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WO2000056865A1 - Histidine kinase - Google Patents

Histidine kinase Download PDF

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Publication number
WO2000056865A1
WO2000056865A1 PCT/US2000/006206 US0006206W WO0056865A1 WO 2000056865 A1 WO2000056865 A1 WO 2000056865A1 US 0006206 W US0006206 W US 0006206W WO 0056865 A1 WO0056865 A1 WO 0056865A1
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Prior art keywords
polypeptide
seq
polynucleotide
isolated
sequence
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PCT/US2000/006206
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English (en)
Inventor
Nicola G. Wallis
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Smithkline Beecham Corporation
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Publication of WO2000056865A1 publication Critical patent/WO2000056865A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention relates to ne lv identified polvnucleotides and polvpeptides. and their production and uses, as well as their variants agonists and antagonists and dieir uses hi particular tin- -mention n-l ⁇ tes to polynucleotides and polypeptides of the histidine kinase familv. as well as their ⁇ anants herein referred to as
  • histidine kinase "histidine kinase polynucleot ⁇ de(s),” and “histidine kinase polypept ⁇ de(s)" as the case ma ⁇ be
  • Staphylococcal genes and gene products as targets for the development of antibiotics
  • the Staphylococci make up a medically important genera of microbes
  • Thev are known to produce two types of disease, invasive and toxigenic Invasive infections are characterized generally by abscess formation effecting both skin surfaces and deep tissues S aureus is the second leading cause of bacteremia in cancer patents Osteomyelitis, septic arthntis.
  • Staphylococcus aureus infections has nsen dramatically in the past few decades This has been attributed to the emergence of multiply antibiotic resistant strains and an increasing population of people with weakened immune systems It is no longer uncommon to isolate Staphylococcus aureus strains that are resistant to some or all of the standard antibiotics This phenomenon has created an unmet medical need and demand for new anti-microbial agents, vaccines, drug screening methods, and diagnostic tests for this organism Moreover, the drug discovery process is currently undergoing a fundamental revolution as it embraces "functional genomics.” that is.
  • the present invention relates to histidine kinase in particular histidine kinase poKpeptides and histidine kinase polynucleotides. recombinant matenals and methods for their production In another aspect the invention relates to methods for using 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 invention relates to diagnostic assays for detecting diseases associated with microbial infections and conditions associated with such infections, such as assays for detecting histidine kinase expression or activity
  • the invention relates to histidine kinase polypeptides and polynucleotides as descnbed in greater detail below
  • the invention relates to polypeptides and polynucleotides of a histidine kinase of Staphylococcus aureus.
  • the invention relates especially to histidine kinase having a nucleotide and amino acid sequences set out in Table 1 as SEQ ID NO 1 and SEQ ID NO 2 respectrv eh Note that sequences recited in the Sequence Listing below as "DNA” represent an exemplification of the invention, since those of ordinary skill will recogmze that such sequences can be usefully employed in polynucleotides in general, including ⁇ bopolynucleotides
  • NCIMB National Collections of Industnal and Marine Bactena Ltd
  • Staphylococcus aureus WCUH29 on deposit
  • the Staphylococcus aureus strain deposit is referred to herein as "d e deposited strain” or as "die DNA of the deposited strain"
  • the deposited stram compnses a full length histidme kinase gene
  • the sequence of the polvnucleotides comp ⁇ sed m the deposited strain, as well as the ammo acid sequence of any polypeptide encoded therebv. are controlling in the event of any conflict with any descnption of sequences herein
  • the deposit of the deposited strain has been made under the terms of the Budapest Treaty on the International Recognition of the Deposit of Micro-organisms for Purposes of Patent Procedure
  • the deposited stram will be irrevocably and without restnction or condition released to the public upon the issuance of a patent
  • the deposited strain is provided merely as convemence to those of skill in the art and is not an admission that a deposit is required for enablement. such as that required under 35 U S C ⁇ 112
  • a license may be required to make, use or sell the deposited strain, and compounds denved therefrom, and no such license is hereby granted
  • an isolated nucleic acid molecule encoding a mature polypeptide expressible by the Staphylococcus aureus WCUH 29 strain, which polypeptide is compnsed in the deposited stram
  • histidme kinase polynucleotide sequences m the deposited strain such as DNA and RNA.
  • ammo acid sequences encoded thereby Also provided by the mvention are histidme kinase polypeptide and polynucleotide sequences isolated from the deposited stram
  • Histidme kinase polypeptide of the mvention is substantially phylogeneticallv related to other proteins of the histidme kinase family
  • polypeptides of Staphylococcus aureus referred to herem as “histidme kinase” and “histidme kinase polypeptides” as well as biologically, diagnosticallv. clinically or therapeutically useful va ⁇ ants thereof, and compositions compnsmg 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 identrt ⁇ to that of SEQ ID NO 2 over the entire length of SEQ ID NO 2, (b) a polypeptide encoded bv an isolated pohnucleotide compnsmg or consisting of a polvnucleotide sequence that has at least 95% identiu e ⁇ en more preferablv at least 97-99% or exact identi to SEQ ID NO 1 o ⁇ er the enure 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 preferabl . at least 97-99% or exact identity, to the ammo acid sequence of SEQ ID NO 2. over the entire length of SEQ ID NO 2
  • polypeptides of die mvention mclude a polypeptide of Table 1 [SEQ ID NO 2] (m particular a mature polypeptide) as well as polypeptides and fragments, particularly those tiiat has a biological acti ⁇ it. of histidme kinase, 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 compnsmg at least 30 ammo acids and more preferably at least 0 ammo acids
  • the mvention also mcludes a polypeptide consisting of or compnsmg a polypeptide of the formula X-(R 1 ) rn -(R 2 )-(R 3 ) n -Y wherein, at the ammo terminus.
  • X is hydrogen, a metal or any other moiety descnbed herem for modified polypeptides.
  • Y is hydrogen, a metal or any other moiety descnbed herem for modified polypeptides.
