0.80 for 80%, 0.85 for 85% etc., and • is the symbol for the multiplication operator, and wherein any non-mteger product of xa and y is rounded down to the nearest integer prior to subtracting it from xa.
"Isolated" means altered "by the hand of man" from its natural state, i e , if it occurs in nature, it has been changed or removed from its original environment, or both For example, a polynucleotide or a polypeptide naturally present in a living organism is not "isolated," but the same polynucleotide or polypeptide separated from the coexisting mateπals of its natural state is "isolated", as the term is employed herein.
"Polynucleotιde(s)" generally refers to any polyπbonucleotide or polydeoxπbonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. "Polynucleotιde(s)" include, without limitation, single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions or single-, double- and tπple-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double- stranded regions, hybπd molecules compπsmg DNA and RNA that may be single-stranded or, more typically, double-stranded, or tπple-stranded regions, or a mixture of single- and double- stranded regions In addition, "polynucleotide" as used herein refers to tπple-stranded regions comprising RNA or DNA or both RNA and DNA. The strands m 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 tπple-hehcal region often is an ohgonucleotide. As used herein, the term "polynucleotιde(s)" also includes DNAs or RNAs as described above that contain one or more modified bases. Thus, DNAs or RNAs with backbones modified for stability or for other reasons are "polynucleotιde(s)" as that term is intended herein. Moreover, DNAs or RNAs comprising unusual bases, such as mosme, or modified bases, such as tπtylated 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 "polynucleotιde(s)" as it is employed herein embraces such chemically, enzymatically or metabohcally modified forms of polynucleotides, as well as the chemical forms of DNA and RNA characteπstic of viruses and cells, including, for example, simple and complex cells. "Polynucleotιde(s)" also embraces short polynucleotides often referred to as ohgonucleotιde(s)
"Polypeptιde(s)" refers to any peptide or protein comprising two or more ammo acids joined to each other by peptide bonds or modified peptide bonds "Polypeptιde(s)" refers to both short chains, commonly referred to as peptides, ohgopeptides and ohgomers and to longer chains generally referred to as proteins. Polypeptides may contain ammo acids other than the 20 gene encoded ammo acids. "Polypeptιde(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 m more detailed monographs, as well as in a voluminous research literature, and they are well known to those of skill m the art. It will be appreciated that the same type of modification may be present in the same or varying degree at several sites in a given polypeptide. Also, a given polypeptide may contain many types of modifications. Modifications can occur anywhere in a polypeptide, including the peptide backbone, the ammo acid side-chains, and the amino or carboxyl termini. Modifications include, for example, acetylation, acylation, ADP-πbosylation, amidahon, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a hpid or lipid deπvative, covalent attachment of phosphotidyhnositol, cross-linking, cychzation, disulfide bond formation, demethylation, formation of covalent cross-lmks, formation of cysteme, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, lodination, methylation, myπstoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, glycosylation, hpid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP-πbosylation, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins, such as arginylation, and ubiquitmation. 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 Modifications1 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., Protein Synthesis Posttranslational Modifications and Aging, Ann. N.Y 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.
"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 m amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence, as discussed below. A typical variant of a polypeptide differs m amino acid sequence from another, reference polypeptide. Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, in many regions, identical. A variant and reference polypeptide may differ in ammo acid sequence by one or more substitutions, additions, deletions m any combination. A substituted or inserted ammo acid residue may or may not be one encoded by the genetic code. A variant of a polynucleotide or polypeptide may be a naturally occurring such as an allehc variant, or it may be a variant that is not known to occur naturally. Non-naturally occuπing variants of polynucleotides and polypeptides may be made by mutagenesis techniques, by direct synthesis, and by other recombinant methods known to skilled artisans.
DESCRIPTION OF THE INVENTION The invention relates to priA polypeptides and polynucleotides as descπbed in greater detail below. In particular, the invention relates to polypeptides and polynucleotides of a pπA of Staphylococcus aureus, which is related by ammo acid sequence homology to pπA in B subtihs (p94461), H influenzae (P44647), E. coh (P17888), R rubrum (P05445), and a low sequence homology to S cerevisiae (p20449) polypeptide. The invention relates especially to priA having the nucleotide and amino acid sequences set out in Table 1 [SEQ ID NO: 1] and Table 1 [SEQ ID NO: 2] respectively, and to the priA nucleotide sequences of the DNA in the deposited strain and amino acid sequences encoded thereby.
TABLE 1
PriA Polynucleotide and Polypeptide Sequences
(A) Sequences from Staphylococcus aureus priA polynucleotide sequence [SEQ ID NO:l]. 5 ' -
TGGCATTTTAAAAATTAAATGAAAAGGGTAAATTTTCAAGGAAAGGGAAAAAAAAGGTAGTATTTAGC
GGCCCAAATTTT
TAGATGACCTAGAAACTAGGTGCCAATAATGATAGCGAAAAGTCATAGTCGATGTCGCGTCGAAGAGC
GTTGACTATAAA TTTGATTATATAATTTCCCGAACAACTCGAATCTGTCATCCAACCTGGTGTGCGTGTAATTGTACCTT
TTGGACCAAGAA
CGATTCAAGGTTATGTAATGGAAGTAACAGCAGAACCTGATGCACAACTTGACGTTTCGAAGTTAAAA
AAAATCATAGAA
