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WO2001032022A1 - YtgP - Google Patents

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Publication number
WO2001032022A1
WO2001032022A1 PCT/US2000/030199 US0030199W WO0132022A1 WO 2001032022 A1 WO2001032022 A1 WO 2001032022A1 US 0030199 W US0030199 W US 0030199W WO 0132022 A1 WO0132022 A1 WO 0132022A1
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WO
WIPO (PCT)
Prior art keywords
polypeptide
seq
polynucleotide
sequence
isolated
Prior art date
Legal status (The legal status 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 status listed.)
Ceased
Application number
PCT/US2000/030199
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English (en)
Inventor
Magdalena Zalacain
Sanjoy Biswas
Martin K. R. Burnham
Stephanie Van Horn
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
SmithKline Beecham Ltd
SmithKline Beecham Corp
Original Assignee
SmithKline Beecham Ltd
SmithKline Beecham Corp
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Publication of WO2001032022A1 publication Critical patent/WO2001032022A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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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)
    • 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
    • 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
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/02—Screening involving studying the effect of compounds C on the interaction between interacting molecules A and B (e.g. A = enzyme and B = substrate for A, or A = receptor and B = ligand for the receptor)

Definitions

  • This invention relates to newly identified polynucleotides and polypeptides, and their production and uses, as well as their vanants, agonists and antagonists, and their uses hi particular, the invention relates to polynucleotides and polypeptides of the ytgP (membrane protein) family, as well as their vanants, herein referred to as "ytgP,” “ytgP polynucleot ⁇ de(s),” and “ytgP polypept ⁇ de(s)" as the case may be
  • 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 charactenzed generally by abscess formation effecting both skin surfaces and deep tissues S aureus is the second leading cause of bacteremia in cancer patients Osteomyelitis, septic arthntis.
  • 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
  • polynucleotides and polypeptides such as the ytgP embodiments of the invention, that have a present benefit of, among other tilings, being useful to screen compounds for antimicrobial activity
  • Such factors are also useful to determine their role in pathogenesis of infection. dysfunction and disease
  • identification and characterization of such factors and their antagonists and agonists to find ways to prevent, ameliorate or correct such infection, dysfunction and disease
  • the present invention relates to ytgP. in particular ytgP polypeptides and ytgP polynucleotides. recombinant matenals and methods for their production
  • the mvention relates to methods for usmg such polypeptides and polynucleotides.
  • the invention relates to methods for identifying agonists and antagonists using the materials provided by the invention, and for treating microbial infections and conditions associated with such infections with the identified agonist or antagonist compounds
  • the mvention relates to diagnostic assays for detectmg diseases associated with microbial infections and conditions associated with such infections, such as assays for detecting ytgP expression or activity
  • the mvention relates to ytgP polypeptides and polynucleotides as descnbed in greater detail below
  • the mvention relates to polypeptides and polynucleotides of a ytgP of Staphylococcus aureus that is related by ammo acid sequence homology to B subtihs ytgP polypeptide
  • the mvention relates especially to ytgP havmg a nucleotide and ammo acid sequences set out m Table 1 as SEQ ID NO 1 and SEQ ID NO 2 respectively
  • sequences recited in the Sequence Listing below as "DNA” represent an exemplification of the invention, since those of ordinary skill will recognize that such sequences can be usefully employed in polynucleotides in general, including ⁇ bopolynucleotides
  • an isolated nucleic acid molecule encoding a mature polypeptide expressible by the Staphylococcus aureus WCUH 29 strain, which polypeptide is compnsed in the deposited stram
  • ytgP polynucleotide sequences m the deposited strain such as DNA and RNA
  • ammo acid sequences encoded thereby Also provided by the mvention are ytgP polypeptide and polynucleotide sequences isolated from the deposited stram
  • YtgP polypeptide of the mvention is substantially phylogenetically related to other proteins of the ytgP (membrane protem) family
  • polypeptides of Staphylococcus aureus referred to herem as "ytgP” and “ytgP polypeptides” as well as biologically, diagnostically, prophylactically, clinically or therapeutically useful variants thereof, and compositions comprising the same
  • the present mvention further provides for an isolated polypeptide that (a) comprises or consists of an ammo acid sequence that has at least 95% identity, most preferably at least 97-99% or exact identity, to that of SEQ ID NO 2 over the entire length of SEQ ID NO 2, (b) a polypeptide encoded by an isolated polynucleotide comprising or consisting of a polynucleotide sequence that has at least 95% identity, even more preferably at least 97-99% or exact identity to SEQ ID NO 1 over the entire length of SEQ ID NO 1, (c) a polypeptide encoded by an isolated polynucleotide comprising or consistmg of a polynucleotide sequence encoding a polypeptide that has at least 95% identity, even more preferably at least 97-99% or exact identity, to the ammo acid sequence of SEQ ID NO 2, over the entire length of SEQ ID N02
  • polypeptides of the mvention include 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 activity of ytgP, and also those that have at least 95% identity to a polypeptide of Table 1 [SEQ ID NO:2] and also include portions of such polypeptides with such portion of the polypeptide generally comprising at least 30 amino acids and more preferably at least 50 amino acids.
  • the invention also includes a polypeptide consisting of or comprising a polypeptide of the formula: X-(R 1 ) m -(R 2 )-(R 3 ) n -Y wherein, at the amino terminus, X is hydrogen, a metal or any other moiety described herein for modified polypeptides, and at the carboxyl terminus, Y is hydrogen, a metal or any other moiety described herein for modified polypeptides, Ri 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 fonns tliereof.
  • R 2 i s oriented so that its amino terminal amino acid residue is at the left, covalently bound to R ⁇ and its carboxy terminal amino acid residue is at the right, covalently bound to R3.
  • Any stretch of amino acid residues denoted by either R ⁇ or R3, where m and/or n is greater than 1, may be either a heteropolymer or a homopolymer, preferably a heteropolymer.
