WO2001023418A1 - YphC - Google Patents

YphC Download PDF

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Publication number
WO2001023418A1
WO2001023418A1 PCT/US2000/025566 US0025566W WO0123418A1 WO 2001023418 A1 WO2001023418 A1 WO 2001023418A1 US 0025566 W US0025566 W US 0025566W WO 0123418 A1 WO0123418 A1 WO 0123418A1
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Prior art keywords
polypeptide
seq
polynucleotide
sequence
tlie
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PCT/US2000/025566
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English (en)
Inventor
Magdalena Zalacain
Sanjoy Biswas
Martin K. R. Burnham
Daniel Sylvester
Damien Mcdevitt
Thomas B. Mathie
Original Assignee
Smithkline Beecham Corporation
Smithkline Beecham Plc
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Publication of WO2001023418A1 publication Critical patent/WO2001023418A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • G01N33/56938—Staphylococcus
    • 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)

Definitions

  • tlie invention relates to newly identified polynucleotides and polypeptides. and their production and uses, as well as their vanants, agonists and antagonists, and their uses
  • tlie invention relates to polynucleotides and polypeptides of the yphC (GTP -binding proteins) famih . as well as their variants, herein referred to as "yphC,” “yphC polynucleot ⁇ de(s),” and “yphC polypept ⁇ de(s)” as tlie case may be
  • Staphylococcal genes and gene products as targets for the development of antibiotics
  • the Staphylococci make up a medicalh important genera of microbes arc known to produce two types of disease, invasive and toxigenic Invasive infections are characterized gcnci lh by abscess fomiation effecting both skin surfaces and deep tissues S aureus is tlie second leading cause of bacteremia in cancer patients Osteomyelitis, septic arthntis, septic thromboplilebitis and acute bacte ⁇ al endocarditis are also relatively common There are at least tliree clinical conditions resultmg from tl e toxigenic properties of Staphylococci Tlie manifestation of these diseases result from tlie actions of exotoxins as opposed to tissue invasion and bacteremia These conditions mclude Staphylococcal food poisoning, scalded skin syndrome and toxic shock syndrome
  • the present invention relates to yphC, in particular yphC polypeptides and yphC pohiiucleotides recombinant matenals and methods for their production
  • tlie invention relates to methods for usmg such polypeptides and polynucleotides.
  • the mvention relates to methods for identifying agonists and antagonists using the materials provided by the invention, and for treating microbial infections and conditions associated with such infections with the identified agonist or antagonist compounds
  • tlie invention relates to diagnostic assays for detecting diseases associated with microbial infections and conditions associated with such infections, such as assays for detecting yphC expression or activits
  • Tlie mvention relates to yphC polypeptides and polynucleotides as descnbed in greater detail below hi particular, tlie mvention relates to polypeptides and polynucleotides of a yphC of Staphyl ⁇ coccus aureus that is related by ammo acid sequence homology to B subtilis yphC polvpeptide Tlie mvention relates especially to yphC having a nucleotide and ammo acid sequences set out m Table 1 as SEQ ID NO 1 and SEQ ID NO 2 respectively Note that sequences recited in the Sequence Listing below as DNA represent an exemplification of the invention, since those of ordinary skill will recognize that such sequences can be usefully employed m polynucleotides m general, including ⁇ bopohnucleotides
  • NCIMB National Collections of Industrial and Marine Bactena Ltd
  • Staphylococcus aureus WCUH29 on deposit
  • the Staphylococcus aureus stram deposit is referred to herem as "tlie deposited stram” or as "the DNA of the deposited stram"
  • the deposited stram compnses a full length yphC gene
  • the sequence of tlie pohnucleotides compnsed the deposited stram, as well as the ammo acid sequence of any pohpeptide encoded thereb are controllmg in the event of any conflict with any desc ⁇ ption of sequences here
  • Tl e deposit of tlie deposited stram has been made under tlie terms of tlie Budapest Treal ⁇ on the International Recognition of the Deposit of Micro-organisms for Purposes of Patent Procedure
  • Tlie deposited stram will be irrevocably and without restnction or condition released to tlie public upon tl e issuance of a patent
  • the deposited stram is provided merely as convenience to tliose of skill in tl e 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 stra
  • pohpeptide is comprised in tlie deposited stram
  • yphC polynucleotide sequences in the deposited stram such as DNA and RNA.
  • ammo acid sequences encoded thereby Also provided by the inv ention are yphC polypeptide and polynucleotide sequences isolated from tlie deposited stram
  • YphC polypeptide of tlie mvention is substantially phylogeneticalh related to other proteins of tlie yphC (GTP-bmdmg proteins) family hi one aspect of tlie mvention there are provided polypeptides of Staphylococcus aureus referred to herem as "yphC” and "yphC polypeptides" as well as biologically, diagnostically. prophylactical . clrnicalh or therapeutically useful vanants thereof, and compositions compnsing tl e same
  • Tlie present mvention furtlier provides for an isolated polypeptide that (a) comprises or consists of an ammo acid sequence that has at least 95% identity, most preferably at least 97-99% or exact kauitit . to that of SEQ ID NO 2 over the entire length of SEQ ID NO 2.
  • polypeptide encoded by an isolated polynucleotide comprising or consisting of a polynucleotide sequence that has at least 95% identity, even more preferably at least 97-99% or exact identity to SEQ ID NO 1 over the entire length of SEQ ID NO 1
  • polypeptide encoded by an isolated polynucleotide comprising or consisting of a polynucleotide sequence encoding a polypeptide that has at least 95% identity, even more preferabh at least 97-99% or exact identity, to tlie amiiio acid sequence of SEQ ID NO 2.
  • polypeptides of tlie mvention mclude a polypeptide of Table 1 [SEQ ID NO 2] (m particular a mature polypeptide) as well as polypeptides and fragments, particularly those that has a biological activitv of yphC, and also those that have at least 95% identity to a polypeptide of Table 1 [SEQ ID NO 2] and also mclude portions of such polypeptides with such portion of the polypeptide generally comprising at least 30 ammo acids and more preferably at least 50 ammo acids
  • Tlie invention also includes a polypeptide consisting of or comprising a polypeptide of tl e fonnula:
  • X is hydrogen, a metal or any other moiety described herein for modified polypeptides, and at the carboxyl te ⁇ rtfnus, Y is hydrogen, a metal or any other moiety described herein for modified polypeptides, R ⁇ and R3 are any amino acid residue or modified amino acid residue, m is an integer between 1 and 1000 or zero, n is an integer between 1 and 1000 or zero, and R 2 is an amino acid sequence of the invention, particularly an amino acid sequence selected from Table 1 or modified forms thereof.
  • R 2 is oriented so that its aniino te ⁇ ninal amino acid residue is at tlie left, covalently bound to Ri and its carboxy terminal amino acid residue is at tlie right, covalently bound to R3.
  • Any stretch of amino acid residues denoted by either Ri or R3, where m and/or 11 is greater than 1, may be either a heteropohnier or a homopolymer, preferably a heteropolymer.
  • Other preferred embodiments of tlie invention are provided where ni is an integer between 1 and 50, 100 or 500, and n is an integer between 1 and 50. 100. or 500.
  • a polypeptide of tlie invention is derived from Staphylococcus aureus, however, it may preferably be obtained from other organisms of tl e same taxonomic genus.
  • a pohpeptide of tlie invention may also be obtained, for example, from organisms of tlie same taxonomic family or order.