GTGAAAGATATAC ACCAGAATTAACATCAGAAT AATAGCTTTAAGTGAGTGGATGGGTTCAACTCA TGTCATTAAACG
TATTTCTATGCTAGAAGTGATGCTTCCAAGTGCTATTAAAGCGAAGTATAAAAAAGCATTTAAGATGA
AAGATGACATAG
AGCTACCTTCAGCTTTATTACAAAAATTTGATAAGCATGGTTACTATTATTATAAAGATGCGCAAAAA
AATAATGAT TT CAATTGCTTATGAAGTTGTTAAAAGATGATATCGTTGAAGAAAAAACGATTCTCACACAAAATATAAC
TAAAAAAACCAA
GCGTGCTGTTCGTGTCATTGAAGGGTATCATCCTGATGAAGTATTAGCTAAGTTGGAGAAAGTTATTA
AACAATACGATT
TGTATGCTTACTTGTCTGAAGAACAACATAAAACAATATTTTTAACTGATATTGAGGATATGGGCTTT TCAAAATCCAGT
TTAGATGGACTTATCAAAAAAGGTTATGTTGAAAAATATGACGCGGTTGTTGAAAGAGACCCATTTAA
AGATCGTGTTTT
CGAACAAGAATCAAAACAGCAATTAACAGAAGACCAATATAAAGCATATGAAGCGATTAAAGCTAAAA
TTGTAAGCCAAG AGCAAGAAACATTTTTACTTCATGGTGTGACGGGATCAGGTAAAACAGAAGTATATTTACAAACGATA
GAAGATGTTTTA
AGCCAAGGAAAACAGGCGATGATGTTAGTTCCTGAAATCGCTCTAACACCGCAAATGGTTTTACGCTT
CAAACGTCGATT
TGGTGATGACGTTGCTGTATTACATTCTGGCTTATCTAATGGGGAACGTTATGATGAGTGGCAAAAAA TTAGGGATGATC GTGCGAGAGTAAGTGTTGGTGCAAGGTCAAGTGTGTTCGCACCTTTCAAAAATTTAGGGTTAATCATC
ATTGATGAAGAA
CATGAATCTACATATAAACAAGAAGATTATCCGAGATATCACGCTAGAGAAATTGCCCAATGGCGAAG
TGAATATCATCA CTGTCCAGTCATTTTAGGAAGTGCAACACCATGTCTTGAAAGTTATGCACGAGCTGAAAAAGACGTTT
ATCATTTGCTAT
CATTACCAAACAGAGTGAACCAACAAGCTTTACCTGAAATTGATATAGTAGACATGCGTGAAGAATTG
AGTGAAGGTAAT
CGGTCAATGTTTTCAAAAGATTTACGTGAAGCCATACAATTAAGATTAGATCGACAGGAACAAGTTGT TTTATTTTTAAA
TCGACGTGGTTATGCATCGTTTATGTTATGTCGGGATTGTGGATATGTACCGCAATGTCCAAACTGTG
ATATTTCATTAA
CGTATCATAAAACGACAGACTTATTAAAATGTCACTATTGTGGTTACCAAGAGACGCCACCGAATCAA
TGTCCAAATTGT GAGAGTGAACACATTCGACAAGTAGGTACTGGTACTCAGAAAGTTGAAGAACTATTGCAACAAGAATT
TGAAGATGCGCG
CATAATTAGGATGGATGTAGATACAACCTCAAAGAAAGGTGCACATGAAAAGTTATTGACTGAATTCG
AAAAAGGTAACG
GTGACATTTTACTAGGTACTCAGATGATTGCGAAAGGATTAGATTATCCAAATATTACTTTAGTTGGT GTGCTGAATGCA
GATACAATGTTAAATTTACCTGATTTTCGGGCGAGCGAACGTACTTATCAACTATTAACGCAAGTGGC
TGGTAGAGCTGG
TCGTCATGAAAAGGCAGGTCAAGTCATCATTCAAACGTATAATCCAGATCATTATTCAATATTGGATG
TTCAAAAAAATG ATTATTTAACATTTTATCGTCAGGAAATGGAATATCGTAAATTAGGAAAGTATCCACCGTATTATTAT
TTGATTAATTTC
ACAATCTCACATAAAGAAATGAAGAAGGTTATGGAAGCATCGCAGCATGTTCATAAAATTTTATTACA
GCATTTAACAGA
AAAAGCGCTTGTACTAGGTCCATCTCCGGCAGCACTTGCGAGAATCAACAATGAATTTAGATTCCAAA TTTTAGTGAAAT
ATAAAAGTGAACCTGGATTATTACAAGCCATTCAGTTTTTAGATGACTATTACCATGAAAAATTTATA
AAAGAAAAATTA
GCATTGAAGATTGATATTGATCCACAGATGATGATGTAACATTACTAATTATTAGAAACAAGTTCAGT
ATGTACGAGTAT TTGAACCAGTGTTTGAATATTTACTTTAAGTACAGAAAAAGGGCAGAATATACAACTGTTAACTATTT
AAATTAGCAGTT
TATATTCTGTCTTTTTATATGGCTTTATAACTTACGTGATTTTGGTTTGATAAGGAATTTATTAGTAT
TTTCATTTACGA
ATTCTGATTTTAATGCAAGCGCCATACTTACTGATGAATAAAAGTACTAATGATTAAACAAACTATTT TTAAATGGCAAA
TTGAAATAGTGCAAATTTTACACTTATAAATTAATGGCTATAAACTATTTTATATCTATCTATGTTTT
AACTACTTAAAT
TATTAGACAAAACAATTTTCTTAAATTATCAATTAAAGTATTTAAAAATATTGATGGTTAATGTAAAA
TATCAATATAAA TAAGTTTTTTAAATTTATGTATGTTTATTTGATTCAAACAAAATAACTTAAGAGGAGAAAGTTTATGA
AAAAGACACTGG
GATGTTTACTTTTAATTATGCTTTTAGTCGTAGCAGGTTGTTCTTTTGGTGGGAATCATAAATTATCA
TCAAAGAAATCA GAAGAATCAAAACAAGAAACTGTAAAAAAAGAATCGGAAGAAGAGAAAGATCCAGATTTAGAGAAATA
TGAAGAAATAGA
GAAGAAAATGAAAGGAATTAAAGATGCGCCATCTCTTGATAAGTTGGATCCATTAATGACAGAAAAGT
CGTTTACGAATA
GTAAAGGGATTCAAGGATGGAAAGATTACAAAGAATTAATGGGTAAAGTGGAACTTGCAGATTATAGA TTTACTAAAGAT
TCAAAAGGATCTTCAATAAAAGATGTTGATGCATTCTTTAAAGGTAAGAAAGGTATAAAAAGGAAAGT
GATTGAAACACA
CGATGATGTAAAACAAGTTGATTATTGGTATGTACATCCAGATGGAAAGAAAATTGGCAATTCAAACA
CACCTGTTTTTT ACGCAGAAATTATGACAAAATATAAAGATGGAAAGTTAGTTTATGCATCAGTCGAACCAGGATCTTAC
GTAATACATAAA
GATGATGCAATTAAATATGACGATTATTCTAAGTTAAAAAAATTAAGCCAGCTAACTAAACTTGATCA
TCCAAAACCAGT
TCCCATATAGCGTAACTCAAATCAAATCTGTCCGAATACCTCTTAACAAGCGTTTCCATTTATGACAC ATGGATCACAGA
ATACTAAAATAATGTTTGCCCGCATTGCCTTATTCAC
(B) PriA polypeptide sequence deduced from the polynucleotide sequence in this table [SEQ ID NO.2].
NH2-
PEQLESVIQPGVRVIVPFGPRTIQGYVMEVTAEPDAQLDVSKLKKIIEVKDIQPELTSELIALSEWMG
STHVIKRISMLE VMLPSAIKAKYKKAFKMKDDIELPSALLQKFDKHGYYYYKDAQKNNDIQLLMKLLKDDIVEEKTILTQ
NITKKTKRAVRV
IEGYHPDEVLAKLEKVIKQYDLYAYLSEEQHKTIFLTDIEDMGFSKSSLDGLIKKGYVEKYDAVVERD
PFKDRVFEQESK
QQLTEDQYKAYEAIKAKIVSQEQETFLLHGVTGSGKTEVYLQTIEDVLSQGKQAMMLVPEIALTPQMV LRFKRRFGDDVA
VLHSGLSNGERYDE QKIRDDRARVSVGARSSVFAPFKNLGLIIIDEEHESTYKQEDYPRYHAREIAQ RSEYHHCPVIL
GSATPCLESYARAEKDVYHLLSLPNRVNQQALPEIDIVDMREELSEGNRSMFSKDLREAIQLRLDRQE
QVVLFLNRRGYA SFMLCRDCGYVPQCPNCDISLTYHKTTDLLKCHYCGYQETPPNQCPNCESEHIRQVGTGTQKVEELLQ
QEFEDARIIRMD
VDTTSKKGAHEKLLTEFEKGNGDILLGTQMIAKGLDYPNITLVGVLNADTMLNLPDFRASERTYQLLT
QVAGRAGRHEKA GQVIIQTYNPDHYSILDVQKNDYLTFYRQEMEYRKLGKYPPYYYLINFTISHKEMKKVMEASQHVHKI
LLQHLTEKALVL GPSPAALARINNEFRFQILVKYKSEPGLLQAIQFLDDYYHEKFIKEKLALKIDIDPQMMM-COOH
(C) Polynucleotide sequence embodiments [SEQ ID NO: l]. χ - (Rl > n-
TGGCATTTTAAAAATTAAATGAAAAGGGTAAATTTTCAAGGAAAGGGAAAAAAAAGGTAGTATTTAGC
GGCCCAAATTTT
TAGATGACCTAGAAACTAGGTGCCAATAATGATAGCGAAAAGTCATAGTCGATGTCGCGTCGAAGAGC GTTGACTATAAA
TTTGATTATATAATTTCCCGAACAACTCGAATCTGTCATCCAACCTGGTGTGCGTGTAATTGTACCTT
TTGGACCAAGAA
CGATTCAAGGTTATGTAATGGAAGTAACAGCAGAACCTGATGCACAACTTGACGTTTCGAAGTTAAAA
AAAATCATAGAA GTGAAAGATATACAACCAGAATTAACATCAGAATTAATAGCTTTAAGTGAGTGGATGGGTTCAACTCA
TGTCATTAAACG
TATTTCTATGCTAGAAGTGATGCTTCCAAGTGCTATTAAAGCGAAGTATAAAAAAGCATTTAAGATGA
AAGATGACATAG
AGCTACCTTCAGCTTTATTACAAAAATTTGATAAGCATGGTTACTATTATTATAAAGATGCGCAAAAA AATAATGATATT
CAATTGCTTATGAAGTTGTTAAAAGATGATATCGTTGAAGAAAAAACGATTCTCACACAAAATATAAC
TAAAAAAACCAA
GCGTGCTGTTCGTGTCATTGAAGGGTATCATCCTGATGAAGTATTAGCTAAGTTGGAGAAAGTTATTA
AACAAT CGATT TGTATGCTTACTTGTCTGAAGAACAACATAAAACAATATTTTTAACTGATATTGAGGATATGGGCTTT
TCAAAATCCAGT
TTAGATGGACTTATCAAAAAAGGTTATGTTGAAAAATATGACGCGGTTGTTGAAAGAGACCCATTTAA
AGATCGTGTTTT
CGAACAAGAATCAAAACAGCAATTAACAGAAGACCAATATAAAGCATATGAAGCGATTAAAGCTAAAA TTGTAAGCCAAG
AGCAAGAAACATTTTTACTTCATGGTGTGACGGGATCAGGTAAAACAGAAGTATATTTACAAACGATA
GAAGATGTTTTA
AGCCAAGGAAAACAGGCGATGATGTTAGTTCCTGAAATCGCTCTAACACCGCAAATGGTTTTACGCTT
CAAACGTCGATT TGGTGATGACGTTGCTGTATTACATTCTGGCTTATCTAATGGGGAACGTTATGATGAGTGGCAAAAAA
TTAGGGATGATC
GTGCGAGAGTAAGTGTTGGTGCAAGGTCAAGTGTGTTCGCACCTTTCAAAAATTTAGGGTTAATCATC
ATTGATGAAGAA
CATGAATCTACATATAAACAAGAAGATTATCCGAGATATCACGCTAGAGAAATTGCCCAATGGCG AG TGAATATCATCA
CTGTCCAGTCATTTTAGGAAGTGCAACACCATGTCTTGAAAGTTATGCACGAGCTGAAAAAGACGTTT
ATCATTTGCTAT CATTACCAAACAGAGTGAACCAACAAGCTTTACCTGAAATTGATATAGTAGACATGCGTGAAGAATTG
AGTGAAGGTAAT
CGGTCAATGTTTTCAAAAGATTTACGTGAAGCCATACAATTAAGATTAGATCGACAGGAACAAGTTGT