  • Other preferred embodiments of the invention are provided where m is an integer between 1 and 50, 100 or 500, and n is an integer between 1 and 50, 100, or 500.
  • a polypeptide of the invention is derived from Staphylococcus aureus, however, it may preferably be obtained from other organisms of the same taxonomic genus.
  • a polypeptide of the invention may also be obtained, for example, from organisms of the same taxonomic family or order.
  • a fragment is a variant polypeptide having an amino acid sequence that is entirely the same as part but not all of any amino acid sequence of any polypeptide of the invention.
  • fragments may be "free-standing,” or comprised within a larger polypeptide of which they form a part or region, most preferably as a single continuous region in a single larger polypeptide.
  • Preferred fragments include, for example, truncation polypeptides having a portion of an amino acid sequence of Table 1 [SEQ ID NO:2], or of variants tliereof. such as a continuous series of residues that includes an amino- and/or carboxyl-terminal amino acid sequence.
  • Degradation forms of the polypeptides of the invention produced by or in a host cell, particularly a 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-forming regions, turn and tiim-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.
  • 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 the mvention may be employed for producing the corresponding full-length polypeptide by peptide synthesis, therefore, these vanants may be employed as intermediates for producmg the full-length polypeptides of the mvention Polynucleotides It is an object of the mvention to provide polynucleotides that encode ⁇ tgP polypeptides, particularly polynucleotides that encode a polypeptide herem designated ytgP
  • the polynucleotide compnses a region encodmg ytgP polypeptides compnsmg a sequence set out in Table 1 [SEQ ID NO 1] that mcludes a full length gene, or a variant thereof
  • This mvention provides that this full length gene is essential to the growth and or survival of an organism that possesses it, such as Staphylococcus aureus
  • isolated nucleic acid molecules encoding and/or expressmg ytgP polypeptides and polynucleotides, particularly Staphylococcus aureus ytgP polypeptides and polynucleotides, including, for example, unprocessed RNAs, nbozyme RNAs, mRNAs, cDNAs, genomic DNAs, B- and Z-DNAs
  • ytgP polypeptides and polynucleotides including, for example, unprocessed RNAs, nbozyme RNAs, mRNAs, cDNAs, genomic DNAs, B- and Z-DNAs
  • Further embodiments of the mvention mclude biologically, diagnostically, prophylactically, clinically or therapeutically useful polynucleotides and polypeptides, and vanants thereof, and compositions compnsmg the same
  • Another aspect of the mvention relates to isolated polynucleotides, including at least one full length gene, that encodes a ytgP polypeptide havmg a deduced ammo acid sequence of Table 1 [SEQ ID NO 2] and polynucleotides closely related thereto and vanants thereof
  • a ytgP polypeptide from Staphylococcus aureus comprising or consisting of an ammo acid sequence of Table 1 [SEQ ID NO 2], or a variant thereof Usmg the information provided herein, such as a polynucleotide sequence set out in Table 1 [SEQ ID NO 2], or a variant thereof Usmg the information provided herein, such as a polynucleotide sequence set out in Table 1 [SEQ ID NO 2]
  • a polynucleotide of the mvention encodmg ytgP polypeptide may be obtained usmg standard cloning and screening methods, such as those for cloning and sequencmg chromosomal DNA fragments from bacte ⁇ a usmg Staphylococcus aureus WCUH 29 cells as starting matenal, followed b ⁇ obtaining a full length clone
  • standard cloning and screening methods such as those for cloning and sequencmg chromosomal DNA fragments from bacte ⁇ a usmg Staphylococcus aureus WCUH 29 cells as starting matenal, followed b ⁇ obtaining a full length clone
  • a polynucleotide sequence of the mvention may be obtained usmg standard cloning and screening methods, such as those for cloning and sequencmg chromosomal DNA fragments from bacte ⁇ a usmg Sta
  • 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 coh or some other suitable host is probed with a radiolabeled ohgonucleotide.
  • Clones carrying DNA identical to that of the probe can then be distinguished using stringent hybridization conditions
  • sequencing primers designed from the o ⁇ gmal polypeptide or polynucleotide sequence it is then possible to extend the polynucleotide sequence m both directions to determine a full length gene sequence
  • sequencing is performed, for example, using denatured double stranded DNA prepared from a plasmid clone Suitable techniques are described by Mamatis, T , F ⁇ tsch.