  • Ri and R3 are any ammo acid residue or modified ammo acid residue, m is an integer between 1 and 1000 or zero, n is an mteger between 1 and 1000 or zero, and R is an ammo acid sequence of the mvention, particularly an ammo acid sequence selected from Table 1 or modified forms thereof In the formula above, R is onented so that its ammo terminal ammo acid residue is at the left, covalently bound to Ri and its carboxy terminal ammo acid residue is at the ⁇ ght, covalently bound to R3 Any stretch of ammo acid residues denoted by either Ri or R3, where m and/or n is greater than 1. may be either a heteropo-ymer or a homopolymer. preferably a heteropolymer Other preferred embodiments of the mvention are provided where m is an mteger between 1 and 50. 100 or 500, and n is an mteger between 1 and 50. 100. or 500
  • a polypeptide of the mvention is de ⁇ ved from Staphylococcus aureus, however, it may preferably be obtained from other organisms of the same taxononuc genus
  • a polypeptide of the mvention may also be obtained, for example, from organisms of the same taxonomic family or order
  • a fragment is a vanant polypeptide having an ammo acid sequence that is entirely the same as part but not all of any ammo acid sequence of any polypeptide of the mvention As with histidme kinase polypeptides. fragments may be "free-standing,” or comp ⁇ sed within a larger polypeptide of which they form a part or region, most preferably as a smgle continuous region in a single larger polypeptide
  • Preferred fragments m include, for example, truncation polypeptides having a portion of an ammo acid sequence of Table 1 [SEQ ID NO 2], or of vanants thereof, such as a continuous senes of residues that mcludes an ammo- and/or carboxyl-termmal ammo acid sequence Degradation forms of the polypeptides of the mvention produced bv or m a host cell, particularly a Staphylococcus aureus.
  • fragments charactenzed bv structural or functional attributes such as fragments Uiat compnse alpha-helix and alpha-helix forming regions, beta-sheet and beta-sheet-formmg regions, turn and turn- forming regions, coil and coil-forming regions. 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 o ⁇ 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 ma ⁇ be employed for producmg the corresponding full-length polypeptide by peptide synthesis, therefore, these vanants mav be employed as intermediates for producmg the full-length polypeptides of the mvention
  • polynucleotide compnses a region encodmg histidme kinase polypeptides compnsmg a sequence set out in Table 1 [SEQ ID NO 1] that mcludes a full length gene, or a variant thereof The Applicants believe 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 encodmg and/or expressmg histidme kinase polypeptides and polynucleotides, particularly Staphylococcus aureus histidme kinase polypeptides and polynucleotides.
  • histidme kinase polypeptides and polynucleotides particularly Staphylococcus aureus histidme kinase polypeptides and polynucleotides.
  • isolated nucleic acid molecules encodmg and/or expressmg histidme kinase polypeptides and polynucleotides, particularly Staphylococcus aureus histidme kinase polypeptides and polynucleotides.
  • unprocessed RNAs nbozyme RNAs including, for example, unprocessed RNAs nbozyme RNAs, mRNAs, c
  • Another aspect of the mvention relates to isolated polynucleotides. including at least one full length gene, that encodes a histidme kinase polypeptide havmg a deduced ammo acid sequence of Table 1 [SEQ ID NO 2] and polynucleotides closely related thereto and vanants thereof
  • a polynucleotide of the mvention encodmg histidme kinase pohpeptide mav be obtained usmg standard cloning and screening methods, such as those for cloning and sequencing chromosomal DNA fragments from bactena usmg Stapnylococcus aureus WCUH 29 cells as starting matenal followed b ⁇ obtainmg a full length clone
  • a polynucleotide sequence of the invention such as a polynucleotide sequence given m Table 1 [SEQ ID NO 1]
  • t ⁇ p ⁇ callv a library of clones of chromosomal DNA of Staphylococcus aureus WCUH 29 m E colt or some other suitable host is probed with a radiolabeled o gonucleotide.
  • Clones carrying DNA identical to that of the probe can then be distinguished using stringent hvbndization conditions B ⁇ sequencing the individual clones thus identified b ⁇ hybridization with sequencing primers designed from the original polypeptide or poh nucleotide sequence it is then possible to extend the polynucleotide sequence in both directions to determine a full length gene sequence Conveniently such sequencing is performed, for example, usmg denatured double stranded DNA prepared from a plasmid clone Suitable techniques are descnbed by Maniatis. T , F ⁇ tsch. E F and Sambrook et al .
  • each DNA sequence set out m Table 1 [SEQ ID NO 1] contains an open reading frame encodmg a protem havmg about the number of ammo acid residues set forth m 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 m the art
  • the 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 encodmg 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, including homologs and orthologs from species other than Staphylococcus aureus. may be obtained by a process that compnses the steps of screening an appropnate library under stringent h ⁇ bndization conditions with a labeled or detectable probe consisting of or compnsmg die sequence of SEQ ID NO 1 or a fragment thereof, and isolating a full-length gene and/or genomic clones compnsmg said polynucleotide sequence
  • the mvention provides a pohnucleotide sequence identical over its entire lengtii to a coding sequence (open reading frame) in Table 1 [SEQ ID NO 1] Also provided by the mvention is a coding sequence for a mature polypeptide or a fragment thereof, by itself as well as a coding sequence for a mature polypeptide or a fragment m reading frame with another coding sequence, such as a sequence encodmg a leader or secretory sequence, a pre-, or pro- or prepro-protem sequence
  • the polynucleotide of die invention mav also compnse at least one non-coding sequence including for example but not limited to at least one non-coding 5 and 3 ' sequence such as the transcnbed but non-translated sequences termination signals (such as rho-dependent and rho-mdependent termination signals), nbosome binding sites kozak sequences sequences that stabilize mRNA.
  • the polynucleotide sequence m ⁇ also compnse additional coding sequence encodmg additional ammo acids.