TTTATTTTTAAA TCGACGTGGTTATGCATCGTTTATGTTATGTCGGGATTGTGGATATGTACCGCAATGTCCAAACTGTG
ATATTTCATTAA
CGTATCATAAAACGACAGACTTATTAAAATGTCACTATTGTGGTTACCAAGAGACGCCACCGAATCAA
TGTCCAAATTGT
GAGAGTGAACACATTCGACAAGTAGGTACTGGTACTCAGAAAGTTGAAGAACTATTGCAACAAGAATT TGAAGATGCGCG
CATAATTAGGATGGATGTAGATACAACCTCAAAGAAAGGTGCACATGAAAAGTTATTGACTGAATTCG
AAAAAGGTAACG
GTGACATTTTACTAGGTACTCAGATGATTGCGAAAGGATTAGATTATCCAAATATTACTTTAGTTGGT
GTGCTGAATGCA GATACAATGTTAAATTTACCTGATTTTCGGGCGAGCGAACGTACTTATCAACTATTAACGCAAGTGGC
TGGTAGAGCTGG
TCGTCATGAAAAGGCAGGTCAAGTCATCATTCAAACGTATAATCCAGATCATTATTCAATATTGGATG
TTCAAAAAAATG
ATTATTTAACATTTTATCGTCAGGAAATGGAATATCGTAAATTAGGAAAGTATCCACCGTATTATTAT TTGATTAATTTC
ACAATCTCACATAAAGAAATGAAGAAGGTTATGGAAGCATCGCAGCATGTTCATAAAATTTTATTACA
GCATTTAACAGA
AAAAGCGCTTGTACTAGGTCCATCTCCGGCAGCACTTGCGAGAATCAACAATGAATTTAGATTCCAAA
TTTTAGTGAAAT ATAAAAGTGAACCTGGATTATTACAAGCCATTCAGTTTTTAGATGACTATTACCATGAAAAATTTATA
AAAGAAAAATTA
GCATTGAAGATTGATATTGATCCACAGATGATGATGTAACATTACTAATTATTAGAAACAAGTTCAGT
ATGTACGAGTAT
TTGAACCAGTGTTTGAATATTTACTTTAAGTACAGAAAAAGGGCAGAATATACAACTGTTAACTATTT AAATTAGCAGTT
TATATTCTGTCTTTTTATATGGCTTTATAACTTACGTGATTTTGGTTTGATAAGGAATTTATTAGTAT
TTTCATTTACGA
ATTCTGATTTTAATGCAAGCGCCATACTTACTGATGAATAAAAGTACTAATGATTAAACAAACTATTT
TTAAATGGCAAA TTGAAATAGTGCAAATTTTACACTTATAAATTAATGGC ATAAACTATTTTATATCTATCTATGTTTT
AACTACTTAAAT
TATTAG CAAAACAATTTTCTTAAATTATCAATTAAAGTATTTAAAAATATTGATGGTTAATGTAAAA
TATCAATATAAA
TAAGTTTTTTAAATTTATGTATGTTTATTTGATTCAAACAAAATAACTTAAGAGGAGAAAGTTTATGA AAAAGACACTGG
GATGTTTACTTTTAATTATGCTTTTAGTCGTAGCAGGTTGTTCTTTTGGTGGGAATCATAAATTATCA
TCAAAGAAATCA
GAAGAATCAAAACAAGAAACTGTAAAAAAAGAATCGGAAGAAGAGAAAGATCCAGATTTAG GAAATA
TGAAGAAATAGA GAAGAAAATGAAAGGAATTAAAGATGCGCCATCTCTTGATAAGTTGGATCCATTAATGACAGAAAAGT
CGTTTACGAATA
GTAAAGGGATTCAAGGATGGAAAGATTACAAAGAATTAATGGGTAAAGTGGAACTTGCAGATTATAGA
TTTACTAAAGAT TCAAAAGGATCTTCAATAAAAGATGTTGATGCATTCTTTAAAGGTAAGAAAGGTATAAAAAGGAAAGT
GATTGAAACACA
CGATGATGTAAAACAAGTTGATTATTGGTATGTACATCCAGATGGAAAGAAAATTGGCAATTCAAACA
CACCTGTTTTTT
ACGCAGAAATTATGACAAAATATAAAGATGGAAAGTTAGTTTATGCATCAGTCGAACCAGGATCTTAC GTAATACATAAA
GATGATGCAATTAAATATGACGATTATTCTAAGTTAAAAAAATTAAGCCAGCTAACTAAACTTGATCA
TCCAAAACCAGT
TCCCATATAGCGTAACTCAAATCAAATCTGTCCGAATACCTCTTAACAAGCGTTTCCATTTATGACAC
ATGGATCAC GA ATACTAAAATAATGTTTGCCCGCATTGCCTTATTCAC
(R 2a)' -
(D) Polypeptide sequence embodiments [SEQ ID NO:2]. x- (Rι) n-
PEQLESVIQPGVRVIVPFGPRTIQGYVMEVTAEPDAQLDVSKLKKIIEVKDIQPELTSELIALSE MG
STHVIKRISMLE
VMLPSAIKAKYKKAFKMKDDIELPSALLQKFDKHGYYYYKDAQKNNDIQLLMKLLKDDIVEEKTILTQ
NITKKTKRAVRV IEGYHPDEVLAKLEKVIKQYDLYAYLSEEQHKTIFLTDIEDMGFSKSSLDGLIKKGYVEKYDAVVERD
PFKDRVFEQESK
QQLTEDQYKAYEAIKAKIVSQEQETFLLHGVTGSGKTEVYLQTIEDVLSQGKQAMMLVPEIALTPQMV
LRFKRRFGDDVA
VLHSGLSNGERYDE QKIRDDRARVSVGARSSVFAPFKNLG IIIDEEHESTYKQEDYPRYHAREIAQ WRSEYHHCPVIL
GSATPCLESYARAEKDVYHLLSLPNRVNQQALPEIDIVDMREELSEGNRSMFSKDLREAIQLRLDRQE
QVVLFLNRRGYA
SFMLCRDCGYVPQCPNCDISLTYHKTTDLLKCHYCGYQETPPNQCPNCESEHIRQVGTGTQKVEELLQ
QEFEDARIIRMD VDTTSKKGAHEKLLTEFEKGNGDILLGTQMIAKGLDYPNITLVGVLNADTMLNLPDFRASERTYQLLT
QVAGRAGRHEKA
GQVIIQTYNPDHYSILDVQKNDYLTFYRQEMEYRK GKYPPYYYLINFTISHKEMKKVMEASQHVHKI
LLQHLTEKALVL GPSPAALARINNEFRFQILVKYKSEPGL QAIQFLDDYYHEKFIKEKLALKIDIDPQMMM- (R2 ) n-Y
Deposited materials A deposit containing a Staphylococcus aureus WCUH 29 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 11 September 1995 and assigned NCIMB
Deposit No. 40771 The Staphylococcus aureus strain deposit is referred to herein as "the deposited strain" or as "the DNA of the deposited strain."
The deposited strain contains the full length priA gene. The sequence of the polynucleotides contained in the deposited strain, as well as the ammo acid sequence of the 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 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. §112.
A license may be required to make, use or sell the deposited strain, and compounds derived therefrom, and no such license is hereby granted. Polypeptides
The polypeptides of the invention include the polypeptide of Table 1 [SEQ ID NO:2] (in particular the mature polypeptide) as well as polypeptides and fragments, particularly those which have the biological activity of priA, and also those which have at least 70% identity to the polypeptide of Table 1 [SEQ ID NO:2] or the relevant portion, preferably at least 80% identity to the polypeptide of Table 1 [SEQ ID NO:2], and more preferably at least 90% similarity (more preferably at least 90% identity) to the polypeptide of Table 1 [SEQ ID NO:2] and still more preferably at least 95% similaπty (still more preferably at least 95% identity) to the polypeptide of Table 1 [SEQ ID NO: 2] and also include portions of such polypeptides with such portion of the polypeptide generally containing at least 30 ammo acids and more preferably at least 50 ammo acids.
The invention also includes polypeptides of the formula set forth in Table 1 (D) wherein, at the amino terminus, X is hydrogen, and at the carboxyl terminus, Y is hydrogen or a metal, Ri and R2 is any amino acid residue, and n is an integer between 1 and 1000. Any stretch of ammo acid residues denoted by either R group, where R is greater than 1, may be either a heteropolymer or a homopolymer, preferably a heteropolymer. A fragment is a vaπant polypeptide having an ammo acid sequence that entirely is the same as part but not all of the ammo acid sequence of the aforementioned polypeptides. As with priA 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, a single larger polypeptide An example of such a fragment is the polypeptide of SEQ ID NO 6
Preferred fragments include, for example, truncation polypeptides having a portion of the ammo acid sequence of Table 1 [SEQ ID NO.2], or of variants thereof, such as a continuous series of residues that includes the ammo terminus, or a continuous series of residues that includes the carboxyl terminus Degradation forms of the polypeptides of the invention m a host cell, particularly a Staphylococcus aureus, 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-formmg regions, turn and turn-forming regions, coil and coil-forming regions, hydrophihc regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions, substrate binding region, and high antigenic index regions.
