WO1999065530A1 - NrdD - Google Patents

NrdD Download PDF

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Publication number
WO1999065530A1
WO1999065530A1 PCT/US1999/012975 US9912975W WO9965530A1 WO 1999065530 A1 WO1999065530 A1 WO 1999065530A1 US 9912975 W US9912975 W US 9912975W WO 9965530 A1 WO9965530 A1 WO 9965530A1
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Prior art keywords
polypeptide
sequence
polynucleotide
seq
identity
Prior art date
Application number
PCT/US1999/012975
Other languages
French (fr)
Inventor
Edwina I. Wilding
Michael T. Black
Christopher M. Traini
Original Assignee
Smithkline Beecham Corporation
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Publication date
Application filed by Smithkline Beecham Corporation filed Critical Smithkline Beecham Corporation
Priority to EP99957023A priority Critical patent/EP1087792A1/en
Priority to JP2000554407A priority patent/JP2002517993A/en
Publication of WO1999065530A1 publication Critical patent/WO1999065530A1/en

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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
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0093—Oxidoreductases (1.) acting on CH or CH2 groups (1.17)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y117/00—Oxidoreductases acting on CH or CH2 groups (1.17)
    • C12Y117/04—Oxidoreductases acting on CH or CH2 groups (1.17) with a disulfide as acceptor (1.17.3)
    • C12Y117/04002—Ribonucleoside-triphosphate reductase (1.17.4.2)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies

Definitions

  • This invention relates to newly identified polynucleotides and polypeptides, and their production and uses, as well as their variants, agonists and antagonists, and their uses
  • the invention relates to polynucleotides and polypeptides of the nrdD (anaerobic ⁇ bonucleotide t ⁇ phosphate reductase) family, as well as their variants, hereinafter referred to as "nrdD,” “nrdD polynucleot ⁇ de(s),” and “nrdD polypept ⁇ de(s)" as the case may be
  • Staphylococcal genes and gene products are particularly preferred to employ Staphylococcal genes and gene products as targets for the development of antibiotics
  • the Staphylococci make up a medically important genera of microbes They are known to produce two types of disease, invasive and toxigenic Invasive infections are 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 exotoxins as opposed to tissue invasion and bacteremia These conditions include Staphylococcal food poisoning, scalded skin syndrome and toxic shock
  • Staphylococcus aureus infections has ⁇ sen 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 which 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
  • nrdD embodiments of the invention that have a present benefit of, among other things, bemg useful to screen compounds for antibiotic activity
  • Such factors are also useful to determine their role in pathogenesis of infection, dysfunction and disease
  • identification and characte ⁇ zation of such factors and their antagonists and agonists to find ways to prevent, ameliorate or correct such infection, dysfunction and disease
  • polypeptides of the invention possess significant amino acid sequence homology to a known nrdD protein
  • the present invention relates to nrdD, m particular nrdD polypeptides and nrdD polynucleotides. recombinant materials and methods for their production
  • the invention relates to methods for using such polypeptides and polynucleotides, including the 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 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 nrdD expression or activity
  • the invention relates to nrdD polypeptides and polynucleotides as desc ⁇ bed in greater detail below
  • the invention relates to polypeptides and polynucleotides of a nrdD of Staphylococcus aureus. which is related by arruno acid sequence homology to nrdD polypeptide
  • the invention relates especially to nrdD having the nucleotide and amino acid sequences set out in Table 1 as SEQ ID NO 1 and SEQ ID NO 2 respectively
  • a deposit containing a Staphylococcus aureus WCUH 29 strain has been deposited with the National Collections of Industrial and Marine Bactena Ltd (herein "NCIMB"), 23 St Machar Dnve, Aberdeen AB2 1RY, Scotland on 11 September 1995 and assigned NCIMB Deposit No 40771, and referred to as Staphylococcus aureus WCUH29 on deposit
  • NCIMB National Collections of Industrial and Marine Bactena Ltd
  • the deposited strain contains the full length nrdD gene
  • the sequence of the polynucleotides contained in the deposited strain, as well as the amino acid sequence of any polypeptide encoded thereby, are controlling in the event of am conflict with any description of sequences herein
  • the deposit of the deposited strain has been made under the terms of the Budapest Treaty on the
  • the strain 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 convenience to those of skill in the art and is not an admission that a deposit is required for enablement, such as that required under 35 U S C ⁇ 112
  • a license may be required to make, use or sell the deposited strain, and compounds de ⁇ ved 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 contained in the deposited strain
  • nrdD polynucleotide sequences in the deposited strain such as DNA and RNA.
  • amino acid sequences encoded thereby Also provided by the invention are nrdD polypeptide and polynucleotide sequences isolated from the deposited stram Polypeptides
  • NrdD polypeptide of the invention is substantially phylogenetically related to other proteins of the nrdD (anaerobic ⁇ bonucleotide t ⁇ phosphate reductase) family
  • nrdD Staphylococcus aureus referred to herein as "nrdD” and “nrdD polypeptides” as well as biologically, diagnostically, prophylactically, clinically or therapeutically useful variants thereof, and compositions comp ⁇ smg the same
  • the present mvention further provides for an isolated polypeptide which (a) comprises or consists of an ammo acid sequence which has at least 70% identit , preferably at least 80% identity, more preferably at least 90% identity, yet more preferably at least 95 % identity, most preferably at least 97-99% or exact identity, to that of SEQ ID NO 2 over the entire length of SEQ ID NO 2,
  • polypeptide encoded by an isolated polynucleotide comprising or consistmg of a polynucleotide sequence which has at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, yet more preferably at least 95% identity, even more preferably at least 97-99% or exact identity to SEQ ID NO 1 over the entire length of SEQ ID NO 1,
  • polypeptide encoded by an isolated polynucleotide comprising or consisting of a polynucleotide sequence encoding a polypeptide which has at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, yet more preferably at least 95% identity, even more preferably at least 97-99% or exact identity, to the amino acid sequence of SEQ ID NO 2, over the entire length of SEQ ID NO 2
  • polypeptides of the mvention include a polypeptide of Table 1 [SEQ ID NO 2] (in particular the mature polypeptide) as well as polypeptides and fragments, particularly those which have the biological activity of nrdD. and also those which have at least 70% identity to a polypeptide of Table 1 [SEQ ID NO l]or the relevant portion, preferably at least 80% identity to a polypeptide of Table 1 [SEQ ID NO 2and more preferably at least 90% identity to a polypeptide of Table 1 [SEQ ID NO 2] and still more preferably at least 95% identity to a polypeptide of Table 1 [SEQ ID NO 2] and also include portions of such polypeptides with such portion of the polypeptide generally containing at least 30 ammo acids and more preferably at least 50 ammo acids
  • the mvention also mcludes a polypeptide consistmg of or comp ⁇ smg a polypeptide of the formula
  • X is hydrogen, a metal or any other moiety described herem for modified polypeptides. and at the carboxyl terminus, 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 mteger between 1 and 1000 or zero, n is an integer between 1 and 1000 or zero, and R 2 is an amino acid sequence of the invention, particularly an amino acid sequence selected from Table 1 or modified forms thereof.
  • R 2 is oriented so that its amino terminal amino acid residue is at the left, covalently bound to R ] and its carboxy terminal amino acid residue is at the right, covalently bound to R3.
  • Any stretch of arnino acid residues denoted by either Ri or R3, where m and or n is greater than 1, may be either a heteropolymer or a homopolymer, preferably a heteropolymer.
  • Other preferred embodiments of the invention are provided where m is an integer between 1 and 50, 100 or 500, and n is an integer between 1 and 50, 100, or 500.
  • a polypeptide of the invention is derived from Staphylococcus aureus, however, it may preferably be obtained from other organisms of the same taxonomic genus.
  • a polypeptide of the invention may also be obtained, for example, from organisms of the same taxonomic family or order.
  • a fragment is a variant polypeptide having an arnino acid sequence that is entirely the same as part but not all of any amino acid sequence of any polypeptide of the invention.
  • fragments may be "free-standing," or comprised within a larger polypeptide of which they form a part or region, most preferably as a single continuous region in a single larger polypeptide.
  • Preferred fragments include, for example, truncation polypeptides having a portion of an amino acid sequence of Table 1 [SEQ ID NO:2], or of variants 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 characterized by structural or functional attributes such as fragments that comprise alpha-helix and alpha-helix forming regions, beta-sheet and beta-sheet-fo ⁇ riing regions, turn and turn-forming regions, coil and coil-forming regions, hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-foiming regions, substrate binding region, and high antigenic index regions.
  • fragments include an isolated polypeptide comprising an amino acid sequence having at least 15, 20, 30, 40, 50 or 100 contiguous amino acids from the amino acid sequence of SEQ ID NO: 2, or an isolated polypeptide comprising an amino acid sequence having at least 15, 20, 30, 40, 50 or 100 contiguous amino acids truncated or deleted from the amino acid sequence of SEQ ID NO: 2.
  • biologically active fragments which are those fragments that mediate activities of nrdD, including those with a similar activity or an improved activity, or with a decreased undesirable activity. Also included are those fragments that are antigenic or immunogenic in an animal, especially in a human. Particularly preferred are fragments comprising receptors or domains of enzymes that confer a function essential for viability of Staphylococcus aureus or the ability to initiate, or maintain cause Disease in an individual, particularly a human. Fragments of the polypeptides of the mvention may be employed for producing the corresponding full- length polypeptide by peptide synthesis, therefore, these variants may be employed as intermediates for producmg the full-length polypeptides of the mvention
  • X or "Xaa” may also be used in describing certain polypeptides of the invention "X” and “Xaa” mean that any of the twenty naturally occurring amino acids may appear at such a designated position in the polypeptide sequence
  • nrdD polypeptides
  • the polynucleotide comprises a region encoding nrdD polypeptides comp ⁇ smg a sequence set out m Table 1 [SEQ ID NO 1] which 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 which possesses it, such as Staphylococcus aureus
  • isolated nucleic acid molecules encoding and or expressmg nrdD polypeptides and polynucleotides, particularly Staphylococcus aureus nrdD polypeptides and polynucleotides, including, for example, unprocessed RNAs, ⁇ bozyme RNAs.
  • mRNAs, cDNAs, genomic DNAs, B- and Z-DNAs Further embodiments of the mvention mclude biologically, diagnostically, prophylactically, clinically or therapeutically useful polynucleotides and polypeptides, and va ⁇ ants thereof, and compositions comp ⁇ smg the same
  • Another aspect of the mvention relates to isolated polynucleotides. including at least one full length gene, that encodes a nrdD polypeptide having a deduced ammo acid sequence of Table 1 [SEQ ID NO 2] and polynucleotides closely related thereto and va ⁇ ants thereof
  • nrdD polypeptide from Staphylococcus aureus comprising or consisting of an amino acid sequence of Table 1 [SEQ ID NO 2 or a variant thereof
  • a polynucleotide of the mvention encoding nrdD polypeptide may be obtained usmg standard cloning and screening methods, such as those for cloning and sequencmg chromosomal DNA fragments from bacte ⁇ a usmg Staphylococcus aureus WCUH 29 cells as startmg mate ⁇ al, followed by obtairung a full length clone
  • a polynucleotide sequence of the invention such as a polynucleotide sequence given in Table 1 [SEQ ID NO 1].
  • 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, preferably a 17-mer or longer, derived from a partial sequence
  • Clones carrying DNA identical to that of the probe can then be distinguished usmg stringent hybridization conditions
  • sequencmg primers designed from the original polypeptide or polynucleotide sequence it is then possible to extend the polynucleotide sequence m both directions to determine a full length gene sequence Conveniently, such sequencing is performed, for example, using denatured double stranded DNA prepared from a plasmid clone Suitable techmques are described by Maniatis, T .
  • each DNA sequence set out m Table 1 [SEQ ID NO 1] contains an open reading frame encoding a protein having 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 in the art
  • the present mvention provides for an isolated polynucleotide comp ⁇ smg or consistmg of
  • a pohnucleotide encoding a polypeptide of the present mvention, mcludmg homologs and orthologs from species other than Staphylococcus aureus may be obtained by a process which comp ⁇ ses the steps of screening an approp ⁇ ate library under stringent hyb ⁇ dization conditions with a labeled or detectable probe consistmg of or comp ⁇ smg the sequence of SEQ ID NO 1 or a fragment thereof, and isolating a full-length gene and/or genomic clones containing said polynucleotide sequence
  • the mvention provides a polynucleotide sequence identical over its entire length to a coding sequence
  • polynucleotide sequence may also contain at least one non-codmg sequence, mcludmg for example, but not limited to at least one non-codmg 5' and 3' sequence, such as the transc ⁇ bed but non-translated sequences, termination signals (such as rho-dependent and rho-independent termination signals), ⁇ bosome binding sites, Kozak sequences, sequences that stabilize mRNA, rntrons, and polyadenylation signals
  • the polynucleotide sequence may also comp ⁇ se additional coding sequence encoding additional
  • a prefe ⁇ ed embodiment of the mvention is a polynucleotide of consistmg of or comp ⁇ smg nucleotide 1 to the nucleotide immediately upstream of or mcludmg nucleotide 1849 set forth m SEQ ID NO 1 of Table 1, both of which encode the nrdD polypeptide
  • the mvention also mcludes a polynucleotide consisting of or comp ⁇ smg a polynucleotide of the formula
  • n is an integer between 1 and 3000 or zero
  • R 2 is a nucleic acid sequence or modified nucleic acid sequence of the invention, particularly a nucleic acid sequence selected from Table 1 or a modified nucleic acid sequence thereof
  • R is oriented so that its 5' end nucleic acid residue is at the left, bound to R ⁇ and its 3' end nucleic acid residue is at the ⁇ ght, bound to R3
  • Any stretch of nucleic acid residues denoted by either R ⁇ and/or R 2 , where m and or n is greater than 1 may be either a heteropolymer or a homopolymer, preferably a heteropolymer
  • X and Y together define a covalent bond
  • the polynucleotide of the above formula is a closed, circular polynucleotide, which can be a double-stranded polynucleotide wherein the formula shows a first strand to which the second
  • polynucleotide of the mvention is de ⁇ ved from Staphylococcus aureus, however, it may preferably be obtained from other organisms of the same taxonomic genus A polynucleotide of the mvention may also be obtained, for example, from organisms of the same taxonomic family or order
  • polynucleotide encoding a polypeptide encompasses polynucleotides that include a sequence encoding a polypeptide of the mvention, particularly a bactenal polypeptide and more particularly a polypeptide of the Staphylococcus aureus nrdD having an ammo acid sequence set out m Table 1
  • polynucleotides that m include a smgle contmuous region or discontinuous regions encoding the polypeptide (for example, polynucleotides interrupted by integrated phage, an integrated insertion sequence, an integrated vector sequence, an mtegrated transposon sequence, or due to
  • RNA editing or genomic DNA reorganization together with additional regions, that also may contain coding and or non-codmg sequences
  • the mvention further relates to va ⁇ ants of the polynucleotides descnbed herem that encode va ⁇ ants of a polypeptide havmg a deduced ammo acid sequence of Table 1 [SEQ ID NO 2] Fragments of a polynucleotides of the mvention may be used, for example, to synthesize full-length polynucleotides of the mvention
  • prefe ⁇ ed embodiments are polynucleotides encoding nrdD va ⁇ ants, that have the ammo acid sequence of nrdD 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, in any combination
  • polynucleotides that are at least 70% identical over their entire length to a polynucleotide encoding nrdD polypeptide havmg an ammo acid sequence set out in Table 1 [SEQ ID NO 2], and polynucleotides that are complementary to such polynucleotides
  • polynucleotides that comp ⁇ se a region that is at least 80% identical over its entire length to a polynucleotide encoding nrdD polypeptide and polynucleotides complementary thereto
  • polynucleotides at least 90% identical over their entire length to the same are particularly prefe ⁇ ed, and among these particularly prefe ⁇ ed polynucleotides, those with at least 95% are especially preferred
  • those with at least 97% are highly prefe ⁇ ed among those with at least 95%, and among these those with at least 98% and at least 99% are particularly highly preferred
  • Prefe ⁇ ed embodiments are polynucleotides encoding polypeptides that retain substantially the same biological function or activity as the mature polypeptide encoded by a DNA of Table 1 [SEQ ID NO 1]
  • polynucleotides that hybndize, particularly under strmgent conditions, to nrdD 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 desc ⁇ bed herem
  • strmgent conditions and “stringent hybndization conditions” mean hybndrzation occumng only if there is at least 95% and preferably at least 97% identity between the sequences
  • strmgent hybridization conditions is overnight incubation at 42°C in a solution comprising 50% formamide, 5x SSC (150mM NaCl, 15mM t ⁇ sodium citrate), 50 mM sodium phosphate (pH7 6).