  • a marker sequence that facilitates purification of a fused polypeptide can be encoded in certain embodiments of the mvention, the marker sequence is a hexa-histidine peptide, as provided m the pQE vector (Qiagen, Inc ) and descnbed m Gentz el al . Proc Natl Acad Set USA 86 821-824 (1989). or an HA peptide tag (Wilson et al . Cell 37 767 (1984).
  • Polynucleotides of the mvention also mclude. but are not limited to, polynucleotides compnsmg a structural gene and its naturally associated sequences that control gene expression
  • a preferred embodiment of the mvention is a polynucleotide of consisting of or compnsmg nucleotide 222 to the nucleotide immediately upstream of or including nucleotide 1259 set forth in SEQ ID NO 1 of Table 1. both of that encode a histidme kinase polypeptide
  • the mvention also mcludes a polynucleotide consisting of or compnsmg a polynucleotide of die formula X-(R 1 ) m -(R 2 )-(R 3 )n-Y wherein, at the 5 1 end of the molecule.
  • X is hydrogen, a metal or a modified nucleotide residue, or together with Y defines a covalent bond, and at the 3' end of the molecule.
  • Y is hydrogen, a metal, or a modified nucleotide residue, or together with X defines the covalent bond
  • each occurrence of R ⁇ and R3 is independently any nucleic acid residue or modified nucleic acid residue
  • m is an mteger 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 mvention, 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 Ri and its 3' end nucleic acid residue is at the nght, bound to R3 Any stretch of nucleic acid residues denoted by either Rj and/or R 2 . where m and/or n is greater than 1.
  • 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 mteger between 1 and 1000. Otiier preferred embodiments of the mvention are provided where m is an mteger between 1 and 50. 100 or 500. and n is an mteger between 1 and 50. 100. or 500
  • a polynucleotide of the mvention is denved from Staphylococcus aureus however, it mav preferably be obtained from other organisms of the same taxonomic genus A. polynucleotide of the invention mav also be obtained, for example from organisms of die same taxonomic family or order
  • polynucleotide encodmg a polypeptide encompasses polvnucleotides that mclude a sequence encodmg a polypeptide of the mvention. particularly a bactenal polypeptide and more particularly a polypeptide of the Staphylococcus aureus histidme kinase havmg an ammo acid sequence set out m Table 1 [SEQ ID NO 2]
  • the term also encompasses polynucleotides that mclude a smgle contmuous region or discontinuous regions encoding the polypeptide (for example, polynucleotides interrupted bv integrated phage.
  • 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 histidme kinase vanants that have the ammo acid sequence of histidme kinase polypeptide of Table 1 [SEQ ID NO 2] in which several, a few. 5 to 10, 1 to 5, 1 to 3, 2, 1 or no ammo acid residues are substituted, modified, deleted and/or added, m any combination Especially preferred among these are silent substitutions, additions and deletions, that do not alter the properties and activities of histidme kinase polypeptide
  • 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 havmg 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.
  • 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 havmg at least 15, 20, 30, 40
  • polynucleotides that are at least 95% or 97% identical over their entire length to a polynucleotide encodmg histidme kinase polypeptide havmg an ammo acid sequence set out m Table 1 [SEQ ID NO 2], and polynucleotides that are complementary to such polvnucleotides Most highly preferred are polvnucleotides that compnse a region tiiat is at least 95% are especially preferred Furthermore, those with at least 97% are highly preferred among those with at least 95%o. and among these those with at least 98% and at least 99% are particularly highly preferred, with at least 99% bemg the more preferred
  • Preferred embodiments are polvnucleotides encodmg polypeptides that retain substantially the same biological function or activit as a mature polvpeptide encoded bv a D A. of Table 1 [SEQ ID NO 1 1 In accordance witii certain preferred embodiments of this invention there arc provided polynucleotides that hybndize. particularly under strmgent conditions to histidme kinase polynucleotide sequences, such as those polynucleotides m Table 1
  • the mvention further relates to polynucleotides that hybndize to the polynucleotide sequences provided herem
  • the mvention especially relates to polynucleotides that hybndize under strmgent conditions to the polynucleotides descnbed herem
  • strmgent conditions and “strmgent hybndization conditions” mean hvb ⁇ dization occurring only if there is at least 95% and preferably at least 97% identity between the sequences
  • strmgent hybndization conditions is overnight incubation at 42°C m a solution compnsmg 50% formamide.
  • 5x SSC 150mM NaCl, 15mM t ⁇ sodium citrate), 50 mM sodium phosphate (pH7 6).
  • 5x Denhardt's solution, 10% dextran sulfate, and 20 micrograms/ml of denatured, sheared salmon sperm DNA, followed by washmg the hybndization support m 0 lx SSC at about 65°C Hybndization and wash conditions are well known and exemplified in Sambrook. et al . Molecular Cloning A Laboratory Manual.
