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WO2000071568A1 - Operon ica staphylococcique - Google Patents

Operon ica staphylococcique Download PDF

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Publication number
WO2000071568A1
WO2000071568A1 PCT/US2000/012253 US0012253W WO0071568A1 WO 2000071568 A1 WO2000071568 A1 WO 2000071568A1 US 0012253 W US0012253 W US 0012253W WO 0071568 A1 WO0071568 A1 WO 0071568A1
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Prior art keywords
polypeptide
seq
polynucleotide
sequence
amino acid
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English (en)
Inventor
Martin Karl Russel Burnham
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SmithKline Beecham Ltd
SmithKline Beecham Corp
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SmithKline Beecham Ltd
SmithKline Beecham Corp
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Publication of WO2000071568A1 publication Critical patent/WO2000071568A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/305—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F)
    • C07K14/31—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F) from Staphylococcus (G)

Definitions

  • This invention relates to newly identified polynucleotides and polypeptides. and their production and uses, as well as their variants, agonists and antagonists, and their uses
  • the invention relates to polynucleotides and polypeptides of an ica operon, as well as their va ⁇ ants, herein referred to as ' ica A, B. C or D," “ica A, B, C or D polynucleot ⁇ de(s),” and “ica A, B, C or D polypept ⁇ de(s)” as the case may be BACKGROUND OF THE INVENTION
  • Staphylococcal genes and gene products are particularly preferred to employ Staphylococcal genes and gene products as targets for the development of antibiotics
  • the Staphylococci make up a medically important genera of microbes They are known to produce two types of disease. e 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 patients Osteomyelitis, septic arthritis, septic thrombophlebitis and acute bacterial endocarditis are also relatively common.
  • Osteomyelitis, septic arthritis, septic thrombophlebitis and acute bacterial endocarditis are also relatively common
  • There are at least three clinical conditions resulting from the toxigenic properties of Staphylococci The manifestation of these diseases result from the actions of exotoxms as opposed to tissue invasion and bacteremia These conditions include Staphylococcal food poisoning, scalded skin syndrome
  • the present invention relates to the ica operon in Staphylococcus aureus Clearly, there exists a need for polynucleotides and polypeptides, such as the ica A, B
  • the present invention relates to ica A, B, C or D, in particular ica A, B, C or D polypeptides and ica A, B, C or D polynucleotides, recombinant mate ⁇ als and methods for their production
  • the invention relates to methods for using such polypeptides and polynucleotides, including treatment of microbial diseases, amongst others
  • the invention relates to methods for identifying agonists and antagonists using the materials provided by the invention, and for treating 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 ica A, B, C or D expression or activity
  • the invention relates to ica A, B, C or D polypeptides and polynucleotides as desc ⁇ bed in greater detail below
  • the invention relates especially to ica A, B, C or D having a nucleotide and ammo acid sequences set out in Table 1 as SEQ ID NO 1 ,3,5.7 or 9 and SEQ ID NO 2,4,6,8 or 10 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
  • a deposit comp ⁇ sing a Staphylococcus aureus WCUH 29 strain has been deposited with the National Collections of Industrial and Marine Bacteria Ltd (herein "NCIMB"), 23 St Machar D ⁇ ve, Aberdeen AB2 1RY, Scotland on 1 1 September 1995 and assigned NCIMB Deposit No 40771, and referred to as Staphylococcus aureus WCUH29 on deposit
  • NCIMB National Collections of Industrial and Marine Bacteria Ltd
  • Staphylococcus aureus WCUH29 on deposit
  • the Staphylococcus aureus strain deposit is referred to herein as "the deposited strain” or as "the DNA of the deposited strain "
  • the deposited strain comprises a full length ica A, B, C or D gene
  • the sequence of the polynucleotides composed in the deposited strain, as well as the amino acid sequence of any polypeptide encoded thereby, are controlling in the event of any conflict with any desc ⁇ ption of sequences herein
  • the deposit of the deposited strain has been made under the terms of the Budapest Treaty on the International Recognition of the Deposit of Micro-organisms for Purposes of Patent Procedure
  • the deposited strain will be irrevocably and without rest ⁇ ction or condition released to the public upon the issuance of a patent
  • the deposited strain is provided merely as convenience to those of skill in the art and is not an admission that a deposit is required for enablement, such as that required under 35 U S C ⁇ 1 12
  • a license may be required to make, use or sell the deposited strain, and compounds de ⁇ ved therefrom, and no such license is hereby granted
  • an isolated nucleic acid molecule encoding a mature polypeptide expressible by the Stapfnlococcus aureus WCUH 29 strain, which polypeptide is comp ⁇ sed in the deposited strain
  • ica A, B, C or D polynucleotide sequences in the deposited strain such as DNA and RNA, and amino acid sequences encoded thereby
  • Polypeptides ica A, B, C or D polypeptide of the invention is substantially phJogenetically related to other proteins of the ica operon family
  • polypeptides of Staphylococcus aureus referred to herein as "ica A, B, C or D” and "ica A, B, C or D polypeptides" as well as biologically, diagnostically, prophylactically, clinically or therapeutically useful variants
  • the present invention further provides for an isolated polypeptide that (a) comprises or consists of an amino acid sequence that has at least 95% identity, most preferably at least 97- 99% or exact identity, to that of SEQ ID NO.2,4,6,8 or 10 over the entire length of SEQ ID NO.2,4,6, 8 or 10; (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 ,3,5,7 or 9 over the entire length of SEQ ID NOJ ,3,5,7or 9; (c) a polypeptide encoded by an isolated polynucleotide comprising or consisting of a polynucleotide sequence encoding a polypeptide that has at least 95% identity, even more preferably at least 97-99% or exact identity, to the amino acid sequence of SEQ ID NO.2,4,6,8 or 10, over the entire length of
  • polypeptides of the invention include a polypeptide of Table 1 [SEQ ID NO:2,4,6,8 or 10] (in particular a mature polypeptide) as well as polypeptides and fragments, particularly those that has a biological activity of ica A, B, C or D, and also those that have at least 95% identity to a polypeptide of Table 1 [SEQ ID NO.2,4,6,8 or 10] and also include portions of such polypeptides with such portion of the polypeptide generally comp ⁇ sing at least 30 amino acids and more preferably at least 50 amino acids
  • the invention also includes a polypeptide consisting of or comp ⁇ sing a polypeptide of the formula X-(R 1 ) m -(R 2 )-(R 3 ) n -Y wherein, at the amino terminus, X is hydrogen, a metal or any other moiety desc ⁇ bed herein for modified polypeptides, and at the carboxyl terminus, Y is hydrogen, a metal or any other moiety desc ⁇ bed herein for modified polypeptides, R j and R are any amino acid residue or modified ammo acid residue, m is an integer between 1 and 1000 or zero, n is an integer between 1 and 1000 or zero, and R 2 is an amino acid sequence of the invention, particularly an amino acid sequence selected from Table 1 or modified forms thereof In the formula above, R 2 is o ⁇ ented so that its amino terminal amino acid residue is at the left, co ⁇ alentl> bound to R ⁇ and its carboxy terminal amino acid residue is at the ⁇ ght.