  • a fragment is a variant polypeptide having an amino acid sequence that is entirely tlie same as part but not all of any amino acid sequence of any polypeptide of tlie invention.
  • fragments may be "free-standing," or comprised vvithin a larger polypeptide of which they fonn a part or region, most preferably as a single continuous region in a single larger polypeptide.
  • Preferred fragments include, for example, truncation polypeptides having a portion of an amino acid sequence of Table 1 [SEQ ID NO:2], or of variants thereof, such as a continuous series of residues that includes an aniino- and/or carboxyl-teiminal amino acid sequence.
  • tlie polypeptides of the invention produced by or in 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-foniiing regions, turn and turn-forming regions, coil and coil-forming regions, hydropliilic regions, hydrophobic regions, alpha amphipatliic regions, beta amphipathic regions, flexible regions, surface-fomiing regions, substrate binding region, and high antigenic index regions.
  • fragments include an isolated polypeptide comprising an amino acid sequence having at least 15, 20, 30, 40, 50 or 100 contiguous amino acids from the amino acid sequence of SEQ ID NO:2, or an isolated polypeptide comprising an amino acid sequence having at least 15, 20, 30, 40, 50 or 100 contiguous amino acids truncated or deleted from the amino acid sequence of SEQ ID NO:2.
  • Fragments of the polypeptides of tlie mvention may be employed for producmg tlie corresponding full-length polypeptide by peptide synthesis, therefore, these vanants may be employed as intermediates for producmg tlie full-length polypeptides of tlie mvention Polynucleotides It is an object of the mvention to provide polynucleotides that encode yphC polypeptides. particularh polynucleotides that encode a polypeptide herem designated yphC
  • tlie polynucleotide compnses a region encoding yphC polypeptides compnsmg a sequence set out in Table 1 [SEQ ID NO 1] that includes a full length gene, or a variant thereof
  • This mvention provides that tins full length gene is essential to tlie giowth and or survival of an organism that possesses it, such as Staphylococcus aureu s
  • isolated nucleic acid molecules encoding and or expressmg yphC polypeptides and polynucleotides.
  • mcludmg. for example, unprocessed RNAs.
  • Further embodiments of tlie mvention mclude biologically diagnostically, prophylactically . clinically or therapeutically useful polynucleotides and polypeptides. and vanants thereof, and compositions compnsmg the same
  • Another aspect of the mvention relates to isolated polynucleotides.
  • mcludmg at least one full length gene, that encodes a yphC polypeptide having a deduced ammo acid sequence of Table 1 [SEQ ID NO 2J and polynucleotides closely related thereto and vanants thereof
  • yphC polypeptide from
  • Staphylococcus aureus comprising or consisting of an amino acid sequence of Table 1 [SEQ ID NO 2] or a variant thereof
  • a polynucleotide of the mvention encoding yphC polypeptide may be obtained usmg standard cloning and screenmg methods, such as tliose for cloning and sequencmg chromosomal DNA fragments from bactena usmg Staphylococcus aureus WCUH 29 cells as starting matenal, followed by obtaining a full length clone
  • a polynucleotide sequence of the invention such as a polynucleotide sequence given in Table 1 [SEQ ID NO 1]
  • typically a library of clones of chromosomal DNA of Staphylococcus aureus WCUH 29 in E colt or some other suitable host is probed with a radiolabeled ohgonucleotide.
  • Clones carrying DNA identical to that of the probe can then be distinguished usmg stringent hybridization conditions
  • sequencing primers designed from the original polypeptide or polynucleotide sequence it is then possible to extend the polynucleotide sequence m both directions to determine a full length gene sequence Conveniently, such sequencing is perfomied. for example, using denatured double stranded DNA prepared from a plasmid clone Suitable techniques are described by Mamatis, T , Fritsch, E F and Sambrook et al , MOLECULAR CLONING, A LABORATORY MANUAL, 2nd Ed .
  • each DNA sequence set out m Table 1 [SEQ ID NO 1] contams an open readmg frame encoding a protem having about the number of ammo acid residues set forth in Table 1 [SEQ ID NO 2] w ith a deduced molecular weight that can be calculated usmg ammo acid residue molecular weight values well known to tliose skilled m the art
  • the polynucleotide of SEQ ID NO 1. betw een nucleotide numbci 1 and the stop codon that begins at nucleotide number 1309 of SEQ ID NO 1.
  • tlie present mvention provides for an isolated polynucleotide comprising or consisting of (a) a polynucleotide sequence that has at least 95% identity, even more preferably at least 97-99% or exact identity to SEQ ID NO 1 over the entire length of SEQ ID NO 1 or the entire length of that portion of SEQ ID NO 1 which encodes SEQ ID NO 2, (b) a polynucleotide sequence encoding a polypeptide that has at least 95% identity, even more preferably at least 97-99% or 100% exact to tlie ammo acid sequence of SEQ ID NO 2, over the entire length of SEQ ID NO 2
  • a polynucleotide encoding a polypeptide of tlie present mvention, mcludmg homologs and ortliologs from species other than Staphylococcus aureus may be obtained by a process that compnses tlie steps of screenm
  • tlie mvention is a codmg sequence for a mature polypeptide or a fragment thereof, by itself as well as a coding sequence for a mature polypeptide or a fragment m readmg frame with another coding sequence, such as a sequence encoding a leader or secretory sequence, a pre-, or pro- or prepro-protern sequence
  • the polynucleotide of die mvention may also comprise at least one non-coding sequence, mcludmg for example, but not limited to at least one non-coding 5 and 3 ' sequence, such as the transcnbed but non-translated sequences, teimination signals (such as rho-dependent and rho-mdependent termination signals), nbosome bmding sites.
  • polynucleotide sequence may also compnse additional codmg sequence encoding additional ammo acids
  • a marker sequence that facilitates punfication of a fused polypeptide can be encoded In certain embodiments of the mvention.
  • die marker sequence is a hexa-histidme peptide, as provided m the pQE vector (Qiagen, Inc ) and descnbed in Gentz et al , Proc Natl Acad Set , USA 86 821-824 (1989), or an HA peptide tag (Wilson et al Cell 37 767 (1984), both of that may be useful m punfymg polypeptide sequence fused to them
  • Polynucleotides of tlie mvention also mclude, but are not limited to, polynucleotides compnsmg a structural gene and its naturalh associated sequences that control gene expression
  • a preferred embodiment of the mvention is a polynucleotide of consisting of or compnsmg nucleotide 1 to tlie nucleotide immediately upstream of or mcludmg nucleotide 1309 set forth in SEQ ID NO 1 of Table 1, both of that encode a yphC polypeptide
  • the mvention also mcludes a polynucleotide consisting of or comprising a pohnucleotide ot die formula
  • X is hydrogen, a metal or a modified nucleotide residue or together with Y defines a covalent bond, and at the 3' end of the molecule.
  • Y is hydrogen a metal or a modified nucleotide residue, or together with X defines the covalent bond
  • each occurrence of R ] and R3 is independently any nucleic acid residue or modified nucleic acid residue
  • m is an integer betw een 1 and 3000 or zero
  • n is an integer between 1 and 3000 or zero
  • R 2 is a nucleic acid sequence or modified nucleic acid sequence of the invention, particularly a nucleic acid sequence selected from Table 1 or a modified nucleic acid sequence thereof
  • R 2 is oriented so that its 5' end nucleic acid residue is at the left, bound to Rj and its 3' end nucleic acid residue is at the right, bound to R3 Any stretch of nucleic acid residues denoted by either R j and/or R , where m and/or n is greater than 1.