  • each polynucleotide set out m Table 1 [SEQ ID NO 1] was discovered m a DNA library denved from Staphylococcus aureus WCUH 29 Moreover, each DNA sequence set out m Table 1 [SEQ ID NO 1] contains an open readmg frame encodmg a protem havmg about the number of ammo acid residues set forth in Table 1 [SEQ ID NO 2] with a deduced molecular weight that can be calculated usmg ammo acid residue molecular weight values well known to those skilled in the art
  • the present mvention provides for an isolated polynucleotide comprising or consistmg 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 that 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 the ammo acid sequence of SEQ ID NO 2, over the entire length of SEQ ID NO 2
  • a polynucleotide encodmg a polypeptide of the present mvention, mcludmg homologs and orthologs from species other than Staphylococcus aureus. may be obtained by a process that comp ⁇ ses the steps of screening an approp ⁇ ate library under stringent hyb ⁇ dization conditions with a labeled or detectable probe consistmg of or comp ⁇ smg the sequence of SEQ ID NO 1 or a fragment thereof, and isolatmg a full-length gene and/or genomic clones comp ⁇ smg said polynucleotide sequence
  • the mvention provides a polynucleotide sequence identical over its entire length to a codmg sequence (open reading frame) m Table 1 [SEQ ID NO 1] Also provided by the mvention is a codmg sequence for a mature polypeptide or a fragment thereof, by itself as well as a codmg sequence for a mature polypeptide or a fragment m readmg frame with another codmg sequence, such as a sequence encodmg a leader or secretoiy sequence, a pre-, or pro- or prepro-protem sequence
  • the polynucleotide of the mvention may also compnse at least one non-coding sequence, mcludmg for example, but not limited to at least one non-coding 5 " and 3' sequence, such as the transc ⁇ bed but non-translated sequences, termination signals (such as rho-dependent and rho-mdependent termination signals), ⁇ bosome binding sites, Kozak sequences, sequences that stabilize mRNA
  • a preferred embodiment of the mvention is a polynucleotide consistmg of or comp ⁇ smg nucleotide 721 to the nucleotide immediately upstream of or mcludmg nucleotide 1660 set fortli m SEQ ID NO 1 of Table 1, both of which encode a ytgP polypeptide
  • the mvention also mcludes a polynucleotide consistmg of or comp ⁇ smg a polynucleotide of the formula
  • Ri and R3 is independently any nucleic acid residue or modified nucleic acid residue
  • m is an integer between 1 and 3000 or zero
  • n is an integer between 1 and 3000 or zero
  • R 2 is a nucleic acid sequence or modified nucleic acid sequence of the invention, particularly a nucleic acid sequence selected from Table 1 or a modified nucleic acid sequence thereof In the polynucleotide formula above, R 2 is oriented so that its 5' end nucleic acid residue is at the left, bound to Ri and its 3' end nucleic acid residue
  • polynucleotide of the mvention is de ⁇ ved from Staphylococcus aureus, however, it may preferably be obtained from other organisms of the same taxonomic genus
  • a polynucleotide of the mvention may also be obtained, for example, from organisms of the same taxonomic family or order
  • polynucleotide encoding a polypeptide encompasses polynucleotides that mclude a sequence encodmg a polypeptide of the mvention, particularly a bactenal polypeptide and more particularly a polypeptide of the Staphylococcus aureus ytgP havmg an ammo acid sequence set out m Table 1 [SEQ ED NO 2]
  • the tenn also encompasses polynucleotides that mclude a smgle contmuous region or discontinuous regions encoding the polypeptide (for example, polyn
  • the mvention further relates to vanants of the polynucleotides descnbed herem that encode vanants of a polypeptide havmg 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 polynucleotides of the mvention
  • polynucleotides encodmg ytgP vanants that have the ammo acid sequence of ytgP polypeptide of Table 1 [SEQ ID NO 2] m which several, a few, 5 to 10, 1 to 5, 1 to 3, 2. 1 or no ammo acid residues are substituted, modified, deleted and/or added, m any combmation
  • Preferred isolated polynucleotide embodiments also mclude polynucleotide fragments, such as a polynucleotide comprising a nuclic acid sequence having at least 15, 20, 30, 40, 50 or 100 contiguous nucleic acids from the polynucleotide sequence of SEQ ID NO: l, or an polynucleotide comprising a nucleic acid sequence having at least 15, 20, 30, 40, 50 or 100 contiguous nucleic acids truncated or deleted from the 5' and/or 3' end of the polynucleotide sequence of SEQ ID NO. l
  • prefened embodiments of the mvention are polynucleotides that are at least 95% or 97% identical over their entire lengtli to a polynucleotide encodmg ytgP polypeptide having an ammo acid sequence set out m Table 1 [SEQ ID NO 2], and polynucleotides that are complementary to such polynucleotides
  • Most highly prefened are polynucleotides that compnse a region that is at least 95% are especially prefened
  • those with at least 97% are highly prefened among those with at least 95%, and among these those with at least 98% and at least 99% are particularly highly preferred, with at least 99% bemg the more prefened
  • Preferred embodiments are polynucleotides encoding polypeptides that retain substantially the same biological function or activity as a mature polypeptide encoded by a DNA of Table 1 [SEQ ID NO 1]
  • the mvention further relates to polynucleotides that hybndize to the polynucleotide sequences provided herem
  • the mvention especially relates to polynucleotides that hybndize under stringent conditions to the polynucleotides descnbed herem
  • a specific example of stringent hybridization conditions is overnight incubation at 42°C m a solution compnsmg 50% formamide, 5x SSC (150mM NaCl, 15mM tnsodium citrate), 50 mM sodium phosphate (pH7 6), 5x Denhardt's solution, 10% dextran sulfate, and 20 micrograms/ml of denatured, sheared salmon sperm DNA, followed by washing the hybridization support in 0 lx SSC at about 65 °C Hybridization and wash conditions are well known and exemplified in Sambrook, et al . Molecular Cloning A Laboratory Manual. Second Edition, Cold Spn
  • the invention also provides a polynucleotide consistmg of or comprising a polynucleotide sequence obtained by screening an appropriate library comprising a complete gene for a polynucleotide sequence set forth m SEQ ID NO 1 under stringent hybridization conditions with a probe having the sequence of said polynucleotide sequence set forth SEQ ID NO 1 or a fragment thereof, and isolating said polynucleotide sequence Fragments useful for obtaining such a polynucleotide include, for example, probes and pnmers fully described elsewhere herein As discussed elsewhere herem regarding polynucleotide assays of the mvention.
  • the polynucleotides of the mvention may be used as a hybndization probe for RNA, cDNA and genomic DNA to isolate full-length cDNAs and genomic clones encodmg ytgP and to isolate cDNA and genomic clones of other genes that have a high identity, particularly high sequence identity, to a ytgP 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 parrs and may have at least 50 nucleotide residues or base pairs
  • Particularly prefened probes will have at least 20 nucleotide residues or base pairs and will have lee than 30 nucleotide residues or base pairs
  • a codmg region of a ytgP gene may be isolated by screening usmg a DNA sequence provided m Table 1 [SEQ ID NO 1] to synthesize an oligonucleotide probe
  • a labeled ohgonucleotide havmg 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 determine which members of the library the probe hybndizes to
  • polynucleotides and polypeptides of the mvention may be employed, for example, as research reagents and mate ⁇ als for discovery of treatments of and diagnostics for diseases, particularly human diseases, as further discussed herem relatmg to polynucleotide assays
  • the 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 determine whether or not the polynucleotides identified herein in whole or in part are transcribed in bacteria in mfected tissue It is recognized that such sequences will also have utility m diagnosis of the stage of infection and type of infection the pathogen has attained
  • the mvention also provides polynucleotides that encode a polypeptide that is a mature protem plus additional ammo or carboxyl-termmal 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 m processmg of a protem from precursor to a mature form, may allow protem transport, may lengthen or shorten protem half-hfe or may facilitate manipulation of a protem for assay or production, among other thmgs
  • the additional ammo acids may be processed
  • a precursor protem, havmg a mature form of the polypeptide fused to one or more prosequences may be an inactive form of the polypeptide When prosequences are removed such mactive precursors generally are activated Some or all of the prosequences may be removed before activation Generalh .