  • the invention also provides a polynucleotide consisting of or comprising a polynucleotide sequence obtamed by screemng an appropnate library compnsmg a complete gene for a polynucleotide sequence set forth in SEQ ID NO 1 under strmgent hybridization conditions with a probe having the sequence of said polynucleotide sequence set forth m SEQ ID NO 1 or a fragment thereof, and isolating said polynucleotide sequence Fragments useful for obtaimng such a polynucleotide include. for example, probes and primers fully descnbed elsewhere herein
  • RNA, cDNA and genomic DNA may be used as a hybndization probe for RNA, cDNA and genomic DNA to isolate full-length cDNAs and genomic clones encodmg histidme kinase and to isolate cDNA and genomic clones of other genes that have a high identity, particularly high sequence identity, to a histidme kinase 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 preferred probes will have at least 20 nucleotide residues or base pairs and will have lee than 30 nucleotide residues or base pairs
  • a coding region of a histidme kinase gene may be isolated by screening us
  • primers designed to anneal within the amplified product typically an adaptor specific primer that anneals further 3' in the adaptor sequence and a gene specific primer that anneals further 5' in the selected gene sequence
  • the products of this reaction can then be analyzed by DNA sequencing and a full-length DNA constructed either by joining the product directly to the existmg DNA to give a complete sequence, or carrying out a separate full- length PCR usmg the new sequence information for the design of the 5' primer
  • polynucleotides and polypeptides of the mvention may be employed, for example, as research reagents and matenals for discovery of treatments of and diagnostics for diseases, particularly human diseases, as further discussed herem relatmg to polynucleotide assays
  • polynucleotides of the invention that are ohgonucleotides denved from a sequence of Table 1 [SEQ ID NOS 1 or 2] may be used in the processes herem as descnbed. but preferably for PCR. to determine whether or not the polynucleotides identified herem in whole or m part are transcribed in bactena m infected tissue It is recogmzed that such sequences will also have 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 that is a mature protem plus additional ammo or carboxyl-terminal ammo acids, or ammo acids tenor 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-life or may facilitate manipulation of a protem for assay or production, among other things As generally is the case in vivo, the additional ammo acids may be processed awav from a mature protem bv cellular enzymes For each and every polynucleotide of the mvention there is provided a polynucleotide complementarv to it It is preferred that these complementary polynucleotides are fullv complementarv to each polynucleotide with which they are complementary
  • a precursor protem havmg a mature form of the polvpeptide fused to one or more prosequences may be an mactive form of the polypeptide When prosequences are removed such mactive precursors generallv are activated Some or all of the prosequences mav be remo ed before activation Generally such precursors are called proproterns
  • Tliese experiments utilize exonuclease digestion or convement restnction sites to cleave coding nucleic acid sequence For example.
  • 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 the mvention, as are polypeptides so encoded
  • a polynucleotide of the mvention may encode a mature protem, a mature protem plus a leader sequence (which 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 preproprotein. that is a precursor to a proprotein. having a leader sequence and one or more prosequences. that generally are removed during processing steps that produce active and mature forms of the polypeptide Vectors, Host Cells, Expression Systems
  • the mvention also relates to vectors that compnse a polynucleotide or polynucleotides of the mvention.
  • Recombinant polypeptides of the present mvention may be prepared by processes well known m those skilled m the art from genetically engmeered 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 engmeered with such expression systems, and to the production of polypeptides of the mvention bv recombinant techniques For recombinant production of the polypeptides of the mvention.
  • host cells can be genetically engmeered 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 in many standard laboratory manuals, such as Davis et al BASIC METHODS IN MOLECULAR BIOLOGY (1986) and Sambrook. et al MOLECULAR CLONING A LABORATORY MANUAL 2nd Ed . Cold Spring Harbor Laboratory Press. Cold Spring Harbor N Y (1989).
  • 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 albicans and Aspergillus. insect cells such as cells of Drosophtla S2 and Spodoptera Sf9, animal cells such as CHO, COS. HeLa. C127. 3T3. BHK, 293. CV-1 and Bowes melanoma cells, and plant cells, such as cells of a gymnosperm or angiosperm A great vanety of expression systems can be used to produce the polypeptides of the mvention
  • Such vectors mclude, among others, chromosomal-, episomal- and vmis-denved vectors, for example, vectors denved from bactenal plasmids, from bactenophage, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses such as baculoviruses, papova viruses, such as SV40. vaccinia viruses, adenoviruses. fowl pox viruses, pseudorabies viruses, picornaviruses and retroviruses.
  • the expression system constructs may compnse control regions that regulate as well as engender expression
  • any system or vector suitable to maintain, propagate or express polynucleotides and/or to express a polypeptide m a host may be used for expression m this regard
  • the appropnate DNA sequence may be inserted mto the expression system by any of a vanety of well-known and routme techmques, such as, for example, those set forth m Sambrook et al , MOLECULAR CLONING, A LABORATORY MANUAL, (supra)
  • Polypeptides of the mvention can be recovered and punfied from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography. phosphocellulose chromatographv hvdrophobic interaction chromatographv affinity chromatography. hydroxylapatite chromatography. and lectm chromatographv Most preferably, high performance liquid chromatography is employed for purification Well known techmques for refolding protem mav be employed to regenerate active conformation when the polvpeptide is denatured during isolation and or purification
  • This mvention is also related to the use of histidme kinase polvnucleotides and polypeptides of the mvention for use as diagnostic reagents Detection of histidme kinase polvnucleotides and/or polypeptides in a eukaryote.
  • a mammal particularly a mammal, and especially a human
  • Polypeptides and polynucleotides for prognosis, diagnosis or other analysis may be obtained from a putative-y infected and or infected individual's bodily matenals
  • Polynucleotides from any of these sources may be used directly for detection or may be amplified enzymatically by usmg PCR or any other amplification technique pnor to analysis RNA, particularly mRNA, cDNA and genomic DNA may also be used m the same ways Using amplification, characterization 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 insertions 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.
  • Pomt mutations can be identified by hybndizmg amplified DNA to labeled histidme kinase polynucleotide sequences Perfectly or significantly matched sequences can be distinguished from imperfectly or more significantly mismatched duplexes by DNase or RNase digestion, for DNA or RNA respectively, or by detecting differences m meltmg temperatures or renaturation kinetics Polynucleotide sequence differences may also be detected bv alterations m the electrophoretic mobility of polynucleotide fragments m gels as compared to a reference sequence This may be earned out with or without denaturing agents Polynucleotide differences may also be detected by direct DNA or RNA sequencmg See, for example, Myers et al .
  • Sequence changes at specific locations also may be revealed by nuclease protection assays, such as RNase. VI and SI protection assay or a chemical cleavage method See, for example, Cotton et al . Proc Nail Acad Sci , USA, 85 4397-4401 (1985)
  • an array of oligonucleotides probes compnsmg histidme kinase nucleotide sequence or fragments thereof can be constructed to conduct efficient screening of. for example genetic mutations, serotype. taxonomic classification or identification Array technology' metiiods are w ell known and have general applicability and can be used to address a vanetv of questions m molecular genetics including 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 mvention. preferably the nucleotide sequence of SEQ ID NO 1. or a fragment thereof . (b) a nucleotide sequence complementary to that of (a) (c) a poh peptide of the present invention, preferably the polypeptide of SEQ ID NO 2 or a fragment thereof or (d) an antibody to a polypeptide of the present invention, preferably to the polvpeptide of SEQ ID NO 2 It will be appreciated that in any such kit, (a), (b).