  • n is an integer between 1 and 50, 100 or 500
  • n is an integer between 1 and 50, 100, or 500
  • a polypeptide of the invention is de ⁇ ved 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 ammo acid sequence of Table 1 [SEQ ID NO 2,4,6,8 or 10]. or of va ⁇ ants thereof, such as a continuous series of residues that includes an amino- and or carboxyl-terminal amino acid sequence
  • Degradation forms of the polypeptides of the invention produced by or in a host cell, particularly a Staphylococcus aureus are also preferred
  • fragments characte ⁇ zed by structural or functional att ⁇ butes such as fragments that comp ⁇ se alpha-helix and alpha-helix forming regions, beta-sheet and beta-sheet-forming regions, turn and turn- forming regions, coil and coil-forming regions, hydrophi c 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 ammo acid sequence having at least 15, 20, 30, 40, 50 or 100 contiguous ammo acids from the ammo acid sequence of SEQ ID NO 2,4,6,8 or 10, or an isolated polypeptide comprising an ammo acid sequence having at least 15, 20, 30, 40, 50 or 100 contiguous amino acids truncated or deleted from the ammo acid sequence of SEQ ID NO 2,4,6,8 or 10
  • Fragments of the polypeptides of the invention may be employed for producing the corresponding full-length polypeptide by peptide synthesis, therefore, these v a ⁇ ants may be employed as intermediates for producing the full-length polypeptides of the invention
  • Polynucleotides It is an object of the invention to provide polynucleotides that encode ica A, B, C or D polypeptides, particularly polynucleotides that encode a polypeptide herein designated ica A, B C or D
  • polynucleotide comp ⁇ ses a region encoding ica A, B, C or D polypeptides comprising a sequence set out in Table 1 [SEQ ID NO: 1]
  • isolated nucleic acid molecules encoding and/or expressing ica A, B, C or D polypeptides and polynucleotides, particularly Staphylococcus aureus ica A, B C or D polypeptides and polynucleotides, including, for example, unprocessed RNAs, ⁇ bozyme RNAs, RNAs, cDNAs, genomic DNAs, B- and Z-DNAs
  • Further embodiments of the invention include biologically, diagnostically, prophylactically, clinically or therapeutical ly useful polynucleotides and polypeptides, and va ⁇ ants thereof, and compositions comp ⁇ sing the same
  • Another aspect of the invention relates to isolated polynucleotides, including at least one full length gene, that encodes a ica A, B, C or D polypeptide having a deduced amino acid sequence of Table 1 [SEQ ID NO 2,4,6,8 or 10] and polynucleotides closely related thereto and va ⁇ ants thereof
  • a ica A, B, C or D polypeptide from Staphylococcus aureus comprising or consisting of an amino acid sequence of Table 1 [SEQ ID NO 2,4,6,8 or 10], or a variant thereof
  • a polynucleotide of the invention encoding ica A, B, C or D polypeptide may be obtained using standard cloning and screening methods, such as those for cloning and sequencing chromosomal DNA fragments from bacteria using Staphylococcus aureus WCUH 29 cells as starting mate ⁇ al, followed by obtaining a full length clone
  • a polynucleotide sequence of the invention such as a polynucleotide sequence given in Table 1 [SEQ ID NO 1 ,3,5,7 or 9]
  • typically a library of clones of chromosomal DNA fragments from bacteria using Staphylococcus aureus WCUH 29 cells as starting mate ⁇ al
  • each DNA sequence set out in Table 1 [SEQ ID NO 13,5,7 or 9] contains an open reading frame encoding a protein having about the number of amino acid residues set forth in Table 1 [SEQ ID NO 2,4,6,8 or 10] with a deduced molecular weight that can be calculated using amino acid residue molecular weight values well known to those skilled in the art
  • the present invention provides for an isolated polynucleotide comprising or consisting of (a) a polynucleotide sequence that has at least 95% identity, even more preferably at least 97-99% or exact identity to SEQ ID NO 1 ,3,5,7 or 9 over the entire length of SEQ ID NO 1 ,3,5,7 or 9, (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 amino acid sequence of SEQ ID NO 2,4,6.8 or 10, over the entire length of SEQ ID NO 2,4,6,8 or 10
  • a polynucleotide encoding a polypeptide of the present invention including homologs and orthologs from species other than Staphylococcus aureus.