  • X and Y together define a covalent bond the polynucleotide of the above formula is a closed, circular polynucleotide, that can be a double-stranded polynucleotide wherein the formula shows a first strand to which the second strand is complementary
  • m and/or n is an integer between 1 and 1000.
  • Other preferred embodiments of die mvention are provided where m is an mteger between 1 and 50. 100 or 500 and n is an integer between 1 and 50, 100, or 500
  • a polynucleotide of tlie mvention is derived from Staphylococcus aureus however, it may preferably be obtamed from other organisms of tlie same taxonoimc genus A pohnucleotide of tlie mvention may also be obtamed, for example, from organisms of the same taxonomic family or order
  • polynucleotide encoding a polypeptide encompasses polynucleotides that mclude a sequence encoding a polypeptide of the mvention, particularly a bactenal polypeptide and more particularly a polypeptide of the Staphylococcus aureus yphC havmg an amino acid sequence set out 111 Table 1 [SEQ ID NO 2]
  • the term also encompasses polynucleotides that mclude a smgle continuous region or discontmuous regions encoding the polypeptide (for example, polynucleotides interrupted by integrated phage an integrated insertion sequence, an integrated vector sequence, an integrated transposon sequence or due to RNA editing or genomic DNA reorganization) together with additional regions, that also may compnse codmg and/or non-coding sequences
  • the mvention further relates to vanants of the polynucleotides descnbed herem that encode vanants of a polypeptide having a deduced ammo acid sequence of Table 1 [SEQ ID NO 2] Fragments of polynucleotides of the mvention may be used, for example, to synthesize full-length pohnuclcotides of tlie mvention Further particularly preferred embodiments are polynucleotides encoding yphC ⁇ anants that hav e die ammo acid sequence of yphC polypeptide of Table 1 [SEQ ID NO 2] in winch several a few 5 to 10 1 to 5. 1 to 3. 2.
  • Preferred isolated polynucleotide embodmients also mclude polynucleotide fragments such as a polynucleotide comprising a nuclic acid sequence having at least 15, 20, 30, 40, 50 or 100 contiguous nucleic acids from the polynucleotide sequence ot SEQ ID NO.
  • polynucleotide comprising a nucleic acid sequence having at least 15, 20, 30, 40, 50 or 100 contiguous nucleic acids truncated or deleted from the 5' and/or 3' end of the polynucleotide sequence ot SEQ ID NO: l .
  • polynucleotides that are at least 95% or 97% identical over dieir entire lengdi to a polynucleotide encoding yphC polypeptide having an ammo acid sequence set out m Table 1 [SEQ ID NO 2], and polynucleotides that are complementary to such polynucleotides
  • polynucleotides tiiat compnse a region that is at least 95% are especially preferred
  • those with at least 97% are highly preferred among diose with at least 95%. and among tiiese diose with at least 98% and at least 99% are particularly highly preferred with at least 99% being the more preferred
  • Preferred embodiments are polynucleotides encoding polypeptides that retain substantially die same biological function or activity as a mature polypeptide encoded by a DNA of Table 1 [SEQ ID NO 1] hi accordance with certain preferred embodiments of this mvention diere are provided polynucleotides that hybndize, particularly under strmgent conditions, to yphC polynucleotide sequences, such as those polynucleotides m Table 1
  • the mvention further relates to polynucleotides that hybndize to tlie polynucleotide sequences provided herem
  • the mvention especially relates to polynucleotides that hybndize under strmgent conditions to the polynucleotides descnbed herem
  • a specific example of stringent hybridization conditions is overnight incubation at 42°C m a solution comprising 50% formamide. 5x SSC (150mM NaCl. 15mM tnsodium citrate), 50 mM sodium phosphate (pH7 6), 5x Denliardt's solution.
  • the invention also provides a polynucleotide consisting of or comprising a pohnucleotide sequence obtained by screening an appropriate library comprising a complete gene for a po nucleotide sequence set forth in SEQ ID NO 1 under stringent hybridization conditions with a probe ha ⁇ mg the sequence of said polynucleotide sequence set forth in SEQ ID NO 1 or a fragment thereof and isolating said polynucleotide sequence Fragments useful for obtaining such a polynucleotide include foi example, probes and primers fully described elsewhere herem As discussed elsewhere herem regarding polynucleotide assays of die mvention. for instance die polynucleotides of the mvention.
  • RNA may be used as a hybndization probe for RNA.
  • cDNA and genomic DNA to isolate full-length cDNAs and genomic clones encoding yphC and to isolate cDNA and genomic clones of other genes that have a high identity, particularly high sequence identity, to a yphC gene
  • Such probes generally will compnse at least 15 nucleotide residues or base pairs
  • such probes will have at least 30 nucleotide residues or base pairs and may have at least 50 nucleotide residues or base pairs
  • Particularh preferred probes will have at least 20 nucleotide residues or base pairs and will have lee tiian 30 nucleotide residues or base pairs
  • a codmg region of a yphC gene may be isolated by screenmg usmg a DNA sequence prov ided in Table 1 [SEQ ID NO 1] to synthesize an ohgonucleotide probe
  • a labeled o gonucleotide having a sequence complementary to that of a gene of the mvention is then used to screen a library of cDNA. genomic DNA or mRNA to detennrne winch members of die library die probe hybndizes to
  • polynucleotides and polypeptides of die mvention may be employ ed. for example, as research reagents and matenals for discovery of treatments of and diagnostics for diseases, particularly human diseases, as further discussed herem relating to polynucleotide assays
  • polynucleotides of the invention that are ohgonucleotides derived from a sequence of Table
  • SEQ ID NOS 1 or 2 may be used in the processes herein as described, but preferably for PCR. to detenume 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 e utility in diagnosis of the stage of infection and type of infection the pathogen has attained
  • the mvention also provides polynucleotides that encode a polypeptide diat is a mature protein plus additional ammo or carboxyl-ter mal ammo acids, or ammo acids mtenor to a mature polypeptide (when a mature form has more than one polypeptide chain, for instance) Such sequences may play a role in processmg of a protem from precursor to a mature form, may allow protein transport, may lengthen or shorten protem half-life or may facilitate manipulation of a protem for assav or production, among otiier thmgs As generally is the case m vivo, die additional amino acids
  • polynucleotide complementan For each and every polynucleotide of the mvention tiiere is provided a polynucleotide complementan to it It is preferred that these complementary polynucleotides are fully complementary to each polynucleotide widi which they are complementary
  • the entire polypeptide encoded by an open readmg frame is often not required for activity Accordmgly, it has become routme in molecular biology to map die boundanes of die pnmary structure required for activity with N-terminal and C-temnnal deletion experiments
  • These experiments utilize exonuclease digestion or convement restnction sites to cleave codmg nucleic acid sequence
  • Promega (Madison, WI) sell an Erase-a-baseTM system that uses Exonuclease III designed to facilitate analysis of the deletion products (protocol avadable at www promega com)
  • the digested endpoints can be repaired (e g .
  • nucleic acid of SEQ ID NO 1 readily provides contiguous fragments of SEQ ID NO 2 sufficient to provide an activity, such as an enzymatic, binding or antibody-inducing activity
  • Nucleic acid sequences encoding such fragments of SEQ ID NO 2 and vanants thereof as descnbed herem are within die mvention. as are polypeptides so encoded
  • a polynucleotide of the mvention may encode a mature protem.
  • a mature protem plus a leader sequence that may be referred to as a preprotem.