  • proproterns As will be recognized, the entire polypeptide encoded by an open readmg frame is often not required for activity Accordingly, it has become routme m molecular biology to map the boundanes of the primary structure required for activity with N-te ⁇ ninal and C-terminal deletion experiments These experiments utilize exonuclease digestion or convenient rest ⁇ ction sites to cleave codmg nucleic acid sequence
  • Promega (Madison, WT) sell an Erase-a-baseTM system that uses Exonuclease III designed to facihtate analysis of the deletion products (protocol available at www promega com)
  • the digested endpomts can be repaired (e g , by ligation to synthetic linkers) to the extent necessary to preserve an open reading frame
  • the nucleic acid of SEQ ID NO 1 readily provides contiguous fragments of SEQ ID NO 2 sufficient to provide an activity, such as an enzymatic, binding or antibody-inducing activity Nucleic
  • portions of the N-termmal and/or C-terminal sequence of a protein can generally be removed without serious consequence to the function of the protem
  • the amount of sequence that can be removed is often quite substantial
  • the nucleic acid cutting and deletion methods used for creating such deletion variants are now quite routine Accordingly, anv contiguous fragment of SEQ ID NO 2 which retams at least 20%, preferably at least 50%.
  • the contiguous fragment compnses at least 70% of the ammo acid residues of SEQ ID NO 2, preferably at least 80%. 90% or 95% of the residues
  • a polynucleotide of the mvention may encode a mature protem, a mature protem plus a leader sequence (that may be refened 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, that is a precursor to a proprotern, havmg a leader sequence and one or more prosequences, that generally are removed dunng processmg steps that produce active and mature forms of the polypeptide
  • the mvention also relates to vectors that compnse a polynucleotide or polynucleotides of the mvention, host cells that are genetically engineered with vectors of the mvention and the production of polypeptides of the mvention by recombinant techniques Cell-free translation systems can also be employed to produce such proteins usmg RNAs de ⁇ ved from the DNA constructs of the mvention
  • Recombinant polypeptides of the present mvention may be prepared by processes well known m those skilled m the art from genetically engineered host cells compnsmg expression systems Accordingly, m a further aspect, the present mvention relates to expression systems that compnse a polynucleotide or polynucleotides of the present mvention, to host cells that are genetically engineered with such expression systems, and to the production of polypeptides of the mvention by recombinant techniques
  • host cells can be genetically engineered to incorporate expression systems or portions thereof or polynucleotides of the mvention
  • Introduction of a polynucleotide mto the host cell can be effected by methods descnbed m many standard laboratory manuals, such as Davis, et al , BASIC METHODS IN MOLECULAR BIOLOGY, (1986) and Sambrook, et al , MOLECULAR CLONING A LABORATORY MANUAL, 2nd Ed , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N Y (1989). such as, calcium phosphate transfection, DEAE-dextran mediated transfection, transvection. micromjection, catiomc hpid-mediated transfection, electroporation, transduction. scrape loading, ballistic mtroduction and infection
  • bacte ⁇ al cells such as cells of streptococci, staphylococci, enterococci E col , streptomyces, cyanobactena, Bacillus subtihs, and Staphylococcus aureus
  • fungal cells such as cells of a yeast, Kluveromyces, Saccharomyces, a basidiomycete, Candida albicans and Aspergillus
  • insect cells such as cells of Drosoph ⁇ a S2 and Spodoptera Sf9
  • animal cells such as CHO, COS, HeLa. C127, 3T3, BHK, 293, CV-1 and Bowes melanoma cells
  • plant cells such as cells of a gymnosperm or angiosperm
  • vectors include, among others, chromosomal-, episomal- and virus-denved vectors, for example, vectors denved from bacte ⁇ al plasmids, from bacte ⁇ ophage, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses such as baculoviruses, papova viruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses, picornaviruses and retroviruses, and vectors denved from combinations thereof, such as those denved from plasmid and bactenophage genetic elements, such as cosmids and phagemids
  • the expression system constructs may compnse control regions that regulate as well as engender expression Generally, any system or vector suitable to mamtam, propagate or express polynucle
  • appropnate secretion signals may be mcorporated mto the expressed polypeptide These signals may be endogenous to the polypeptide or they may be heterologous signals
  • Polypeptides of the mvention can be recovered and punfied from recombinant cell cultures by well-known methods mcludmg ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic mteraction chromatography, affinity chromatography, hydroxylapatite chromatography, and lectin chromatography Most preferably, high performance liquid chromatography is employed for punfication
  • Well known techniques for refolding protem may be employed to regenerate active conformation when the polypeptide is denatured dunng isolation and or punfication
  • This mvention is also related to the use of ytgP polynucleotides and polypeptides of the mvention for use as diagnostic reagents Detection of ytgP polynucleotides and or polypeptides m a eukaryote, particularly a mammal, and especially a human will provide a diagnostic method for diagnosis of disease staging of disease or response of an infectious organism to drugs Eukaryotes, particularly mammals, and especially humans, particularly those infected or suspected to be infected with an organism compnsmg the ytgP gene or protem, may be detected at the nucleic acid or ammo acid level by a va ⁇ ety of well known techniques as well as by methods provided herem
  • Polypeptides and polynucleotides for prognosis, diagnosis or other analysis may be obtained from a putatively infected and/or infected individual's bodily matenals
  • Polynucleotides from any of these sources, particularly DNA or RNA may be used directly for detection or may be amplified enzymatically by usmg PCR or any other amplification techmque pnor to analysis RNA.