  • telomere sequence may be obtained by a process that comp ⁇ ses the steps of screening an approp ⁇ ate library under st ⁇ ngent hyb ⁇ dization conditions with a labeled or detectable probe consisting of or comp ⁇ sing the sequence of SEQ ID NO 1,3,5,7 or 9 or a fragment thereof, and isolating a full-length gene and or genomic clones comp ⁇ sing said polynucleotide sequence
  • the invention provides a polynucleotide sequence identical over its entire length to a coding sequence (open reading frame) in Table 1 [SEQ ID NO 1 ,3,5,7 or 9] Also provided by the invention is a coding sequence for a mature polypeptide or a fragment thereof, by itself as well as a coding sequence for a mature polypeptide or a fragment in reading frame with another coding sequence, such as a sequence encoding a leader or secretory sequence, a pre-, or pro- or prepro-protein sequence
  • the polynucleotide of the invention may also comp ⁇ se at least one non-coding sequence, including for example, but not limited to at least one non-coding 5' and 3' sequence, such as the transcribed but non-translated sequences, termination signals (such as rho- dependent and rho-independent termination signals), ribosome binding sites, Kozak sequences, sequences that stabilize mRNA, introns, and polyadenylation signals
  • a marker sequence that facilitates pu ⁇ fication of a fused polypeptide can be encoded.
  • the marker sequence is a hexa-histidine peptide, as provided in the pQE vector (Qiagen, Inc ) and desc ⁇ bed in Gentz et al , Proc. Natl. Acad.
  • Polynucleotides of the invention also include, but are not limited to, polynucleotides comprising a structural gene and its naturally associated sequences that control gene expression
  • the invention also includes a polynucleotide consisting of or comp ⁇ sing a polynucleotide of the formula
  • R 2 is oriented so that its 5 ' end nucleic acid residue is at the left, bound to R j and its 3 " end nucleic acid residue
  • a polynucleotide of the invention is de ⁇ ved from Staphylococcus aureus, however, it may preferably be obtained from other organisms of the same taxonomic genus
  • a polynucleotide of the invention may also be obtained, for example, from organisms of the same taxonomic family or order
  • polynucleotide encoding a polypeptide encompasses polynucleotides that include a sequence encoding a polypeptide of the invention, particularly a bacte ⁇ al polypeptide and more particularly a polypeptide of the Staphylococcus aureus ica A, B, C or D having an amino acid sequence set out in Table 1 [SEQ ID NO 2,4,6.8 or 10]
  • the term also encompasses polynucleotides that include a single continuous region or discontinuous regions encoding the polypeptide (for example, polynucleotides interrupted by integrated phage, an integrated insertion sequence, an integrated vector sequence, an integrated transposon sequence, or due to RNA editing or genomic DNA reorganization) together with additional regions, that also may comp ⁇ se coding and/or non-coding sequences
  • the invention further relates to va ⁇ ants of the polynucleotides desc ⁇ bed herein that encode va ⁇ ants of a polypeptide having a
  • polynucleotides encoding ica A, B, C or D variants, that have the amino acid sequence of ica A, B, C or D polypeptide of Table 1 [SEQ ID NO 2,4,6,8 or 10] in which several, a few, 5 to 10, 1 to 5, 1 to 3, 2, 1 or no amino acid residues are substituted, modified, deleted and/or added, in any combination Especially preferred among these are silent substitutions, additions and deletions, that do not alter the properties and activities of ica A, B, C or D polypeptide
  • Preferred isolated polynucleotide embodiments also include 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 1,3,5,7 or 9, or an polynucleotide comprising a nucleic acid sequence having at least 15, 20, 30, 40, 50 or 100 contiguous nucleic acid
  • polynucleotides that are at least 95% or 97% identical over their entire length to a polynucleotide encoding ica A, B. C or D polypeptide having an amino acid sequence set out in Table 1 [SEQ ID NO 2,4,6,8 or 10], and polynucleotides that are complementary to such polynucleotides
  • polynucleotides that comp ⁇ se a region that is at least 95% are especially preferred
  • those with at least 97% are highly preferred among those with at least 957c, and among these those with at least 98% and at least 99% are particularly highly preferred, with at least 99% being the more preferred
  • Preferred embodiments are polynucleotides encoding polypeptides that retain substantially the same biological function or activity as a mature polypeptide encoded by a DNA of Table 1 [SEQ ID NO 1,3,5,7 or 9]
  • polynucleotides that hybridize, particularly under st ⁇ ngent conditions, to ica A, B, C or D polynucleotide sequences, such as those polynucleotides in Table 1
  • the invention further relates to polynucleotides that hyb ⁇ dize to the polynucleotide sequences provided herein
  • the invention especially relates to polynucleotides that hyb ⁇ dize under st ⁇ ngent conditions to the polynucleotides described herein
  • the terms "st ⁇ ngent conditions” and “st ⁇ ngent hybridization conditions” mean hyb ⁇ dization occurring only if there is at least 95% and preferably at least 97% identity between the sequences
  • a specific example of stringent hybridization conditions is overnight incubation at 42°C in a solution comp ⁇ sing 50% formamide, 5x SSC ( 1 0mM NaCl, 15mM t ⁇ sodium citrate), 50 raM 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
  • the invention also provides a polynucleotide consisting of or comprising a polynucleotide sequence obtained by screening an appropriate library comprising a complete gene for a polynucleotide sequence set forth in SEQ ID NO 13,5,7 or 9 under stringent hybridization conditions with a probe having the sequence of said polynucleotide sequence set forth in SEQ ID NO 1 ,3,5,7 or 9 or a fragment thereof, and isolating said polynucleotide sequence Fragments useful for obtaining such a polynucleotide include, for example, probes and primers fully described elsewhere herein As discussed elsewhere herein regarding polynucleotide assays of the invention, for instance, the polynucleotides of the invention, may be used as a hybridization probe for RNA, cDNA and genomic DNA to isolate full-length cDNAs and genomic clones encoding ica A, B, C or D and to isolate cDNA and genomic clones of other genes that have a high identity
  • a coding region of a ica A, B, C or D gene may be isolated by screening using a DNA sequence provided in Table 1 [SEQ ID NO 1 ,3,5,7 or 9] to synthesize an o gonucleotide probe
  • a labeled ohgonucleotide having a sequence complementary to that of a gene of the invention is then used to screen a library of cDNA, genomic DNA or mRNA to determine which members of the library the probe hybridizes to
  • polynucleotides and polypeptides of the invention may be employed, for example, as research reagents and mate ⁇ als for discovery of treatments of and diagnostics for diseases, particularly human diseases, as further discussed herein relating to polynucleotide assays
  • polynucleotides of the invention that are ohgonucleotides derived from a sequence of Table 1 [SEQ ID NOS 1 ,2,3,4.5,6,7,8.9 or 10] 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 intected tissue It is recognized that such sequences will also have utility in diagnosis of the stage of infection and type of infection the pathogen has attained
  • the invention also provides polynucleotides that encode a polypeptide that is a mature protein plus additional amino or carboxyl-terminal amino acids, or ammo acids te ⁇ or to a mature polypeptide (when a mature form has more than one polypeptide chain, for instance) Such sequences may play a role in processing of a protein from precursor to a mature form, may allow protein transport, may lengthen or shorten protein half-life or may facilitate manipulation of a protein for assay or production