  • the invention also provides a polynucleotide consistmg of or comprising a polynucleotide sequence obtained by screening an appropriate library containing the complete gene for a polynucleotide sequence set forth in SEQ ID NO 1 under stringent hybridization conditions with a probe havmg the sequence of said polynucleotide sequence set forth in SEQ ID NO 1 or a fragment thereof, and isolating said polynucleotide sequence Fra
  • the polynucleotides of the mvention may be used as a hybndization probe for RNA, cDNA and genomic DNA to isolate full-length cDNAs and genomic clones encoding nrdD and to isolate cDNA and genomic clones of other genes that have a high identity, particularly high sequence identity, to the nrdD gene
  • Such probes generally will comp ⁇ se at least 15 nucleotide residues or base pairs
  • such probes will have at least 30 nucleotide residues or base pairs and may have at least 50 nucleotide residues or base pairs
  • Particularly prefe ⁇ ed 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 nrdD gene may be isolated by screening usmg a DNA sequence provided m Table 1 [SEQ ID NO 1] to synthesize an oligonucleotide probe
  • a labeled oligonucleotide havmg a sequence complementary to that of a gene of the mvention is then used to screen a library of cDNA, genomic DNA or rnRNA to determine which members of the library the probe hybndizes to
  • polynucleotides and polypeptides of the mvention may be employed, for example, as research reagents and mate ⁇ als for discovery of treatments of and diagnostics for diseases, particularly human diseases, as further discussed herem relating to polynucleotide assays
  • [SEQ ID NOS 1 or 2] may be used m the processes herein as described, but preferably for PCR, to determine whether or not the polynucleotides identified herein m whole or m part are transc ⁇ bed in bacteria in infected tissue It is recognized that such sequences will also have utility m diagnosis of the stage of infection and type of infection the pathogen has attained
  • the mvention also provides polynucleotides that encode a polypeptide that is the mature protem plus additional ammo or carboxyl-teiminal ammo acids, or ammo acids mtenor to the mature polypeptide (when the mature form has more than one polypeptide chain, for instance)
  • Such sequences may play a role in processmg of a protem from precursor to a mature form, may allow protem transport, may lengthen or shorten protem half- life or may facilitate manipulation of a protem for assay or production, among other things
  • the additional am o acids may be processed away from the mature protem
  • a precursor protem, havmg a mature form of the polypeptide fused to one or more prosequences may be an mactive form of the polypeptide When prosequences are removed such mactive precursors generally are activated Some or all of the prosequences may be removed before activation Generally, such precursors are called proproteins
  • N means that any of the four DNA or RNA nucleotides may appear at such a designated position m the DNA or RNA sequence, except it is preferred that N is not a nucleic acid that when taken m combination with adjacent nucleotide positions, when read m the correct reading frame, would have the effect of generating a premature termination codon in such reading frame
  • a polynucleotide of the mvention may encode a mature protem, a mature protem plus a leader sequence (which mav be refe ⁇ ed to as a preprotem). a precursor of a mature protem havmg one or more prosequences that are not the leader sequences of a preprotem, or a preproprotem.
  • the mvention also relates to vectors that comp ⁇ se a polynucleotide or polynucleotides of the mvention, host cells that are genetically engineered with vectors of the mvention and the production of polypeptides of the mvention by recombinant techniques Cell-free translation systems can also be employed to produce such proteins usmg RNAs de ⁇ ved from the DNA constructs of the mvention
  • Recombinant polypeptides of the present mvention may be prepared by processes well known m those skilled m the art from genetically engmeered host cells comp ⁇ smg expression systems Accordingly, in a further aspect, the present mvention relates to expression systems which compnse a polynucleotide or polynucleotides of the present mvention, to host cells which are genetically engmeered with such expression systems, and to the production of polypeptides of the mvention by recombinant techmques
  • 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 m many standard laboratory manuals, such as Davis, et al , BASIC METHODS IN MOLECULAR BIOLOGY, (1986) and Sambrook, et al , MOLECULAR CLONING A LABORATORY MANUAL, 2nd Ed , Cold Spring Harbor Laboratory Press Cold Spring Harbor, N Y (1989).
  • calcium phosphate transfection DEAE-dextran mediated transfection.
  • bacte ⁇ al cells such as cells of streptococci, staphylococci. enterococci E coh, streptomyces. cyanobactena. Bacillus subtths, and Staphylococcus aureus fungal cells, such as cells of a yeast, Kluveromyces, Saccharomyces, a basidiomycete, Candida albicans and Aspergillus.
  • insect cells such as cells of Drosoph ⁇ a S2 and Spodoptera Sf9
  • animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, 293, CV-1 and Bowes melanoma cells
  • plant cells such as cells of a gvmnosperm or angiosperm
  • vectors include, among others, chromosomal-, episomal- and virus-denved vectors, for example, vectors denved from bacte ⁇ al plasmids, from bacte ⁇ ophage. from transposons, from yeast episomes.
  • the expression system constructs may contain control regions that regulate as well as engender expression Generally, any system or vector suitable to maintain, propagate or express polynucleotides and/or to express a polypeptide m a host may be used for expression m this regard
  • the approp ⁇ ate DNA sequence may be inserted mto the expression system by any of a va ⁇ ety of well-known and routme techmques.
  • Polypeptides of the mvention can be recovered and purified from recombinant cell cultures by well-known methods mcludmg ammonium sulfate or ethanol precipitation, acid extraction, amon or cation exchange chromatography, phosphocellulose chromatography, hydrophobic mteraction chromatography, affinity chromatography, hydroxylapatite chromatography, and lectm chromatography Most preferably, high performance liquid chromatography is employed for purification
  • Well known techmques for refolding protem may be employed to regenerate active conformation when the polypeptide is denatured during isolation and or purification
  • This mvention is also related to the use of nrdD polynucleotides and polypeptides of the mvention for use as diagnostic reagents Detection of nrdD polynucleotides and or polypeptides m a eukaryote, particularly a mammal, and especially a human, will provide a diagnostic method for diagnosis of disease, staging of disease or response of an infectious orgamsm to drugs Eukaryotes, particularly mammals, and especially humans, particularly those infected or suspected to be infected with an orgamsm comp ⁇ smg the nrdD gene or protem may be detected at the nucleic acid or ammo acid level by a va ⁇ ety of well known techniques as well as by methods provided herem
  • Polypeptides and polynucleotides for prognosis, diagnosis or other analysis may be obtained from a putatively infected and/or infected mdividual's bodily mate ⁇ als
  • Polynucleotides from any of these sources, particularly DNA or RNA may be used directly for detection or may be amplified enzymatically by usmg PCR or any other amplification technique p ⁇ or to analysis RNA, particularly mRNA, cDNA and genomic DNA may also be used m the same ways Usmg amplification, characte ⁇ zation of the species and stram of infectious or resident orgamsm present m an individual, may be made by an analysis of the genotype of a selected polynucleotide of the orgamsm Deletions and insertions can be detected by a change m size of the amphfied product m compa ⁇ son to a genotype of a reference sequence selected from a related orgamsm, preferably a different species of the
  • 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 Natl Acad Set , USA, 85 4397-4401 (1985)
  • an a ⁇ ay of oligonucleotides probes compnsmg nrdD nucleotide sequence or fragments thereof can be constructed to conduct efficient screening of, for example, genetic mutations, serotype, taxonomic classification or identification
  • Anay technology methods are well known and have general applicability and can be used to address a vanety of questions m molecular genetics mcludmg gene expression, genetic linkage, and genetic va ⁇ abihty (see, for example. Chee et al , Science, 274 610 (1996))
  • the present mvention relates to a diagnostic kit which compnses
  • polypeptide of the present invention preferably the polypeptide of SEQ ID NO 2 or a fragment thereof, or
  • kits an antibod ⁇ to a polypeptide of the present invention, preferably to the polypeptide of SEQ ID NO 2
  • a kit will be of use in diagnosmg a disease or susceptibility to a Disease, among others
  • This mvention also relates to the use of polynucleotides of the present mvention as diagnostic reagents Detection of a mutated form of a polynucleotide of the mvention, preferable, SEQ ID NO 1, which is associated with a disease or pathogenicity will provide a diagnostic tool that can add to.
  • Organisms particularly infectious organisms, carrying mutations in such polynucleotide may be detected at the polynucleotide level by a vanety of techmques, such as those descnbed elsewhere herem
  • the nucleotide sequences of the present mvention are also valuable for orgamsm chromosome identification
  • the sequence is specifically targeted to, and can hybndize with, a particular location on an organism's chromosome, particularly to a Staphylococcus aureus chromosome
  • the mapping of relevant sequences to chromosomes according to the present mvention may be an important step m co ⁇ elating those sequences with pathogenic potential and or an ecological mche of an orgamsm and or drug resistance of an organism, as ell as the essentiality of the gene to the orgamsm
  • the physical position of the sequence on the chromosome can be co ⁇ elated with genetic map data Such data may be found on-lme m a sequence database
  • the relationship between genes and diseases that have been mapped to the same chromosomal region are then identified through known genetic methods, for example, through linkage analysis (coinhe ⁇ tance of physically adjacent genes) or
  • the mvention also includes p ⁇ mers of the formula
  • X is hydrogen, a metal or a modified nucleotide residue, and at the 3' end of the molecule.
  • Y is hydrogen, a metal or a modified nucleotide residue
  • Rj and R3 are any nucleic acid residue or modified nucleotide residue
  • m is an mteger between 1 and 20 or zero
  • n is an mteger between 1 and 20 or zero
  • R is a pnmer sequence of the mvention, particularly a p ⁇ mer sequence selected from Table 2
  • R is o ⁇ ented so that its 5' end nucleotide residue is at the left, bound to Ri and its 3' end nucleotide residue is at the nght, bound to R3
  • Any stretch of nucleic acid residues denoted by either R group, where m and/or n is greater than 1, may be either a heteropolymer or a homopolymer, preferably a heteropolymer bemg complementary to a region of a polynucleotide of Table 1
  • m and/or n is
  • the mvention 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 among other things, amplifying nrdD DNA and/or RNA isolated from a sample de ⁇ ved from an individual, such as a bodily matenal
  • the primers may be used to amplify a polynucleotide isolated from an infected mdividual, such that the polynucleotide may then be subject to vanous techmques for elucidation of the polynucleotide sequence In this way, mutations in the polynucleotide sequence may be detected and used to diagnose and/or prognose the infection or its stage or course, or to serotype and/or classify the infectious agent
  • the mvention further provides a process for diagnosing, disease, preferably bacterial infections, more preferably infections caused by Staphylococcus aureus, compnsmg determining from a sample denved 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 nrdD polynucleotide can be measured using any on of the methods well known in the art for the quantitation of polynucleotides.
  • a diagnostic assay in accordance with the mvention for detectmg over-expression of mdD 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 mdD polypeptide, m a sample de ⁇ ved from a host, such as a bodily mate ⁇ al, are well-known to those of skill m the art
  • Assay techmques that can be used to determine levels of a mdD polypeptide, m a sample de ⁇ ved from a host, such as a bodily mate ⁇ al, are well-known to those of skill m the art
  • Such assay methods mclude radioimmunoassays, competitive-binding assays, Western Blot analysis, antibody sandwich assays, antibody detection and ELISA assays Differential Expression
  • the polynucleotides and polynucleotides of the mvention may be used as reagents for differential screening methods
  • differential screening and differential display methods There are many differential screening and differential display methods known m the art in which the polynucleotides and polypeptides of the mvention may be used
  • the differential display technique is described by Chuang et al , J Bactenol 175 2026-2036 (1993) This method identifies those genes which are expressed in an organism by identifying mRNA present using randomly- primed RT-PCR By comparing pre-mfection and post infection profiles, genes up and down regulated during infection can be identified and the RT-PCR product sequenced and matched to ORF "unknowns " In Vivo Expression Technology (IVET) is described by Camilli et al , Proc Nat'l Acad Sci USA 91 2634-2638 (1994) IVET identifies genes up-regulated durmg infection when compared to laboratory cultivation, implying an important role in infection ORFs identified
  • RT-PCR may also be used to analyze gene expression patterns
  • messenger RNA is isolated from bacterial infected tissue, e g , 48 hour munne lung infections, and the amount of each mRNA species assessed by reverse transcription of the RNA sample primed with random hexanucleotides followed by PCR with gene specific primer pairs
  • the determination of the presence and amount of a particular mRNA species by quantification of the resultant PCR product provides information on the bacterial genes which are transcribed in the infected tissue Analysis of gene transcription can be carried out at different times of infection to gam a detailed knowledge of gene regulation in bacterial pathogenesis allowing for a clearer understanding of which gene products represent targets for screens for antibacterials
  • the bacterial mRNA preparation need not be free of mammalian RNA This allows the investigator to carry out a simple and quick RNA preparation from infected tissue to obtain bacterial mRNA
  • the polynucleotides of the invention may be used as components of polynucleotide arrays, preferably high density arrays or grids These high density arrays are particularly useful for diagnostic and prognostic purposes
  • a set of spots each comprising a different gene, and further comprising a polynucleotide or polynucleotides of the invention may be used for probing, such as usmg hybridization or nucleic acid amplification, using a probes obtained or derived from a bodily sample, to determine the presence of a particular polynucleotide sequence or related sequence m an mdividual
  • Such a presence may indicate the presence of a pathogen, particularly Staphylococcus aureus, and may be useful in diagnosing and/or prognosmg disease or a course of disease
  • a grid compnsing a number of variants of the polynucleotide sequence of SEQ ID NO 1 are preferred Also preferred is a comprising a number of variants of
  • Antibodies The polypeptides and polynucleotides of the mvention or vanants thereof, or cells expressmg the same can be used as lmmunogens to produce antibodies immunospecific for such polypeptides or polynucleotides respectively
  • antibodies against nrdD polypeptides or polynucleotides Antibodies generated against the polypeptides or polynucleotides of the mvention can be obtained by admmistenng the polypeptides and or polynucleotides of the mvention, or epitope-bea ⁇ ng fragments of either or both, analogues of either or both, or cells expressmg either or both, to an animal, preferably a nonhuman, usmg routme protocols
  • any technique known in the art that provides antibodies produced by continuous cell lme cultures can be used Examples mclude va ⁇ ous techniques, such as those m Kohler.
  • phage display technology may be utilized to select antibody genes with binding activities towards a polypeptide of the invention either from repertoires of PCR amplified v-genes of lymphocytes from humans screened for possessing anti-nrdD or from naive hbranes (McCafferty, et al , (1990). Nature 348, 552-554. Marks, et al , (1992) Biotechnology 10, 779-783) The affimty of these antibodies can also be improved by. for example, chain shuffling (Clackson et al , (1991) Nature 352
  • the above-desc ⁇ bed antibodies may be employed to isolate or to identify' clones expressmg the polypeptides or polynucleotides of the mvention to punfy the polypeptides or polynucleotides by, for example, affinity chromatography
  • antibodies agamst nrdD-polypeptide or mdD-polynucleotide may be employed to treat infections, particularly bacte ⁇ al infections
  • Polypeptide variants include antigemcally, epitopically or immunologically equivalent vanants form a particular aspect of this invention
  • a polypeptide or polynucleotide of the invention such as an antigemcally or immunologically equivalent derivative or a fusion protein of the polypeptide is used as an antigen to immunize a mouse or other animal such as a rat or chicken
  • the fusion protem may provide stability to the polypeptide
  • the antigen may be associated, for example by conjugation, with an lmmunogemc earner protem for example bovine serum albumin, keyhole limpet haemocyanm or tetanus toxoid
  • a multiple antigenic polypeptide comprising multiple copies of the polypeptide, or an antigemcally or immunologically equivalent polypeptide thereof may be sufficiently antigenic to improve lmmunogenicity so as to obviate the use of a earner
  • the antibody or variant thereof is modified to make it less lmmunogemc in the individual
  • the antibody may most preferably be "humanized,” where the comphmentanty determining region or regions of the hybridoma-de ⁇ ved antibody has been transplanted into a human monoclonal antibody, for example as described m Jones et al (1986), Nature
  • a polynucleotide of the mvention for therapeutic or prophylactic purposes, in particular genetic immunization
  • a polynucleotide of the mvention for therapeutic or prophylactic purposes, in particular genetic immunization
  • a polynucleotide of the invention in genetic immunization will preferably employ a suitable delivery method such as direct injection of plasmid DNA into muscles (Wolff et al , Hum Mol
  • Polypeptides and polynucleotides of the mvention may also be used to assess the binding of small molecule substrates and ligands m. for example, cells, cell-free preparations, chemical hbranes, and natural product mixtures These substrates and ligands may be natural substrates and ligands or may be structural or functional mimetics See, e g .