Also preferred are biologically active fragments which are those fragments that mediate activities of pπA, including those with a similar activity or an improved activity, or with a decreased undesirable activity Also included are those fragments that are antigenic or immunogenic in an animal, especially in a human Particularly preferred are fragments comprising receptors or domains of enzymes that confer a function essential for viability of Staphylococcus aureus or the ability to initiate, or maintain cause disease in an individual, particularly a human
Variants that are fragments of the polypeptides of the invention may be employed for producing the corresponding full-length polypeptide by peptide synthesis; therefore, these vaπants may be employed as intermediates for producing the full-length polypeptides of the invention. An example of such a fragment is the polynucleotide of SEQ ID NO: 5. Polynucleotides
Another aspect of the invention relates to isolated polynucleotides that encode the priA polypeptide having the deduced ammo acid sequence of Table 1 [SEQ ID NO.2] and polynucleotides closely related thereto and variants thereof
Using the information provided herein, such as the polynucleotide sequence set out in Table 1 [SEQ ID NO:l], a polynucleotide of the invention encoding priA polypeptide may be obtained using standard cloning and screening methods, such as those for cloning and sequencing chromosomal DNA fragments from bacteria using Staphylococcus aureus WCUH 29 cells as starting material, followed by obtaining a full length clone. For example, to obtain a polynucleotide sequence of the invention, such as the sequence given in Table 1 [SEQ ID NO.l], typically a library of clones of chromosomal DNA of Staphylococcus aureus WCUH 29 in E coh or some other suitable host is probed with a radiolabeled ohgonucleotide, preferably a 17-mer or longer, derived from a partial sequence. Clones carrying DNA identical to that of the probe can then be distinguished using stringent conditions. By sequencing the individual clones thus identified with sequencing primers designed from the original sequence it is then possible to extend the sequence in both directions to determine the full gene sequence Conveniently, such sequencing is performed using denatured double stranded DNA prepared from a plasmid clone Suitable techniques are described by Mamatis, T., Fπtsch, E.F. and Sambrook et al, MOLECULAR CLONING, A LABORATORY MANUAL, 2nd Ed , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989) (see m particular Screening By Hybridization 1 90 and Sequencing Denatured Double-Stranded DNA Templates 13.70) Illustrative of the invention, the polynucleotide set out in Table 1 [SEQ ID NO.l] was discovered in a DNA library derived from Staphylococcus aureus WCUH 29
The DNA sequence set out in Table 1 [ SEQ ID NO 1] contains an open reading frame encoding a protein having about or exactly the number of ammo acid residues set forth in Table 1 [SEQ ID NO:2] with a deduced molecular weight that can be calculated using ammo acid residue molecular weight values well known in the art. The open reading frame may be correlated with the polypeptide of Table 1 [SEQ ID NO: 2] by using a codon chart well known in the art or any algoπthm for detecting open reading frames, such as the known methods of Framesearch (Wisconsin Package of the Genetics Computer Group (GCG) software), TBLASTN (Blast software from NCBI), and FASTA (TFASTX of Pearson & Lipman; PNAS 85. 2444 (1988).
PπA of the invention is structurally related to other proteins of the pπmosome family, as shown by the results of sequencing the DNA encoding priA of the deposited strain The protein exhibits greatest homology to priA in B subtihs (p94461) protein among known proteins. PπA of Table 1 [SEQ ID NO 2] has about 61% identity (I e, 250/410) over its entire length and about 80.7% similarity (1 e , 331/410) over its entire length with the ammo acid sequence of the pπA in B subtihs (p94461) polypeptide.
The invention provides a polynucleotide sequence identical over its entire length to the coding sequence in Table 1 [SEQ ID NO.l]. Also provided by the invention is the coding sequence for the mature polypeptide or a fragment thereof, by itself as well as the coding sequence for the mature polypeptide or a fragment in reading frame with other coding sequence, such as those encoding a leader or secretory sequence, a pre-, or pro- or prepro- protem sequence. The polynucleotide may also contain non-codmg sequences, including for example, but not limited to non-codmg 5' and 3' sequences, such as the transcπbed, non- translated sequences, termination signals, πbosome binding sites, sequences that stabilize mRNA, mtrons, polyadenylation signals, and additional coding sequence which encode additional ammo acids. For example, a marker sequence that facilitates purification of the 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 described in Gentz et al, Proc. Natl Acad. Sci , USA 86- 821-824 (1989), or an HA tag (Wilson et al, Cell 37- 767 (1984). Polynucleotides of the invention also include, but are not limited to, polynucleotides comprising a structural gene and its naturally associated sequences that control gene expression. A preferred embodiment of the invention is the polynucleotide comprising nucleotide set forth in SEQ ID NO: 1 of Table 1 which encodes the pπA polypeptide.
The invention also includes polynucleotides of the formula set forth in Table 1 (C) wherein, at the 5' end of the molecule, X is hydrogen, and at the 3' end of the molecule, Y is hydrogen or a metal, Ri and R2 is any nucleic acid residue, and n is an integer between 1 and 1000. Any stretch of nucleic acid residues denoted by either R group, where R is greater than 1 , may be either a heteropolymer or a homopolymer, preferably a heteropolymer.
The term "polynucleotide encoding a polypeptide" as used herein encompasses polynucleotides that include a sequence encoding a polypeptide of the invention, particularly a bacteπal polypeptide and more particularly a polypeptide of the Staphylococcus aureus priA having the ammo acid sequence set out in Table 1 [SEQ ID NO:2]. The term also encompasses polynucleotides that include a single continuous region or discontinuous regions encoding the polypeptide (for example, interrupted by integrated phage or an insertion sequence or editing) together with additional regions, that also may contain coding and/or non-codmg sequences. The invention further relates to vaπants of the polynucleotides described herein that encode for variants of the polypeptide having the deduced ammo acid sequence of Table 1 [SEQ ID NO.2]. Vaπants that are fragments of the polynucleotides of the invention may be used to synthesize full-length polynucleotides of the invention. Further particularly preferred embodiments are polynucleotides encoding priA vaπants, that have the ammo acid sequence of priA polypeptide of Table 1 [SEQ ID NO:2] in which several, a few, 5 to 10, 1 to 5, 1 to 3, 2, 1 or no ammo acid residues are substituted, deleted or added, in any combination. Especially preferred among these are silent substitutions, additions and deletions, that do not alter the properties and activities of pπA.
Further preferred embodiments of the invention are polynucleotides that are at least 70%) identical over their entire length to a polynucleotide encoding pπA polypeptide having the amino acid sequence set out in Table 1 [SEQ ID NO:2], and polynucleotides that are complementary to such polynucleotides. Alternatively, most highly preferred are polynucleotides that comprise a region that is at least 80% identical over its entire length to a polynucleotide encoding pπA polypeptide of the deposited strain and polynucleotides complementary thereto. In this regard, polynucleotides at least 90% identical over their entire length to the same are particularly preferred, and among these particularly preferred polynucleotides, those with at least 95% are especially preferred Furthermore, those with at least 97% are highly preferred among those with at least 95%, and among these those with at least 98%o and at least 99% are particularly highly preferred, with at least 99% being the more preferred.
Preferred embodiments are polynucleotides that encode polypeptides that retain substantially the same biological function or activity as the mature polypeptide encoded by the DNA of Table 1 [SEQ ID NO: 1].
The invention further relates to polynucleotides that hybridize to the herein above- described sequences. In this regard, the invention especially relates to polynucleotides that hybπdize under stπngent conditions to the herein above-described polynucleotides. As herein used, the terms "stringent conditions" and "stringent hybridization conditions" mean hybridization will occur only if there is at least 95% and preferably at least 97% identity between the sequences. An example of stringent hybridization conditions is overnight incubation at 42°C m 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 denatured, sheared salmon sperm DNA, followed by washing the hybridization support in O.lx SSC at about 65°C. Hybridization and wash conditions are well known and exemplified in Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (1989), particularly Chapter 11 therein. The invention also provides a polynucleotide consisting essentially of a polynucleotide sequence obtainable by screening an appropriate library containing the complete gene for a polynucleotide sequence set forth in SEQ ID NO: l under stringent hybridization conditions with a probe having the sequence of said polynucleotide sequence set forth m SEQ ID NO:l or a fragment thereof; and isolating said DNA sequence. Fragments useful for obtaining such a polynucleotide include, for example, probes and primers described elsewhere herein.
As discussed additionally herein regarding polynucleotide assays of the invention, for instance, polynucleotides of the invention as discussed above, may be used as a hybridization probe for RNA, cDNA and genomic DNA to isolate full-length cDNAs and genomic clones encoding priA and to isolate cDNA and genomic clones of other genes that have a high sequence similarity to the priA gene. Such probes generally will compπse at least 15 bases. Preferably, such probes will have at least 30 bases and may have at least 50 bases. Particularly preferred probes will have at least 30 bases and will have 50 bases or less. For example, the coding region of the priA gene may be isolated by screening using the DNA sequence provided in SEQ ID NO. 1 to synthesize an ohgonucleotide probe. A labeled ohgonucleotide 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. 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 disease, particularly human disease, as further discussed herein relating to polynucleotide assays.
Polynucleotides of the invention that are ohgonucleotides derived from the sequences of SEQ ID NOS.1 and/or 2 may be used in the processes herein as described, but preferably for PCR, to determine whether or not the polynucleotides identified herein in whole or in part are transcribed in bacteria in infected tissue. It is recognized that such sequences 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 may encode a polypeptide that is the mature protein plus additional ammo or carboxyl-termmal ammo acids, or ammo acids interior to the mature polypeptide (when the mature form has more than one polypeptide chain, for instance). Such sequences may play a role in processing of a protein from precursor to a mature form, may allow protein transport, may lengthen or shorten protein half-life or may facilitate manipulation of a protein for assay or production, among other things. As generally is the case in vivo, the additional amino acids may be processed away from the mature protein by cellular enzymes.
A precursor protein, having the mature form of the polypeptide fused to one or more prosequences may be an inactive form of the polypeptide. When prosequences are removed such inactive precursors generally are activated. Some or all of the prosequences may be removed before activation. Generally, such precursors are called proprotems.