  • a precursor protein, having a mature form of the polypeptide fused to one or more prosequences may be an inactive form of the polypeptide When prosequences are removed such inactive precursors generally are activated Some or all of the prosequences may be removed before activation Generally, such precursors are called proproteins
  • a polynucleotide of the invention may encode a mature protein, a mature protein plus a leader sequence (which may be referred to as a preprotein), a precursor of a mature protein having one or more prosequences that are not the leader sequences of a preprotein, or a 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
  • the invention also relates to vectors that comp ⁇ se a polynucleotide or polynucleotides of the invention, host cells that are genetically engineered with vectors of the invention and the production of polypeptides of the invention by recombinant techniques
  • Cell-free translation systems can also be employed to produce such proteins using RNAs de ⁇ ved from the DNA constructs of the invention
  • Recombinant polypeptides of the present invention may be prepared by processes well known in those skilled in the art from genetically engineered host cells comp ⁇ sing expression systems
  • the present invention relates to expression systems that comp ⁇ se a polynucleotide or polynucleotides of the present invention, to host cells that are genetically engineered with such expression systems, and to the production of polypeptides of the invention by recombinant techniques
  • host cells can be genetically engineered to incorporate expression systems or portions thereof or polynucleotides of the invention
  • Introduction of a polynucleotide into the host cell can be effected by methods desc ⁇ bed in many standard laboratory manuals, such as Davis, et al , BASIC METHODS IN MOLECULAR BIOLOGY, (1986) and Sambrook, et al , MOLECULAR CLONING A LABORATORY MANUAL.
  • encoding 2nd Ed , Cold Sp ⁇ ng Harbor Laboratory Press, Cold Spring Harbor, N Y (1989), such as, calcium phosphate transfection, DEAE-dextran mediated transfection, transvection, microinjection, cationic lipid-mediated transfection, electroporation. transduction, scrape loading, ballistic introduction and infection
  • Representative examples of approp ⁇ ate hosts include bacterial cells such as cells of streptococci, staphylococci. enterococci E coh, streptomyces, cyanobacte ⁇ a. Bacillus subttlis, and Staplnlococcus aureus, fungal cells, such as cells of a yeast.
  • insect cells such as cells of Drosophila S2 and Spodoptera Sf9.
  • animal cells such as CHO. COS. HeLa. C 127, 3T3, BHK, 293, CV- 1 and Bowes melanoma cells, and plant cells, such as cells of a gymnosperm or angiosperm
  • vectors include, among others, chromosomal-, episomal- and virus-de ⁇ ved vectors, for example, vectors de ⁇ ved from bacterial plasmids, from bacteriophage, 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 derived from combinations thereof, such as those derived from plasmid and bacte ⁇ ophage genetic elements, such as cosmids and phagemids
  • the expression system constructs may comprise control regions that regulate as well as engender expression Generally, any system or vector suitable to maintain, propagate or express polynucleotides and or to express a polypeptide in
  • approp ⁇ ate secretion signals may be incorporated into the expressed polypeptide These signals may be endogenous to the polypeptide or they may be heterologous signals
  • Polypeptides of the invention can be recovered and pu ⁇ fied from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography, and lectin chromatography Most preferably, high performance liquid chromatography is employed for pu ⁇ fication Well known techniques for refolding protein may be employed to regenerate active conformation when the polypeptide is denatured du ⁇ ng isolation and or purification Diagnostic, Prognostic, Serotyping and Mutation Assays
  • This invention is also related to the use of ica A B C or D polynucleotides and polypeptides of the invention for use as diagnostic reagents Detection of ica A, B C or D polynucleotides and/or polypeptides in a eukaryote, particularly a mammal and especially a human, will provide a diagnostic method for diagnosis of disease staging of disease or response of an infectious organism to drugs Eukaryotes, particularly mammals, and especially humans particularly those infected or suspected to be infected with an organism comp ⁇ sing the ica A, B, C or D gene or protein, may be detected at the nucleic acid or amino acid level by a va ⁇ ety of well known techniques as well as by methods provided herein Polypeptides and polynucleotides for prognosis, diagnosis or other analysis may be obtained from a putatively infected and/or infected individual s bodily mate ⁇ als Polynucleotides from any of these sources particularly DNA or RNA
  • an array of ohgonucleotides probes comp ⁇ sing ica A, B, C or D 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 va ⁇ ety of questions in molecular genetics including gene expression, genetic linkage, and genetic variability (see, for example, Chee et al , Science, 274 610 ( 1996))
  • the present invention relates to a diagnostic kit that comprises
  • kits (a) a polynucleotide of the present invention, preferably the nucleotide sequence of SEQ ID NO 1 ,3,5,7 or 9, 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 2,4,6,8 or 10 or a fragment thereof, or (d) an antibody to a polypeptide of the present invention, preferably to the polypeptide of SEQ ID NO 2,4,6,8 OR 10
  • kit may comprise a substantial component
  • Such a kit will be of use in diagnosing a disease or susceptibility to a Disease, among others
  • This invention also relates to the use of polynucleotides of the present in ⁇ ention as diagnostic reagents Detection of a mutated form of a polynucleotide of the invention, preferable, SEQ ID NO 1,3,5,7 or 9, that is associated with a disease or pathogenicity will provide a diagnostic tool that can add to, or define, a diagnosis of a disease, a prognosis of a course of disease, a determination of a stage of disease, or a susceptibility to a disease, that results from under-expression, over-expression or altered expression of the polynucleotide
  • Organisms, particularly infectious organisms, carrying mutations in such polynucleotide may be detected at the polynucleotide level by a variety of techniques, such as those described elsewhere herein
  • the differences in a polynucleotide and/or polypeptide sequence between organisms possessing a first phenotype and organisms possessing a different, second different phenotype can also be determined If a mutation is observed in some or all organisms possessing the first phenotype but not in any organisms possessing the second phenotype, then the mutation is likely to be the causative agent of the first phenotype
  • RNA from an organism carrying mutations or polymorphisms (alle c va ⁇ ations) in a polynucleotide and/or polypeptide of the invention may also be detected at the polynucleotide or polypeptide level by a variety of techniques, to allow for serotyping, for example
  • RT-PCR can be used to detect mutations in the RNA It is particularly preferred to use RT-PCR in conjunction with automated detection systems, such as, for example, GeneScan RNA, cDNA or genomic DNA may also be used for the same purpose, PCR
  • PCR pnmers complementary to a polynucleotide encoding ica A, B, C or D polypeptide can be used to identify and analyze mutations
  • the invention further provides these primers with 1 , 2, 3 or 4 nucleotides removed from the 5' and/or the 3' end These p ⁇ mers may be used for.