  • the present mvention provides for a method of screening compounds to identify those which stimulate or which inhibit the function of a polypeptide or polynucleotide of the mvention, as well as related polypeptides and polynucleotides
  • agomsts or antagonists may be employed for therapeutic and prophylactic purposes for such Diseases as herembefore mentioned
  • Compounds may be identified from a vanety of sources, for example, cells, cell-free preparations, chemical hbranes, and natural product mixtures Such agomsts, antagonists or inhibitors so-identified may be natural or modified
  • 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 protem 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
  • these screening methods may test whether the candidate compound results m a signal generated b> activation or inhibition of 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 polypeptide and/or constitutively expressed polypeptides and polynucleotides may be employed in screening methods for inverse agonists or inhibitors, m the absence of an agonist or inhibitor, by testing whether the candidate compound results m inhibition of activation of the polypeptide or polynucleotide, as the case may be
  • the screemng methods may simply comprise the steps of mixing a candidate compound with a solution containing a polypeptide or polynucleotide of the present invention, to form a mixture, measuring nrdD polypeptide and/or polynucleotide activity
  • polypeptides and antibodies that bind to and/or interact with a polypeptide of the present invention may also be used to configure screening methods for detecting the effect of added compounds on the production of mRNA and/or polypeptide in cells
  • an ELISA assay may be constructed for measuring secreted or cell associated levels of polypeptide usmg monoclonal and polyclonal antibodies by standard methods known in the art This can be used to discover agents which may inhibit or enhance the production of polypeptide (also called antagonist or agonist, respectively) from suitably manipulated cells or tissues
  • the mvention also provides a method of screening compounds to identify those which enhance
  • nrdD polypeptides or polynucleotides particularly those compounds that are bactenstatic and/or bacte ⁇ cidal
  • the method of screening may mvolve high-throughput techmques
  • a synthetic reaction mix 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 any thereof, compnsmg mdD polypeptide and a labeled substrate or hgand of such polypeptide is mcubated m the absence or the presence of a candidate molecule that may be a nrdD agomst or antagonist
  • the ability of the candidate molecule to agonize or antagonize the nrdD polypeptide is reflected m decreased binding of the labeled hgand or decreased production of product from such substrate Molecules that bmd gratuitously, z e , without mducmg the effects of nr
  • 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 mclude. but are not limited to, hgand binding and crosshnking assays m which the polypeptide is labeled with a radioactive isotope (for instance, 1 * "I), chemically modified (for instance, biot ylated), or fused to a peptide sequence suitable for detection or purification, and incubated with a source of the putative receptor (e g .
  • a radioactive isotope for instance, 1 * "I
  • chemically modified for instance, biot ylated
  • Fluorescence energy transfer may also be used characterize small molecules that interfere with the formation of nrdD polypeptide d mers, turners, tetramers or higher order structures, or structures formed by nrdD polypeptide bound to another polypeptide NrdD 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
  • nrdD polypeptide can be coupled to a sensor chip at low site density such that covalently bound molecules will be monome ⁇ c Solution protem can then passed over the mdD polypeptide -coated surface and specific binding can be detected m 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 equihbnum bindmg constants for nrdD polypeptide self-association as well as an association of nrdD polypeptide and another polypeptide or small molecule
  • a scintillation proximity assay may be used to characterize the interaction between an association of nrdD polypeptide with another nrdD polypeptide or a different polypeptide mdD polypeptide can be coupled to a scmtillation-filled bead Addition of radio-labeled nrdD polypeptide results in binding where the radioactive source molecule is m close proximity to the scintillation fluid Thus, signal is emitted upon nrdD polypeptide bmdmg and compounds that prevent nrdD polypeptide self-association or an association of nrdD polypeptide and another polypeptide or small molecule will diminish signal
  • ICS biosensors have been described by AMBRI (Australian Membrane Biotechnology Research Institute) They couple the self-association of macromolecules to the closing of gramacidm- facihtated ion channels m suspended membrane bilayers and hence to a measurable change m the admittance (similar to impedence) of the biosensor This approach is linear over six decades of admittance change and is ideally suited for large scale, high through-put screenmg of small molecule combinatorial libraries.
  • methods for identifying compounds which 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 bmdmg to or other interaction between the compound and the polypeptide and or polynucleotide to assess the bmdmg to
  • an assay for nrdD agomsts is a competitive assay that combines mdD and a potential agomst with nrdD-binding molecules, recombinant nrdD bmdmg molecules, natural substrates or ligands. or substrate or hgand mimetics, under approp ⁇ ate conditions for a competitive inhibition assay mdD can be labeled, such as by radioactivity or a colo ⁇ met ⁇ c compound, such that the number of mdD molecules bound to a bmdmg molecule or converted to product can be determined accurately to assess the effectiveness of the potential antagonist
  • Potential antagonists mclude. among others, small orgamc molecules, peptides, polypeptides and antibodies that bmd to a polynucleotide and or polypeptide of the mvention and thereby inhibit or extinguish its activity or expression
  • Potential antagonists also may be small orgamc molecules, a peptide, a polypeptide such as a closely related protem or antibody that bmds the same sites on a bmdmg molecule, such as a bmdmg molecule, without inducing nrdD-rnduced activities, thereby preventmg the action or expression of mdD polypeptides and or polynucleotides by excluding mdD polypeptides and or polynucleotides from bmdmg
  • Potential antagonists m include a small molecule that bmds to and occupies the bmdmg site of the polypeptide thereby preventmg bmdmg to cellular bmdmg molecules, such that normal biological activity is prevented
  • small molecules include but are not limited to small orgamc molecules, peptides or peptide-hke molecules
  • Other potential antagonists m include antisense molecules (see Okano, J Neurochem 56 560 (1991) OLIGODEOXYNUCLEOTIDES AS ANTISENSE INHIBITORS OF GENE EXPRESSION, CRC Press.
  • Preferred potential antagonists m include compounds related to and vanants of mdD
  • Other examples of potential polypeptide antagonists m include antibodies or, m some cases, ohgonucleotides or proteins which are closely related to the ligands, substrates, receptors, enzymes, etc , as the case may be. of the polypeptide, e g .
  • Certam of the polypeptides of the mvention are biomimetics.
  • functional mimetics of the natural mdD polypeptide These functional mimetics may be used for. among other things, antagonizing the activity of mdD polypeptide or as a antigen or lmmunogen m a manner desc ⁇ bed elsewhere herem
  • Functional mimetics of the polypeptides of the mvention mclude but are not limited to truncated polypeptides For example, preferred functional mimetics mclude.
  • Polynucleotides encoding each of these functional mimetics may be used as expression cassettes to express each mimetic polypeptide It is prefe ⁇ ed that these cassettes compnse 5' and 3' rest ⁇ ction sites to allow for a convement means to hgate the cassettes together when desired It is further prefe ⁇ ed that these cassettes compnse gene expression signals known in the art or desc ⁇ bed elsewhere herem
  • the present invention relates to a screening kit for identifying agomsts, antagonists, ligands receptors, substrates, enzymes, etc for a polypeptide and/or polynucleotide of the present invention, or compounds which decrease or enhance the production of such polypeptides and/or polynucleotides , which comprises
  • polypeptide and/or polynucleotide of the present invention may also be used in a method for the structure-based design of an agonist, antagonist or inhibitor of the polypeptide and/or polynucleotide, by
  • the present mvention provides methods of treatmg abnormal conditions such as, for instance, a Disease, related to either an excess of. an under-expression of, an elevated activity of. or a decreased activity of mdD polypeptide and or polynucleotide
  • the present invention relates to genetically engineered soluble fusion proteins comprising a polypeptide of the present mvention, or a fragment thereof, and vanous portions of the constant regions of heavy or light chains of immunoglobulms of various subclasses (IgG, IgM, IgA, IgE) Preferred as an lmmunoglobulm is the constant part of the heavy chain of human IgG, particularly IgGl, where fusion takes place at the hmge region
  • the Fc part can be removed simply by incorporation of a cleavage sequence which can be cleaved with blood clotting factor Xa
  • this invention relates to processes for the preparation of these fusion protems by genetic engineering, and to the use thereof for drug screenmg. diagnosis and therapy
  • a further aspect of the invention also relates to polynucleotides encoding such fusion proteins Examples of fusion prote technology can be found in International Patent Application Nos W094/29458 and W094/2
  • expression of the gene encoding endogenous mdD polypeptide can be inhibited using expression blocking techniques
  • This blocking may be targeted against any step m gene expression, but is preferably targeted against transcription and/or translation
  • An examples of a known technique of this sort mvolve the use of antisense sequences, either internally generated or separately administered (see, for example. O'Connor, J Neurochem (1991) 56 560 in Ohgodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press. Boca Raton, FL (1988))
  • ohgonucleotides which 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 o gomers can be administered per se or the relevant ohgomers 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 encodmg 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 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 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, m particular gram positive and/or gram negative bacteria, to eukaryotic, preferably mammalian, extracellular matrix proteins on in-dwelling devices or to extracellular matrix proteins in wounds, to block mdD protem-mediated mammalian cell invasion by.
  • mdD agomsts and antagonists preferably bacte ⁇ static or bactencidal agomsts and antagonists
  • the antagonists and agomsts of the mvention may be employed, for instance, to prevent, inhibit and or treat diseases
  • Hehcobacter pylori (herein "H pylori”) bactena infect the stomachs of over one-third of the world's population causing stomach cancer, ulcers, and gastritis (International Agency for Research on Cancer (1994) Schistosomes, Liver Flukes and Hehcobacter Pylori (International Agency for Research on Cancer. L ⁇ on. France, http //www uicc ch ecp/ecp2904 htm)
  • the International Agency for Research on Cancer recently recognized a cause-and-effect relationship between H pylori and gastric adenocarcinoma.
  • Preferred antimicrobial compounds of the invention agonists and antagonists of nrdD 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 carcinoma Such treatment should also prevent, inhibit and/or cure gastric ulcers and gastritis Vaccines
  • mvention products, compositions and methods for assessmg mdD expression, treatmg disease, assaying genetic vanation, and administering a nrdD polypeptide and/or polynucleotide to an orgamsm to raise an lmmunological response against a bactena. especially a Staphylococcus aureus bactena
  • Another aspect of the mvention relates to a method for inducing an immunological response in an individual, particularly a mammal which comprises inoculating the individual with mdD polynucleotide and or polypeptide.
  • Yet another aspect of the invention relates to a method of mducmg immunological response in an individual which comprises delivering to such mdividual a nucleic acid vector, sequence or ⁇ bozyme to direct expression of mdD polynucleotide and/or polypeptide, or a fragment or a vanant thereof, for expressing mdD polynucleotide and/or polypeptide, or a fragment or a variant thereof in vivo m order to induce an immunological response, such as, to produce antibody and/ or T cell immune response, including, for example, cytokine-producing T cells or cytotoxic T cells, to protect said individual, preferably a human, from disease, whether that disease is already established withm the mdividual or not
  • an immunological response such as, to produce antibody and/ or T cell immune response, including, for example, cytokine-producing T cells or cytotoxic T cells, to protect said individual, preferably a human, from disease, whether that disease is already established
  • a further aspect of the invention relates to an immunological composition that when introduced into an individual, preferably a human, capable of having induced within it an immunological response, mduces an immunological response in such individual to a mdD polynucleotide and or polypeptide encoded therefrom, wherein the composition comprises a recombinant mdD polynucleotide and/or polypeptide encoded therefrom and/or comprises DNA and/or RNA which encodes and expresses an antigen of said mdD polynucleotide.
  • fused recombinant protein preferably further comprises an antigenic co-protein, such as hpoprotein D from Hemoph ⁇ us influenzae, Glutathione-S-transferase (GST) or beta-galactosidase or any other relatively large co-protem which solubihzes the protem and facilitates production and purification thereof
  • co-protein may act as an adjuvant in the sense of providing a generalized stimulation of the immune system of
  • polynucleotide or particular fragments thereof which have been shown to encode non-va ⁇ able regions of bacterial cell surface proteins, in polynucleotide constructs used in such genetic immunization experiments in animal models of infection with Staphylococcus aureus
  • Such experiments will be particularly useful for identifying protem epitopes able to provoke a prophylactic or therapeutic immune response It is believed that this approach will allow for the subsequent preparation of monoclonal antibodies of particular value, de ⁇ ved from the requisite organ of the animal successfully resisting or clearmg infection, for the development of prophylactic agents or therapeutic treatments of bacterial mfection, particularly Staphylococcus aureus infection, in mammals, particularly humans
  • a polypeptide of the invention may be used as an antigen for vaccmation of a host to produce specific antibodies which protect against invasion of bactena. for example by blocking adherence of bacteria to damaged tissue
  • tissue damage mclude wounds in skm or connective tissue caused, for example, by mechanical, chemical, thermal or radiation damage or by implantation of indwelling devices, or wounds in the mucous membranes, such as the mouth, throat, mammary glands, urethra or vagina
  • the invention also includes a vaccine formulation which comprises an lmmunogenic recombinant polypeptide and/or polynucleotide of the invention together with a suitable carrier, such as a pharmaceutically acceptable earner Since the polypeptides and polynucleotides may be broken down in the stomach, each is preferably administered parenterally, mcludmg, for example, administration that is subcutaneous, intramuscular, intravenous, or intradermal
  • mcludmg for example, administration that is subcutaneous, intramuscular, intravenous, or intradermal
  • Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti- oxidants.
  • the formulations may be presented in unit- dose or multi-dose containers, for example, sealed ampoules and vials and may be stored m a freeze-d ⁇ ed condition requiring only the addition of the sterile liquid carrier immediately prior to use
  • the vaccine formulation may also include adjuvant systems for enhancing the lmmunoge city of the formulation, such as oil-m water systems and other systems known m the art The dosage will depend on the specific activity of the vaccine and can be readily determined by routine experimentation
  • compositions for purposes of compositions, kits and administration
  • compositions comp ⁇ smg a mdD polynucleotide and/or a mdD polypeptide for administration to a cell or to a multicellular orgamsm
  • the mvention also relates to compositions compnsrng a polynucleotide and/or a polypeptides discussed herem or their agomsts or antagonists
  • the polypeptides and polynucleotides of the mvention may be employed m combination with a non-ste ⁇ le or stenle earner or earners for use with cells, tissues or orgamsms, such as a pharmaceutical earner suitable for administration to an mdividual Such compositions compnse.