  • kits will be of use m diagnosing 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 the mvention. preferable. SEQ ID NO 1.
  • 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 orgamsms. carrying mutations m such polynucleotide may be detected at the polynucleotide level by a vanety of techmques, such as those descnbed elsewhere herem
  • a polynucleotide and/or polypeptide sequence between orgamsms possessmg a first phenotype and organisms possessmg a different, second different phenotype can also be determined If a mutation is observed m some or all orgamsms possessing the first phenotype but not in any orgamsms possessmg 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 techmques, to allow for serotyping, for example
  • RT-PCR can be used to detect mutations m the RNA It is particularly preferred 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 histidme kinase polypeptide can be used to identify and analyze mutations.
  • the mvention further provides these primers with 1, 2, 3 or 4 nucleotides removed from the 5' and/or the 3' end These primers may be used for.
  • 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 tins vvav. mutations m the polynucleotide sequence mav 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 diagnosing, disease, preferably bacterial infections, more preferably infections caused by Staphylococcus aureus. comprising determining from a sample derived from an individual such as a bodily material an increased level of expression of polynucleotide havmg a sequence of Table 1 [SEQ ID NO 1 ] Increased or decreased expression of a histidme kinase polynucleotide can be measured using any on of the methods ell knovvn in the art tor the quantitation of polynucleotides. such as, for example, amplification. PCR. RT-PCR. RNase protection.
  • a diagnostic assay m accordance with the mvention for detectmg over-expression of histidme kinase polypeptide compared to normal 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 histidme kinase polypeptide.
  • Assay techmques that can be used to determine levels of a histidme kinase polypeptide.
  • Such assay methods mclude radioimmunoassays, competitive-binding assays, Western Blot analysis. antibody sandwich assays, antibody detection and ELISA assays
  • Polypeptides and polynucleotides of the mvention may also be used to assess the binding of small molecule substrates and hgands in. for example, cells, cell-free preparations, chemical hbranes. and natural product mixtures
  • substrates and gands 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, including 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 Accordingly, m a further aspect, the present mvention provides for a method of screening compounds to identify those that agonize 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 vanety of sources, for example, cells, cell-free preparations, chemical hbranes.
  • agonists and antagonists so-identified may be natural or modified substrates, hgands. receptors, enzymes, etc , as the case mav be. of histidme kinase polypeptides and polynucleotides. or mav be structural or functional mimetics thereof (see Coligan et al . Current Protocols in Immunology 1 (2) Chapter 5 ( 1991 ))
  • the screemng methods mav simply measure the binding of a candidate compound to the polypeptide or polynucleotide. or to cells or membranes bearmg the polypeptide or polynucleotide. or a fusion protem of the polypeptide bv means of a label directly or indirectly associated with the candidate compound Alternativ elv the screenmg method may involve competition with a labeled competitor Further, these screenmg methods mav test whether the candidate compound results in a signal generated by activation or inhibition of the polvpeptide or polynucleotide.
  • the cells compnsmg the polypeptide or polynucleotide Inhibitors of activation are generally assayed m the presence of a known ago st and the effect on activation by the agonist bv the presence of the candidate compound is observed
  • Constitutively active polypeptide and/or constitutively expressed polypeptides and polynucleotides may be employed in screemng methods for inverse agomsts, m the absence of an agonist or antagonist, by testing whether the candidate compound results in inhibition of activation of the polypeptide or polynucleotide, as the case may be Further, the screemng methods may simply compnse the steps of mixing a candidate compound with a solution compnsmg a polypeptide or polynucleotide of the present invention, to form a mixture, measuring histidme kinase polvpeptide and/or polynucleotide activity in the mixture, and comparing the histid
  • polypeptides and antibodies that bmd to and/or mteract with a polypeptide of the present mvention may also be used to configure screemng methods for detectmg the effect of added compounds on the production of mRNA and/or polypeptide m cells
  • an ELISA assay may be constructed for measu ⁇ ng secreted or cell associated levels of polypeptide usmg monoclonal and polvclonal antibodies bv standard methods known in the art This can be used to discover agents that may inhibit or enhance the production of polypeptide (also called antagonist or agonist, respectively) from suitably manipulated cells or tissues
  • the mvention also provides a method of screenmg compounds to identify those that enhance (agomst) or block (antagonist) the action of histidme kinase polypeptides or polvnucleotides particularly those compounds that are bacte ⁇ static and/or bacte ⁇ cidal
  • the method of screening mav mvolve high- throughput techmques For example, to screen for agomsts or antagonists, a synthetic reaction mix a cellular compartment, such as a membrane, cell envelope or cell wall, or a preparation of anv thereof compnsmg histidme kinase polypeptide and a labeled substrate or ligand of such polypeptide is mcubated m the absence or the presence of a candidate molecule that may be a histidme kinase agomst or antagonist The ability of the candidate molecule to agonize or antagonize the histidme kinase polypeptide is reflected in decreased binding
  • e . widiout inducing the effects of histidme kinase polvpeptide are most likelv to be good antagonists Molecules that bmd well and. as the case may- be. mcrease the rate of product production from substrate, mcrease signal transduction. or mcrease chemical channel activ ⁇ tv are agomsts Detection of the rate or level of. as the case may be. production of product -from substrate, signal transduction. or chemical channel activity may be enhanced by usmg a reporter system. Reporter systems tiiat mav be useflil in this regard include but are not limited to colonmetric. labeled substrate converted into product, a reporter gene that is responsive to changes in histidine kinase 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, ligand binding and crosslinking assays in which the polypeptide is labeled with a radioactive isotope (for instance, ⁇ 5 ⁇ chemically modified (for instance, biotinylated), or fused to a peptide sequence suitable for detection or purification, and incubated with a source of the putative receptor (e g.. cells, cell membranes, cell supernatants.