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, which is a precursor to a proprotem, having a leader sequence and one or more prosequences, which generally are removed during processing steps that produce active and mature forms of the polypeptide. Vectors, host cells, expression The invention also relates to vectors that comprise 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 derived from the DNA constructs of the invention. For recombinant production, 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 descπbed 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 Spπng Harbor Laboratory Press, Cold Spπng Harbor, N.Y. (1989), such as, calcium phosphate transfection, DEAE-dextran mediated transfection, transvection, micromjection, catiomc hpid-mediated transfection, electroporation, transduction, scrape loading, ballistic introduction and infection.
Representative examples of appropπate hosts include bacterial cells, such as streptococci, staphylococci, enterococci E coli, streptomyces and Bacillus subtihs cells; fungal cells, such as yeast cells and Aspergillus cells; insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, 293 and Bowes melanoma cells; and plant cells. A great variety of expression systems can be used to produce the polypeptides of the invention. Such vectors include, among others, chromosomal, episomal and virus-derived vectors, e.g. , vectors derived from bacterial plasmids, from bacteπophage, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses such as baculoviruses, papova viruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors derived from combinations thereof, such as those derived from plasmid and bacteπophage genetic elements, such as cosmids and phagemids. The expression system constructs may contain 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 m this regard The appropriate DNA sequence may be inserted into the expression system by any of a variety of well-known and routine techniques, such as, for example, those set forth in Sambrook et al, MOLECULAR CLONING, A LABORATORY MANUAL, (supra). For secretion of the translated protein into the lumen of the endoplasmic reticulum, into the peπplasmic space or into the extracellular environment, appropriate secretion signals may be incorporated into the expressed polypeptide. These signals may be endogenous to the polypeptide or they may be heterologous signals.
Polypeptides of the invention can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography, and lectin chromatography. Most preferably, high performance liquid chromatography is employed for purification. Well known techniques for refolding protein may be employed to regenerate active conformation when the polypeptide is denatured duπng isolation and or purification. Diagnostic Assays
This invention is also related to the use of the priA polynucleotides of the invention for use as diagnostic reagents. Detection of priA in a eukaryote, particularly a mammal, and especially a human, will provide a diagnostic method for diagnosis of a disease. Eukaryotes (herein also "individuals)"), particularly mammals, and especially humans, particularly those infected or suspected to be infected with an organism comprising the pπA gene may be detected at the nucleic acid level by a vaπety of techniques. Nucleic acids for diagnosis may be obtained from an infected individual's cells and tissues, such as bone, blood, muscle, cartilage, and skin Genomic DNA may be used directly for detection or may be amplified enzymatically by using PCR or other amplification technique prior to analysis RNA or cDNA may also be used in the same ways Using amplification, characterization of the species and strain of prokaryote present in an individual, may be made by an analysis of the genotype of the prokaryote gene Deletions and insertions can be detected by a change m size of the amplified product in comparison to the genotype of a reference sequence Point mutations can be identified by hybridizing amplified DNA to labeled priA polynucleotide sequences Perfectly matched sequences can be distinguished from mismatched duplexes by RNase digestion or by differences m melting temperatures DNA sequence differences may also be detected by alterations in the electrophoretic mobility of the DNA fragments in gels, with or without denaturing agents, or by direct DNA 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 and SI protection or a chemical cleavage method See, e g , Cotton et al , Proc Natl Acad Sci , USA, 85 4397-4401 (1985)
Cells carrying mutations or polymorphisms in the gene of the invention may also be detected at the DNA level by a variety of techniques, to allow for serotypmg, for example For example, RT-PCR can be used to detect mutations It is particularly preferred to used RT- PCR in conjunction with automated detection systems, such as, for example, GeneScan RNA or cDNA may also be used for the same purpose, PCR or RT-PCR As an example, PCR pπmers complementary to a nucleic acid encoding priA can be used to identify and analyze mutations Examples of representative primers are shown below in Table 2
_
Table 2
Primers for amplification of priA polynucleotides SEQ ID NO PRIMER SEQUENCE
3 5'-TGCGAGAGTAAGTGTTGGTCCA-3'
4 5'-TTTACGATATTCCATTTCCTGACG-3' The invention further provides these primers with 1 , 2, 3 or 4 nucleotides removed from the 5' and/or the 3' end. These primers may be used for, among other things, amplifying priA DNA isolated from a sample derived from an individual. The pπmers may be used to amplify the gene isolated from an infected individual such that the gene may then be subject to various techniques for elucidation of the DNA sequence. In this way, mutations in the DNA sequence may be detected and used to diagnose infection and to serotype and/or classify the infectious agent.
The invention further provides a process for diagnosing, disease, preferably bacterial infections, more preferably infections by Staphylococcus aureus, and most preferably disease, such as, infections of the upper respiratory tract (e.g., otitis media, bacteπal tracheitis, acute epiglottitis, thyroiditis), lower respiratory (e.g., empyema, lung abscess), cardiac (e.g , infective endocarditis), gastrointestinal (e.g., secretory diarrhoea, splenic absces, retropeπtoneal abscess), CNS (e.g., cerebral abscess), eye (e.g., blepharitis, conjunctivitis, keratitis, endophthalmitis, preseptal and orbital celluhtis, darcryocystitis), kidney and uπnary tract (e.g., epididymitis, mtrarenal and peπnephπc absces, toxic shock syndrome), skin (e.g., impetigo, follicuhtis, cutaneous abscesses, celluhtis, wound infection, bacterial myositis) bone and joint (e.g., septic arthritis, osteomyelitis), comprising determining from a sample derived from an individual a increased level of expression of polynucleotide having the sequence of Table 1 [SEQ ID NO: 1]. Increased or decreased expression of pπA polynucleotide can be measured using any on of the methods well known m the art for the quantation of polynucleotides, such as, for example, amplification, PCR, RT-PCR, RNase protection, Northern blotting and other hybridization methods.
In addition, a diagnostic assay in accordance with the invention for detecting over- expression of priA protein 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 pπA protein, in a sample derived from a host are well-known to those of skill in the art. Such assay methods include radioimmunoassays, competitive-binding assays, Western Blot analysis and ELISA assays. Antibodies The polypeptides of the invention or vaπants thereof, or cells expressing them can be used as an immunogen to produce antibodies lmmunospecific for such polypeptides. "Antibodies" as used herein includes monoclonal and polyclonal antibodies, chimeπc, single chain, simianized antibodies and humanized antibodies, as well as Fab fragments, including the products of an Fab lmmunolglobulm expression library. Antibodies generated against the polypeptides of the invention can be obtained by administering the polypeptides or epitope-bearmg fragments, analogues or cells to an animal, preferably a nonhuman, using routine protocols. For preparation of monoclonal antibodies, any technique known in the art that provides antibodies produced by continuous cell line cultures can be used. Examples include various techniques, such as those in Kohler, G. and Milstem, C, Nature 256 495-497 (1975); Kozbor et al , Immunology Today 4 72 (1983); Cole et al, pg. 77-96 in MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc. (1985).
Techniques for the production of single chain antibodies (U.S. Patent No. 4,946,778) can be adapted to produce single chain antibodies to polypeptides of this invention. Also, transgenic mice, or other organisms such as other mammals, may be used to express humanized antibodies.
Alternatively phage display technology may be utilized to select antibody genes with binding activities towards the polypeptide either from repertoires of PCR amplified v- genes of lymphocytes from humans screened for possessing anti-pπA or from naive libraries (McCafferty, J. et al, (1990), Nature 348, 552-554; Marks, J. et al, (1992) Biotechnology 10, 779-783). The affinity of these antibodies can also be improved by chain shuffling (Clackson, T. et al., (1991) Nature 352, 624-628).
If two antigen binding domains are present each domain may be directed against a different epitope - termed 'bispecific' antibodies.
The above-descπbed antibodies may be employed to isolate or to identify clones expressing the polypeptides to purify the polypeptides by affinity chromatography.
Thus, among others, antibodies against pπA- polypeptide may be employed to treat infections, particularly bacteπal infections and especially 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, retropeπtoneal abscess), CNS (e.g., cerebral abscess), eye (e.g., blepharitis, conjunctivitis, keratitis, endophthalmitis, preseptal and orbital celluhtis, darcryocystitis), kidney and uπnary tract (e.g., epididymitis, mtrarenal and peπnephπc absces, toxic shock syndrome), skm (e.g., impetigo, follicuhtis, cutaneous abscesses, celluhtis, wound infection, bacterial myositis) bone and joint (e.g., septic arthπtis, osteomyelitis).
Polypeptide variants include antigemcally, epitopically or immunologically equivalent variants that form a particular aspect of this invention. The term "antigemcally equivalent derivative" as used herein encompasses a polypeptide or its equivalent which will be specifically recognized by certain antibodies which, when raised to the protein or polypeptide according to the invention, interfere with the immediate physical interaction between pathogen and mammalian host. The term "immunologically equivalent derivative" as used herein encompasses a peptide or its equivalent which when used in a suitable formulation to raise antibodies in a vertebrate, the antibodies act to interfere with the immediate physical interaction between pathogen and mammalian host.
The polypeptide, such as an antigemcally or immunologically equivalent derivative or a fusion protein thereof is used as an antigen to immunize a mouse or other animal such as a rat or chicken. The fusion protein may provide stability to the polypeptide. The antigen may be associated, for example by conjugation, with an immunogenic carrier protein for example bovine serum albumin (BSA) or keyhole limpet haemocyanm (KLH). Alternatively a multiple antigenic peptide comprising multiple copies of the protein or polypeptide, or an antigemcally or immunologically equivalent polypeptide thereof may be sufficiently antigenic to improve lmmunogenicity so as to obviate the use of a carrier.