  • earners may mclude, but are not limited to, salme, buffered salme. dextrose, water, glycerol. ethanol and combmations thereof
  • the formulation should suit the mode of administration
  • the mvention further relates to diagnostic and pharmaceutical packs and kits compnsrng one or more containers filled with one or more of the ingredients of the aforementioned compositions of the mvention
  • Polypeptides, polynucleotides and other compounds of the mvention may be employed alone or m conjunction with other compounds, such as therapeutic compounds
  • compositions may be admmistered in any effective, convement manner mcludmg, for instance, admmistration by topical, oral, anal, vaginal, mtravenous, mtrape ⁇ toneal. intramuscular, subcutaneous, mtranasal or lntradermal routes among others
  • the active agent may be administered to an mdividual as an mjectable composition, for example as a sterile aqueous dispersion, preferably isotomc
  • composition may be formulated for topical application for example m the form of ointments, creams, lotions, eye ointments, eye drops, ear drops, mouthwash, impregnated dressings and sutures and aerosols, and may contain appropriate conventional additives, including, for example, preservatives, solvents to assist drug penetration, and emollients in omtments and creams
  • topical formulations may also contain compatible conventional carriers, for example cream or ointment bases, and ethanol or oleyl alcohol for lotions
  • Such carriers may constitute from about 1% to about 98% by weight of the formulation, more usually they will constitute up to about 80% by weight of the formulation
  • the present mvention provides for pharmaceutical compositions compnsrng a therapeutically effective amount of a polypeptide and/or polynucleotide, such as the soluble form of a polypeptide and or polynucleotide of the present mvention, agonist or antagonist peptide or small molecule compound, in combination with a pharmaceutically acceptable earner or excipient
  • a pharmaceutically acceptable earner or excipient Such earners mclude, but are not limited to, salme. buffered salme.
  • the mvention further relates to pharmaceutical packs and kits compnsrng one or more containers filled with one or more of the ingredients of the aforementioned compositions of the mvention
  • Polypeptides, polynucleotides and other compounds of the present mvention may be employed alone or in conjunction with other compounds, such as therapeutic compounds
  • the composition will be adapted to the route of admmistration, for instance by a systemic or an oral route
  • Prefe ⁇ ed forms of systemic admmistration m clude injection, typically by mtravenous injection
  • Other injection routes such as subcutaneous, intramuscular, or mtrape ⁇ toneal
  • Alternative means for systemic admmistration m include transmucosal and transdermal admmistration usmg penetrants such as bile salts or fusidic acids or other detergents
  • a polypeptide or other compounds of the present mvention can be employed alone or in conjunction with other compounds, such as therapeutic compounds
  • the composition will
  • the daily dosage level of the active agent will be from 0 01 mg/kg to 10 mg/kg, typically around 1 mg/kg
  • the physician in any event will determine the actual dosage which will be most suitable for an mdividual and will vary with the age, weight and response of the particular individual
  • the above dosages are exemplary of the average case There can. of course, be individual instances where higher or lower dosage ranges are merited, and such are within the scope of this mvention
  • In-dwellmg devices include surgical implants, prosthetic devices and catheters, l e , devices that are introduced to the body of an individual and remain in position for an extended time
  • Such devices mclude. for example, artificial jomts, heart valves, pacemakers, vascular grafts, vascular catheters, cerebrospmal fluid shunts, urinary catheters, continuous ambulatory pentoneal dialysis (CAPD) catheters
  • composition of the mvention may be administered by injection to achieve a systemic effect agamst relevant bacteria shortly before insertion of an in-dwelling device Treatment may be continued after surgery during the m-body time of the device
  • composition could also be used to broaden pe ⁇ operative cover for any surgical technique to prevent bacterial wound infections, especially Staphylococcus aureus wound infections
  • compositions of this invention may be used generally as a wound treatment agent to prevent adhesion of bacteria to matrix protems exposed in wound tissue and for prophylactic use m dental treatment as an alternative to, or in conjunction with, antibiotic prophylaxis
  • composition of the invention may be used to bathe an indwelling device immediately before insertion
  • the active agent will preferably be present at a concentration of 1 ⁇ g/ml to lOmg/ml for bathing of wounds or indwelling devices
  • a vaccine composition is conveniently in mjectable form
  • Conventional adjuvants may be employed to enhance the immune response
  • a suitable unit dose for vaccination is 0 5-5 microgram kg of antigen, and such dose is preferably administered 1-3 times and with an interval of 1-3 weeks With the mdicated dose range, no adverse toxicological effects will be observed with the compounds of the invention which would preclude their administration to suitable individuals
  • Sequence Databases Sequences in a Tangible Medium, and Algorithms
  • Polynucleotide and polypeptide sequences form a valuable information resource with which to determme their 2- and 3-d ⁇ mens ⁇ onal structures as well as to identify further sequences of similar homology
  • These approaches are most easily facilitated by storing the sequence in a computer readable medium and then usmg the stored data m a known macromolecular structure program or to search a sequence database usmg well known searching tools, such as GCC
  • polynucleotide and polypeptide sequences of the invention are particularly useful as components in databases useful for search analyses as well as in sequence analysis algonthms
  • sequence databases Sequences in a Tangible Medium, and Algorithms
  • polynucleotide of the invention and polynucleotide sequence of the invention mean any detectable chemical or physical characteristic of a polynucleotide of the invention that is or may be reduced to or stored in a tangible medium, preferably a computer readable form For example, chromatographic scan data or peak data, photographic data or scan data therefrom, called bases, and mass spectrographic data
  • polypeptide of the invention and polypeptide sequence of the invention mean any detectable chemical or physical characteristic of a polypeptide of the mvention that is or may be reduced to or
  • a computer based method for performing homology identification This method comprises the steps of providing a polynucleotide sequence comprising the sequence a polynucleotide of the invention in a computer readable medium, and comparing said polynucleotide sequence to at least one polynucleotide or polypeptide sequence to identify homology
  • a computer based method is also provided for performing homology identification, said method comprising the steps of providing a polypeptide sequence comprising the sequence of a polypeptide of the mvention in a computer readable medium, and comparing said polypeptide sequence to at least one polynucleotide or polypeptide sequence to identify homology
  • a computer based method is still further provided for polynucleotide assembly, said method comprising the steps of providing a first polynucleotide sequence comprising the sequence of a polynucleotide of the invention in a computer readable medium, and screening for at least one overlapping region between said first polynucleotide sequence and a second polynucleotide sequence
  • a further embodiment of the invention provides a computer based method for performing homology identification, said method comprising the steps of providing a polynucleotide sequence comprising the sequence of a polynucleotide of the invention in a computer readable medium, and comparing said polynucleotide sequence to at least one polynucleotide or polypeptide sequence to identify homology
  • a further embodiment of the invention provides a computer based method for performing homology identification, said method comprising the steps of providing a polypeptide sequence compnsrng the sequence of a polypeptide of the mvention in a computer readable medium, and comparing said polypeptide sequence to at least one polynucleotide or polypeptide sequence to identify homology
  • a further embodiment of the invention provides a computer based method for polynucleotide assembly, said method comprising the steps of providing a first polynucleotide sequence comprising the sequence of a polynucleotide of the invention in a computer readable medium, and screening for at least one overlapping region between said first polynucleotide sequence and a second polynucleotide sequence
  • a computer readable medium having stored thereon a member selected from the group consisting of a polynucleotide comprising the sequence of SEQ ID NO 1 , a polypeptide comprising the sequence of SEQ ID NO 2, a set of polynucleotide sequences wherein at least one of said sequences compnses the sequence of SEQ ID NO 1 , a set of polypeptide sequences wherein at least one of said sequences comprises the sequence of SEQ ID NO 2.
  • a data set representing a polynucleotide sequence comprising the sequence of SEQ ID NO 1 a data set representing a polynucleotide sequence encoding a polypeptide sequence compnsrng the sequence of SEQ ID NO 2
  • a polynucleotide comprising the sequence of SEQ ID NO 1 a polypeptide compnsrng the sequence of SEQ ID NO 2
  • a set of polynucleotide sequences wherein at least one of said sequences comprises the sequence of SEQ ID NO 1
  • a set of polypeptide sequences wherein at least one of said sequences comprises the sequence of SEQ ID NO 2.
  • a further preferred embodiment of the invention provides a computer based method for performing homology identification, said method comprising the steps of providing a polynucleotide sequence comprising the sequence of SEQ ID NO 1 m a computer readable medium, and comparing said polynucleotide sequence to at least one polynucleotide or polypeptide sequence to identify homology
  • a still further preferred embodiment of the invention provides a computer based method for performing identification, said method comprising the steps of providmg a polypeptide sequence comprising the sequence of SEQ ID NO 2 in a computer readable medium, and comparing said polypeptide sequence to at least one polynucleotide or polypeptide sequence to identify homology
  • a further embodiment of the invention provides a computer based method for polynucleotide assembly, said method comprising the steps of providing a first polynucleotide sequence comprising the sequence of SEQ ID NO 1 in a computer readable medium, and screening for at least one overlappmg region between said first polynucleotide sequence and a second polynucleotide sequence
  • a further embodiment of the invention provides a computer based method for performing homology identification, said method comprising the steps of providing a polynucleotide sequence comprising the sequence of SEQ ID NO 1 in a computer readable medium, and comparing said polynucleotide sequence to at least one polynucleotide or polypeptide sequence to identify homology
  • a further embodiment of the mvention provides a computer based method for performing homology identification, said method comprising the steps of providing a polypeptide sequence compnsrng the sequence of SEQ ID NO 2 in a computer readable medium, and comparing said polypeptide sequence to at least one polyn
  • a further embodiment of the invention provides a computer based method for polynucleotide assembly, said method comprising the steps of providing a first polynucleotide sequence comprising the sequence of SEQ ID NO 1 in a computer readable medium, and screemng for at least one overlappmg region between said first polynucleotide sequence and a second polynucleotide sequence
  • ant ⁇ body( ⁇ es) as used herein includes polyclonal and monoclonal antibodies, chime ⁇ c. single chain, and humanized antibodies, as well as Fab fragments, including the products of an Fab or other lmmunoglobulin expression library
  • Antigemcally equivalent de ⁇ vat ⁇ ve(s) as used herem encompasses a pohpeptide, polynucleotide. or the equivalent of either which will be specifically recognized b ⁇ certain antibodies which, when raised to the protem, polypeptide or polynucleotide according to the mvention. interferes with the immediate physical interaction between pathogen and mammalian host
  • Bispecific antibody( ⁇ es) means an antibody comprising at least two antigen bmdmg domains, each domain directed against a different epitope
  • Bodily mate ⁇ al(s) means any matenal denved from an mdividual or from an orgamsm infecting, infesting oi inhabiting an mdividual.
  • mcludmg 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 autopsy mate ⁇ als
  • D ⁇ sease(s) means any disease caused by or related to infection by a bactena. mcludmg , for example, disease, such as.
  • infections of the upper respiratory tract e g , otitis media, bacte ⁇ al tracheitis, acute epiglottitis, thyroiditis.
  • lower respiratory e g , empyema, lung abscess
  • cardiac e g , infective endocarditis
  • gastrointestinal e g , secretory dia ⁇ hoea. splemc absces, retrope ⁇ toneal abscess
  • CNS e g , cerebral abscess
  • eye e g . blephantis.
  • conjunctivitis conjunctivitis, keratitis, endophthalmitis, preseptal and orbital celluhtis, darcryocystitis), kidney and urinary tract (e g . epididymitis, rntrarenal and pennephnc absces, toxic shock syndrome), skin (e g , impetigo, follicu tis, cutaneous abscesses, celluhtis, wound infection, bacte ⁇ al myositis) bone and joint (e.g , septic arthntis, osteomyelitis)
  • Fusion protem(s) refers to a protein encoded by two, often unrelated, fused genes or fragments thereof
  • EP-A-0464 discloses fusion proteins comprising vanous portions of constant region of lmmunoglobuhn molecules together with another human protem or part thereof
  • employing an lmmunoglobuhn Fc region as a part of a fusion protein is advantageous for use m therapy and diagnosis resulting in. for example, improved pharmacokinetic properties [see, e g . EP-A 0232262]
  • “Host cell(s)” is a cell which has been transformed or transfected, or is capable of transformation or transfection by an exogenous polynucleotide sequence
  • Identity is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as the case may be, as determined by comparing the sequences
  • identity also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences
  • Identity can be readily calculated by known methods, including but not limited to those described m (Computational Molecular Biology Lesk. A M . ed . Oxford University Press. New York, 1988, Biocomputtng Informatics and Genome Projects Smith, D W ed Academic Press, New York. 1993. Computer Analysis of Sequence Data. Part I.
  • Methods to determine identity are designed to give the largest match between the sequences tested Moreover, methods to determine identity are codified m publicly available computer programs
  • Computer program methods to determine identity between two sequences include, but are not limited to, the GCG program package (Devereux, J , et al , Nucleic Acids Research 12(1) 387 (1984)), BLASTP, BLASTN. and FASTA (Altschul, S F et al , J Molec Bwl 215 403-410 (1990)
  • the BLAST X program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S , et al , NCBI NLM NIH Bethesda, MD 20894. Altschul, S , et al , J Mol Bwl 215 403-410 (1990)
  • the well known Smith Waterman algorithm may also be used to determine identity
  • Parameters for polypeptide sequence companson include the following
  • Parameters for polynucleotide companson include the following
  • Polynucleotide embodiments further mclude an isolated polynucleotide comprising a polynucleotide sequence having at least a 50, 60, 70. 80. 85. 90. 95, 97 or 100% identity to the reference sequence of SEQ ID NO 1. wherein said polynucleotide sequence may be identical to the reference sequence of SEQ ID NO 1 or ma ⁇ mclude 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.
  • alterations may occur at the 5' or 3' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among the nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence, and wherein said number of nucleotide alterations is determined by multiplying the total number of nucleotides SEQ ID NO: 1
  • n n is the number of nucleotide alterations.
  • x n is the total number of nucleotides in SEQ ID NO 1
  • y is 0 50 for 50%, 0 60 for 60%, 0 70 for 70%, 0 80 for 80%, 0 85 for 85%, 0 90 for 90%, 0 95 for 95%, 0 97 for 97% or 1 00 for 100%
  • • is the symbol for the multiplication operator, and wherein any non-integer product of x n and y is rounded down to the nearest integer prior to subtractmg it from x n
  • Alterations of a polynucleotide sequence encoding the polypeptide of SEQ ID NO 2 may create nonsense, missense or frameshift mutations in this coding sequence and alter the polypeptide encoded by the polynucleotide following such alterations
  • a polynucleotide sequence of the present invention may be identical to the reference sequence of SEQ ID NO 1. that is it may be 100% identical, or it may include up to a certain mteger number of nucleic acid alterations as compared to the reference sequence such that the percent identity is less than 100% identity
  • Such alterations are selected from the group consisting of at least one nucleic acid deletion, substitution, including transition and transversion, or insertion, and w herem said alterations may occur at the 5' or 3' terminal positions of the reference polynucleotide sequence or anywhere between those terminal positions, mterspersed either individually among the nucleic acids in the reference sequence or m one or more contiguous groups withm the reference sequence
  • the number of nucleic acid alterations for a given percent identity is determined by multiplying the total number of nucleic acids in SEQ ID NO 1 by the integer defining the percent identity divided by 100 and then subtracting that product from said total number of nucleic acids m SEQ ID NO 1. or
  • n n is the number of nucleic acid alterations
  • x n is the total number of nucleic acids m SEQ ID NO 1
  • y is, for instance 0 70 for 70%, 0 80 for 80%, 0 85 for 85% etc
  • • is the symbol for the multiplication operator, and wherein any non-mteger product of x n and y is rounded down to the nearest mteger prior to subtracting it from x n
  • Polypeptide embodiments further include an isolated polypeptide compnsrng a polypeptide having at least a 50,60, 70, 80, 85, 90, 95, 97 or 100% identity to a polypeptide reference sequence of SEQ ID NO 2, wherein said polypeptide sequence may be identical to the reference sequence of SEQ ID NO 2 or may include up to a certain integer number of ammo acid alterations as compared to the reference sequence, wherein said alterations are selected from the group consisting of at least one amino acid deletion, substitution, including conservative and non-conservative substitution, or insertion, and wherein said alterations may occur at the amino- or carboxy-termmal positions of the reference polypeptide sequence or anywhere between those terminal positions, interspersed either individually among the amino acids m the reference sequence or in one or more contiguous groups within the reference sequence, and wherein said number of amino acid alterations is determined by multiplying the total number of ammo acids in SEQ ID NO 2 by the mteger defining the percent identity divided by 100 and then subtracting that product from said
  • n a is the number of ammo acid alterations
  • x a is the total number of amino acids in SEQ ID NO 2.