  • a radioactive isotope for instance, ⁇ 5 ⁇ 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 biophy sical techniques such as surface plasmon resonance and spectroscopy These screening methods also be used to identify agonists and antagonists of the polypeptide that compete with the binding of the polvpeptide 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 Protem complexes, such as formed by histidme kinase polypeptide associating with another histidine kinase polypeptide or other polypeptide. Labeled to compnse a fluorescently-labeled molecule will have higher polarization values than a fluorescently labeled monomeric protein. It is preferred that this method be used to characte ⁇ ze small molecules that disrupt polypeptide complexes. Fluorescence energy transfer may also be used characte ⁇ ze small molecules that interfere with the formation of histidine kinase polypeptide dimers.
  • histidme kinase polypeptide bound to another polypeptide can be labeled w ith both a donor and acceptor fluorophore pon mixmg 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 dimenzation will inhibit fluorescence energy transfer
  • Histidme kinase 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 ov er the histidme kinase polypeptide -coated surface and specific binding can be detected in real-time bv monito ⁇ ng the change m resonance angle caused bv a change in local refractive index
  • This technique can be used to characte ⁇ ze the effect of small molecules on kmetic rates and equihb ⁇ um binding constants for histidine kinase polvpeptide self-association as well as an association of histidine kinase polvpeptide and another polv peptide or bmall molecu
  • a scintillation proximity assay mav be used to characterize the interaction between an association of histidme kinase polypeptide with another histidine kinase polvpeptide or a different polvpeptide Histidine kinase polypeptide can be coupled to a scmtillation-filled bead Addition of radio-labeled histidine kmase polypeptide results in binding where the radioactive source molecule is in close proximity to the scintillation fluid Thus, signal is emitted upon histidme kinase polypeptide bindmg and compounds that prevent histidine kinase polypeptide self-association or an association of histidine kinase polypeptide and another polypeptide or small molecule will dimmish signal
  • mvention diere In other embodiments of the mvention diere are provided methods for identifying compounds that bmd to or otherwise mteract 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 binding to or other interaction between the compound and the polypeptide and/or polynucleotide to assess the binding to or other interaction with the compound, such binding or interaction preferably bemg associated with a second component capable of providing a detectable signal m response to the binding or interaction of the polypeptide and/or polynucleotide with the compound, and determining whether the compound bmds to or otherwise interacts with and activates or inhibits an activity or expression of the polypeptide and/or polynucleotide by detecting the presence or absence of a signal generated from the binding or mteraction of the compound with
  • Histidme kinase can be labeled, such as by radioactivity or a colonmetnc compound, such that the number of histidme kinase molecules bound to a binding molecule or converted to product can be determmed accurately to assess the effectiveness of the potential antagomst
  • a polypeptide and/or polynucleotide of the present mv ention may also be used m a method for the structure-based design of an agonist or antagonist of the polypeptide and/or polynucleotide. by (a) determining in the first instance the three- dimensional structure of the polypeptide and/or polynucleotide. or complexes thereof, (b) deducing the three-dimensional structure for the likely reactive s ⁇ te(s). binding s ⁇ te(s) or mot ⁇ f(s) of an agonist or antagomst.
  • the present mvention provides methods of treating abnormal conditions such as. for instance, a Disease, related to either an excess of. an under-expression of, an elevated activity of, or a decreased activity of histidme kinase polypeptide and/or polynucleotide If the expression and/or activity of the polypeptide and/or polynucleotide is m excess, several approaches are available One approach comprises administering to an individual m need thereof an inhibitor compound (antagomst) as herem descnbed. optionally m combination with a pharmaceutically acceptable earner, m an amount effective to inhibit the function and or expression of the polypeptide and/or polynucleotide. such as.
  • oligonucleotides that form triple helices with the gene can be supplied (see toi example. Lee et al Nucleic Acids Res (1979) 6 3073, Coonev et al Science ( 1988) 241 456 Dervan et al , Science (1991) 251 1360) These ohgomers can be administered per se or the relevant ohgomers can be expressed in vivo
  • Each of the polynucleotide sequences provided herem may be used in the discovery and development of antibacterial compounds
  • the encoded protein upon expression, can be used as a target for the screening of antibacterial drugs
  • the polynucleotide sequences encoding the amino terminal regions of the encoded protein 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 codmg sequence of interest
  • the invention also provides the use of the polypeptide, polynucleotide. agonist or antagonist of the invention to interfere with the initial phy sical interaction between a pathogen or pathogens and a eukaryotic.
  • the molecules of the mvention may be used' in the prevention of adhesion of bacteria, in particular gram positive and/or gram negative bacte ⁇ a. to eukaryotic. preferably' mammalian, extracellular matrix proteins on in-dwelling devices or to extracellular matrix proteins in wounds, to block bacterial adhesion between eukaryotic, preferably mammalian, extracellular matrix proteins and bacterial histidine kinase proteins that mediate tissue damage and/or. to block the normal progression of pathogenesis in infections initiated other than by the implantation of in-dwelling devices or by other surgical techniques .
  • histidine kinase agonists and antagonists preferably bacteristatic or bactericidal agonists and antagonists.
  • the antagonists and agomsts of the invention may be employed, for instance, to prevent, inhibit and/or treat diseases.
  • Hehcobacter pylon herein "H pylon” bacteria infect the stomachs of over one-third of the world's population causing stomach cancer, ulcers, and gastritis (International Agency for Research on
  • H. pylori a group I (definite) carcinogen
  • Preferred antimicrobial compounds of the invention agonists and antagonists of histidine kinase polypeptides and/or polynucleotides found using screens provided by the invention, or known in the art.
  • particularly narrow-spectrum antibiotics should be useful in the treatment of H. pylori infection.
  • Such treatment should decrease the advent of H. pylori -induced cancers, such as gastrointestinal carcmoma.
  • Such treatment should also prevent, inhibit and/or cure gastric ulcers and gastritis.