  • a precursor of a mature protem havmg one or more prosequences that are not the leader sequences of a preprotem, or a preproprotem.
  • diat is a precursor to a proprotem, havmg a leader sequence and one or more prosequences, tiiat generally are removed dunng processing steps that produce active and mature forms of die polypeptide Vectors, Host Cells, Expression Systems
  • the mvention also relates to vectors that compnse a polynucleotide or polynucleotides of the mvention.
  • host cells that are genetically engmeered with vectors of the mvention and die production of polypeptides of die mvention by recombinant techniques
  • Cell-free translation systems can also be emplov ed to produce such protems usmg RNAs denved from die DNA constructs of die mvention
  • Recombinant polypeptides of die present mvention may be prepared by processes well known in diose skilled in die art from genetically engmeered host cells compnsmg expression systems Accordmgly.
  • the present mvention relates to expression systems that compnse a polynucleotide or polynucleotides of the present mvention, to host cells that are genetically engmeered with such expression systems, and to the production of polypeptides of the mvention by recombinant techniques For recombinant production of the polypeptides of the mvention.
  • host cells can be genetically engmeered to incorporate expression systems or portions diereof or polynucleotides of die mvention Introduction of a polynucleotide mto the host cell can be effected by mediods descnbed in many standard laboratory manuals, such as Davis, et al .
  • appropnate hosts include bactenal cells, such as cells of streptococci staphylococci, enterococci E colt, streptomyces, cyanobactena, Bacillus subtilis. and Staphylococcus aureus, fungal cells, such as cells of a yeast. Kluveromyces, Saccharomyces, a basidiomycete. Candida a ⁇ bicans and Aspergillus , insect cells such as cells of Drosoph ⁇ a S2 and Spodoptera Sf9.
  • bactenal cells such as cells of streptococci staphylococci, enterococci E colt, streptomyces, cyanobactena, Bacillus subtilis. and Staphylococcus aureus
  • fungal cells such as cells of a yeast. Kluveromyces, Saccharomyces, a basidiomycete.
  • Candida a ⁇ bicans and Aspergillus insect cells such as cells of Drosoph ⁇ a S2 and Spo
  • Such vectors mclude, among others, chromosomal-, episomal- and virus-derived vectors, for example v ectors denved from bactenal plasnnds. from bactenophage, from transposons from yeast episomes from insertion elements, from yeast chromosomal elements, from viruses such as baculovirases. papova ⁇ I ⁇ USCS. such as SV40.
  • vaccinia viruses adenoviruses. fowl pox viruses, pseudorabies viruses, piconiavirases and retroviruses. and vectors denved from combinations thereof, such as those denved from plasnnd and bactenophage genetic elements, such as cosmids and phagemids Tlie expression system constructs niav compnse control regions that regulate as well as engender expression
  • any sv stem or vector suitable to maintain, propagate or express polynucleotides and/or to express a polypeptide m a host mav be used for expression m this regard
  • the appropnate DNA sequence may be inserted mto die expression s ⁇ stem by any of a vanety of well-known and routme techmques. such as, for example, tiiose set forth m Sambrook el al . MOLECULAR CLONING, A LABORATORY MANUAL, (supra)
  • Polypeptides of the mvention can be recovered and punfied from recombinant cell cultures by well-known methods mcludmg ammomum sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic mteraction chromatograph . affimty chromatography, hydroxylapatite chromatography. and lec ⁇ n cliromatography Most preferabh high performance liquid chromatography is employed for punfication
  • Well known teclnnques for refoldmg protem may be employed to regenerate active conformation when die polypeptide is denatured during isolation and or purification
  • This mvention is also related to the use of yphC polynucleotides and polypeptides of die invention for use as diagnostic reagents Detection of yphC polynucleotides and/or polypeptides in a eukaryote particularly a mammal, and especially a human, will provide a diagnostic metiiod for diagnosis of disease staging of disease or response of an infectious organism to drugs Eukaryotes.
  • Polypeptides and polynucleotides for prognosis, diagnosis or otiier analysis may be obtamed from a putatively mfected and/or infected mdividual's bodily matenals
  • Polynucleotides from anv of tiiese sources particularly DNA or RNA. may be used directly for detection or may be amplified enzymaticallv bv usmg PCR or any otiier amplification technique pnor to analysis RNA.
  • mRNA cDNA and genomic DNA may also be used m tlie same ways Usmg amplification, characte ⁇ zation of die species and strain of infectious or resident organism present m an individual, may be made by an analysis of tlie genotype of a selected polynucleotide of the organism
  • Deletions and insertions can be detected by a change m size of die amplified product m companson to a genotype of a reference sequence selected from a related orgamsm preferably a different species of the same genus or a different stram of die same species
  • Point mutations can be identified by hybndizmg amplified DNA to labeled yphC polynucleotide sequences Perfecth or significantly matched sequences can be distinguished from imperfectly or more significanth mismatched duplexes by DNase or RNase digestion, for DNA or RNA respectively, or by detectmg differences m melting temperatures or renaturation kmetics Polynucleotide sequence differences
  • an array of ohgonucleotides probes compnsmg yphC nucleotide sequence or fragments thereof can be constructed to conduct efficient screenmg of. for example, genetic mutations serotype.
  • taxonomic classification or identification Array technology metiiods are well known and have general applicability and can be used to address a vanety of questions m molecular genetics mcludmg gene expression, genetic linkage, and genetic vanabihty (see, for example, Chee et al , Science, 274 610 (1996))
  • the present mvention relates to a diagnostic kit that comprises (a) a polynucleotide of the present invention, preferably the nucleotide sequence of SEQ ID NO 1. or a fragment thereof , (b) a nucleotide sequence complementary to that of (a), (c) a pohpeptide of the present mvention, preferably the polypeptide of SEQ ID NO 2 or a fragment thereof, or (d) an antibody to a polypeptide of the present mvention, preferably to the polypeptide of SEQ ID NO 2
  • a), (b), (c) or (d) may comprise a substantial component
  • Such a kit will be of use in diagnosmg a disease or susceptibility to a Disease, among others
  • This mvention also relates to the use of polynucleotides of the present mvention as diagnostic reagents Detection of a mutated form of a polynucleotide of die mvention,
  • SEQ ID NO 1. that is associated with a disease or pathogenicity will provide a diagnostic tool tiiat can add to. or define, a diagnosis of a disease, a prognosis of a course of disease, a determination of a stage of disease, or a susceptibility to a disease, that results from under-expression. over-expression or altered expression of die pohnucleotide Organisms, particularly infectious organisms, carrying mutations such polynucleotide mav be detected at die polynucleotide level by a vanety of techmques. such as tiiose descnbed elsewhere herein
  • the differences in a polynucleotide and or polypeptide sequence between organisms possessing a first phenotype and organisms possessing a different, second different phenotype can also be determined If a mutation is observed m some or all organisms possessing the first phenot pe but not in any organisms possessing the second phenotype. then the mutation is likely to be the causativ e agent of the first phenotype
  • a polynucleotide and/or polypeptide of the mvention may also be detected at the polynucleotide or polypeptide level by a vanetv of techmques. to allow for serotyping, for example
  • RT-PCR can be used to detect mutations in the RNA It is particularly preferred to use RT-PCR m conjunction with automated detection svstems. such as, for example. GeneScan RNA.
  • cDNA or genonnc DNA may also be used for tlie same purpose PCR As an example.