  • y is 0 50 for 50%, 0 60 for 60%, 0 70 for 70%, 0 80 for 80%, 0 85 for 85%, 0 90 for 90%, 0 95 for 95%, 0 97 for 97% or 1 00 for 100%
  • • is the symbol for the multiplication operator, and wherein any non-integer product of x a and y is rounded down to the nearest integer prior to subtractmg it from x a
  • a polypeptide sequence of the present invention may be identical to the reference sequence of SEQ ID NO 2, that is it may be 100% identical, or it may mclude up to a certain integer number of amino acid alterations as compared to the reference sequence such that the percent identity is less than 100% identity
  • Such alterations are selected from the group consistmg 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-terminal positions of the reference polypeptide sequence or anywhere between those terminal positions, interspersed either individually among the amino acids in the reference sequence or in one or more contiguous groups within the reference sequence
  • the number of ammo acid alterations for a given % identity is determined by multiplying the total number of ammo acids m SEQ ID NO 2 by the integer defining the percent identity divided by 100 and then subtracting that product from said total number of amino acids in SEQ ID NO 2, or
  • n a is the number of amino acid alterations
  • x a is the total number of amino acids in SEQ ID NO 2
  • y is. for instance 0 70 for 70%. 0 80 for 80%, 0 85 for 85% etc
  • • is the symbol for the multiplication operator, and wherem any non-mteger product of x a and y is rounded down to the nearest mteger prior to subtracting it from x a
  • “Immunologically equivalent de ⁇ vat ⁇ ve(s)” as used herein encompasses a polypeptide, poh nucleotide or the equivalent of either which when used in a suitable formulation to raise antibodies in a ⁇ ertebrate.
  • Immunospecific means that charactenstic of an antibody whereby it possesses substantially greater affinity for the polypeptides of the mvention or the polynucleotides of the mvention than its affimty for other related polypeptides or polynucleotides respectively, particularly those polypeptides and polynucleotides in the p ⁇ or art "Ind ⁇ v ⁇ dual(s)” means a multicellular eukaryote. mcludmg. but not limited to a metazoan, a mammal, an ovid, a bovid. a simian, a primate, and a human
  • Isolated means altered “by the hand of man” from its natural state, / , if it occurs m nature, it has been changed or removed from its onginal environment, or both
  • a polynucleotide or a polypeptide naturally present m a Irving orgamsm is not “isolated,” but the same polynucleotide or polypeptide separated from the coexistmg matenals of its natural state is “isolated", as the term is employed herem
  • a polynucleotide or polypeptide that is mtroduced mto an orgamsm by transformation, genetic manipulation or by any other recombinant method is "isolated” even if it is still present in said orgamsm, which orgamsm may be living or non-living
  • Organ ⁇ sm(s) means a (I) prokaryote, mcludmg but not limited to, a member of the genus Streptococcus Staphylococcus, Bordetella, Corynebactenum, Mycobacterium, Neissena, Haemophilus, Actinomycetes Streptomycetes, Nocardia, Enterobacter, Yersinia, Fancisella, Pasturella, Moraxella, Acmetobacter Erysipelothnx, Branhamella, Actinobacillus, Streptobacillus, Listena, Calymmatobactenum, Brucella, Bacillus, Clostndium, Treponema, Eschench a, Salmonella, Kleibsiella, Vibrio, Proteus, Erwinia, Borrelia, Leptospira, Spirillum, Campylobacter, Shigella, Legionella, Pseudomonas
  • mclud g but not limited to, a protozoan, a fungus, a member of the genus Saccharomyces, Kluveromyces, or Candida, and a member of the species Saccharomyces cenv seae, Kluveromyces lactis, or Candida albicans
  • Polynucleot ⁇ de(s) generally refers to any polynbonucleoti.de or polydeoxynbonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA "Polynucleotide(s)” mclude, without limitation, smgle- and double-stranded DNA.
  • DNA that is a mixture of single- and double-stranded regions or single-, double- and t ⁇ ple-stranded regions, smgle- and double-stranded RNA, and RNA that is mixture of s gle- and double-stranded regions
  • hybnd molecules comp ⁇ smg DNA and RNA that may be single-stranded or, more typically, double-stranded, or t ⁇ ple-stranded regions, or a mixture of smgle- and double-stranded regions
  • polynucleotide refers to tnple-stranded regions comp ⁇ smg RNA or DNA or both RNA and DNA
  • the strands m such regions may be from the same molecule or from different molecules
  • the regions may mclude all of one or more of the molecules, but more typically mvolve only a region of some of the molecules
  • One of the molecules of a tnple-he cal region often is an oligonucleotide As used herem,
  • Polypept ⁇ de(s) refers to any peptide or protem comp ⁇ smg two or more amino acids jomed to each other b ⁇ peptide bonds or modified peptide bonds
  • Polypeptide(s) refers to both short chains, commonly refe ⁇ ed to as peptides o gopeptides and ohgomers and to longer chains generally refe ⁇ ed to as proteins Polypeptides may contam ammo acids other than the 20 gene encoded ammo acids
  • Polypeptide(s)” mclude those modified either by natural processes, such as processmg and other post-translational modifications, but also by chemical modification techmques Such modifications are well desc ⁇ bed m 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 varying degree at several sites m a given polypeptide Also, a given polypeptide may contam
  • Modifications mclude, for example, acetylation, acylation. ADP- nbosylation. amidation. covalent attachment of flavrn, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide de ⁇ vative, covalent attachment of a pid or lipid denvative, covalent attachment of phosphotidy nositol cross-linking, cvchzation, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteme. formation of pyroglutamate. formylation.
  • gamma- carboxylation GPI anchor formation hydroxylation, lodination, methylation, mynstoylation, oxidation, proteolytic processmg, phosphorylation, prenylation, racemization, glycosylation, hpid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP-nbosylation. selenoylation, sulfation. transfer-RNA mediated addition of ammo acids to protems, such as arginylation, and ubiquitination See, for instance, PROTEINS - STRUCTURE AND MOLECULAR PROPERTIES, 2nd E .
  • Polypeptides may be branched or cyclic, with or without branching Cyclic, branched and branched circular polypeptides may result from post-translational natural processes and may be made by entirely synthetic methods, as well
  • Recombinant expression system(s) refers to expression systems or portions thereof or polynucleotides of the mvention mtroduced or transformed mto a host cell or host cell lysate for the production of the polynucleotides and polypeptides of the mvention
  • “Subtraction set” is one or more, but preferably less than 100, polynucleotides compnsrng at least one polynucleotide 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 m the nucleotide sequence of the variant may or may not alter the ammo acid sequence of a polypeptide encoded by the reference polynucleotide Nucleotide changes may result in amino acid substitutions, additions, deletions, fusion proteins and truncations in the polypeptide encoded by the reference sequence, as discussed below
  • a typical variant of a polypeptide differs m amino acid sequence from another, reference Generally, differences are limited so that the sequences of the reference polypeptide and the vanant are closely similar overall and.
  • a variant and reference polypeptide may differ in amino acid sequence by one or more substitutions, additions, deletions m 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 mclude va ⁇ ants of each of the polypeptides of the mvention. that is polypeptides that vary from the referents by conservative ammo acid substitutions, whereby a residue is substituted b ⁇ 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.
  • va ⁇ ants among Asn and Gin, and among the basic residues Lys and Arg or aromatic residues Phe and Tyr Particularly prefe ⁇ ed are va ⁇ ants m which several. 5-10, 1-5, 1-3 1-2 or 1 ammo acids are substituted, deleted, or added in any combination
  • a vanant of a polynucleotide or polypeptide may be a naturally occurring such as an allelic variant, or it ma% be a variant that is not known to occur naturally
  • Non-naturally occurring 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 having a DNA sequence given m Table 1 [SEQ ID NO 1] was obtained from a library of clones of chromosomal DNA of Staphylococcus aureus in E cob
  • the sequencing data from two or more clones containmg overlapping Staphylococcus aureus DNAs was used to construct the contiguous DNA sequence in SEQ ID NO 1 Libraries may be prepared by routine methods, for example Methods 1 and 2 below
  • Total cellular DNA is isolated from Staphylococcus aureus WCUH 29 according to standard procedures and size-fractionated by either of two methods
  • Method 1 Total cellular DNA is mechanically sheared by passage through a needle in order to size- fractionate according to standard procedures
  • DNA fragments of up to l lkbp in size are rendered blunt by treatment with exonuclease and DNA polymerase. and EcoRI linkers added Fragments are hgated mto the vector Lambda ZapII that has been cut with EcoRI.
  • 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 mto library vectors (e g . Rsal, Pall, Alul, Bshl235I), and such fragments are size-fractionated according to standard procedures EcoRI linkers are hgated to the DNA and the fragments then hgated into the vector Lambda ZapII that have been cut with EcoRI, the library packaged by standard procedures, and E coli infected with the packaged library
  • the library is amplified by standard procedures

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Abstract

The invention provides nrdD polypeptides and polynucleotides encoding nrdD polypeptides and methods for producing such polypeptides by recombinant techniques. Also provided are methods for utilizing nrdD polypeptides to screen for antibacterial compounds.

Description

NrdD
FIELD OF THE INVENTION
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 In particular, the invention relates to polynucleotides and polypeptides of the nrdD (anaerobic πbonucleotide tπphosphate reductase) family, as well as their variants, hereinafter referred to as "nrdD," "nrdD polynucleotιde(s)," and "nrdD polypeptιde(s)" as the case may be
BACKGROUND OF THE INVENTION
It is particularly preferred to employ Staphylococcal genes and gene products as targets for the development of antibiotics The Staphylococci make up a medically important genera of microbes They are known to produce two types of disease, invasive and toxigenic Invasive infections are 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 There are at least three clinical conditions resulting from the toxigenic properties of Staphylococci The manifestation of these diseases result from the actions of exotoxins as opposed to tissue invasion and bacteremia These conditions include Staphylococcal food poisoning, scalded skin syndrome and toxic shock syndrome
The frequency of Staphylococcus aureus infections has πsen dramatically in the past 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 which 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, high throughput genome- or gene-based biology This approach is rapidly superseding earlier approaches based on "positional cloning" and other methods Functional genomics relies heavily on the various tools of bioinformatics to identify gene sequences of potential interest from the many molecular biology databases now available as well as from other sources There is a continuing and significant need to identify and characteπze further genes and other polynucleotides sequences and their related polypeptides. as targets for drug discovery Clearly, there exists a need for polynucleotides and polypeptides. such as the nrdD embodiments of the invention, that have a present benefit of, among other things, bemg useful to screen compounds for antibiotic activity Such factors are also useful to determine their role in pathogenesis of infection, dysfunction and disease There is also a need for identification and characteπzation of such factors and their antagonists and agonists to find ways to prevent, ameliorate or correct such infection, dysfunction and disease
Certain of the polypeptides of the invention possess significant amino acid sequence homology to a known nrdD protein
SUMMARY OF THE INVENTION
The present invention relates to nrdD, m particular nrdD polypeptides and nrdD polynucleotides. recombinant materials and methods for their production In another aspect, the invention relates to methods for using such polypeptides and polynucleotides, including the treatment of microbial diseases, amongst others In a further aspect, 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 compounds In a still further aspect, the invention relates to diagnostic assays for detecting diseases associated with microbial infections and conditions associated with such infections, such as assays for detecting nrdD expression or activity
Various changes and modifications within the spiπt and scope of the disclosed invention will become readily apparent to those skilled in the art from reading the following descriptions and from reading the other parts of the present disclosure
DESCRIPTION OF THE INVENTION
The invention relates to nrdD polypeptides and polynucleotides as descπbed in greater detail below In particular, the invention relates to polypeptides and polynucleotides of a nrdD of Staphylococcus aureus. which is related by arruno acid sequence homology to nrdD polypeptide The invention relates especially to nrdD having the nucleotide and amino acid sequences set out in Table 1 as SEQ ID NO 1 and SEQ ID NO 2 respectively
TABLE 1 NrdD Polynucleotide and Polypeptide Sequences
(A) Staphylococcus aureus nrdD polynucleotide sequence [SEQ ID NO 1] 5 ' -ATGAATCAAATCGAAGAAGCATTAACGGGTTTGATTTCTAAAGATCCTGC TATTGTTAACGAAAATGCCAACAAAGATAGTGATACATTTTCAACAATGA
GAGATTTAACAGCAGGTATCGTTTCTAAATCTTACGCATTAAATTATTTA
TTACCAAAGCACGTTGCAGATGCACATCAAAGAGGGGACATACATTTTCA
CGACTTAGATTATCATCCATTCCAACCGTTAACAAACTGTTGTTTAATAG ATGCTAAAAATATGCTACATAATGGATTTGAAATAGGCAACGCGAATGTA
ACTTCACCAAAATCAATACAAACTGCATCAGCGCAGCTTGTACAAATTAT
AGCCAATGTTTCCAGCAGTCAATATGGTGGCTGTACGGTTGACCGCGTTG
ACGAATTACTTAGTACATATGCACGACATAATGAAGAACAACATAGAAAT
ATCGCAAAGCAATTTGTCAAAGAATCTGAAATTGATCGTTATGTTGATCA ACAAGTCACTAAAGACATCAATGATGCGATTGAAAGTCTAGAATATGAAA
TTAATACCTTATATACATCTAATGGACAGACACCTTTTGTAACATTAGGA
TTCGGCTTAGGTACAGATCATTTAAGTCGCAAAATTCAACAAGCTATCTT
AAATACTCGTATCAAAGGCTTAGGAAAAGACCGCACGACAGCGATTTTCC
CAAAACTTGTATTTTCAATTAAAAAAGGAACCAACTTTAGTCCGCAAGAT CCGAACTATGACATTAAACAACTAGCATTAAAGTGTTCAACGAAACGTAT
GTATCCAGATATTTTAAATTATGACAAACTCGTAGAAATATTAGGTGATT
TCAAAGCGCCAATGGGTTGTCGTTCATTTTTACCAAGTTGGAAAGATGCG
GAAGGTCATTTTGAAAATAATGGTCGTTGTAATCTTGGTGTTGTTACACT
TAATTTACCTAGAATGGCATTAGAATCTGCCGGTAATATGACGAAATTCT GGGAAATCTTTTATGAACGTATCGATGTGTTACATGATGCATTACTTTAT
CGTATAAATCGTTTGAAAGATGCTGTACCGAATAACGCACCAATTTTATA
TAAAAGTGGCGCTTTTAACTATAAATTAAAAGAAACAGATGATGTTGCTG
AGTTATTTAAAAATAAACGTGCAACGATTTCAATGGGCTATATAGGGTTG
TATGAAACAGCTACTGTTTTCTATGGTCCAGACTGGGAAACATCTCAAGA AGCAAAAGCATTTACGCTTGAAATTCTTAAAGAAATGAAACGTTATCAAA
CGAAATGGACAGAATTATATGACATTTGGTTCAGTATTTACAGTACGCCG
AGTGAATCGCTAACGGATCGTTTTTGTCGTTTAGACCAAGAGAGATTTGG
AGATATTAAAGACATTACAGATAAAGGATATTATCAAAACTCTTTCCATT
ATGATGTACGTAAAGATGTTACACCTTTTGAAAAGTTAGATTTTGAAAAA GATTATCCTTATTATGCGAGTGGTGGTTTCATTCACTATTGTGAGTATCC
GAAATTGCAACACAATTTGAAAGCACTAGAAGCGGTATGGGACTACTCTT
ATGACAAAGTTGGTTACTTAGGTACAAATATTCCGATTGATCATTGTTAT
GAATGTGATTACGATGGAGATTTTGAAGCAACTGAAAAAGGATTTAAATG
CCCGAACTGTGGCAATGATAATCCTAAAACAGTTGATGTCGTTAAACGAA CATGTGGTTACTTAGGCAATCCAGTTCAACGTCCAGTAATTAAAGGCCGT
CATAAAGAAATTTGCGCACGAGTAAAACATATGAAAGCGCCTAAAGAATGA
(B) Staphylococcus aureus nrdD polypeptide sequence deduced from a polynucleotide sequence in this table [SEQ ID NO:2].