  • mRNA, cDNA and genomic DNA may also be used in the same ways Usmg amplification, charactenzation of the species and stram of infectious or resident organism present m an individual, may be made by an analysis of the genotype of a selected polynucleotide of the organism Deletions and msertions can be detected by a change m size of the amplified product m companson to a genotype of a reference sequence selected from a related organism, preferably a different species of the same genus or a different stram of the same species Pomt mutations can be identified by hyb ⁇ dizmg amplified DNA to labeled ytgP polynucleotide sequences Perfectlj or significantly matched sequences can be distinguished from imperfectly or more significantly mismatched duplexes by DNase or RNase digestion, for DNA or RNA respectively, or by detectmg differences in meltmg temperatures or renaturation kinetics Polynucleotide sequence differences may also be
  • an anay of oligonucleotides probes comp ⁇ smg ytgP nucleotide sequence or fragments thereof can be constructed to conduct efficient screening of, for example, genetic mutations, serotype, taxonomic classification or identification
  • Array technology methods are well known and have general applicability and can be used to address a vanety of questions m molecular genetics mcludmg gene expression, genetic linkage, and genetic vanabi ty (see, for example, Chee et al , Science, 274 610 (1996))
  • the present invention relates to a diagnostic kit that comprises (a) a polynucleotide of the present invention, preferably the nucleotide sequence of SEQ ID NO 1, or a fragment thereof , (b) a nucleotide sequence complementary to that of (a), (c) a polypeptide of the present invention, preferably the polypeptide of SEQ ID NO 2 or a fragment thereof, or (d) an antibod) to a polypeptide of the present invention, preferably to the polypeptide of SEQ ID NO 2
  • 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 polypeptide of the present invention, preferably the polypeptide of SEQ ID NO 2 or a fragment thereof, or (d) an antibod) to a polypeptide of the present invention, preferably to the polypeptid
  • This mvention also relates to the use of polynucleotides of the present mvention as diagnostic reagents Detection of a mutated form of a polynucleotide of the mvention, preferable.
  • SEQ ID NO 1 that is associated with a disease or pathogenicity will provide a diagnostic tool that can add to, or define, a diagnosis of a disease, a prognosis of a course of disease, a determination of a stage of disease, or a susceptibility to a disease, that results from under-expression, over-expression or altered expression of the polynucleotide
  • Organisms, particularly infectious organisms, carrying mutations m such polynucleotide may be detected at the polynucleotide level by a va ⁇ ety of techniques, such as those descnbed elsewhere herem
  • 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 phenotype but not in any orgamsms possessing the second phenotype, then the mutation is likely to be the causative 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 vanety of techniques, to allow for serotypmg, for example
  • RT-PCR can be used to detect mutations m the RNA It is particularly prefened to use RT-PCR m conjunction with automated detection systems, such as, for example, GeneScan RNA, cDNA or genomic DNA may also be used for the same purpose.
  • PCR primers complementary to a polynucleotide encodmg ytgP polypeptide can be used to identify and analyze mutations
  • the mvention further provides these p ⁇ mers with 1, 2, 3 or 4 nucleotides removed from the 5' and/or the 3' end
  • These primers may be used for, among other thmgs, amphfymg ytgP DNA and/or RNA isolated from a sample denved from an individual, such as a bodily matenal
  • the primers may be used to amplify a polynucleotide isolated from an infected individual, such that the polynucleotide may then be subject to vanous techmques for elucidation of the polynucleotide sequence In this way, mutations in the polynucleotide sequence may be detected and used to diagnose and/or prognose the infection or its stage or course, or to serotype and or classify the infectious agent
  • the mvention further provides a process for diagnosmg, disease, preferably bacterial infections, more preferably infections caused by Staphylococcus aureus, comprising determmmg from a sample denved from an mdividual, 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 ytgP polynucleotide can be measured using any on of the methods well known in the art for the quantitation of polynucleotides, such as, for example, amplification, PCR, RT-PCR, RNase protection, Northern blotting, spectrometry and other hybridization methods.
  • a diagnostic assay m accordance with the mvention for detectmg over-expression of ytgP polypeptide compared to no ⁇ nal control tissue samples may be used to detect the presence of an infection, for example Assay techmques that can be used to determine levels of a
  • Polypeptides and polynucleotides of the mvention may also be used to assess the binding of small molecule substrates and ligands in, for example, cells, cell-free preparations, chemical bra ⁇ es, and natural product mixtures
  • substrates and ligands may be natural substrates and ligands or may be structural or functional mimetics See, e g , Coligan et al , Current Protocols in Immunology 1(2) Chapter 5 (1991)
  • Polypeptides and polynucleotides of the present mvention are responsible for many biological functions, mcludmg many disease states, m particular the Diseases herem mentioned It is therefore desirable to devise screening methods to identify compounds that agonize (e g , stimulate) or that antagonize (e g , ⁇ nh ⁇ b ⁇ t) the function of the polypeptide or polynucleotide Accordmgly, m a further aspect, the present mvention provides for a method of screening compounds to identify those that agomze or that antagonize the function of a polypeptide or polynucleotide of the mvention, as well as related polypeptides and polynucleotides In general, agonists or antagonists (e g , inhibitors) may be employed for therapeutic and prophylactic purposes for such Diseases as herem mentioned Compounds may be identified from a va ⁇ ety of sources, for example, cells, cell-free preparations, chemical bra ⁇ es, and natural product mixture
  • polypeptides, polypeptides and antibodies that bind to and/or interact with a polypeptide of the present invention may also be used to configure screening methods for detecting the effect of added compounds on the production of mRNA and/or polypeptide in cells
  • an ELISA assay may be constructed for measuring secreted or cell associated levels of polypeptide usmg 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 agomst, respectively) from suitably manipulated cells or tissues
  • the mvention also provides a method of screemng compounds to identify those that enhance (agonist) or block (antagonist) the action of ytgP polypeptides or polynucleotides, particularly those compounds that are bactenstatic and/or bactencidal
  • the method of screening may mvolve high-throughput techniques
  • a synthetic reaction mix for agonists or antagonists, a synthetic reaction mix, a cellular compartment, such as a membrane, cell envelope or cell wall, or a preparation of any thereof, compnsmg ytgP polypeptide and a labeled substrate or gand of such polypeptide is mcubated m the absence or the presence of a candidate molecule that may be a ytgP agomst or antagonist
  • the ability of the candidate molecule to agonize or antagonize the ytgP polypeptide is reflected in decreased binding of the labeled ligand or decreased production of product from such substrate Molecules that bmd gratuit
  • Fluorescence energy transfer may also be used characterize small molecules that interfere with the formation of ytgP polypeptide dimers, t ⁇ mers, tetramers or higher order structures, or structures formed by ytgP polypeptide bound to another polypeptide
  • YtgP polypeptide can be labeled with both a donor and acceptor fluorophore Upon mixing of the two labeled species and excitation of the donor fluorophore.