  • PCR primers complementary to a polynucleotide encodmg yphC pohpeptide can be used to identify and analyze mutations
  • the mvention further provides tiiese pnmers with 1. 2. 3 or 4 nucleotides removed from tlie 5' and/or the 3' end These pnmers may be used for, among otiier tilings, amplifying yphC DNA and/or RNA isolated from a sample denved from an mdividual, such as a bodily matenal
  • the pnmers may be used to amplify a polynucleotide isolated from an infected mdividual.
  • tlie polynucleotide may then be subject to vanous techmques for elucidation of the polynucleotide sequence hi tins wav mutations m the polynucleotide sequence may be detected and used to diagnose and/or prognose tlie infection or its stage or course, or to serotype and/or classify the infectious agent
  • the mvention further provides a process for diagnosmg. disease, preferably bacterial infections. more preferably infections caused by Staphylococcus aureus.
  • a sample derived from an individual comprising deteniunmg from a sample derived from an individual, such as a bodily material, an increased level of expression of polynucleotide havmg a sequence of Table 1 [SEQ ID NO 1]
  • Increased or decreased expression of a yphC polynucleotide can be measured usmg any on of the methods well known in the art for the quantitation of polynucleotides, such as, for example, amplification, PCR, RT-PCR. RNase protection Northern blotting, spectrometry and other hybndization methods
  • a diagnostic assay m accordance with the mvention for detecting over-expression of yphC polypeptide compared to normal control tissue samples may be used to detect die presence of an infection, for example Assay techmques that can be used to determine levels of a yphC polypeptide. in a sample denved from a host, such as a bodily matenal, are well-known to those of skill m the art
  • Assay techmques that can be used to determine levels of a yphC polypeptide. in a sample denved from a host, such as a bodily matenal, are well-known to those of skill m the art
  • Such assay methods mclude radioirnmunoassays, competitive-bmdmg assays. Western Blot analysis, antibodv sandwich assays, antibody detection and ELISA assays
  • Polypeptides and polynucleotides of die mvention may also be used to assess die binding of small molecule substrates and hgands in, for example, cells, cell-free preparations, chemical branes and natural product mixtures These substrates and hgands may be natural substrates and hgands or may be structural or functional mimetics See, e g , Coligan et al , Current Protocols in Immunology 1 (2) Chapter 5 (1991) Polypeptides and polynucleotides of the present mvention are responsible for many biological functions, mcludmg many disease states, in particular the Diseases herem mentioned It is tiierefore desirable to devise screening methods to identify compounds that agonize (e g .
  • die present mvention provides for a method of screenmg compounds to identify those that agomze or that antagonize die function of a polypeptide or polynucleotide of the mvention. as well as related polypeptides and polynucleotides
  • agonists or antagonists e g .
  • Compounds may be employed ed for therapeutic and prophylactic purposes for such Diseases as herem mentioned
  • Compounds may be identified from a ⁇ arietv of sources, for example, cells, cell-free preparations, chenncal hbranes and natural product mixtures
  • Such agonists and antagonists so-identified may be natural or modified substrates, hgands, receptors, enzvmes. etc as the case may be, of yphC polypeptides and polynucleotides. or may be stnictural or functional nnmetics thereof (see Coligan et al , Current Protocols n Immunology 1(2) Chapter 5 (1991))
  • the screening methods may simply measure the binding of a candidate compound to the polypeptide or polynucleotide, or to cells or membranes bearing the polypeptide or polynucleotide or a fusion protein of the polypeptide by means of a label directly or indirectly associated with the candidate compound Alternatively, the screening method may mvolve competition with a labeled competitor
  • these screening methods may test whether the candidate compound results m a signal generated by activation or inhibition of the polypeptide or polynucleotide.
  • usmg detection systems appropriate to the cells compnsmg the polypeptide or polynucleotide Inhibitors of activation are generally assav ed in the presence of a known agonist and the effect on activation by the agonist by the presence of the candidate compound is observed
  • Constitutively active polypeptide and/or constitutively expressed polypeptides and polynucleotides may be employed in screening methods for inverse agonists, in the absence of an agonist or antagonist, by testing whether the candidate compound results m inhibition of activation of the polypeptide or polynucleotide.
  • the screening methods may simply compnse the steps of mixing a candidate compound with a solution comprising a polypeptide or polynucleotide of the present invention, to form a mixture, measuring yphC polypeptide and/or polynucleotide activity m the mixture, and companng the yphC polypeptide and or polynucleotide activity of tlie mixture to a standard Fusion proteins, such as those made from Fc portion and yphC polypeptide.
  • Tlie polynucleotides, polypeptides and antibodies that bind to and or interact with a polv peptide of the present invention may also be used to configure screening methods for detecting the effect of added compounds on the production of mRNA and/or polypeptide in cells
  • an ELISA assay may be constructed for measuring secreted or cell associated levels of polypeptide using monoclonal and polyclonal antibodies by standard methods known m the art This can be used to discover agents that may inhibit or enhance the production of polypeptide (also called antagonist or agonist, respectively) from suitably manipulated cells or tissues
  • the mvention also provides a method of screening compounds to identify tiiose that enhance (agonist) or block (antagonist) the action of yphC polypeptides or polynucleotides.
  • tliose compounds that are bactenstatic and/or bactencidal
  • the method of screenmg may mvolve high-throughput techmques
  • a synthetic reaction mix to screen for agonists or antagonists, a synthetic reaction mix.
  • a cellular compartment such as a membrane, cell envelope or cell wall, or a preparation of any thereof, compnsmg yphC pohpeptide and a labeled substrate or hgand of such polypeptide is mcubated m the absence or the presence of a candidate molecule that may be a yphC agomst or antagonist
  • the ability of the candidate molecule to agonize or antagonize tlie yphC polypeptide is reflected in decreased binding of tlie labeled hgand or decreased production of product from such substrate Molecules that bind gratuitously, i e .
  • Molecules that bind well and. as die case mav be mcrease tlie rate of product production from substrate, mcrease signal transduction. or mcrease chemical channel activity are agonists Detection of the rate or level of, as tlie case may be, production of product from substrate, signal transduction, or chemical channel activity may be enhanced by usmg a reporter s stem Reporter systems that may be useful m tins regard mclude but are not limited to colonmetnc. labeled substrate converted mto product, a reporter gene that is responsive to changes in yphC polynucleotide or polypeptide activity, and binding assays known m the art
  • Polypeptides of the invention may be used to identify membrane bound or soluble receptors, if any, for such polypeptide, through standard receptor binding techniques known in the art These techniques include, but are not limited to, hgand binding and crosshnkmg assays in which the polypeptide is labeled with a radioactive isotope (for instance, -- ⁇ l). chemically modified (for instance, biotinylated), or fused to a peptide sequence suitable for detection or purification, and mcubated with a source of the putative receptor (e g . cells, cell membranes, cell supernatants.
  • a radioactive isotope for instance, -- ⁇ l
  • chemically modified for instance, biotinylated
  • a source of the putative receptor e g . cells, cell membranes, cell supernatants.