NH -MNQIEEALTG I SKDPAIVNENANKDSDTFSTMRDLTAGIVSKSYALNYL LPKHVADAHQRGDIHFHDLDYHPFQPLTNCCLIDAKNMLHNGFEIGNANV TSPKSIQTASAQLVQIIANVSSSQYGGCTVDRVDELLSTYARHNEEQHRN IAKQFVKESEIDRYVDQQVTKDINDAIESLEYEINTLYTSNGQTPFVTLG FGLGTDH SRKIQQAILNTRIKGLGKDRTTAIFPKLVFSIKKGTNFSPQD PNYDIKQLALKCSTKRMYPDILNYDKLVEILGDFKAPMGCRSFLPS KDA EGHFENNGRCNLGWTLNLPRMALESAGN TKFWEIFYERIDVLHDALLY RINRLKDAVPNNAPILYKSGAFNYKLKETDDVAELFKNKRATISMGYIGL YETATVFYGPD ETSQEAKAFTLEILKEMKRYQTKWTELYDI FSIYSTP SESLTDRFCRLDQERFGDIKDITDKGYYQNSFHYDVRKDλTPFEKLDFEK DYPYYASGGFIHYCEYPKLQHNLKALEAV DYSYDKVGYLGTNIPIDHCY ECDYDGDFEATEKGFKCPNCGNDNPKTVDWKRTCGYLGNPVQRPVIKGR HKEICARVKHMKAPKE -COOH
Deposited materials
A deposit containing a Staphylococcus aureus WCUH 29 strain has been deposited with the National Collections of Industrial and Marine Bactena Ltd (herein "NCIMB"), 23 St Machar Dnve, Aberdeen AB2 1RY, Scotland on 11 September 1995 and assigned NCIMB Deposit No 40771, and referred to as 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 contains the full length nrdD gene The sequence of the polynucleotides contained in the deposited strain, as well as the amino acid sequence of any polypeptide encoded thereby, are controlling in the event of am conflict with any description of sequences herein The deposit of the deposited strain has been made under the terms of the Budapest Treaty on the
International Recognition of the Deposit of Micro-organisms for Purposes of Patent Procedure The strain will be irrevocably and without restnction or condition released to the public upon the issuance of a patent The deposited strain is provided merely as convenience to those of skill in the art and is not an admission that a deposit is required for enablement, such as that required under 35 U S C §112 A license may be required to make, use or sell the deposited strain, and compounds deπved therefrom, and no such license is hereby granted
In one aspect of the invention there is provided an isolated nucleic acid molecule encoding a mature polypeptide expressible by the Staphylococcus aureus WCUH 29 strain, which polypeptide is contained in the deposited strain Further provided by the invention are nrdD polynucleotide sequences in the deposited strain, such as DNA and RNA. and amino acid sequences encoded thereby Also provided by the invention are nrdD polypeptide and polynucleotide sequences isolated from the deposited stram Polypeptides
NrdD polypeptide of the invention is substantially phylogenetically related to other proteins of the nrdD (anaerobic πbonucleotide tπphosphate reductase) family
In one aspect of the invention there are provided polypeptides of Staphylococcus aureus referred to herein as "nrdD" and "nrdD polypeptides" as well as biologically, diagnostically, prophylactically, clinically or therapeutically useful variants thereof, and compositions compπsmg the same
Among the particularly preferred embodiments of the invention are variants of nrdD polypeptide encoded by naturally occurring alleles of the nrdD gene
The present mvention further provides for an isolated polypeptide which (a) comprises or consists of an ammo acid sequence which has at least 70% identit , preferably at least 80% identity, more preferably at least 90% identity, yet more preferably at least 95 % identity, most preferably at least 97-99% or exact identity, to that of SEQ ID NO 2 over the entire length of SEQ ID NO 2,
(b) a polypeptide encoded by an isolated polynucleotide comprising or consistmg of a polynucleotide sequence which has at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, yet more preferably at least 95% identity, even more preferably at least 97-99% or exact identity to SEQ ID NO 1 over the entire length of SEQ ID NO 1,
(c) a polypeptide encoded by an isolated polynucleotide comprising or consisting of a polynucleotide sequence encoding a polypeptide which has at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, yet more preferably at least 95% identity, even more preferably at least 97-99% or exact identity, to the amino acid sequence of SEQ ID NO 2, over the entire length of SEQ ID NO 2
The polypeptides of the mvention include a polypeptide of Table 1 [SEQ ID NO 2] (in particular the mature polypeptide) as well as polypeptides and fragments, particularly those which have the biological activity of nrdD. and also those which have at least 70% identity to a polypeptide of Table 1 [SEQ ID NO l]or the relevant portion, preferably at least 80% identity to a polypeptide of Table 1 [SEQ ID NO 2and more preferably at least 90% identity to a polypeptide of Table 1 [SEQ ID NO 2] and still more preferably at least 95% identity to a polypeptide of Table 1 [SEQ ID NO 2] and also include portions of such polypeptides with such portion of the polypeptide generally containing at least 30 ammo acids and more preferably at least 50 ammo acids
The mvention also mcludes a polypeptide consistmg of or compπsmg a polypeptide of the formula
X-(R1)m-(R2)-(R3)n-Y wherein, at the ammo terminus. X is hydrogen, a metal or any other moiety described herem for modified polypeptides. and at the carboxyl terminus, 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 mteger between 1 and 1000 or zero, n is an integer between 1 and 1000 or zero, and R2 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, R2 is oriented so that its amino terminal amino acid residue is at the left, covalently bound to R] and its carboxy terminal amino acid residue is at the right, covalently bound to R3. Any stretch of arnino acid residues denoted by either Ri or R3, where m and or n is greater than 1, may be either a heteropolymer or a homopolymer, preferably a heteropolymer. Other preferred embodiments of the invention are provided where m is an integer between 1 and 50, 100 or 500, and n is an integer between 1 and 50, 100, or 500.
It is most preferred that a polypeptide of the invention is derived from Staphylococcus aureus, however, it may preferably be obtained from other organisms of the same taxonomic genus. A polypeptide of the invention may also be obtained, for example, from organisms of the same taxonomic family or order.
A fragment is a variant polypeptide having an arnino acid sequence that is entirely the same as part but not all of any amino acid sequence of any polypeptide of the invention. As with nrdD polypeptides, fragments may be "free-standing," or comprised within a larger polypeptide of which they form a part or region, most preferably as a single continuous region in a single larger polypeptide. Preferred fragments include, for example, truncation polypeptides having a portion of an amino acid sequence of Table 1 [SEQ ID NO:2], or of variants thereof, such as a continuous series of residues that includes an 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. Further preferred are fragments characterized by structural or functional attributes such as fragments that comprise alpha-helix and alpha-helix forming regions, beta-sheet and beta-sheet-foπriing regions, turn and turn-forming regions, coil and coil-forming regions, hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-foiming regions, substrate binding region, and high antigenic index regions.
Further preferred fragments include an isolated polypeptide comprising an amino acid sequence having at least 15, 20, 30, 40, 50 or 100 contiguous amino acids from the amino acid sequence of SEQ ID NO: 2, or an isolated polypeptide comprising an amino acid sequence having at least 15, 20, 30, 40, 50 or 100 contiguous amino acids truncated or deleted from the amino acid sequence of SEQ ID NO: 2.
Also preferred are biologically active fragments which are those fragments that mediate activities of nrdD, including those with a similar activity or an improved activity, or with a decreased undesirable activity. Also included are those fragments that are antigenic or immunogenic in an animal, especially in a human. Particularly preferred are fragments comprising receptors or domains of enzymes that confer a function essential for viability of Staphylococcus aureus or the ability to initiate, or maintain cause Disease in an individual, particularly a human. Fragments of the polypeptides of the mvention may be employed for producing the corresponding full- length polypeptide by peptide synthesis, therefore, these variants may be employed as intermediates for producmg the full-length polypeptides of the mvention
In addition to the standard single and triple letter representations for ammo acids, the term "X" or "Xaa" may also be used in describing certain polypeptides of the invention "X" and "Xaa" mean that any of the twenty naturally occurring amino acids may appear at such a designated position in the polypeptide sequence
Polynucleotides
It is an object of the mvention to provide polynucleotides that encode nrdD polypeptides. particularly polynucleotides that encode the polypeptide herem designated nrdD
In a particularly preferred embodiment of the mvention the polynucleotide comprises a region encoding nrdD polypeptides compπsmg a sequence set out m Table 1 [SEQ ID NO 1] which 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 which possesses it, such as Staphylococcus aureus As a further aspect of the mvention there are provided isolated nucleic acid molecules encoding and or expressmg nrdD polypeptides and polynucleotides, particularly Staphylococcus aureus nrdD polypeptides and polynucleotides, including, for example, unprocessed RNAs, πbozyme RNAs. mRNAs, cDNAs, genomic DNAs, B- and Z-DNAs Further embodiments of the mvention mclude biologically, diagnostically, prophylactically, clinically or therapeutically useful polynucleotides and polypeptides, and vaπants thereof, and compositions compπsmg the same
Another aspect of the mvention relates to isolated polynucleotides. including at least one full length gene, that encodes a nrdD polypeptide having a deduced ammo acid sequence of Table 1 [SEQ ID NO 2] and polynucleotides closely related thereto and vaπants thereof
In another particularly preferred embodiment of the invention there is a nrdD polypeptide from Staphylococcus aureus comprising or consisting of an amino acid sequence of Table 1 [SEQ ID NO 2 or a variant thereof
Usmg the information provided herem, such as a polynucleotide sequence set out m Table 1 [SEQ ID NO 1], a polynucleotide of the mvention encoding nrdD polypeptide may be obtained usmg standard cloning and screening methods, such as those for cloning and sequencmg chromosomal DNA fragments from bacteπa usmg Staphylococcus aureus WCUH 29 cells as startmg mateπal, followed by obtairung a full length clone For example, to obtain a polynucleotide sequence of the invention, such as a polynucleotide sequence given in Table 1 [SEQ ID NO 1]. typically a library of clones of chromosomal DNA of Staphylococcus aureus WCUH 29 m E colt or some other suitable host is probed with a radiolabeled o gonucleotide, preferably a 17-mer or longer, derived from a partial sequence Clones carrying DNA identical to that of the probe can then be distinguished usmg stringent hybridization conditions By sequencing the individual clones thus identified by hybridization with sequencmg primers designed from the original polypeptide or polynucleotide sequence it is then possible to extend the polynucleotide sequence m both directions to determine a full length gene sequence Conveniently, such sequencing is performed, for example, using denatured double stranded DNA prepared from a plasmid clone Suitable techmques are described by Maniatis, T . Fπtsch, E F and Sambrook et al , MOLECULAR CLONING, A LABORATORY MANUAL, 2nd Ed . Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989) (see in particular Screening By Hybridization 1 90 and Sequencmg Denatured Double-Stranded DNA Templates 13 70) Direct genomic DNA sequencing may also be perfoπned to obtain a full length gene sequence Illustrative of the mvention. each polynucleotide set out m Table 1 [SEQ ID NO 1] was discovered m a DNA library deπved from Staphylococcus aureus WCUH 29
Moreover, each DNA sequence set out m Table 1 [SEQ ID NO 1] contains an open reading frame encoding a protein having 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 in the art The polynucleotide of SEQ ID NO 1, between nucleotide number 1 and the stop codon which begins at nucleotide number 1849 ofSEQ ID NO 1, encodes the polypeptide of SEQ ID NO 2 In a further aspect, the present mvention provides for an isolated polynucleotide compπsmg or consistmg of
(a) a polynucleotide sequence which has at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, yet more preferably 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 ,
(b) a polynucleotide sequence encoding a polypeptide which has at least 70% identity . preferably at least 80% identity, more preferably at least 90% identity, yet more preferably at least 95 % identity, even more preferably at least 97-99% or 100% exact, to the am o acid sequence of SEQ ED NO 2. over the entire length of SEQ ID NO 2 A pohnucleotide encoding a polypeptide of the present mvention, mcludmg homologs and orthologs from species other than Staphylococcus aureus, may be obtained by a process which compπses the steps of screening an appropπate library under stringent hybπdization conditions with a labeled or detectable probe consistmg of or compπsmg the sequence of SEQ ID NO 1 or a fragment thereof, and isolating a full-length gene and/or genomic clones containing said polynucleotide sequence The mvention provides a polynucleotide sequence identical over its entire length 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 encoding a leader or secretory sequence, a pre-, or pro- or prepro-protern sequence The polynucleotide of the mvention may also contain at least one non-codmg sequence, mcludmg for example, but not limited to at least one non-codmg 5' and 3' sequence, such as the transcπbed but non-translated sequences, termination signals (such as rho-dependent and rho-independent termination signals), πbosome binding sites, Kozak sequences, sequences that stabilize mRNA, rntrons, and polyadenylation signals The polynucleotide sequence may also compπse additional coding sequence encoding additional ammo acids For example, a marker sequence that facilitates puπfication of the fused polypeptide can be encoded In certam embodiments of the mvention, the marker sequence is a hexa- histidine peptide, as provided m the pQE vector (Qiagen, Ine ) and descπbed m Gentz et al , Proc Natl Acad Set , USA 86 821-824 (1989), or an HA peptide tag (Wilson et al , Cell 37 767 (1984), both of which may be useful m punfying polypeptide sequence fused to them Polynucleotides of the mvention also include, but are not limited to. polynucleotides compπsmg a structural gene and its naturally associated sequences that control gene expression
A prefeπed embodiment of the mvention is a polynucleotide of consistmg of or compπsmg nucleotide 1 to the nucleotide immediately upstream of or mcludmg nucleotide 1849 set forth m SEQ ID NO 1 of Table 1, both of which encode the nrdD polypeptide
The mvention also mcludes a polynucleotide consisting of or compπsmg a polynucleotide of the formula
X-(Rι)m-(R2)-(R3)n-Y wherein, at the 5' 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 integer between 1 and 3000 or zero . n is an integer between 1 and 3000 or zero, and R2 is a nucleic acid sequence or modified nucleic acid sequence of the invention, particularly a nucleic acid sequence selected from Table 1 or a modified nucleic acid sequence thereof In the polynucleotide formula above, R is oriented so that its 5' end nucleic acid residue is at the left, bound to R\ and its 3' end nucleic acid residue is at the πght, bound to R3 Any stretch of nucleic acid residues denoted by either R^ and/or R2, where m and or n is greater than 1 , may be either a heteropolymer or a homopolymer, preferably a heteropolymer Where, in a preferred embodiment, X and Y together define a covalent bond, the polynucleotide of the above formula is a closed, circular polynucleotide, which can be a double-stranded polynucleotide wherein the formula shows a first strand to which the second strand is complementary In another preferred embodiment m and/or n is an integer between 1 and 1000. 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
It is most prefeπed that a polynucleotide of the mvention is deπved from Staphylococcus aureus, however, it may preferably be obtained from other organisms of the same taxonomic genus A polynucleotide of the mvention may also be obtained, for example, from organisms of the same taxonomic family or order The term "polynucleotide encoding a polypeptide" as used herem encompasses polynucleotides that include a sequence encoding a polypeptide of the mvention, particularly a bactenal polypeptide and more particularly a polypeptide of the Staphylococcus aureus nrdD having 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 by integrated phage, an integrated insertion sequence, an integrated vector sequence, an mtegrated transposon sequence, or due to
RNA editing or genomic DNA reorganization) together with additional regions, that also may contain coding and or non-codmg sequences
The mvention further relates to vaπants of the polynucleotides descnbed herem that encode vaπants of a polypeptide havmg a deduced ammo acid sequence of Table 1 [SEQ ID NO 2] Fragments of a polynucleotides of the mvention may be used, for example, to synthesize full-length polynucleotides of the mvention
Further particularly prefeπed embodiments are polynucleotides encoding nrdD vaπants, that have the ammo acid sequence of nrdD 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, in any combination
Especially preferred among these are silent substitutions, additions and deletions, that do not alter the properties and activities of nrdD polypeptide
Further preferred embodiments of the mvention are polynucleotides that are at least 70% identical over their entire length to a polynucleotide encoding nrdD polypeptide havmg an ammo acid sequence set out in Table 1 [SEQ ID NO 2], and polynucleotides that are complementary to such polynucleotides Alternatively, most highly preferred are polynucleotides that compπse a region that is at least 80% identical over its entire length to a polynucleotide encoding nrdD polypeptide and polynucleotides complementary thereto In this regard, polynucleotides at least 90% identical over their entire length to the same are particularly prefeπed, and among these particularly prefeπed polynucleotides, those with at least 95% are especially preferred Furthermore, those with at least 97% are highly prefeπed among those with at least 95%, and among these those with at least 98% and at least 99% are particularly highly preferred, with at least 99% bemg the more prefeπed
Prefeπed embodiments are polynucleotides encoding polypeptides that retain substantially the same biological function or activity as the mature polypeptide encoded by a DNA of Table 1 [SEQ ID NO 1] In accordance with certam prefeπed embodiments of this mvention there are provided polynucleotides that hybndize, particularly under strmgent conditions, to nrdD polynucleotide sequences, such as those polynucleotides m Table 1