  • fluorescence energy transfer can be detected by observing fluorescence of the acceptor Compounds that block dime ⁇ zation will inhibit fluorescence energy transfer
  • YtgP polypeptide can be coupled to a sensor chip at low site density such that covalently bound molecules will be monome ⁇ c Solution protein can then passed over the ytgP polypeptide -coated surface and specific bindmg can be detected in real-time by monitoring the change in resonance angle caused by a change in local refractive index
  • This technique can be used to characterize the effect of small molecules on kinetic rates and equilibrium binding constants for ytgP polypeptide self-association as well as an association of ytgP polypeptide and another polypeptide or small molecule
  • a scintillation proximity assay may be used to characterize the interaction between an association of ytgP polypeptide with another ytgP polypeptide or a different polypeptide
  • YtgP polypeptide can be coupled to a scmtillation-filled bead Addition of radio-labeled ytgP polypeptide results in bmdmg where the radioactive source molecule is in close proximity to the scintillation fluid
  • signal is emitted upon ytgP polypeptide bindmg and compounds that prevent ytgP polypeptide self-association or an association of ytgP polypeptide and another polypeptide or small molecule will dimmish signal
  • identifying compounds that bmd to or otherwise interact with and inhibit or activate an activity or expression of a polypeptide and/or polynucleotide of the mvention compnsmg contacting a polypeptide and/or polynucleotide of the mvention with a compound to be screened under conditions to permit bmdmg to or other mteraction between the compound and the polypeptide and or polynucleotide to assess the bmdmg to or other mteraction with the compound, such bmdmg or mteraction preferably bemg associated with a second component capable of providmg a detectable signal m response to the bmdmg or mteraction of the polypeptide and/or polynucleotide with the compound, and determining whether the compound bmds to or otherwise mteracts with and activates or inhibits an activity or expression of the polypeptide and/or polynu
  • an assay for ytgP agonists is a competitive assaj that combines ytgP and a potential agomst with ytgP-bmdmg molecules, recombinant ytgP bmdmg molecules, natural substrates or ligands, or substrate or ligand mimetics, under appropnate conditions for a competitive inhibition assay
  • YtgP can be labeled, such as by radioactivity or a colonmetnc compound, such that the number of ytgP molecules bound to a bmdmg molecule or converted to product can be determined accurately to assess the effectiveness of the potential antagonist
  • a polypeptide and/or polynucleotide of the present invention may also be used in a method for the structure-based design of an agonist or antagonist of the polypeptide and or polynucleotide, by (a) determining in the first instance the three- dimensional structure of the polypeptide and
  • the present mvention provides methods of treatmg abnormal conditions such as. for instance, a Disease, related to either an excess of, an under-expression of, an elevated activity of. or a decreased activity of ytgP polypeptide and/or polynucleotide
  • expression of the gene encoding endogenous ytgP polypeptide can be inhibited usmg expression blockmg techmques
  • This blockmg may be targeted against any step in gene expression, but is preferably targeted against transcription and/or translation
  • An examples of a known techmque of this sort mvolve the use of antisense sequences, either internally generated or separately administered (see, for example, O'Connor, J Neurochem (1991) 56 560 m Ohgodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, FL (1988))
  • ohgonucleotides that form t ⁇ ple helices with the gene can be supplied (see, for example, Lee et al , Nucleic Acids Res (1979) 6 3073, Cooney et al , Science (1988) 241 456, Dervan et al , Science (1991) 251 1360)
  • These o gomers can be administered per se or the relevant ohgomers
  • the invention also provides the use of the polypeptide, polynucleotide. agonist or antagonist of the invention to interfere with the initial physical interaction between a pathogen or pathogens and a eukaryotic, preferably mammalian, host responsible for sequelae of infection
  • the molecules of the invention may be used in the prevention of adhesion of bacteria, in particular gram positive and or gram negative bacteria, to eukaryotic, preferably mammalian, extracellular matrix proteins on m-dwelling devices or to extracellular matrix proteins in wounds, to block bacterial adhesion between eukaryotic, preferably mammalian, extracellular matrix proteins and bacte ⁇ al ytgP protems that mediate tissue damage and/or.