  • the p ⁇ mers may be used to amplify a polynucleotide isolated from an infected individual, such that the polynucleotide may then be subject to va ⁇ ous techniques for elucidation of the polynucleotide sequence In this way, mutations in the polynucleotide sequence may be detected and used to diagnose and or prognose the infection or its stage or course, or to serotype and/or classify the infectious agent
  • the invention further provides a process for diagnosing, disease, preferably bacterial infections, more preferably infections caused by Staplnlococcus aureus. comprising determining from a sample derived from an individual, such as a bodily material, an increased level of expression of polynucleotide having a sequence of Table 1 [SEQ ID NO.1 ,3.5,7 or 9] Increased or decreased expression of a ica A, B, C or D polynucleotide can be measured using any on of the methods well known in the art for the quantitation of polynucleotides, such as, for example, amplification, PCR, RT-PCR, RNase protection, Northern blotting, spectrometry and other hybridization methods
  • a diagnostic assay in accordance with the invention for detecting over-expression of ica A, B, C or D polypeptide compared to normal control tissue samples may be used to detect the presence of an infection, for example Assay techniques that can be used to determine levels of a ica A, B, C or D polypeptide, in a sample derived from a host, such as a bodily material, are well-known to those of skill in the art Such assay methods include radioimmunoassays, competitive-binding assays, Western Blot analysis, antibody sandwich assays, antibody detection and ELISA assays.
  • Polypeptides and polynucleotides of the invention may also be used to assess the binding of small molecule substrates and gands in, for example, cells, cell-free preparations, chemical libraries, and natural product mixtures
  • substrates and hgands may be natural substrates and hgands or may be structural or functional mimetics See, e g , Coligan et al , Current Protocols in Immunology 1(2) • Chapter 5 ( 1991 )
  • Polypeptides and polynucleotides of the present invention are responsible for many biological functions, including many disease states, in particular the Diseases herein mentioned It is therefore desirable to devise screening methods to identify compounds that agonize (e.g., stimulate) or that antagonize (e g .inhibit) the function of the polypeptide or polynucleotide Accordingly, in a further aspect, the present invention provides for a method of screening compounds to identify those that agonize or that an
  • inhibitors may be employed for therapeutic and prophylactic purposes for such Diseases as herein mentioned
  • Compounds ma> be identified from a va ⁇ ety of sources, for example, cells, cell-free preparations, chemical libraries, and natural product mixtures
  • Such agonists and antagonists so-identified may be natural or modified substrates, hgands, receptors, enzymes, etc .
  • the screening methods may simply measure the binding of a candidate compound to the polypeptide or polynucleotide, or to cells or membranes bearing the polypeptide or polynucleotide, or a fusion protein of the polypeptide by means of a label directly or indirectly associated with the candidate compound Alternatively, the screening method may involve competition with a labeled competitor Further, these screening methods may test whether the candidate compound results in a signal generated by activation or inhibition of the polypeptide or polynucleotide, using detection systems appropriate to the cells comprising the polypeptide or polynucleotide Inhibitors of activation are generally assayed in the presence of a known agonist and the effect on activation by the agonist by the presence of the candidate compound is observed Constitutively active polypeptid
  • 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 using monoclonal and polyclonal antibodies by standard methods known in the art This can be used to discover agents that may inhibit or enhance the production of polypeptide (also called antagonist or agonist, respectiveh ) from suitably manipulated cells or tissues
  • the invention also provides a method of screening compounds to identify those that enhance (agonist) or block (antagonist) the action of ica A, B, C or D polypeptides or polynucleotides, particularly those compounds that are bacte ⁇ static and/or bacte ⁇ cidal
  • the method of screening may involve high-throughput techniques
  • a synthetic reaction mix, a cellular compartment, such as a membrane, cell envelope or cell wall, or a preparation of any thereof, comprising ica A, B, C or D polypeptide and a labeled substrate or hgand of such polypeptide is incubated in the absence or the presence of a candidate molecule that may be a ica A, B, C or D agonist or antagonist
  • the ability of the candidate molecule to agonize or antagonize the ica A, B, C or D polypeptide is reflected in decreased binding of the labeled gand or decreased production of product from such substrate Molecules that bind gratuitously, t
  • Polypeptides of the invention may be used to identify membrane bound or soluble receptors, if any, for such polypeptide, through standard receptor binding techniques known in the art These techniques include, but are not limited to, hgand binding and crosslinkmg assays in which the polypeptide is labeled with a radioactive isotope (for instance, !