Preferably, the antibody or variant thereof is modified to make it less immunogenic in the individual. For example, if the individual is human the antibody may most preferably be "humanized"; where the comphmentaπty determining regιon(s) of the hybπdoma-deπved antibody has been transplanted into a human monoclonal antibody , for example as described m Jones, P. et al. (1986), Nature 321, 522-525 or Tempest et al.,(1991) Biotechnology 9, 266-273.
The use of a polynucleotide of the invention in genetic immunization will preferably employ a suitable delivery method such as direct injection of plasmid DNA into muscles (Wolff et al., Hum Mol Genet 1992, 1 :363, Manthorpe et al., Hum. Gene Ther. 1963:4, 419), delivery of DNA complexed with specific protein caπiers (Wu et al., J Biol Chem 1989: 264,16985), coprecipitation of DNA with calcium phosphate (Benvemsty & Reshef, PNAS USA, 1986:83,9551), encapsulation of DNA in various forms of hposomes (Kaneda et al , Science 1989:243,375), particle bombardment (Tang et al , Nature 1992, 356: 152, Eisenbraun et al , DNA Cell Biol 1993, 12:791) and in vivo infection using cloned retroviral vectors (Seeger et al., PNAS USA 1984:81,5849). Antagonists and agonists - assays and molecules
Polypeptides of the invention may also be used to assess the binding of small molecule substrates and hgands in, for example, cells, cell-free preparations, chemical hbraπes, and natural product mixtures. These substrates and hgands may be natural substrates and hgands or may be structural or functional mimetics. See, e g , Cohgan et al , Current Protocols in Immunology 1(2) Chapter 5 (1991).
The invention also provides a method of screening compounds to identify those which enhance (agonist) or block (antagonist) the action of priA polypeptides or polynucleotides, particularly those compounds that are bacteπostatic and/or bacteriocidal. The method of screening may involve high-throughput techniques. For example, to screen for agonists or antagoists, a synthetic reaction mix, a cellular compartment, such as a membrane, cell envelope or cell wall, or a preparation of any thereof, comprising priA polypeptide and a labeled substrate or ligand of such polypeptide is incubated in the absence or the presence of a candidate molecule that may be a priA agonist or antagonist The ability of the candidate molecule to agonize or antagonize the priA polypeptide is reflected in decreased binding of the labeled ligand or decreased production of product from such substrate. Molecules that bind gratuitously, i e., without inducing the effects of priA polypeptide are most likely to be good antagonists Molecules that bind well and increase the rate of product production from substrate are agonists. Detection of the rate or level of production of product from substrate may be enhanced by using a reporter system Reporter systems that may be useful in this regard include but are not limited to coloπmetπc labeled substrate converted into product, a reporter gene that is responsive to changes in priA polynucleotide or polypeptide activity, and binding assays known in the art. Another example of an assay for priA antagonists is a competitive assay that combines priA and a potential antagonist with pπA-bindmg molecules, recombinant priA binding molecules, natural substrates or hgands, or substrate or ligand mimetics, under appropriate conditions for a competitive inhibition assay PπA can be labeled, such as by radioactivity or a coloπmetπc compound, such that the number of priA molecules bound to a binding molecule or converted to product can be determined accurately to assess the effectiveness of the potential antagonist.
Potential antagonists include small organic molecules, peptides, polypeptides and antibodies that bind to a polynucleotide or polypeptide of the invention and thereby inhibit or extinguish its activity Potential antagonists also may be small organic molecules, a peptide, a polypeptide such as a closely related protein or antibody that binds the same sites on a binding molecule, such as a binding molecule, without inducing pπA-induced activities, thereby preventing the action of priA by excluding pπA from binding.
Potential antagonists include a small molecule that binds to and occupies the binding site of the polypeptide thereby preventing binding to cellular binding molecules, such that normal biological activity is prevented. Examples of small molecules include but are not limited to small organic molecules, peptides or peptide-hke molecules Other potential antagonists include antisense molecules (see Okano, J Neurochem 56 560 (1991); OLIGODEOXYNUCLEOTIDES AS ANTISENSE INHIBITORS OF GENE EXPRESSION, CRC Press, Boca Raton, FL (1988), for a description of these molecules). Preferred potential antagonists include compounds related to and vanants of priA.
Each of the DNA sequences provided herein may be used in the discovery and development of antibacterial compounds. The encoded protein, upon expression, can be used as a target for the screening of antibacterial drugs Additionally, the DNA sequences encoding the ammo terminal regions of the encoded protein or Shine-Delgarno or other translation facilitating sequences of the respective mRNA can be used to construct antisense sequences to control the expression of the coding sequence of interest
The invention also provides the use of the polypeptide, polynucleotide or inhibitor of the invention to interfere with the initial physical interaction between a pathogen and 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 bacteria, to mammalian extracellular matrix proteins on m-dwellmg devices or to extracellular matrix proteins in wounds; to block priA protem-mediated mammalian cell invasion by, for example, initiating phosphorylation of mammalian tyrosme kmases (Rosenshme et al, Infect Immun (50:2211 (1992); to block bacterial adhesion between mammalian extracellular matrix proteins and bacteπal pπA proteins that mediate tissue damage and, to block the normal progression of pathogenesis m infections initiated other than by the implantation of m-dwelhng devices or by other surgical techniques.
The antagonists and agonists of the invention may be employed, for instance, to inhibit and treat 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, retropeπtoneal abscess), CNS (e.g., cerebral abscess), eye (e.g., blephaπtis, conjunctivitis, keratitis, endophthalmitis, preseptal and orbital celluhtis, darcryocystitis), kidney and uπnary tract (e.g., epididymitis, mtrarenal and peπnephπc absces, toxic shock syndrome), skin (e.g., impetigo, follicuhtis, cutaneous abscesses, celluhtis, wound infection, bacterial myositis) bone and joint (e.g., septic arthritis, osteomyelitis).
Helicobacter pylori (herein H pylori) bacteria mfect 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/ecp2904.htm). Moreover, the international Agency for Research on Cancer recently recognized a cause-and-effect relationship between H pylori and gastric adenocarcinoma, classifying the bacterium as a Group I (definite) carcinogen. Preferred antimicrobial compounds of the invention (agonists and antagonists of priA) found using screens provided by the invention, particularly broad-spectrum antibiotics, should be useful in the treatment of H pylori infection. Such treatment should decrease the advent of H pylori- d ced cancers, such as gastrointestinal carcinoma Such treatment should also cure gastric ulcers and gastritis. Vaccines
Another aspect of the invention relates to a method for inducing an immunological response in an individual, particularly a mammal which comprises inoculating the individual with priA, or a fragment or variant thereof, adequate to produce antibody and/ or T cell immune response to protect said individual from infection, particularly bacterial infection and most particularly Staphylococcus aureus infection Also provided are methods whereby such immunological response slows bacterial replication Yet another aspect of the invention relates to a method of inducing immunological response in an individual which comprises delivering to such individual a nucleic acid vector to direct expression of pπA, or a fragment or a variant thereof, for expressing priA, or a fragment or a variant thereof in vivo in order to induce an immunological response, such as, to produce antibody and/ or T cell immune response, including, for example, cytokine-producmg T cells or cytotoxic T cells, to protect said individual from disease, whether that disease is already established withm the individual or not One way of administering the gene is by accelerating it into the desired cells as a coating on particles or otherwise. Such nucleic acid vector may comprise DNA, RNA, a modified nucleic acid, or a DNA/RNA hybrid.
A further aspect of the invention relates to an immunological composition which, when introduced into an individual capable or having induced withm it an immunological response, induces an immunological response in such individual to a pπA or protein coded therefrom, wherein the composition comprises a recombinant priA or protein coded therefrom comprising DNA which codes for and expresses an antigen of said pπA or protein coded therefrom. The immunological response may be used therapeutically or prophylactically and may take the form of antibody immunity or cellular immunity such as that arising from CTL or CD4+ T cells. A pπA polypeptide or a fragment thereof may be fused with co-protein which may not by itself produce antibodies, but is capable of stabilizing the first protein and producing a fused protein which will have immunogenic and protective properties. Thus fused recombinant protein, preferably further comprises an antigenic co-protem, such as hpoprotein D from Hemophilus influenzae, Glutathione-S-transferase (GST) or beta- galactosidase, relatively large co-protems which solubihze the protein and facilitate production and purification thereof. Moreover, the co-protem may act as an adjuvant in the sense of providing a generalized stimulation of the immune system. The co-protem may be attached to either the ammo or carboxy terminus of the first protein. Provided by this invention are compositions, particularly vaccine compositions, and methods comprising the polypeptides or polynucleotides of the invention and immunostimulatory DNA sequences, such as those described in Sato, Y. et al. Science 273: 352 (1996).