  • mate ⁇ al(s) means any matenal denved from an individual or from an organism infecting, infesting or inhabiting an individual, including but not limited to. cells, tissues and waste, such as. bone blood, serum, cerebrospinal fluid, semen, saliva, muscle, cartilage, organ tissue, skin, urine stool or autopsv matenals
  • D ⁇ sease(s) means any disease caused bv or related to infection by a bactena. including . for example, disease, such as. infections of the upper respiratory tract (e g . otitis media, bactenal tracheitis. acute epiglottitis.
  • loyyer respiratory e g . empvema lung abscess
  • cardiac ⁇ s. g infective endocarditis gastromtestmal
  • gastromtestmal e g . secretory dia ⁇ hoea. splemc absces retropentoneal abscess
  • CNS e g cerebral abscess
  • eve e g . blephantis conjunctivitis, keratitis endophthalrrutis preseptal and orbital celluhtis. darcryocystitis
  • kidney and urinary tract e g . epididvm-tis. mtrarenal and penneph ⁇ c absces.
  • “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 m the art. is a relationship between two or more polvpeptide sequences or two or more polynucleotide sequences, as the case may be, as determmed 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 descnbed in (Computational Molecular Biology, Lesk, A M . ed . Oxford University Press. New York. 1988, Biocomputing Informatics and Genome Projects. Smith. D W . ed , Academic Press. New York, 1993. Computer Analysis of Sequence Data. Part I. Griffin. A M . and Griffin. H G . eds . Humana Press. New Jersey. 1994, Sequence Analysis in Molecular Biology, von Heinje. G . Academic Press. 1987.
  • the BLAST X program is publicly available from NCBI and other sources (BLAST Manual. Altschul. S . et al , NCBI NLM NIH Bethesda, MD 20894. Altschul. S . et al . J Mol Biol 215 403-410 (1990)
  • the w ell known Smith Waterman algorithm may also be used to determine identity Parameters for polypeptide sequence comparison include the following Algorithm Needleman and Wunsch. J Mol Biol 48 443-453 (1970)
  • Polynucleotide embodiments further mclude an isolated polynucleotide compnsmg a polynucleotide sequence havmg 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 mclude up to a certain integer number of nucleotide alterations as compared to the reference sequence, herem said alterations are selected from the group consisting of at least one nucleotide deletion, substitution, including transition and transversion.
  • alterations mav 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 m 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 in SEQ ID NO 1 bv the mteger 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 ⁇ ⁇ n ( x n • y), wherein 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%.
  • Polypeptide embodiments further include an isolated polypeptide comprising a polypeptide havmg at least a 95. 97 or 100% identity to a polypeptide reference sequence of SEQ ID NO 2.
  • said polypeptide sequence may be identical to the reference sequence of SEQ ID NO 2 or may include up to a certain integer number of ammo acid alterations as compared to the reference sequence, wherein said alterations are selected from the group consisting of at least one ammo acid deletion, substitution, including conservative and non-conservative substitution, or insertion, and wherein said alterations may occur at the ammo- or carboxy-termmal positions of the reference polypeptide sequence or anywhere between those terminal positions, interspersed either individually among the ammo acids in the reference sequence or in one or more contiguous groups within the reference sequence, and wherein said number of amino acid alterations is determined bv multiply ing the total number of amino acids in SEQ ID NO 2 bv the integer defining the percent identity dn ided b ⁇ 100 and then subtracting that product from said total
  • n a is the number of amino acid alterations
  • x a is the total number of amino acids in SEQ ID NO 2.
  • y is 0 95 for 95%. 0 97 for 97% or 1 00 for 100%. and • is the symbol for the multiplication operator, and where any non-mteger product of x a and y is rounded down to the nearest integer prior to subtracting it from x a
  • “Indrv ⁇ dual(s)” means a multicellular eukaryote, including, but not limited to a metazoan. a mammal, an ovid. a bov d. a simian, a primate, and a human
  • Isolated means altered “by the hand of man” from its natural state. i e . if it occurs m nature, it has been changed or removed from its ongmal environment, or both
  • a polynucleotide or a polypeptide naturally present in a livmg orgamsm is not isolated but die same poK nucltotidL or polypeptide separated from the coexisting matenals of its natural state is "isolated” as die tenn is employed herem ⁇ loreov er.
  • a polynucleotide or polypeptide that is introduced mto an orgamsm bv transformation genetic manipulation or by any- other recombinant method is "isolated” even if it is still present in said organism, which organism may be living or non-living.
  • Organism(s) means a (i) prokaryote, including but not limited to. a member of the genus Streptococcus. Staphylococcus. Bordetella. Corynebacterium. Mycobacterium. Neisse a. Haemophilus. Actinomycetes. Streptomycetes. Nocardia. Enterobacter, Yersinia, Fancisella. Pasturella. Moraxella. Acinetobacter, Erysipelothrix. Branhamella, Actinobacillus. Streptobacillus. Listeria. Calymmatobacterium. Brucella, Bacillus. Clostridium, Treponema. Escherichia. Salmonella.
  • Streptococcus agalactiae Streptococcus faecalis, Streptococcus faecium.
  • Streptococcus clurans Neisseria gonorrheae. Neisseria meningitidis. Staphylococcus aureus. Staphylococcus epidermidis. Corynebacterium diptheriae. Gardnerella vaginalis, Mycobacterium tuberculosis. Mycobacterium bovis. Mycobacterium ulcerans, Mycobacterium leprae, Actinomyctes israelii, Listeria monocytogenes.
  • Bordetella pertusis Bordatella parapertusis, Bordetella bronchiseptica, Escherichia coli, Shigella dysenteriae, Haemophilus influenzae, Haemophilus aegyptius, Haemophilus parain luenzae, Haemophilus ducreyi, Bordetella, Salmonella typhi, Citrobacter freundii, Proteus mirabilis, Proteus vulgaris, Yersinia pestis. Kleibsiella pneumoniae, Serratia marcessens, Serratia liquefaciens, Vibrio cholera.
  • a protozoan a fungus, a member of the genus Saccharomyces, Kluveromyces. or Candida, and a member of the species Saccharomyces ceriviseae, Kluveromyces lactis, or Candida albicans.