The mvention further relates to polynucleotides that hybndize to the polynucleotide sequences provided herem In this regard, the mvention especially relates to polynucleotides that hybndize under strmgent conditions to the polynucleotides descπbed herem As herem used, the terms "strmgent conditions" and "stringent hybndization conditions" mean hybndrzation occumng only if there is at least 95% and preferably at least 97% identity between the sequences A specific example of strmgent hybridization conditions is overnight incubation at 42°C in a solution comprising 50% formamide, 5x SSC (150mM NaCl, 15mM tπsodium citrate), 50 mM sodium phosphate (pH7 6). 5x Denhardt's solution, 10% dextran sulfate. and 20 micrograms/ml of denatured, sheared salmon sperm DNA, followed by washing the hybridization support in 0 lx SSC at about 65°C Hybridization and wash conditions are well known and exemplified in Sambrook, et al , Molecular Cloning A Laboratory Manual, Second Edition, Cold Spnng Harbor, N Y , (1989), particularly Chapter 11 therein Solution hybridization may also be used with the polynucleotide sequences provided by the invention The invention also provides a polynucleotide consistmg of or comprising a polynucleotide sequence obtained by screening an appropriate library containing the complete gene for a polynucleotide sequence set forth in SEQ ID NO 1 under stringent hybridization conditions with a probe havmg the sequence of said polynucleotide sequence set forth in SEQ ID NO 1 or a fragment thereof, and isolating said polynucleotide sequence Fragments useful for obtaining such a polynucleotide include, for example, probes and primers fully described elsewhere herein
As discussed elsewhere herem regarding polynucleotide assays of the mvention, for instance, the polynucleotides of the mvention, may be used as a hybndization probe for RNA, cDNA and genomic DNA to isolate full-length cDNAs and genomic clones encoding nrdD and to isolate cDNA and genomic clones of other genes that have a high identity, particularly high sequence identity, to the nrdD gene Such probes generally will compπse at least 15 nucleotide residues or base pairs Preferably, such probes will have at least 30 nucleotide residues or base pairs and may have at least 50 nucleotide residues or base pairs Particularly prefeπed 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 nrdD gene may be isolated by screening usmg a DNA sequence provided m Table 1 [SEQ ID NO 1] to synthesize an oligonucleotide probe A labeled oligonucleotide havmg a sequence complementary to that of a gene of the mvention is then used to screen a library of cDNA, genomic DNA or rnRNA to determine which members of the library the probe hybndizes to
There are several methods available and well known to those skilled in the art to obtain full- length DNAs. or extend short DNAs, for example those based on the method of Rapid Amplification of cDNA ends (RACE) (see. for example. Frohman, et al . PNAS USA 85 8998-9002. 1988) Recent modifications of the technique, exemplified by the Marathon™ technology (Clontech Laboratories Ine ) for example, have significantly simplified the search for longer cDNAs In the Marathon™ technology, cDNAs have been prepared from mRNA extracted from a chosen tissue and an 'adaptor' sequence ligated onto each end Nucleic acid amplification (PCR) is then carried out to amplify the "missing" 5' end of the DNA using a combination of gene specific and adaptor specific oligonucleotide primers The PCR reaction is then repeated using "nested" primers, that is, primers designed to anneal withm 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 known gene sequence) The products of this reaction can then be analyzed by DNA sequencing and a full-length DNA constructed either b> joining the product directly to the existing DNA to give a complete sequence, or carrying out a separate full-length PCR using the new sequence information for the design of the 5' primer
The polynucleotides and polypeptides of the mvention may be employed, for example, as research reagents and mateπals for discovery of treatments of and diagnostics for diseases, particularly human diseases, as further discussed herem relating to polynucleotide assays The polynucleotides of the invention that are ohgonucleotides deπved from a sequence of Table 1
[SEQ ID NOS 1 or 2] may be used m the processes herein as described, but preferably for PCR, to determine whether or not the polynucleotides identified herein m whole or m part are transcπbed in bacteria in infected tissue It is recognized that such sequences will also have utility m diagnosis of the stage of infection and type of infection the pathogen has attained The mvention also provides polynucleotides that encode a polypeptide that is the mature protem plus additional ammo or carboxyl-teiminal ammo acids, or ammo acids mtenor to the mature polypeptide (when the mature form has more than one polypeptide chain, for instance) Such sequences may play a role in processmg of a protem from precursor to a mature form, may allow 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 am o acids may be processed away from the mature protem by cellular enzymes
For each and every polynucleotide of the mvention there is provided a polynucleotide complementary to it It is prefeπed that these complementary polynucleotides are fully complementary to each polynucleotide with which the\ are complementary
A precursor protem, havmg a mature form of the polypeptide fused to one or more prosequences may be an mactive form of the polypeptide When prosequences are removed such mactive precursors generally are activated Some or all of the prosequences may be removed before activation Generally, such precursors are called proproteins
In addition to the standard A. G, C. T/U representations for nucleotides. the term "N" may also be used in describing certain polynucleotides of the invention "N" means that any of the four DNA or RNA nucleotides may appear at such a designated position m the DNA or RNA sequence, except it is preferred that N is not a nucleic acid that when taken m combination with adjacent nucleotide positions, when read m the correct reading frame, would have the effect of generating a premature termination codon in such reading frame
In sum. a polynucleotide of the mvention may encode a mature protem, a mature protem plus a leader sequence (which mav be refeπed to as a preprotem). a precursor of a mature protem havmg one or more prosequences that are not the leader sequences of a preprotem, or a preproprotem. which is a precursor to a proprotein, having a leader sequence and one or more prosequences, which generally are removed during processing steps that produce active and mature forms of the polypeptide Vectors, Host Cells, Expression Systems The mvention also relates to vectors that compπse a polynucleotide or polynucleotides of the mvention, host cells that are genetically engineered with vectors of the mvention and the production of polypeptides of the mvention by recombinant techniques Cell-free translation systems can also be employed to produce such proteins usmg RNAs deπved from the DNA constructs of the mvention
Recombinant polypeptides of the present mvention may be prepared by processes well known m those skilled m the art from genetically engmeered host cells compπsmg expression systems Accordingly, in a further aspect, the present mvention relates to expression systems which compnse a polynucleotide or polynucleotides of the present mvention, to host cells which are genetically engmeered with such expression systems, and to the production of polypeptides of the mvention by recombinant techmques
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 m many standard laboratory manuals, such as Davis, et al , BASIC METHODS IN MOLECULAR BIOLOGY, (1986) and Sambrook, et al , MOLECULAR CLONING A LABORATORY MANUAL, 2nd Ed , Cold Spring Harbor Laboratory Press Cold Spring Harbor, N Y (1989). such as, calcium phosphate transfection, DEAE-dextran mediated transfection. transvection, micromjection, catiomc lipid-mediated transfection, electroporation, transduction. scrape loading, ballistic mtroduction and infection
Representative examples of appropπate hosts mclude bacteπal cells, such as cells of streptococci, staphylococci. enterococci E coh, streptomyces. cyanobactena. Bacillus subtths, 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 Drosophύa 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 gvmnosperm or angiosperm
A great vaπety of expression systems can be used to produce the polypeptides of the mvention Such vectors mclude, among others, chromosomal-, episomal- and virus-denved vectors, for example, vectors denved from bacteπal plasmids, from bacteπophage. from transposons, from yeast episomes. from insertion elements, from yeast chromosomal elements, from viruses such as baculovrruses, papova viruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses, picornavrruses and retroviruses, and vectors deπved from combinations thereof, such as those deπved from plasmid and bacteπophage genetic elements, such as cosmids and phagemids The expression system constructs may contain control regions that regulate as well as engender expression Generally, any system or vector suitable to maintain, propagate or express polynucleotides and/or to express a polypeptide m a host may be used for expression m this regard The appropπate DNA sequence may be inserted mto the expression system by any of a vaπety of well-known and routme techmques. such as, for example, those set forth m Sambrook et al , MOLECULAR CLONING, A LABORATORY MANUAL, (supra) In recombinant expression systems m eukaryotes, for secretion of a translated protem mto the lumen of the endoplasmic reticulum, mto the penplasmic space or mto the extracellular environment, appropπate secretion signals may be incorporated mto the expressed polypeptide These signals may be endogenous to the polypeptide or they may be heterologous signals
Polypeptides of the mvention can be recovered and purified from recombinant cell cultures by well- known methods mcludmg ammonium sulfate or ethanol precipitation, acid extraction, amon or cation exchange chromatography, phosphocellulose chromatography, hydrophobic mteraction chromatography, affinity chromatography, hydroxylapatite chromatography, and lectm chromatography Most preferably, high performance liquid chromatography is employed for purification Well known techmques for refolding protem may be employed to regenerate active conformation when the polypeptide is denatured during isolation and or purification
Diagnostic, Prognostic, Serotyping and Mutation Assays
This mvention is also related to the use of nrdD polynucleotides and polypeptides of the mvention for use as diagnostic reagents Detection of nrdD polynucleotides and or polypeptides m a eukaryote, particularly a mammal, and especially a human, will provide a diagnostic method for diagnosis of disease, staging of disease or response of an infectious orgamsm to drugs Eukaryotes, particularly mammals, and especially humans, particularly those infected or suspected to be infected with an orgamsm compπsmg the nrdD gene or protem may be detected at the nucleic acid or ammo acid level by a vaπety of well known techniques as well as by methods provided herem
Polypeptides and polynucleotides for prognosis, diagnosis or other analysis may be obtained from a putatively infected and/or infected mdividual's bodily mateπals Polynucleotides from any of these sources, particularly DNA or RNA, may be used directly for detection or may be amplified enzymatically by usmg PCR or any other amplification technique pπor to analysis RNA, particularly mRNA, cDNA and genomic DNA may also be used m the same ways Usmg amplification, characteπzation of the species and stram of infectious or resident orgamsm present m an individual, may be made by an analysis of the genotype of a selected polynucleotide of the orgamsm Deletions and insertions can be detected by a change m size of the amphfied product m compaπson to a genotype of a reference sequence selected from a related orgamsm, preferably a different species of the same genus or a different stram of the same species Point mutations can be identified hybπdizmg amplified DNA to labeled nrdD 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 detectmg differences m meltmg temperatures or renaturation k etics Polynucleotide sequence differences may also be detected by 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 . Science, 230 1242 (1985) 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 Natl Acad Set , USA, 85 4397-4401 (1985)
In another embodiment, an aπay of oligonucleotides probes compnsmg nrdD nucleotide sequence or fragments thereof can be constructed to conduct efficient screening of, for example, genetic mutations, serotype, taxonomic classification or identification Anay technology methods are well known and have general applicability and can be used to address a vanety of questions m molecular genetics mcludmg gene expression, genetic linkage, and genetic vaπabihty (see, for example. Chee et al , Science, 274 610 (1996))
Thus m another aspect, the present mvention relates to a diagnostic kit which compnses
(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 polypeptide of the present invention, preferably the polypeptide of SEQ ID NO 2 or a fragment thereof, or
(d) an antibod} to a polypeptide of the present invention, preferably to the polypeptide of SEQ ID NO 2 It will be appreciated that in any such kit, (a), (b), (c) or (d) may comprise a substantial component Such a kit will be of use in diagnosmg a disease or susceptibility to a Disease, among others This mvention also relates to the use of polynucleotides of the present mvention as diagnostic reagents Detection of a mutated form of a polynucleotide of the mvention, preferable, SEQ ID NO 1, which 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 susceptibihty to a disease, which 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 vanety of techmques, such as those descnbed elsewhere herem
The nucleotide sequences of the present mvention are also valuable for orgamsm chromosome identification The sequence is specifically targeted to, and can hybndize with, a particular location on an organism's chromosome, particularly to a Staphylococcus aureus chromosome The mapping of relevant sequences to chromosomes according to the present mvention may be an important step m coπelating those sequences with pathogenic potential and or an ecological mche of an orgamsm and or drug resistance of an organism, as ell as the essentiality of the gene to the orgamsm Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be coπelated with genetic map data Such data may be found on-lme m a sequence database The relationship between genes and diseases that have been mapped to the same chromosomal region are then identified through known genetic methods, for example, through linkage analysis (coinheπtance of physically adjacent genes) or matmg studies, such as by conjugation The differences m a polynucleotide and or polypeptide sequence between organisms possessing a first phenotype and organisms possessing a different, second different phenotype can also be determined If a mutation is observed m some or all organisms possessing the first phenotype but not in any organisms possessing the second phenotype, then the mutation is likely to be the causative agent of the first phenotype Cells from an orgamsm carrying mutations or polymorphisms (allelic vanations) in a polynucleotide and or polypeptide of the mvention may also be detected at the polynucleotide or polypeptide level by a vanety of techniques, to allow for serotyprng, for example For example, RT-PCR can be used to detect mutations in the RNA It is particularly prefeπed 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 As an example. PCR primers complementary to a polynucleotide encoding mdD polypφtide can be used to identify and analyze mutations Examples of representative primers are shown below m Table 2
Table 2 Primers for amplification of nrdD polynucleotides SEQ ID NO PRIMER SEQUENCE
3 5'-ATGAATCAAATCGAAGAAGC-3'
4 5'-TCATTCTTTAGGCGCTTTCA-3'
The mvention also includes pπmers of the formula
X-(Rι)m-(R2)-(R3)n-Y wherein, at the 5' end of the molecule, X is hydrogen, a metal or a modified nucleotide residue, and at the 3' end of the molecule. Y is hydrogen, a metal or a modified nucleotide residue, Rj and R3 are any nucleic acid residue or modified nucleotide residue, m is an mteger between 1 and 20 or zero , n is an mteger between 1 and 20 or zero, and R is a pnmer sequence of the mvention, particularly a pπmer sequence selected from Table 2 In the polynucleotide formula above R is oπented so that its 5' end nucleotide residue is at the left, bound to Ri and its 3' end nucleotide residue is at the nght, bound to R3 Any stretch of nucleic acid residues denoted by either R group, where m and/or n is greater than 1, may be either a heteropolymer or a homopolymer, preferably a heteropolymer bemg complementary to a region of a polynucleotide of Table 1 In a prefeπed embodiment m and/or n is an mteger between 1 and 10. The mvention further provides these primers with 1, 2. 3 or 4 nucleotides removed from the 5' and/or the 3' end These pπmers may be used for among other things, amplifying nrdD DNA and/or RNA isolated from a sample deπved from an individual, such as a bodily matenal The primers may be used to amplify a polynucleotide isolated from an infected mdividual, such that the polynucleotide may then be subject to vanous techmques for elucidation of the polynucleotide sequence In this way, mutations in the polynucleotide sequence may be detected and used to diagnose and/or prognose the infection or its stage or course, or to serotype and/or classify the infectious agent
The mvention further provides a process for diagnosing, disease, preferably bacterial infections, more preferably infections caused by Staphylococcus aureus, compnsmg determining from a sample denved 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 nrdD 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 In addition, a diagnostic assay in accordance with the mvention for detectmg over-expression of mdD 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 mdD polypeptide, m a sample deπved from a host, such as a bodily mateπal, are well-known to those of skill m the art Such assay methods mclude radioimmunoassays, competitive-binding assays, Western Blot analysis, antibody sandwich assays, antibody detection and ELISA assays Differential Expression