Also, provided by this invention are methods using the described polynucleotide or particular fragments thereof which have been shown to encode non-variable regions of bacterial cell surface proteins in DNA constructs used in such genetic immunization experiments in animal models of infection with Staphylococcus aureus will be particularly useful for identifying protein epitopes able to provoke a prophylactic or therapeutic immune response. It is believed that this approach will allow for the subsequent preparation of monoclonal antibodies of particular value from the requisite organ of the animal successfully resisting or clearing infection for the development of prophylactic agents or therapeutic treatments of bacteπal infection, particularly Staphylococcus aureus infection, m mammals, particularly humans
The polypeptide may be used as an antigen for vaccination of a host to produce specific antibodies which protect against invasion of bacteria, for example by blocking adherence of bacteria to damaged tissue. Examples of tissue damage include wounds in skin or connective tissue caused, e.g., by mechanical, chemical or thermal damage or by implantation of indwelling devices, or wounds in the mucous membranes, such as the mouth, mammary glands, urethra or vagina. The invention also includes a vaccine formulation which comprises an immunogenic recombinant protein of the invention together with a suitable carrier. Since the protein may be broken down in the stomach, it is preferably administered parenterally, including, for example, administration that is subcutaneous, intramuscular, intravenous, or mtradermal. Formulations suitable for parenteral administration include aqueous and non- aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteπostats and solutes which render the formulation lnsoto c with the bodily fluid, preferably the blood, of the individual; and aqueous and non-aqueous sterile suspensions which may include suspending agents or thickening agents The formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampules and vials and may be stored m a freeze-dπed condition requiring only the addition of the sterile liquid carrier immediately prior to use The vaccine formulation may also include adjuvant systems for enhancing the lmmunogenicity of the formulation, such as oil-m water systems and other systems known in the art. The dosage will depend on the specific activity of the vaccine and can be readily determined by routine experimentation.
While the invention has been described with reference to certain priA protein, it is to be understood that this covers fragments of the naturally occurring protein and similar proteins with additions, deletions or substitutions which do not substantially affect the immunogenic properties of the recombinant protein Compositions, kits and administration
The invention also relates to compositions compπsing the polynucleotide or the polypeptides discussed above or their agonists or antagonists. The polypeptides of the invention may be employed in combination with a non-sterile or sterile carrier or earners for use with cells, tissues or organisms, such as a pharmaceutical carrier suitable for administration to a subject. Such compositions comprise, for instance, a media additive or a therapeutically effective amount of a polypeptide of the invention and a pharmaceutically acceptable earner or excipient. Such earners may include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol and combinations thereof The formulation should suit the mode of administration. The invention further relates to diagnostic and pharmaceutical packs and kits comprising one or more containers filled with one or more of the ingredients of the aforementioned compositions of the invention.
Polypeptides and other compounds of the invention may be employed alone or in conjunction with other compounds, such as therapeutic compounds.
The pharmaceutical compositions may be administered m any effective, convenient manner including, for instance, administration by topical, oral, anal, vaginal, intravenous, mtrapeπtoneal, intramuscular, subcutaneous, mtranasal or mtradermal routes among others.
In therapy or as a prophylactic, the active agent may be administered to an individual as an mjectable composition, for example as a sterile aqueous dispersion, preferably lsotomc. Alternatively the composition may be formulated for topical application for example in the form of ointments, creams, lotions, eye ointments, eye drops, ear drops, mouthwash, impregnated dressings and sutures and aerosols, and may contain appropriate conventional additives, including, for example, preservatives, solvents to assist drug penetration, and emollients in ointments and creams Such topical formulations may also contain compatible conventional carriers, for example cream or ointment bases, and ethanol or oleyl alcohol for lotions. Such caπiers may constitute from about 1% to about 98% by weight of the formulation, more usually they will constitute up to about 80% by weight of the formulation. For administration to mammals, and particularly humans, it is expected that the daily dosage level of the active agent will be from 0 01 mg/kg to 10 mg/kg, typically around 1 mg/kg The physician m any event will determine the actual dosage which will be most suitable for an individual and will vary with the age, weight and response of the particular individual. The above dosages are exemplary of the average case There can, of course, be individual instances where higher or lower dosage ranges are merited, and such are withm the scope of this invention.
In-dwelling devices include surgical implants, prosthetic devices and catheters, i.e., devices that are introduced to the body of an individual and remain in position for an extended time Such devices include, for example, artificial joints, heart valves, pacemakers, vascular grafts, vascular catheters, cerebrospinal fluid shunts, urinary catheters, continuous ambulatory peritoneal dialysis (CAPD) catheters.
The composition of the invention may be administered by injection to achieve a systemic effect against relevant bacteria shortly before insertion of an in-dwelling device Treatment may be continued after surgery during the m-body time of the device. In addition, the composition could also be used to broaden peπoperative cover for any surgical technique to prevent bacterial wound infections, especially Staphylococcus aureus wound infections.
Many orthopaedic surgeons consider that humans with prosthetic joints should be considered for antibiotic prophylaxis before dental treatment that could produce a bacteremia. Late deep infection is a serious complication sometimes leading to loss of the prosthetic joint and is accompanied by significant morbidity and mortality. It may therefore be possible to extend the use of the active agent as a replacement for prophylactic antibiotics in this situation. In addition to the therapy described above, the compositions of this invention may be used generally as a wound treatment agent to prevent adhesion of bacteria to matrix proteins exposed in wound tissue and for prophylactic use in dental treatment as an alternative to, or m conjunction with, antibiotic prophylaxis. Alternatively, the composition of the invention may be used to bathe an indwelling device immediately before insertion The active agent will preferably be present at a concentration of lμg/ml to lOmg/ml for bathing of wounds or indwelling devices.
A vaccine composition is conveniently in mjectable form. Conventional adjuvants may be employed to enhance the immune response. A suitable unit dose for vaccination is 0.5-5 microgram/kg of antigen, and such dose is preferably administered 1-3 times and with an interval of 1-3 weeks. With the indicated dose range, no adverse toxicological effects will be observed with the compounds of the invention which would preclude their administration to suitable individuals
Each reference disclosed herein is incorporated by reference herein in its entirety Any patent application to which this application claims priority is also incorporated by reference herein m its entirety.
EXAMPLES
The examples below are earned out using standard techniques, which are well known and routine to those of skill in the art, except where otherwise described in detail. The examples are illustrative, but do not limit the invention. Example 1 Strain selection, Library Production and Sequencing
The polynucleotide having the DNA sequence given in SEQ ID NO:l was obtained from a library of clones of chromosomal DNA of Staphylococcus aureus in E. coli. The sequencing data from two or more clones containing overlapping Staphylococcus aureus DNAs was used to construct the contiguous DNA sequence m SEQ ID NO: l . Libraries may be prepared by routine methods, for example: Methods 1 and 2 below.
Total cellular DNA is isolated from Staphylococcus aureus WCUH 29 according to standard procedures and size-fractionated by either of two methods. Method 1
Total cellular DNA is mechanically sheared by passage through a needle in order to size-fractionate according to standard procedures. DNA fragments of up to l lkbp in size are rendered blunt by treatment with exonuclease and DNA polymerase, and EcoRI linkers added. Fragments are hgated into the vector Lambda ZapII that has been cut with EcoRI, the library packaged by standard procedures and E coli infected with the packaged library. The library is amplified by standard procedures Method 2 Total cellular DNA is partially hydrolyzed with a one or a combination of restriction enzymes appropriate to generate a series of fragments for cloning into library vectors (e.g., Rsal, Pall, Alul, Bshl235I), and such fragments are size-fractionated according to standard procedures. EcoRI linkers are hgated to the DNA and the fragments then hgated into the vector Lambda ZapII that have been cut with EcoRI, the library packaged by standard procedures, and E coli infected with the packaged library. The library is amplified by standard procedures. Example 2 PriA Characterization
Replication of the lagging strand of DNA is mediated by a multiprotein complex composed of proteins n , priA, n", dnaT, dnaB, dnaC, and dnaG. This complex is referred to as a pπmosome. Purified pπA has ATPase, hehcase, translocase, and pπmosome assembly activities (Lee EH, Masai H, Allen GC Jr, Kornberg A, Proc Natl Acad Sci USA 1990 Jun, 87 (12)- 4620-4624) (Sandier SJ, Samra HS, Clark AJ Genetics 1996 May;143(l):5-13). This gene may be essential in recombination and DNA repair since it binds to D-loops, interacts with recG and has hehcase activity (McGlynn P, Al-Deib AA, Liu J, Marians KJ, Lloyd RG J Mol Biol 1997 Jul 11 ;270(2):212-221) The 3'-5' DNAhehcase activity of priA inhibits recombination (Al-Deib AA, Mahdi AA, Lloyd RG Bacteπol 1996 Dec;178(23):6782-6789). Insertion mutants of priA are Rec- and UVS which is a function of its pπmosome assembly function (Sandier SJ, Samra HS, Clark AJ Genetics 1996 May;143(l):5-13). Null mutants of priA are also defective in transductional and conjugational recombination (Kogoma T, Cadwell GW, Barnard KG, Asai T J Bacteπol 1996 Mar;178(5): 1258-1264). Null mutants can be suppressed by mutations mapping to dnaC.