  • ytgP agomsts and antagomsts preferably bactenstatic or bactencidal agomsts and antagomsts
  • the antagomsts and agomsts of the mvention may be employed, for instance, to prevent, inhibit and or treat diseases
  • Antagomsts of the mvention mclude, among others, small organic molecules, peptides, polypeptides and antibodies that bmd to a polynucleotide and/or polypeptide of the mvention and thereby inhibit or extinguish its activity or expression
  • Antagomsts also may be small organic molecules, a peptide, a polypeptide such as a closely related protem or antibody that bmds the same sites on a bmdmg molecule, such as a bmdmg molecule, without mducmg ytgP-mduced activities, thereby preventing the action or expression of ytgP polypeptides and/or polynucleotides by excludmg ytgP polypeptides and/or polynucleotides from bm
  • Antagomsts of the mvention also mclude a small molecule that bmds to and occupies the bmdmg site of the polypeptide thereby preventmg bmdmg to cellular bmdmg molecules, such that normal biological activity is prevented
  • small molecules include but are not limited to small organic molecules.
  • Other antagomsts mclude antisense molecules (see Okano. J Neurochem 56 560 (1991), OLIGODEOXYNUCLEOTIDES AS ANTISENSE INHIBITORS OF GENE EXPRESSION.
  • Prefened antagomsts m clude compounds related to and vanants of ytgP
  • polypeptide antagomsts m include antibodies or, m some cases, ohgonucleotides or proteins that are closely related to the ligands, substrates, receptors, enzymes, etc , as the case may be. of the polypeptide. e g . a fragment of the ligands, substrates, receptors, enzymes, etc , or small molecules that bmd to the polypeptide of the present mvention but do not elicit a response, so that the activity of the polypeptide is prevented
  • 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 Agency 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 adenocarcinoma, classifying the bacterium as a Group I (definite) carcinogen
  • Preferred antimicrobial compounds of the invention agonists and antagonists of ytgP polypeptides and/or polynucleotides found usmg screens provided by the invention, or known in the art, particularly narrow-spectrum antibiotics, should be useful m the treatment of H pylon infection Such treatment should decrease the advent of H pylor
  • Bodily mate ⁇ al(s) means any matenal denved from an mdividual or from an organism 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, skm urine, stool or autopsy matenals
  • D ⁇ sease(s) means any disease caused by or related to mfection by a bacte ⁇ a. mcludmg .
  • disease such as, infections of the upper respiratory tract (e g , otitis media, bactenal tracheitis, acute epiglottitis, thyroiditis), lower respiratory (e g , empyema, lung abscess), cardiac (e g , infective endocarditis), gastrointestinal (e g . secretory diarrhoea, splenic absces, retropentoneal abscess), CNS (e g , cerebral abscess), eye (e g .
  • infections of the upper respiratory tract e g , otitis media, bactenal tracheitis, acute epiglottitis, thyroiditis
  • lower respiratory e g , empyema, lung abscess
  • cardiac e g , infective endocarditis
  • gastrointestinal e g . secretory diarrhoea, splenic absces, retropentoneal abscess
  • CNS
  • blephantis conjunctivitis, keratitis, endophthalmitis, preseptal and orbital cellu tis, darcryocystitis
  • kidney and urinary tract e g , epididymitis, rntrarenal and penneph ⁇ c absces, toxic shock syndrome
  • skin e g , impetigo, folhculitis, cutaneous abscesses, celluhtis, wound infection, bactenal myositis
  • bone and jomt e g , septic arthritis, osteomyelitis
  • “Host cell(s)” is a cell that has been introduced (e g , transformed or transfected) or is capable of introduction (e g , transformation or transfection) by an exogenous polynucleotide sequence
  • Identity 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
  • 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, 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, mcludmg transition and transversion.
  • nucleotide alterations may occur at the 5' or 3' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among the nucleotides m the reference sequence or in one or more contiguous groups within the reference sequence, and wherem said number of nucleotide alterations is determined by multiplying the total number of nucleotides m 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
  • 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, wherem said polypeptide sequence may be identical to the reference sequence of SEQ ID NO 2 or may include up to a certain integer
  • n a is the number of am o acid alterations
  • x a is the total number of amino acids m 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-mteger product of x a and y is rounded down to the nearest integer prior to subtracting it from x a
  • “Ind ⁇ v ⁇ dual(s)" means a multicellular eukaryote, mcludmg, but not limited to a metazoan, a mammal, an ovid, a bovid, a simian, a primate, and a human
  • Isolated means altered “by the hand of man” from its natural state, z e . if it occurs m nature, it has been changed or removed from its o ⁇ gmal environment, or both
  • a polynucleotide or a polypeptide naturally present m a Houston organism is not “isolated,” but the same polynucleotide or polypeptide separated from the coexistmg matenals of its natural state is “isolated", as the term is employed herem
  • a polynucleotide or polypeptide that is mtroduced mto an organism by transfonnation. genetic manipulation or by any other recombinant method is "isolated” even if it is still present m said organism, which organism may be living or non-living
  • Organ ⁇ sm(s) means a (I) prokaryote. mcludmg but not limited to. a member of the genus Streptococcus, Staphylococcus, Bordetella, Corynebacterium, Mycobactenum, Neissena, Haemophilus Actinomycetes, Streptomycetes, Nocardia, Enterobacter, Yersinia, Fancisella Pasturella, Moraxella, Acmetobacter, Erys ⁇ elothnx, Branhamella, Actinobac ⁇ lus, Streptobacillus, Listena, Calymmatobacterium, Brucella, Bacillus, Clostndium, Treponema, Eschenchia, Salmonella, Kleibsiella, Vibno, Proteus, Erwinia, Borreha, Leptospira, Spirillum, Campylobacter, Shigella, Legwnella, Pseudomon
  • Polynucleotide(s) generally refers to any polynbonucleotide or polydeox) ⁇ bonucleot ⁇ de. that ma) be unmodified RNA or DNA or modified RNA or DNA
  • Polynucleotide(s)" mclude, without 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, hybnd molecules compnsmg DNA and RNA that may be single-stranded or, more typically, double-stranded, or tnple-stranded regions, or a mixture of smgle- and double-stranded regions hi addition, "polynucleotide” as used herem refers to tnple-stranded regions comp ⁇ s
  • Polypept ⁇ de(s) refers to any peptide or protem compnsmg two or more ammo acids jomed to each other by peptide bonds or modified peptide bonds
  • Polypeptide(s) refers to both short chains, commonly refened to as peptides, o gopeptides and ohgomers and to longer chains generally refened to as proteins
  • Polypeptides may compnse ammo acids other than the 20 gene encoded ammo acids
  • Polypept ⁇ de(s)” mclude those modified either by natural processes, such as processmg and other post-translational modifications, but also by chemical modification techmques Such modifications are well descnbed m basic texts and m more detailed monographs, as well as in a volummous 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 m the same or varying degree at several sites m a given polypeptide Also, a given polypeptide may compns
  • amidation covalent attachment of flavm, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide de ⁇ vative, covalent attachment of a hpid or pid de ⁇ vative, covalent attachment of phosphotidylmositol, cross-lmkmg. cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteme, formation of pyroglutamate, formylation, gamma-carboxylation. GPI anchor formation, hydroxylation, lodmation, methylation.