  • the fluorescence polarization value for a fluorescently-tagged molecule depends on the rotational correlation time or tumbling rate
  • Protein complexes such as formed by ica A, B, C or D polypeptide associating with another ica A, B, C or D polypeptide or other polypeptide, labeled to comprise a fluorescently-labeled molecule will have higher polarization values than a fluorescently labeled monomeric protein It is preferred that this method be used to characterize small molecules that disrupt polypeptide complexes
  • Fluorescence energy transfer may also be used characterize small molecules that interfere with the formation of ica A, B, C or D polypeptide dimers, trimers, tetramers or higher order structures, or structures formed by ica A, B, C or D polypeptide bound to another polypeptide ica A, B, C or D 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
  • ica A, B, C or D polypeptide self-association can be coupled to a sensor chip at low site density such that covalently bound molecules will be monomeric Solution protein can then passed over the ica A, B, C or D polypeptide -coated surface and specific binding can be detected in real-time by monitoring the change in resonance angle caused by a change in local refractive index
  • This technique can be used to characterize the effect of small molecules on kinetic rates and equilibrium binding constants for ica A, B, C or D polypeptide self-association as well as an association of ica A, B, C or D polypeptide and another polypeptide or small molecule
  • a scintillation proximity assay may be used to characterize the interaction between an association of ica A, B, C or D polypeptide with another ica A, B, C or D polypeptide or a different polypeptide ica A, B, C or D polypeptide can be coupled to a scintillation-filled bead Addition of radio-labeled ica A, B, C or D polypeptide results in binding where the radioactive source molecule is in close proximity to the scintillation fluid Thus, signal is emitted upon ica A, B, C or D polypeptide binding and compounds that prevent ica A, B, C or D polypeptide self-association or an association of ica A, B, C or D polypeptide and another polypeptide or small molecule will diminish signal
  • an assay for ica A, B, C or D agonists is a competitive assay that combines ica A, B, C or D and a potential agonist with ica A, B, C or D-bindmg molecules, recombinant ica A, B, C or D binding molecules, natural substrates or hgands, or substrate or hgand mimetics, under appropriate conditions for a competitive inhibition assay ica A.
  • B, C or D can be labeled, such as by radioactivity or a colo ⁇ met ⁇ c compound, such that the number of ica A, B, C or D molecules bound to a binding molecule or converted to product can be determined accurately to assess the effectiveness of the potential antagonist
  • a polypeptide and/or polynucleotide of the present invention may also be used in a method for the structure-based design of an agonist or antagonist of the polypeptide and/or polynucleotide, by (a) determining in the first 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 antagonist, (c) synthesizing candidate compounds that are predicted to bind to or react with the deduced binding s ⁇ te(s), reactive s ⁇ te(s), and/or mot ⁇ f(s), and (d) testing whether the candidate compounds are indeed agonists or antagonists It will be further appreciated that this will normally be an iterative process, and this iterative process may be performed using automated and computer-controlled steps
  • the present invention 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 ica A, B C or D polypeptide and/or polynucleotide
  • One approach comprises admi ste ⁇ ng to an individual in need thereof an inhibitor compound (antagonist) as herein desc ⁇ bed optionally in combination with a pharmaceutically acceptable carrier, in an amount effective to inhibit the function and/or expression of the polypeptide and/or polynucleotide, such as, for example, by blocking the binding of hgands, substrates, receptors, enzymes, etc , or by inhibiting a second signal, and thereby alleviating the abnormal condition
  • soluble forms of the polypeptides still capable of binding the hgand, substrate, enzymes receptors, etc in competition with endogenous polypeptide and/or polynucleotide may be administered Typical examples of such competitors include fragments of the ica A, B, C or D polypeptide and/or polypeptide
  • D polypeptide can be inhibited using expression blocking techniques This blocking may be targeted against any step in gene expression, but is preferably targeted against transcription and/or translation
  • An examples of a known technique of this sort involve the use of antisense sequences, either internally generated or separately administered (see, for example, O'Connor, J Neurochem (1991 ) 56 560 in Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, FL ( 1988))
  • ohgonucleotides that form triple helices with the gene can be supplied (see, for example, Lee et al Nucleic Acids Res (1979) 6 3073, Cooney et al Science ( 1988) 241 456, Dervan et al Science (1991 ) 251 1360)
  • These ohgomers can be administered per se or the relevant o gomers can be expressed in vivo
  • polynucleotide sequences provided herein may be used in the discovery and development of antibacterial compounds
  • the encoded protein upon expression, can be used as a target for the screening of antibacterial drugs
  • polynucleotide sequences encoding the amino terminal regions of the encoded protein or Shine-Delgarno or other translation facilitating sequences of the respective mRNA can be used to construct antisense sequences to control the expression of the coding sequence of interest
  • the invention also provides the use of the polypeptide, polynucleotide, agonist or antagonist of the invention to interfere with the initial physical interaction between a pathogen or pathogens and a eukaryotic, preferably mammalian, host responsible for sequelae of infection
  • the molecules of the invention may be used in the prevention of adhesion of bacteria in particular gram positive and/or gram negative bacteria, to eukary otic, preferably mammalian, extracellular matrix proteins on in-dwelling devices or to extracellular matrix proteins in wounds, to block bacterial adhesion between eukary otic, preferably mammalian, extracellular matrix proteins and bacterial ica A, B, C or D 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
  • ica A, B C or D agonists and antagonists preferably bacte ⁇ static or bactericidal agonists and antagonists
  • the antagonists and agonists of the invention may be employed, for instance, to prevent, inhibit and/or treat diseases
  • H pylori' Helicobacter pylon bacteria infect the stomachs of over one-third of the world's population causing stomach cancer, ulcers, and gastritis (International Agenc for Research on Cancer (1994) Schisto omes, Liver Flukes and Helicobacter Pylon
  • H pylori a cause-and-effect relationship between H pylori and gastric adenocarcinoma, classifying the bacterium as a Group I (definite) carcinogen
  • Preferred antimicrobial compounds of the invention agonists and antagonists of ica A, B, C or D 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 pv/o ⁇ -induced cancers, such as gastrointestinal carcinoma
  • Such treatment should also prevent, inhibit and/or cure gastric ulcers and gastritis
  • Bodily mate ⁇ al(s) means any mate ⁇ al de ⁇ ved 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, u ⁇ ne, stool or autopsy mate ⁇ als
  • D ⁇ sease(s) means any disease caused by or related to infection by a bacte ⁇ a, including , for example, disease, such as, infections of the upper respiratory tract (e g , otitis media, bacterial tracheitis, acute epiglottitis, thyroiditis), lower respiratory (e g , empyema.