Example 3 PriA Polynucleotide and Polypeptide Sequence Embodiments [SEQ ID NO: 5] aattagggatgatcgtgcgagagtaagtgttggtccaagtccaagtgtgttcgcaccttc caaaaatttagggttaatcatcattgatgaagaacatgaatctacatataaacaaggaga ttatccgagatatcacgctagagaaattgcccaatggcgaagtgaatatcatcactgtcc agtcattttaggaagtgcaacaccatgtcttgaaagttatgcacgagctgaaaaagacgt ttatcatttgctatcattaccaaacagagtgaaccaacaagctttacctgaaattgatat agtagacatgcgtgaagaattgagtgaaggtaatcggtcaatgttttcaaaagatttacg tgaagccatacaattaagattagatcgacaggaacaagttgttttatttttaaatcgacg tggttatgcatcgtttatgttatgtcgggattgtggatatgtaccgcaatgtccaaactg tgatatttcattaacgtatcatnaaaacgacagacttattaaaatgtcactattgtggtt accaagagacgccaccgaatcaatgtccaaattgtgagagtgaacacattcgacaagtag gtactggtactcagaaagttgaagaactattgcaacaagaatttgaagatgcgcgcataa ttaggatggatgtagatacaacctcaaagaaaggtgcacatgaaaagttattgactgaat tcgaaaaaggtaacggtgacattttactaggtactcagatgattgcgaaaggattagatt atccaaatattactttagttggtgtgctgaatgcagatacaatgttaaatttacctgatt ttcgggcgagcgaacgtacttatcaactattaacgcaagtggctggtagagctggtcgtc atgaaaaggcaggtcaagtcatcattcaaacg ataatccagatcattattcaatattgg atgttcaaaaaaatgattatttaacattttatcgtcaggaaatggaatatcgtaaattag gaaagtatccaccgtattattatttgattaatttcacaatctcacataaagaaatgaaga aggttatggaagcatcgcagcatgttcataaaattttattacagcatttaacagaaaaag cggttgtactaggtccatctccggcagcacttgcgagaatcaacaatgaatttagattcc aaattttagtgaaatataaaagtgaacctggattattacaagccattcagtttttagatg actattaccatgaaaaatttataaaagaaaaattagcattgaagattgatattgatccac agatgatgatgtaacattactaattattagaaacaagttcagtatgtacgagtatttgaa ccagtgtttgaatatttactttaagtacagaaaaagggcagaatatacaactgttaacta tttaaattagcagtttatattctgtctttttatatggctttataacttacgtgattttgg tttgataaggaatttattagtattttcatttacgaattctgattttaatgcaagcgccat acttactgatgaataaaagtactaatgattaaacaaactatttttaaatggcaaattgaa atagtgcaaattttacacttataaattaatggctataaactattttatatctatctatgt tttaactacttaaattattagacaaaacaattttcttaaattatcaattaaagtatttaa aaatattgatggttaatgtaaaatatcaatataaataagttttttaaatttatgtatgtt tatttgattcaaacaaaataacttaagaggagaaagtttatgaaaaagacactgggatgt ttacttttaattatgcttttagtcgtagacaggttgttcttttgggtgggaatcataaat tatcatcaaagaaatcagaagaatcaaaacaagaaactgtaaaaaaagaatcggaagaag agaaagatccagatttagagaaatatgaagaaatagagaagaaaatgaaaggaattaaag atgcgccatctcttgataagttggatccattaatgacagaaaagtcgtttacgaatagta aagggattcaaggatggaaagattacaaagaattaatgggtaaagtggaacttgcagatt atagatttactaaagattcaaaaggatcttcaataaaagatgttgatgcattcttcaaag gtaagaaaggtataaaaaggaaagtgattgaaacacacgatgatgtaaaacaagttgatt attggtatgtagatccagatggaaagaaaattggcaattcaaacacacctgttttttacg cagaaattatgacaaaatataaagatggaaagttagtttatgcatcagtcgaaccaggat cttacgtaatacataaagatgatgcaattaaatatgacgattattctaagttaaaaaaat taatccagctaactaaacttgatcatcccaaacca tcccata
[SEQ ID NO: 6]
IRDDRARVSVGPSPSVFAPSKNLGLIIIDEΞHESTYKQGDYPRYHAREIAQWRSΞYHHCP VILGSATPCLESYARAEKDVYHLLSLPNRVNQQALPEIDIVDMRΞELSEGNRSMFSKDLR
EAIQLRLDRQEQWLFLNRRGYASFMLCRDCGYVPQCPNCDISLTYHXNDR IKCHYCGYQΞTPPNQC PNCESEHIRQVGTGTQKVEELLQQEFEDARIIRMDVDTTSKKGA
HEKLLTEFEKGNGDILLGTQMIAKGLDYPNITLVGVLNADTMLNLPDFRASERTYQLLTQ VAGRAGRHEKAGQVIIQTYNPDHYSILDVQKNDYLTFYRQEMEYRKLGKYPPYYYLINFT ISHKEMKKVMEASQHVHKILLQHLTEKAWLGPSPAALARINNEFRFQILVKYKSEPGLL QAIQFLDDYYHEKFIKEKLALKIDIDPQMMM

Claims

What is claimed is:
1. An isolated polynucleotide comprising a polynucleotide sequence selected from the group consisting of:
(a) a polynucleotide having at least a 70% identity to a polynucleotide encoding a polypeptide comprising the ammo acid sequence of SEQ ID NO:2;
(b) a polynucleotide having at least a 70% identity to a polynucleotide encoding the same mature polypeptide expressed by the pπA gene contained in the Staphylococcus aureus of the deposited strain;
(c) a polynucleotide encoding a polypeptide comprising an ammo acid sequence which is at least 70% identical to the ammo acid sequence of SEQ ID NO.2;
(d) a polynucleotide which is complementary to the polynucleotide of (a), (b) or (c); and
(e) a polynucleotide comprising at least 15 sequential bases of the polynucleotide of (a), (b) or (c).
2. The polynucleotide of Claim 1 wherein the polynucleotide is DNA.
3. The polynucleotide of Claim 1 wherein the polynucleotide is RNA.
4. The polynucleotide of Claim 2 comprising the nucleic acid sequence set forth m SEQ ID NO. l.
5. The polynucleotide of Claim 2 comprising nucleotide open reading from set forth in SEQ ID NO: 1.
6. The polynucleotide of Claim 2 which encodes a polypeptide comprising the ammo acid sequence of SEQ ID NO:2.
7. A vector compnsmg the polynucleotide of Claim 1.
8. A host cell compnsmg the vector of Claim 7.
9. A process for producing a polypeptide comprising: expressing from the host cell of Claim 8 a polypeptide encoded by said DNA.
10. A process for producing a priA polypeptide or fragment comprising culturmg a host of claim 8 under conditions sufficient for the production of said polypeptide or fragment.
11. A polypeptide comprising an ammo acid sequence which is at least 70% identical to the ammo acid sequence of SEQ ID NO:2.
12. A polypeptide comprising an ammo acid sequence as set forth in SEQ ID NO:2.
13. An antibody against the polypeptide of claim 11
14. An antagonist which inhibits the activity or expression of the polypeptide of claim 11.
15. A method for the treatment of an individual m need of priA polypeptide comprising: administering to the individual a therapeutically effective amount of the polypeptide of claim 11.
16. A method for the treatment of an individual having need to inhibit priA polypeptide comprising: administering to the individual a therapeutically effective amount of the antagonist of Claim 14.
17. A process for diagnosing a disease related to expression or activity of the polypeptide of claim 11 m an individual comprising.
(a) determining a nucleic acid sequence encoding said polypeptide, and/or
(b) analyzing for the presence or amount of said polypeptide in a sample derived from the individual.
18. A method for identifying compounds which interact with and inhibit or activate an activity of the polypeptide of claim 11 comprising: contacting a composition comprising the polypeptide with the compound to be screened under conditions to permit interaction between the compound and the polypeptide to assess the interaction of a compound, such interaction being associated with a second component capable of providing a detectable signal m response to the interaction of the polypeptide with the compound; and determining whether the compound interacts with and activates or inhibits an activity of the polypeptide by detecting the presence or absence of a signal generated from the interaction of the compound with the polypeptide.
19 A method for inducing an immunological response m a mammal which comprises inoculating the mammal with priA polypeptide of claim 11, or a fragment or variant thereof, adequate to produce antibody and/or T cell immune response to protect said animal from disease.
20. A method of inducing immunological response m a mammal which comprises delivering a nucleic acid vector to direct expression of priA polypeptide of claim 11 , or fragment or a variant thereof, for expressing said priA polypeptide, or a fragment or a variant thereof in vivo m order to induce an immunological response to produce antibody and/ or T cell immune response to protect said animal from disease.
PCT/US1998/017324 1997-08-25 1998-08-21 NOVEL priA WO1999010012A1 (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
EP98942175A EP1009443A4 (en) 1997-08-25 1998-08-21 priA POLYPEPTIDES
JP2000507401A JP2001513337A (en) 1997-08-25 1998-08-21 New priA

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US5693997P 1997-08-25 1997-08-25
US60/056,939 1997-08-25

Publications (1)

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WO1999010012A1 true WO1999010012A1 (en) 1999-03-04

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JP (1) JP2001513337A (en)
WO (1) WO1999010012A1 (en)

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WO1997030070A1 (en) * 1996-02-20 1997-08-21 Smithkline Beecham Corporation Novel compounds

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MCGLYNN P., ET AL.: "THE DNA REPLICATION PROTEIN PRIA AND THE RECOMBINATION PROTEIN RECG BIND D-LOOPS.", JOURNAL OF MOLECULAR BIOLOGY, ACADEMIC PRESS, UNITED KINGDOM, vol. 270., no. 02., 11 July 1997 (1997-07-11), United Kingdom, pages 212 - 221., XP002915375, ISSN: 0022-2836, DOI: 10.1006/jmbi.1997.1120 *
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114981293A (en) * 2021-01-29 2022-08-30 Cj第一制糖株式会社 Novel initiator assembly protein variants and method for producing L-lysine using the same

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