  • Polynucleotide(s) generally refers to any polyribonucleotide or polydeoxyribonucleotide. that may be unmodified RNA or DNA or modified RNA or DNA.
  • Polynucleotide(s) include, without limitation, single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions or single-, double- and triple-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or. more typically, double-stranded, or triple-stranded regions, or a mixture of single- and double-stranded regions.
  • polynucleotide refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA.
  • the strands in such regions may be from the same molecule or from different molecules.
  • the regions may include all of one or more of the molecules, but more typically involve only a region of some of the molecules
  • One of the molecules of a tnple-he cal region often is an ohgonucleotide
  • polynucleot ⁇ de(s) also mcludes DNAs or RNAs as descnbed above that compnse one or more modified bases Thus.
  • DNAs or RNAs with backbones modified for stability or for other reasons are "polynucleot ⁇ de(s)" as that term is intended herem Moreover. DNAs or RNAs compnsmg unusual bases, such as inosine. or modified bases, such as tntylated bases, to name just two examples, are polynucleotides as die term is used herem It will be appreciated that a great vanety of modifications have been made to DNA and RNA that serve many useful purposes known to those of skill m die art
  • the term "polynucleot ⁇ de(s)" as it is employed herem embraces such chemically, enzymaticallv or metabohcallv modified forms of polynucleotides, as well as the chemical forms of DNA and RNA charactenstic of viruses and cells, mcludmg, for example, simple and complex cells
  • Polynucleot ⁇ de(s) also embraces short polynucleotides often refened to as o
  • Polypeptide(s) refers to any peptide or protem compnsmg two or more ammo acids jomed to each other by peptide bonds or modified peptide bonds
  • Polypept ⁇ de(s) refers to both short chains, commonly refened to as peptides.
  • Polypeptides may compnse ammo acids other than die 20 gene encoded ammo acids "Polypept ⁇ de(s)" mclude those modified either by natural processes, such as processmg and odier 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 will be appreciated that the same type of modification may be present m the same or varymg degree at several sites m a given polypeptide Also, a given polypeptide mav compnse many types of modifications Modifications can occur anywhere m a polypeptide.
  • Modifications mclude. for example, acetylation. acylation. ADP- ⁇ bosylation. amidation. covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide denvative. covalent attachment of a hpid or hpid denvative. covalent attachment of phosphotidylinositol, cross-linking, cyc zation. disulfide bond formation, demethylation.
  • Recombinant expression svstem(s) refers to expression systems or portions thereof or polynucleotides of the mvention mtroduced or transformed into a host cell or host cell lvsate tor the production of the polynucleotides and polypeptides of the mvention
  • ⁇ 'Va ⁇ ant(s) " ' as the term is used herein, is a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide respectively, but retains essential properties
  • a typical v ariant of a polynucleotide differs m nucleotide sequence from another, reference polynucleotide Changes in the nucleotide sequence of the variant mav or may not alter the amino acid sequence of a polvpeptide 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, as discussed below
  • a typical variant of a polypeptide differs in ammo acid sequence from another, reference polypeptide Generally, differences are limited so that the sequences of the reference polypeptide and the vanant are closely similar overall and.
  • a va ⁇ ant and reference polypeptide may differ in ammo acid sequence by one or more substitutions, additions, deletions in any combination
  • a substituted or inserted amino acid residue may or mav not be one encoded by the genetic code
  • the present invention also includes mclude variants of each ot tl polypeptides of the mvention. that is polypeptides that vary from the referents by conserv ativ e ammo acid substitutions, wherebv a residue is substituted bv another with like charactenstics Typical such substitutions are among Ala. Val. Leu and He. among Ser and Thr. among the acidic residues Asp and Glu. among Asn and Gin, and among the basic residues Lys and Arg.
  • vanants m which several. 5-10. 1-5. 1-3. 1-2 or 1 ammo acids are substituted, deleted, or added m anv combmation
  • a variant of a polynucleotide or polypeptide mav be a naturally occurring such as an allehc vanant. or it may be a variant that is not known to occur naturally
  • Non- naturally occur ⁇ ng vanants of polynucleotides and polypeptides may be made by mutagenesis techniques, by direct synthesis, and by other recombinant methods known to skilled artisans EXAMPLES
  • Example 1 Strain selection, Library Production and Sequencing
  • the polynucleotide havmg 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 coh
  • the sequencing data from two or more clones comprising overlapping Staphylococcus aureus DNAs was used to construct the contiguous DNA sequence in SEQ ID NO 1 Libraries mav be prepared bv routine methods toi example
  • Total cellular DNA is isolated from Staphylococcus aureus WCUH 29 according to standard procedures and size-fractionated bv either of two methods
  • Method 1 Total cellular DNA is mechanically sheared by passage through a needle in order to size- fractionate accordmg 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 ligated into the vector Lambda ZapII that has been cut with EcoRI.
  • the library packaged by standard procedures and E coh infected with the packaged library The library is amplified by standard procedures
  • Total cellular DNA is partially hydrolyzed with a one or a combination of restriction enzvmes 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 ligated to the DNA and the fragments then ligated into the vector Lambda ZapII that have been cut with EcoRI, the hbraiy packaged by standard procedures, and E coh infected with the packaged library
  • the library is amplified by standard procedures

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Abstract

Cette invention, qui a trait à des polypeptides d'histidine kinase ainsi qu'à des polynucléotides codant ces polypeptides d'histidine kinase, concerne également des procédés de production desdits polypeptides par le biais de techniques de recombinaison. Elle porte, de surcroît, sur des méthodes d'utilisation des polypeptides d'histidine kinase pour cribler des composés antibactériens
PCT/US2000/006206 1999-03-22 2000-03-09 Histidine kinase WO2000056865A1 (fr)

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WO2001055397A1 (fr) * 2000-01-28 2001-08-02 Keiichi Hiramatsu NOUVEAUX POLYPEPTIDES VraS ET VraR

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