  • upper respiratory tract e g , otitis media, bacterial tracheitis, acute epiglottitis, thyroiditis
  • lower respiratory e g , empyema.
  • lung abscess cardiac (e g , infective endocarditis), gastrointestinal (e g secretory diarrhoea, splenic absces, retrope ⁇ toneal abscess), CNS (e g , cerebral abscess), eye (e g blepha ⁇ tis, conjunctivitis, keratitis, endophthalmitis, preseptal and orbital celluhtis darcryocystitis), kidney and u ⁇ nary tract (e g , epididymitis, intrarenal and pe ⁇ neph ⁇ c absces toxic shock syndrome), skin (e g , impetigo, folhcuhtis, cutaneous abscesses, celluhtis, wound infection, bacte ⁇ al myositis) bone and joint (e g , septic arth ⁇ tis, osteomyelitis)
  • cardiac e g , infective endocarditis
  • gastrointestinal
  • “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 compa ⁇ ng the sequences
  • identity also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences
  • Identity' can be readily calculated by known methods, including but not limited to those described in (Computational Molecular Biology, Lesk, A M , ed , Oxford University Press, New York, 1988, Biocomputing Informatics and Genome Projects, Smith, D W , ed , Academic Press, New York, 1993, Computer Analysis of Sequence Data, Part I, G ⁇ ffm,
  • 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 ,3,5,7 or 9, wherein said polynucleotide sequence may be identical to the reference sequence of SEQ ID NO 1 ,3,5,7 or 9 or may include up to a certain integer number of nucleotide alterations as compared to the reference sequence, wherein said alterations are selected from the group consisting of at least one nucleotide deletion, substitution, including transition and transversion, or insertion, and wherein said alterations may occur at the 5 ' or 3' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among the nucleotides in the reference sequence or in one or more contiguous groups within the reference
  • 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 2,4,6,8 or 10, wherein said polypeptide sequence may be identical to the reference sequence
  • n a is the number of amino acid alterations
  • x a is the total number of amino acids in SEQ ID NO 2,4,6,8 or 10
  • y is 0 95 for 957c, 0 97 for 977c or 1 00 for 1007c
  • • is the symbol for the multiplication operator, and wherein any non-integer product of x a and y is rounded down to the nearest integer prior to subtracting it from x a
  • “Ind ⁇ v ⁇ dual(s)” means a multicellular eukaryote. including, but not limited to a metazoan, a mammal, an ovid, a bovid, a simian, a primate, and a human "Isolated” means altered “by the hand of man” from its natural state, i e , if it occurs in nature, it has been changed or removed from its onginal environment, or both
  • a polynucleotide or a polypeptide naturally present in a living organism is not “isolated/' but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is "isolated", as the term is employed herein
  • a polynucleotide or polypeptide that is introduced into an organism by transformation, genetic manipulation or by any other recombinant method is "isolated” even if it is still present in said organism, which organism may be living or non-living
  • Organ ⁇ sm(s) means a (I) prokaryote, including but not limited to, a member of the genus Streptococcus, Staphylococcus. Bordetella. Mycobacterium, Neisseria, Haemoph ⁇ us, Actinomvcetes, Streptomycetes, Nocardia, Enterobacter, Yersima, Fanctsella, Pasturella, Moraxella, Acinetobacter, Erys ⁇ elothrix, Branhamella, Actinobacillus, Streptobacillus, Listeria, Calvmmatobactertum, Brucella, Bacillus. Clostndium, Treponema, Escherichia, Salmonella.
  • Streptococcus faecahs Streptococcus faecahs. Streptococcus faecium, Streptococcus durans, Neisseria gonorrheae, Neisseria memngitidis, Staphylococcus aureus, Staphylococcus epidermidis, Corynebacterium diptheriae, Gardnerella vaginahs, Mycobacterium tuberculosis, Mycobacterium bovts, Mycobacterium ulcerans, Mycobacterium leprae.
  • Actmomyctes tsraelii Listeria monocytogenes, Bordetella pertusis, Bordatella parapertusis Bordetella bronchisepttca, Escherichia coh, Shigella dysenteriae.
  • Haemophdus tnfluenzae Haemoph ⁇ us aegyptius, Haemophilus parainfluenzae, Haemophdus ducre ⁇ , Bordetella, Salmonella tvphi, Citrobacter freundii, Proteus mirabilis, Proteus vulgans, Yersima pestis, Kleibsiella pneumoniae, Serratta marcessens, Serratia hquefaciens, Vibrio cholera. Shigella dvsenteru, Shigella flexnen.
  • an archaeon including but not limited to Archaebacter
  • a unicellular or filamentous eukaryote including but not limited to, a protozoan, a fungus, a member of the genus Saccharomvces, Kluveromyces, or Candida, and a member of the species Saccharonnces cenviseae, Kluveromyces lactis, or Candida albicans
  • Polynucleot ⁇ de(s) generallv refers to an poly ⁇ bonucleotide or polydeoxy ⁇ bonucleotide, that may be unmodified RNA or DNA or modified RNA or DNA
  • Polynucleot ⁇ de(s) include, without limitation, single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions or single-, double- and triple-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybnd molecules comp ⁇ sing DNA and RNA that ma be single-stranded or, more typically, double-stranded, or tnple-stranded regions, or a mixture of single- and double- stranded regions
  • polynucleotide as used herein refers to triple-stranded regions comp ⁇ sing RNA or DNA or both RNA and DNA The strands in such regions may be from the same
  • the regions may include all of one or more of the molecules, but more typically involve only a region of some of the molecules.