  • tissue extracts, bodily materials Other methods include biophysical techniques such as surface plasmon resonance and spectroscopy* These screening methods may also be used to identify agonists and antagonists of the polypeptide that compete with the binding of the polypeptide to its receptor(s). if any Standard methods for conducting such assays are well understood in the art
  • the fluorescence polarization value for a fluorescently-tagged molecule depends on the rotational correlation time or tumbling rate
  • Protein complexes such as fonned by yphC pol peptide associating with another yphC polypeptide or other polypeptide, labeled to comprise a fluorescenth - labeled molecule will have higher polarization values than a fluorescently labeled monomc ⁇ c protein It is preferred that this method be used to characterize small molecules that dismpt pohpeptide complexes
  • Fluorescence energy transfer may also be used characterize small molecules that interfere with the formation of yphC polypeptide dimers. trimers. tetramers or higher order structures or structures formed by yphC polypeptide bound to another polypeptide YphC polypeptide can be labeled with both a donor and acceptor fluorophore Upon mixing of the two labeled species and excitation of the donor fluorophore, fluorescence energy transfer can be detected by observing fluorescence of the acceptor Compounds that block dime ⁇ zation will inhibit fluorescence energv transfer
  • yphC polypeptide self-association can be used to monitor the effect of small molecules on y phC polypeptide self-association as well as an association of yphC polypeptide and another polypeptide or small molecule
  • yphC polypeptide can be coupled to a sensor chip at low site density such that covalently bound molecules will be monome ⁇ c Solution protem can then passed over the yphC polypeptide -coated surface and specific binding can be detected in real-time by monitoring the change m resonance angle caused by a change in local refractive index
  • This technique can be used to characterize the effect of small molecules on kinetic rates and equilibrium binding constants for v phC polypeptide self-association as well as an association of yphC polypeptide and another polypeptide or small molecule
  • a scintillation proximity assay may be used to characterize the interaction between an association of yphC polypeptide with another yphC polypeptide or a different polypeptide yph
  • a polypeptide and or polynucleotide of the present mvention may also be used in a method for the structure-based design of an agonist or antagonist of the polypeptide and/or polynucleotide, by (a) determining in the first instance the three- dimensional structure of the polypeptide and or poly-nucleotide. or complexes thereof (b) deducing the three-dimensional structure for the likely reactive s ⁇ te(s), binding s ⁇ te(s) or mot ⁇ f(s) of an agonist or antagonist, (c) synthesizing candidate compounds that are predicted to bmd to or react with the deduced bmding s ⁇ te(s).
  • tlie present mvention provides metiiods of treating abnoniial conditions such as for instance, a Disease, related to either an excess of, an under-expression of, an elevated activity of. or a decreased activity of yphC polypeptide and or polynucleotide
  • soluble forms of the polypeptides still capable of binding the hgand. substrate, enzymes, receptors etc m competition with endogenous polypeptide and/or polynucleotide may be administered Tv pical examples of such competitors include fragments of the yphC polypeptide and/or po peptide
  • expression of the gene encoding endogenous yphC pohpeptide can be inhibited using expression blocking techniques
  • This blocking may be targeted against any step in gene expression, but is preferably targeted against transcription and or translation
  • An examples of a known technique of this sort involve the use of antisense sequences, either internally generated or separateh administered (see, for example, O'Connor. J Neurochem (1991) 56 560 m Ohgodeoxvnucleotides as Antisense Inhibitors of Gene Expression, CRC Press. Boca Raton. FL (1988))
  • ohgonucleotides that fonn triple helices with the gene can be supplied (see. for example.
  • polynucleotide sequences provided herein may be used in the discov en and development of antibacterial compounds
  • the encoded protein upon expression, can be used as a target for the screening of antibacterial drugs
  • polynucleotide sequences encoding the ammo terminal regions of the encoded protem or Shme-Delgarno or other translation facilitating sequences of the respective mRNA can be used to construct antisense sequences to control the expression of the coding sequence of interest
  • the invention also provides the use of the polypeptide, polynucleotide, agonist or antagonist of the invention to interfere with the initial physical interaction between a pathogen or pathogens and a eukaryotic. preferably mammalian, host responsible for sequelae of infection
  • the molecules of the mvention may be used in the prevention of adhesion of bacteria, in particular gram positive and/or gram negative bacteria, to eukaryotic, preferably mammalian, extracellular matrix proteins on m-dwellmg devices or to extracellular matrix proteins in wounds to block bacterial adhesion between eukaryotic, preferably mammalian, extracellular matrix proteins and bacterial yphC proteins that mediate tissue damage and/or, to block the nonnal progression of pathogenesis in infections initiated other than by the implantation of in-dwelling devices or by other surgical techniques hi accordance with yet another aspect of the mvention.
  • yphC agonists and antagonists preferably bactenstatic or bactencidal
  • the antagonists and agonists of die mvention may be employed, for instance to prev ent inhibit and/or treat diseases
  • Antagonists of the mvention include, among otiiers, small organic molecules, pcptides polypeptides and antibodies that bmd to a polynucleotide and/or polypeptide of the invention and thereby inhibit or extinguish its activity or expression
  • Antagonists also may be small organic molecules a peptide a polypeptide such as a closely related protem or antibody that bmds tlie same sites on a bmdmg molecule such as a bmdmg molecule, without mducmg yphC-mduced activities, tiiereby preventmg die action or expression of yphC polypeptides and/or polynucleotides by excluding yphC polypeptides and/or pohnucleotides from bmdmg
  • Antagonists of tlie mvention also mclude a small molecule that bmds to and occupies die binding site of tlie polypeptide thereby preventmg bmdmg to cellular bmdmg molecules, such that nonnal biological activity is prevented
  • small molecules include but are not limited to small organic molecules peptides or peptide-like molecules
  • Otiier antagonists mclude antisense molecules (see Okano ./ New ochem 56 560 (1991).
  • OLIGODEOXYNUCLEOTIDES AS ANTISENSE INHIBITORS OF GENE EXPRESSION CRC Press. Boca Raton, FL (1988).
  • Preferred antagonists m include compounds related to and vanants of yphC
  • Other examples of polypeptide antagonists m include antibodies or, m some cases, oligonucleotides or proteins that are closely related to the hgands. substrates, receptors, enzymes, etc . as tlie case may be. of the polypeptide. e g , a fragment of tlie hgands.
  • Small molecules of the invention preferably have a molecular weight below 2.000 daltons more preferably between 300 and 1,000 daltons. and most preferably between 400 and 700 daltons It is preferred that these small molecules are organic molecules
  • Hehcobacter pylori (herein "H pylori”) bacteria mfect the stomachs of over one-third of the world's population causing stomach cancer, ulcers, and gastritis (International Agencv for Research on Cancer (1994) Schistosomes, Liver Flukes and Hehcobacter 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 pylon and gastric adenocarcmoma, classifying the bacterium as a Group I (definite) carcinogen
  • Preferred antimicrobial compounds of the mvention (agomsts and antagonists of yphC pohpeptides and/or polynucleotides) found using screens provided by the invention, or known in the art particularly * narrow-spectrum antibiotics, should be useful in the treatment of H pylori infection Such
  • “Bodily mate ⁇ al(s) means any matenal denved from an mdividual or from an orgamsm infecting infesting or inhabiting an mdividual.
  • mcludmg but not limited to. cells, tissues and waste, such as bone blood serum, cerebrospmal fluid, semen, saliva, muscle, cartilage, organ tissue sl ⁇ n unne stool or autopsv matenals
  • D ⁇ sease(s) means any disease caused by or related to mfection by a bactena. mcludmg for example, disease, such as, infections of the upper respiratory tract (e g . otitis media, bactenal tracheitis.