  • Polypeptides may be branched or cyclic, with or without branching Cyclic, branched and branched circular polypeptides may result from post- translational natural processes and may be made by entirely synthetic methods, as well
  • Recombinant expression system(s) refers to expression systems or portions tliereof or polynucleotides of the mvention mtroduced or transformed mto a host cell or host cell lysate for the production of the polynucleotides and polypeptides of the mvention "Va ⁇ ant(s)" as the term is used herem, is a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide respectively, but retains essential properties
  • a typical variant of a polynucleotide differs in nucleotide sequence from another, reference polynucleotide Changes in the nucleotide sequence of the variant may or may not alter the ammo acid sequence of a polypeptide encoded by the reference polynucleotide Nucleotide changes may result in amino acid substitutions, additions, deletions, fusion proteins and truncations m the polypeptide encoded by the reference sequence,
  • a variant of a polynucleotide or polypeptide may be a naturally occurring such as an allelic variant, or it may be a variant that is not known to occur naturall ⁇
  • Non-naturally occurring variants of polynucleotides and polypeptides may be made by mutagenesis teclimques, by direct synthesis, and by other recombinant methods known to skilled artisans EXAMPLES
  • the polynucleotide having a DNA sequence given in Table 1 [SEQ ID NO 1] was obtained from a library of clones of chromosomal DNA of Staphylococcus aureus in E cob
  • the sequencing data from two or more clones comprising overlapping Staphylococcus aureus DNAs was used to construct the contiguous DNA sequence m SEQ ID NO 1 Libraries may be prepared by routme methods, for example Methods 1 and 2 below
  • Total cellular DNA is mechanically sheared by passage through a needle m order to size- fractionate according to standard procedures
  • DNA fragments of up to 1 lkbp in size are rendered blunt by treatment with exonuclease and DNA polymerase, and EcoRI linkers added Fragments are hgated mto the vector Lambda ZapII that has been cut with EcoRI, the library packaged by standard procedures and E co infected with the packaged hbrar)
  • the library is amplified by standard procedures
  • Total cellular DNA is partially hydrolyzed with a one or a combmation 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 EcoRJ. the library packaged by standard procedures, and E co infected with the packaged hbrarv The library is amplified by standard procedures Example 2 ytgP Characterization
  • the ytgP gene is expressed during infection of Staphylococcus aureus WCUH29 in a pyelonephritis infection model
  • Necrotic fatty tissue from a kidney from a seven day pyelonephritis infection of Staphylococcus aureus WCUH29 in the mouse is efficiently disrupted and processed m the presence of acid phenol and detergent to provide a mixture of animal and bacterial RNA
  • the resultant total RNA is free of DNA and protem (including RNAases and DNAases)
  • the optimal conditions for disruption and processing to give high yields of bacterial mRNA with transcripts of long length are followed by 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 into 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 sihca/ceramic matrix (BIO 101)
  • 1 ml of extraction reagents FastRNA reagents, BIOIOI
  • FastRNA reagents BIOIOI
  • the tubes are shaken in a reciprocating shaker (FastPrep FP120, BIOIOI) at 6000 rpm for 20-120 sec
  • the crude RNA preparation is extracted with chloroform/isoa ⁇ vs 1 alcohol and precipitated with DEPC-treated/Isopropanol Precipitation Solution (BIOIOI) RNA preparations are stored
  • RNA was precipitated with 5 microhters of 3 M NaOAc and 200 microhters 100% EtOH, and pelleted by centrifiigation at 12.000g for 10 minutes
  • the RNA is pelleted (12,000g for 10 mm ), washed with 75% ethanol (v/v in 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 withm one week
  • PCR reactions are set up on ice in 0 2ml tubes by addmg 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 1 OmM initial concentration, and 5 microhtres cDNA PCR reactions are run on a Perkm Elmer GeneAmp PCR System 9600 as follows 2 minutes at
  • RT/PCR controls may include +/- reverse transc ⁇ ptase reactions, 16S rRNA primers or DNA specific primer pairs designed to produce PCR products from non-transcribed Streptococcus pneumoniae 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 uninformative Of those which give the correct size product with DNA PCR two classes are distinguished in RT/PCR 1 Genes which are not transcribed in vivo reproducib fail to give a product in RT/PCR, and 2 Genes which are transcribed m vivo reproducibly give the correct size product in RT/PCR and show a stronger signal in the +RT samples than the signal (if at all present) in -RT controls

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Abstract

L'invention concerne des polypeptides ytgP et des polynucléotides codant les polypeptides ytgP ainsi que des procédés de fabrication de ces polypeptides et polynucléotides par procédés recombinants. Elle concerne aussi des procédés pour utiliser ces polypeptides ytgP pour cribler des composés antibactériens.
PCT/US2000/030199 1999-11-04 2000-11-02 YtgP Ceased WO2001032022A1 (fr)

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Citations (1)

* 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

Patent Citations (1)

* 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

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