  • One of the molecules of a t ⁇ ple-hehcal region often is an ohgonucleotide
  • the term "polynucleot ⁇ de(s)" also includes DNAs or RNAs as described above that comp ⁇ se one or more modified bases.
  • DNAs or RNAs with backbones modified for stability or for other reasons are "polynucleot ⁇ de(s)" as that term is intended herein Moreover, DNAs or RNAs compnsmg unusual bases, such as inosine, or modified bases, such as t ⁇ tylated bases, to name just two examples, are polynucleotides as the term is used herein It will be appreciated that a great va ⁇ ety of modifications have been made to DNA and RNA that serve many useful purposes known to those of skill in the art
  • the term "polynucleot ⁇ de(s)” as it is employed herein embraces such chemically, enzymatically or metabohcally modified forms of polynucleotides, as well as the chemical forms of DNA and RNA charactenstic of viruses and cells, including, for example, simple and complex cells "Polynucleot ⁇ de(s)” also embraces short polynucleotides often referred to as ol ⁇ gonu
  • Polypept ⁇ de(s) refers to any peptide or protein comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds
  • Polypept ⁇ de(s) refers to both short chains, commonly referred to as peptides, ohgopeptides and ohgomers and to longer chains generally referred to as proteins
  • Polypeptides may comp ⁇ se amino acids other than the 20 gene encoded am o acids
  • Polypept ⁇ de(s)” include those modified either by natural processes, such as processing and other post-translational modifications, but also by chemical modification techniques Such modifications are well descnbed in basic texts and in more detailed monographs, as well as in a voluminous research literature, and they are well known to those of skill in the art It will be appreciated that the same type of modification may be present in the same or varying degree at several sites in a given polypeptide Also, a given polypeptide may comprise many types of modifications Modifications can occur anywhere in a polypeptide, including the a
  • Recombinant expression system(s) refers to expression systems or portions thereof or polynucleotides of the invention introduced or transformed into a host cell or host cell lysate for the production of the polynucleotides and polypeptides of the invention
  • Va ⁇ ant(s) is a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide respectively, but retains essential properties
  • a typical vanant 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 amino acid sequence of a polypeptide encoded by the reference polynucleotide Nucleotide changes may result in amino acid substitutions, additions, deletions, fusion proteins and truncations in the polypeptide encoded by the reference sequence, as discussed below.
  • a typical variant of a polypeptide differs in amino acid sequence from another, reference polypeptide Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, in many regions, identical
  • a variant and reference polypeptide may differ in amino acid sequence by one or more substitutions, additions, deletions in any combination
  • a substituted or inserted amino acid residue may or may not be one encoded by the genetic code
  • the present invention also includes include vanants of each of the polypeptides of the invention, that is polypeptides that vary from the referents by conservative amino acid substitutions, whereby a residue is substituted by another with like characte ⁇ stics Typical such substitutions are among Ala, Val, Leu and He, among Ser and Thr, among the acidic residues Asp and Glu.
  • variants among Asn and Gin; and among the basic residues Lys and Arg, or aromatic residues Phe and Tyr Particularly preferred are variants in which several, 5-10, 1-5, 1-3, 1-2 or 1 amino acids are substituted, deleted, or added in any combination.
  • 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 naturally
  • Non-naturally occurring va ⁇ ants of polynucleotides and polypeptides may be made by mutagenesis techniques, by direct synthesis, and by other recombinant methods known to skilled artisans.
  • the polynucleotide having a DNA sequence given in Table 1 [SEQ ID NO.1,3,5.7 and 9] was obtained from a library of clones of chromosomal DNA of Staphylococcus aureus in E. coli
  • 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: 1,3,5,7 and 9.
  • Libraries may be prepared by routine methods, for example Methods 1 and 2 below Total cellular DNA is isolated from Staphylococcus aureus WCUH 29 according to standard procedures and size-fractionated by either of two methods Method 1
  • Total cellular DNA is mechanically sheared by passage through a needle in order to size-fractionate according to standard procedures.
  • DNA fragments of up to 1 lkbp in 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 coli infected with the packaged library The library is amplified by standard procedures Method 2
  • Total cellular DNA is partially hydrolyzed with a one or a combination of restriction enzymes appropriate to generate a series of fragments for cloning into library vectors (e g , Rsal, Pall, Alul, Bshl235I), and such fragments are size-fractionated according to standard procedures EcoRI linkers are ligated to the DNA and the fragments then ligated into the vector Lambda ZapII that have been cut with EcoRI, the library packaged by standard procedures, and E coli infected with the packaged library The library is amplified by standard procedures

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Abstract

L'invention concerne des polypeptides ica A, B, C ou D et des polynucléotides codant les polypeptides ica A, B, C ou D ainsi que des procédés de fabrication de ces polypeptides par des techniques recombinantes. Elle concerne aussi des procédés pour utiliser les polypeptides ica A, B, C ou D dans la recherche de composés antibactériens.
PCT/US2000/012253 1999-05-04 2000-05-04 Operon ica staphylococcique Ceased WO2000071568A1 (fr)

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Publication number Priority date Publication date Assignee Title
US8729013B2 (en) 2004-08-26 2014-05-20 The University Of Western Ontario Methods of inhibiting staphylobactin-mediated iron uptake in S. aureus

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

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
CRAMTON ET AL.: "The intercellular adhesion (ica) locus is present in Staphylocopccus aureus is required for biofilm formation", INFECT. IMMUN.,, vol. 67, no. 10, October 1999 (1999-10-01), pages 5427 - 5433, XP002931379 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8729013B2 (en) 2004-08-26 2014-05-20 The University Of Western Ontario Methods of inhibiting staphylobactin-mediated iron uptake in S. aureus

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