  • mtrarenal and perrnephnc absces. toxic shock syndrome skin (e g , impetigo, follicuhtis. cutaneous abscesses, celluhtis, ound infection, bactenal myositis) bone and jomt (e g , septic arth ⁇ tis, osteomyelitis)
  • “Host cell(s)” is a cell that has been introduced (e g . transformed or transfected) or is capable of introduction (e g . transformation or transfection) by an exogenous polynucleotide sequence "Identity,” as known in the art, is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as the case may be, as determined by comparing the sequences hi tlie 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
  • Polynucleotide embodiments further include an isolated polynucleotide comprising a polynucleotide sequence having at least a 95, 97 or 100% identity to the reference sequence of SEQ ID NO 1.
  • 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, mcludmg 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 withm the reference sequence, and wherein said number of nucleotide alterations is determined by multiplying the total number of nucleotides in SEQ ID NO 1 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 1.
  • y is 0 95 for 95%, 0 97 for 97% or 1 00 for 100%. and • is the symbol for the multiplication operator, and wherein any non-integer product of x 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 m this coding sequence and therebv alter the polypeptide encoded by the polynucleotide following such alterations
  • Polypeptide embodiments further include an isolated polypeptide comprising a polypeptide having at least a 95, 97 or 100% identity to a polypeptide reference sequence of SEQ ID 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 amino acid alterations as compared to the reference sequence, wherein said alterations are selected from the group consisting of at least one amino acid deletion, substitution, including conservative and non-conservative substitution, or insertion and wherein said alterations may occur at the amino- or carboxy-tenninal positions of the reference polypeptide sequence or anywhere between those terminal positions, interspersed either mdividualh among the ammo acids in the reference sequence or in one or more contiguous groups ithin the reference sequence, and wherein said number of amino acid alterations is determined by multiplying the total number of amino acids in SEQ ID NO:2 by the integer defining the percent identity divided by 100 and then subtracting that product from said total number of amino acids in SEQ ID NO.2. or.
  • n a is the number of amino acid alterations
  • x a is the total number of ammo acids in SEQ ID NO:2
  • y is 0.95 for 95%, 0.97 for 97% or 1.00 for 100%.
  • • is the symbol for the multiplication operator, and wherein any non-integer product of x a and y is rounded down to the nearest integer prior to subtracting it from x a .
  • “Individual(s)” means a multicellular eukaryote. mcludmg. but not limited to a metazoan. a mammal, an ovid. a bovid, a simian, a primate, and a human
  • Isolated means altered “by tlie 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 livmg organism is not “isolated.” but tlie same polynucleotide or polypeptide separated from die coexisting materials of its natural state is “isolated", as die term is employed herem
  • a polynucleotide or polypeptide that is introduced mto an orgamsm by transformation, genetic manipulation or by any other recombinant method is "isolated” even if it is still present in said organism. which orgamsm may be living or non-living.
  • Organism(s) means a (i) prokaryote. including but not limited to. a member of the genus
  • Streptococcus Staphylococcus, Bordetella, Coi ⁇ nebactenum, Mycobacterium, Neissena.
  • Streptococcus pyogenes Streptococcus pyogenes, Streptococcus agalacUae, Streptococcus faecalis, Streptococcus faecium, Streptococcus durans.
  • Neissena gonorrheae Neissena meningitidis, Staphylococcus aureus, Staphylococcus epidermidis Corynebactenum dipthenae, Gardnerella vagma s, Mycobactenum tuberculosis, Mycobacterium bovis Mycobactenum ulcerans, Mycobacterium leprae, Actinomyctes israehi, Listena monocytogenes, Bordetella pertusis, Bordatella parapertusis, Bordetella bronchiseptica, Eschenchia coll, Shigella dysentenae Haemophilus influenzae, Haemophilus aegy
  • Polynucleotide(s) generally refers to any polynbonucleotide or polydeoxynbonucleotide.
  • tiiat may be unmodified RNA or DNA or modified RNA or DNA
  • Polynucleot ⁇ de(s)" mclude witiiout limitation smgle- and double-stranded DNA, DNA that is a mixture of smgle- and double-stranded regions or single- double- and tnple-stranded regions, smgle- and double-stranded RNA. and RNA that is mixture of smgle- and double-stranded regions, hyb ⁇ d molecules compnsmg DNA and RNA that may be single-stranded or.
  • polynucleotide refers to tnple-stranded regions compnsmg RNA or DNA or both RNA and DNA
  • Tlie regions may mclude all of one or more of die molecules, but more typically mvolve only a region of some of die molecules
  • One of die molecules of a tnple-helical region often is an ohgonucleotide As used herem.
  • polynucleot ⁇ de(s) also mcludes DNAs or RNAs as descnbed above that compnse one or more modified bases
  • DNAs or RNAs with backbones modified for stability or for other reasons are “polynucleot ⁇ de(s)” as that term is intended herem
  • DNAs or RNAs compnsmg unusual bases such as mosme, or modified bases, such as tntylated bases, to name just two examples are polynucleotides as the tenn is used herem
  • a great vanety of modifications have been made to DN A and RNA that serve many useful purposes known to tiiose of skill m tlie art
  • the tenn "polynucleot ⁇ de(s)” as it is employed herem embraces such chemically, enzymatically or metabolically modified fonns of polynucleotides, as well as the chemical forms of DNA and RNA char
  • Polypept ⁇ de(s) refers to any peptide or protem compnsmg two or more amino acids joined to each other by peptide bonds or modified peptide bonds
  • Polypept ⁇ de(s) refers to botii short chains commonly referred to as peptides, ohgopeptides and oligomers and to longer chains generally referred to as proteins
  • Polypeptides may compnse ammo acids other than the 20 gene encoded ammo acids
  • Polypept ⁇ de(s)” m clude those modified either by natural processes, such as processing and otiier post-translational modifications, but also by chemical modification techmques Such modifications are well descnbed 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 wdl be appreciated that the same type of modification may be present in the same or varying degree at several sites m a given polypeptide Also, a given polypeptide may comp
  • mcludmg die peptide backbone, the ammo acid side-chains, and die ammo or carboxyl tennmi Modifications mclude for example acetylation, acylation. ADP-nbosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide denvative. covalent attachment of a lipid or lipid denvative, covalent attachment of phosphotidylmositol, cross-linkmg. cyclization. disulfidc bond fomiation, demetiiylation, fonnation of covalent cross-links, formation of cysteine.
  • fomiation of pyroglutamate fo ⁇ nylation. gamma-carboxy lation. GPI anchor fonnation. hydroxy lation. lod ation. mediylation, mynstoylation, oxidation, proteolytic processmg, phosphorylation. preny lation. racennzation glycosylation, hpid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hy droxy lation and ADP-nbosylation, selenoylation. sulfation, transfer-RNA mediated addition of ammo acids to proteins such as arginylation. and ubiquitination See, for instance.
  • Recombinant expression system(s) refers to expression systems or portions thereof or polynucleotides of die mvention introduced or transformed mto a host cell or host cell h sate for die production of die polynucleotides and polypeptides of die mvention "Va ⁇ ant(s)" as the tenn 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 m the nucleotide sequence of the variant may or may not alter the ammo acid sequence of a pol peptide encoded by the reference polynucleotide Nucleotide changes may result in ammo acid substitutions additions, deletions, fusion protems and truncations in the polypeptide encoded by the reference sequence, as discussed
  • a substituted or inserted amino acid residue may or may not be one encoded by the genetic code
  • the present mvention also mcludes mclude vanants of each of die polypeptides of the mvention. that is polypeptides that vary from the referents by conservative ammo acid substitutions, whereby a residue is substituted by another with like charactenstics Typical such substitutions are among Ala, Val, Leu and lie, among Ser and Thr. among the acidic residues Asp and Glu among Asn and Gin, and among the basic residues Lys and Arg, or aromatic residues Phe and Tyr Particularly preferred are vanants m which several.
  • 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-naturalh occurring variants of polynucleotides and polypeptides may be made by mutagenesis techniques bv direct synthesis, and bv other recombinant methods known to skilled artisans EXAMPLES
  • Tlie examples below are earned out using standard teclnnques that are well known and routine to tiiose of skill in tlie art. except where otiierwise descnbed in detail Tlie examples are lllustrativ e but do not limit the mvention
  • the polynucleotide having a DNA sequence given m Table 1 [SEQ ID NO 1] was obtained from a library of clones of chromosomal DNA of Staphylococcus aureus in E coh
  • Total cellular DNA is mechanically sheared by passage through a needle in order to size- fractionate according to standard procedures
  • DNA fragments of up to 1 lkbp m size are rendered blunt by treatment with exonuclease and DNA polymerase, and EcoRI linkers added Fragments are hgated mto the vector Lambda ZapII that has been cut with EcoRI.
  • the library packaged by standard procedures and E coh infected with the packaged library The library is amplified by standard procedures
  • Total cellular DNA is partially hydrolyzed with a one or a combination of restriction enzv mes 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 mto the vector Lambda ZapII that have been cut with EcoRI, the library packaged by standard procedures, and E coh infected with the packaged library The library is amplified by standard procedures Example 2 yphC Characterization
  • the yphC gene is expressed during infection of Staphylococcus aureus WCUH29 in a thight lession and pyelonephritis infection models
  • Necrotic fatty tissue from a four day groin infection or kidney from a seven day pyelonephritis infection of Staphylococcus aureus WCUH29 in the mouse is efficiently disrupted and processed in the presence of acid phenol and detergent to provide a mixture of animal and bacterial RNA
  • acid phenol and detergent to provide a mixture of animal and bacterial RNA
  • changes in the population of bacterial mRNA is minimized
  • the resultant total RNA is free of DNA and protein (including RNAases and DNAases)
  • the optimal conditions for disruption and processmg to give high yields of bacterial mRNA with transcripts of long length are follow ed bv reverse transcribing the resulting mRNA to cDNA and amplified with ORF-specific primers for a bacterial gene known to be expressed constitutively and at low copy number in Staphylococcus aureus WCUH29
  • Infected tissue samples in 2-ml cyro-strorage tubes, are removed from -80°C storage mto a dry ice ethanol bath In a microbiological safety cabinet the samples are disrupted up to eight at a time while the remaining samples are kept frozen m the dry ice ethanol bath
  • 50-100 mg of the tissue is transfered to a FastRNA tube containing a silica/ceramic matrix (BIO 101)
  • 1 ml of extraction reagents FastRNA reagents, BIO 101
  • the tubes are shaken m a reciprocating shaker (FastPrep FP120, B1O101) at 6000 rpm for 20-120 sec
  • the crude RNA preparation is extracted with cliloroform/isoamyl alcohol, and precipitated with DEPC-treated Isopropanol Precipitation Solution (BIO101) RNA preparations
  • DNA was removed from 50 microgram samples of RNA by a 30 mmute treatment at 37°C with 10 units of RNAase-free DNAasel (GeneHunter) in the buffer supplied in a final volume of 57 microhters
  • RNA w was inactivated and removed by phenol chloroform extraction RNA w as precipitated with 5 microhters of 3 M NaOAc and 200 microhters 100% EtOH. and pelleted bv cent ⁇ fugation at 12,000g for 10 minutes
  • the RNA is pelleted (12,000g for 10 mm ). washed with 75% ethanol (v/v m DEPC-treated water), air-dried for 5-10 mm. and resuspended in 10-20 microhters of DEPC-treated water RNA yield is quantitated by OD260 after 1 1000 dilution of the cleaned RNA sample RNA is stored at -80°C if necessary and reverse-transcribed within one week
  • PCR reactions are set up on ice in 0 2ml tubes by adding the following components 43 microhtres PCR Master Mix (Advanced Biotechnologies Ltd ). 1 microhtre PCR primers (optimally 18-25 basepairs m length and designed to possess similar annealing temperatures), each primer at lOmM initial concentration, and 5 microhtres cDNA PCR reactions are ran on a Perkm Elmer GeneAmp PCR Sy stem 9600 as follows 2 minutes at
  • PCR products are conveniently labelled by the use of a labelled PCR primer (e g labelled at the 'end with a dve)
  • a suitable aliquot of the PCR product is ran out on a polyacrylamide sequencing gel and its presence and quantity detected using a suitable gel scanning system (e g ABI P ⁇ sniTM 377 Sequencer using GeneScanTM software as supplied by Perkin Elmer)
  • RT/PCR controls may mclude +/- reverse transc ⁇ ptase reactions, 16S rRNA primers or DNA specific pnmer pairs designed to produce PCR products from non-transcribed Streptococcus pneumomae 0100993 genomic sequences
  • Primer pairs which fail to give the predicted sized product in either DNA PCR or RT/PCR are PCR failures and as such are uninfonnative Of those which give the correct size product with DNA PCR two classes are distinguished m RT/PCR 1 Genes which are not transcribed m vivo reproducibly fail to give a product in RT/PCR, and 2 Genes which are transcribed in vivo reproducibly give the correct size product in RT/PCR and show a stronger signal m the +RT samples than the signal (if at all present) in -RT controls
  • Example 3 The yphC gene is essential for S. aureus in vitro growth.
  • allelic replacement cassette was generated using PCR technology
  • the cassette consisted of a pair of 500bp chromosomal DNA fragments flanking an erythromycm resistance gene
  • the chromosomal DNA sequences are the 500bp preceding and following the DNA sequence encoding the yphC gene contained in Seq ID NO 1
  • the cassette was digested and cloned into a pBluesc ⁇ ptIIKS(+) derivative containing the tetracyclme resistance gene (tetK)
  • the resulting plasnnd was introduced into RN4220 by electroporation with selection for erythromycin resistance (Em ⁇ -) Tetracy clme resistant (Tc ⁇ -) clones were examined for target specific plasnnd integration by diagnostic PCR with various primer pairs based on plasnnd and locus specific sequences Plasmid cointegrants were transferred back to either RN4220 or NCTC8325 by phage-11 transduction for a second cross-over recombination

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Abstract

L'invention concerne des polypeptides yphC et des polynucléotides codant pour lesdits polypeptides yphC, ainsi que des procédés de production desdits polypeptides par des techniques de recombinaison. L'invention concerne également des procédés de criblage de composés antibactériens faisant intervenir ces polypeptides yphC.
PCT/US2000/025566 1999-09-28 2000-09-19 YphC WO2001023418A1 (fr)

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US40696899A 1999-09-28 1999-09-28
US09/406,968 1999-09-28

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WO2001023418A1 true WO2001023418A1 (fr) 2001-04-05

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0786519A2 (fr) * 1996-01-05 1997-07-30 Human Genome Sciences, Inc. Polynucléotides et séquences de Staphylococcus aureus
WO2000014200A2 (fr) * 1998-09-09 2000-03-16 Millennium Pharmaceuticals, Inc. Genes bacteriens essentiels et leur utilisation

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0786519A2 (fr) * 1996-01-05 1997-07-30 Human Genome Sciences, Inc. Polynucléotides et séquences de Staphylococcus aureus
WO2000014200A2 (fr) * 1998-09-09 2000-03-16 Millennium Pharmaceuticals, Inc. Genes bacteriens essentiels et leur utilisation

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