The polynucleotides and polynucleotides of the mvention may be used as reagents for differential screening methods There are many differential screening and differential display methods known m the art in which the polynucleotides and polypeptides of the mvention may be used For example, the differential display technique is described by Chuang et al , J Bactenol 175 2026-2036 (1993) This method identifies those genes which are expressed in an organism by identifying mRNA present using randomly- primed RT-PCR By comparing pre-mfection and post infection profiles, genes up and down regulated during infection can be identified and the RT-PCR product sequenced and matched to ORF "unknowns " In Vivo Expression Technology (IVET) is described by Camilli et al , Proc Nat'l Acad Sci USA 91 2634-2638 (1994) IVET identifies genes up-regulated durmg infection when compared to laboratory cultivation, implying an important role in infection ORFs identified by this technique are implied to have a significant role in infection establishment and/or maintenance In this technique random chromosomal fragments of target organism are cloned upstream of a promoter-less recombinase gene m a plasmid vector This construct is introduced mto the target organism which carries an antibiotic resistance gene flanked by resolvase sites Growth in the presence of the antibiotic removes from the population those fragments cloned mto the plasmid vector capable of supporting transcnption of the recombinase gene and therefore have caused loss of antibiotic resistance The resistant pool is introduced mto a host and at various times after infection bacteria may be recovered and assessed for the presence of antibiotic resistance The chromosomal fragment carried by each antibiotic sensitive bactenum should carry a promoter or portion of a gene normally upregulated duπng infection Sequencing upstream of the recombinase gene allows identification of the up regulated gene
RT-PCR may also be used to analyze gene expression patterns For RT PCR usmg the polynucleotides of the invention, messenger RNA is isolated from bacterial infected tissue, e g , 48 hour munne lung infections, and the amount of each mRNA species assessed by reverse transcription of the RNA sample primed with random hexanucleotides followed by PCR with gene specific primer pairs The determination of the presence and amount of a particular mRNA species by quantification of the resultant PCR product provides information on the bacterial genes which are transcribed in the infected tissue Analysis of gene transcription can be carried out at different times of infection to gam a detailed knowledge of gene regulation in bacterial pathogenesis allowing for a clearer understanding of which gene products represent targets for screens for antibacterials Because of the gene specific nature of the PCR primers employed it should be understood that the bacterial mRNA preparation need not be free of mammalian RNA This allows the investigator to carry out a simple and quick RNA preparation from infected tissue to obtain bacterial mRNA species which are very short lived m the bactenum (in the order of 2 minute halflives) Optimally the bacterial mRNA is prepared from infected munne lung tissue by mechanical disruption in the presence of TRlzole (GIBCO-BRL) for very short periods of time, subsequent processing according to the manufacturers of TRlzole reagent and DNAase treatment to remove contaminating DNA Preferably the process is optimized by finding those conditions which give a maximum amount of Staphylococcus aureus 16S πbosomal RNA as detected by probing Northerns with a suitably labeled sequence specific oligonucleotide probe Typically a 5' dye labeled pnmer is used in each PCR primer pair in a PCR reaction which is terminated optimally between 8 and 25 cycles The PCR products are separated on 6% polyacrylamide gels with detection and quantification using GeneScanner (manufactured by ABI)
Gridding and Polynucleotide Subtraction
Methods have been described for obtaining information about gene expression and identity using so called "high density DNA arrays" or grids See, e g , M Chee et al , Science, 274 610-614 (1996) and other references cited therein Such gnddmg assays have been employed to identify certain novel gene sequences, referred to as Expressed Sequence Tags (EST) (Adams et a . Science, 252 1651- 1656 (1991)) A variety of techniques have also been descπbed for identifying particular gene sequences on the basis of their gene products For example, see International Patent Application No W091/07087, published May 30. 1991 In addition, methods have been described for the amplification of desired sequences For example, see International Patent Application No WO 91/17271. published November 14, 1991
The polynucleotides of the invention may be used as components of polynucleotide arrays, preferably high density arrays or grids These high density arrays are particularly useful for diagnostic and prognostic purposes For example, a set of spots each comprising a different gene, and further comprising a polynucleotide or polynucleotides of the invention, may be used for probing, such as usmg hybridization or nucleic acid amplification, using a probes obtained or derived from a bodily sample, to determine the presence of a particular polynucleotide sequence or related sequence m an mdividual Such a presence may indicate the presence of a pathogen, particularly Staphylococcus aureus, and may be useful in diagnosing and/or prognosmg disease or a course of disease A grid compnsing a number of variants of the polynucleotide sequence of SEQ ID NO 1 are preferred Also preferred is a comprising a number of variants of a polynucleotide sequence encoding the polypeptide sequence of SEQ ID NO 2
Antibodies The polypeptides and polynucleotides of the mvention or vanants thereof, or cells expressmg the same can be used as lmmunogens to produce antibodies immunospecific for such polypeptides or polynucleotides respectively
In certam prefeπed embodiments of the mvention there are provided antibodies against nrdD polypeptides or polynucleotides Antibodies generated against the polypeptides or polynucleotides of the mvention can be obtained by admmistenng the polypeptides and or polynucleotides of the mvention, or epitope-beaπng fragments of either or both, analogues of either or both, or cells expressmg either or both, to an animal, preferably a nonhuman, usmg routme protocols For preparation of monoclonal antibodies, any technique known in the art that provides antibodies produced by continuous cell lme cultures can be used Examples mclude vaπous techniques, such as those m Kohler. G and Milstem, C , Nature 256 495-497 (1975), Kozbor et al . Immunology Today 4 72 (1983). Cole et al , pg 77-96 m MONOCLONAL ANTIBODIES AND CANCER THERAPY. Alan R Liss, Ine (1985)
Techmques for the production of single cham antibodies (U S Patent No 4.946,778) can be adapted to produce single cham antibodies to polypeptides or polynucleotides of this mvention Also, transgenic mice, oi other orgamsms such as other mammals, may be used to express humanized antibodies immunospecific to the polypeptides or polynucleotides of the mvention
Alternatively, phage display technology may be utilized to select antibody genes with binding activities towards a polypeptide of the invention either from repertoires of PCR amplified v-genes of lymphocytes from humans screened for possessing anti-nrdD or from naive hbranes (McCafferty, et al , (1990). Nature 348, 552-554. Marks, et al , (1992) Biotechnology 10, 779-783) The affimty of these antibodies can also be improved by. for example, chain shuffling (Clackson et al , (1991) Nature 352
628)
The above-descπbed antibodies may be employed to isolate or to identify' clones expressmg the polypeptides or polynucleotides of the mvention to punfy the polypeptides or polynucleotides by, for example, affinity chromatography
Thus among others, antibodies agamst nrdD-polypeptide or mdD-polynucleotide may be employed to treat infections, particularly bacteπal infections
Polypeptide variants include antigemcally, epitopically or immunologically equivalent vanants form a particular aspect of this invention A polypeptide or polynucleotide of the invention, such as an antigemcally or immunologically equivalent derivative or a fusion protein of the polypeptide is used as an antigen to immunize a mouse or other animal such as a rat or chicken The fusion protem may provide stability to the polypeptide The antigen may be associated, for example by conjugation, with an lmmunogemc earner protem for example bovine serum albumin, keyhole limpet haemocyanm or tetanus toxoid Alternatively, a multiple antigenic polypeptide comprising multiple copies of the polypeptide, or an antigemcally or immunologically equivalent polypeptide thereof may be sufficiently antigenic to improve lmmunogenicity so as to obviate the use of a earner
Preferably, the antibody or variant thereof is modified to make it less lmmunogemc in the individual For example, if the individual is human the antibody may most preferably be "humanized," where the comphmentanty determining region or regions of the hybridoma-deπved antibody has been transplanted into a human monoclonal antibody, for example as described m Jones et al (1986), Nature
321 522-525 or Tempest et al , (1991) Biotechnology 9, 266-273
In accordance with an aspect of the invention, there is provided the use of a polynucleotide of the mvention for therapeutic or prophylactic purposes, in particular genetic immunization Among the particularh prefeπed embodiments of the mvention aie naturally occurring allelic vanants of nrdD polynucleotides and polypeptides encoded thereby
The use of a polynucleotide of the invention in genetic immunization will preferably employ a suitable delivery method such as direct injection of plasmid DNA into muscles (Wolff et al , Hum Mol
Genet (1992) 1 363. Manthorpe et al , Hum Gene Ther (1983) 4 419), delivery of DNA complexed vλith specific protein carriers (Wu et al . J Biol Chem (1989) 264 16985), coprecipitation of DNA with calcium phosphate (Benvemsty & Reshef, PNAS USA, (1986) 83 9551), encapsulation of DNA m various forms of hposomes (Kaneda et al , Science (1989) 243 375), particle bombardment (Tang et al ,
Nature (1992) 356 152, Eisenbraun et al , DNA Cell Bwl (1993) 12 791) and in vivo infection usmg cloned retroviral vectors (Seeger et al . PNAS USA (1984) 81 5849) Antagonists and Agonists - Assays and Molecules Polypeptides and polynucleotides of the mvention may also be used to assess the binding of small molecule substrates and ligands m. for example, cells, cell-free preparations, chemical hbranes, and natural product mixtures These substrates and ligands may be natural substrates and ligands or may be structural or functional mimetics See, e g . Coligan et al . Current Protocols in Immunology 1(2) Chapter 5 (1991) Polypeptides and polynucleotides of the present mvention are responsible for many biological functions, mcludmg many disease states, m particular the Diseases herembefore mentioned It is therefore desirable to devise screening methods to identify compounds which stimulate or which inhibit 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 which stimulate or which inhibit the function of a polypeptide or polynucleotide of the mvention, as well as related polypeptides and polynucleotides In general, agomsts or antagonists may be employed for therapeutic and prophylactic purposes for such Diseases as herembefore mentioned Compounds may be identified from a vanety of sources, for example, cells, cell-free preparations, chemical hbranes, and natural product mixtures Such agomsts, antagonists or inhibitors so-identified may be natural or modified substrates, ligands, receptors, enzymes, etc , as the case may be, of mdD polypeptides and polynucleotides, or may be structural or functional mimetics thereof (see Coligan et al , Current Protocols in Immunology 1(2) Chapter 5 (1991))
The screening methods may simply measure the binding of a candidate compound to the polypeptide or polynucleotide, or to cells or membranes bearing the polypeptide or polynucleotide, or a fusion protem 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 m a signal generated b> 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 polypeptide and/or constitutively expressed polypeptides and polynucleotides may be employed in screening methods for inverse agonists or inhibitors, m the absence of an agonist or inhibitor, by testing whether the candidate compound results m inhibition of activation of the polypeptide or polynucleotide, as the case may be Further, the screemng methods may simply comprise the steps of mixing a candidate compound with a solution containing a polypeptide or polynucleotide of the present invention, to form a mixture, measuring nrdD polypeptide and/or polynucleotide activity in the mixture, and comparing the nrdD polypeptide and/or polynucleotide activity of the mixture to a standard Fusion proteins, such as those made from Fc portion and nrdD polypeptide, as hereinbefore described, can also be used for high-throughput screening assays to identify antagomsts of the polypeptide of the present invention, as well as of phylogenetically and and/or functionally related polypeptides (see D Bennett et al , J Mol Recognition. 8 52-58 (1995), and K Johanson et al , J Biol Chem, 270(16) 9459-9471 (1995))
The polynucleotides, 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 For example, an ELISA assay may be constructed for measuring secreted or cell associated levels of polypeptide usmg monoclonal and polyclonal antibodies by standard methods known in the art This can be used to discover agents which may inhibit or enhance the production of polypeptide (also called antagonist or agonist, respectively) from suitably manipulated cells or tissues The mvention also provides a method of screening compounds to identify those which enhance
(agonist) or block (antagonist) the action of nrdD polypeptides or polynucleotides, particularly those compounds that are bactenstatic and/or bacteπcidal The method of screening may 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 any thereof, compnsmg mdD polypeptide and a labeled substrate or hgand of such polypeptide is mcubated m the absence or the presence of a candidate molecule that may be a nrdD agomst or antagonist The ability of the candidate molecule to agonize or antagonize the nrdD polypeptide is reflected m decreased binding of the labeled hgand or decreased production of product from such substrate Molecules that bmd gratuitously, z e , without mducmg the effects of nrdD polypeptide are most likely to be good antagonists Molecules that b d well and, as the case may be, mcrease the rate of product production from substrate, mcrease signal transduction, or mcrease chemical channel activity 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 that may be useful m this regard mclude but are not limited to colonmetnc, labeled substrate converted mto product, a reporter gene that is responsive to changes m mdD 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 mclude. but are not limited to, hgand binding and crosshnking assays m which the polypeptide is labeled with a radioactive isotope (for instance, 1*"I), chemically modified (for instance, biot ylated), 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, tissue extracts, bodily materials) Other methods include biophysical techmques such as surface plasmon resonance and spectroscopy These screening methods may also be used to identify agonists and antagonists of the polypeptide which compete with the binding of the polypeptide to its receptor(s), if any Standard methods for conducting such assays are well understood in the art The fluorescence polarization value for a fluorescently-tagged molecule depends on the rotational correlation time or tumbling rate Protein complexes, such as formed b\ nrdD polypeptide associating with another nrdD polypeptide or other polypeptide, labeled to compπse a fluorescently- labeled molecule will have higher polarization values than a fluorescently labeled monomeπc protem 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 nrdD polypeptide d mers, turners, tetramers or higher order structures, or structures formed by nrdD polypeptide bound to another polypeptide NrdD 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 dimeπzation will inhibit fluorescence energy transfer
Surface plasmon resonance can be used to monitor the effect of small molecules on mdD polypeptide self-association as well as an association of nrdD polypeptide and another polypeptide or small molecule nrdD polypeptide can be coupled to a sensor chip at low site density such that covalently bound molecules will be monomeπc Solution protem can then passed over the mdD polypeptide -coated surface and specific binding can be detected m 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 equihbnum bindmg constants for nrdD polypeptide self-association as well as an association of nrdD polypeptide and another polypeptide or small molecule
A scintillation proximity assay may be used to characterize the interaction between an association of nrdD polypeptide with another nrdD polypeptide or a different polypeptide mdD polypeptide can be coupled to a scmtillation-filled bead Addition of radio-labeled nrdD polypeptide results in binding where the radioactive source molecule is m close proximity to the scintillation fluid Thus, signal is emitted upon nrdD polypeptide bmdmg and compounds that prevent nrdD polypeptide self-association or an association of nrdD polypeptide and another polypeptide or small molecule will diminish signal
ICS biosensors have been described by AMBRI (Australian Membrane Biotechnology Research Institute) They couple the self-association of macromolecules to the closing of gramacidm- facihtated ion channels m suspended membrane bilayers and hence to a measurable change m the admittance (similar to impedence) of the biosensor This approach is linear over six decades of admittance change and is ideally suited for large scale, high through-put screenmg of small molecule combinatorial libraries In other embodiments of the mvention there are provided methods for identifying compounds which 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 bmdmg to or other interaction between the compound and the polypeptide and or polynucleotide to assess the bmdmg to or other mteraction with the compound, such bmdmg or mteraction preferably being associated with a second component capable of providing a detectable signal m response to the bmdmg or mteraction of the polypeptide and or polynucleotide with the compound, and determining whether the compound bmds to or otherwise interacts with and activates or inhibits an activity or expression of the polypeptide and/or polynucleotide by detectmg the presence or absence of a signal generated from the bmdmg or mteraction of the compound with the polypeptide and/or polynucleotide
Another example of an assay for nrdD agomsts is a competitive assay that combines mdD and a potential agomst with nrdD-binding molecules, recombinant nrdD bmdmg molecules, natural substrates or ligands. or substrate or hgand mimetics, under appropπate conditions for a competitive inhibition assay mdD can be labeled, such as by radioactivity or a coloπmetπc compound, such that the number of mdD molecules bound to a bmdmg molecule or converted to product can be determined accurately to assess the effectiveness of the potential antagonist
Potential antagonists mclude. among others, small orgamc molecules, peptides, polypeptides and antibodies that bmd to a polynucleotide and or polypeptide of the mvention and thereby inhibit or extinguish its activity or expression Potential antagonists also may be small orgamc molecules, a peptide, a polypeptide such as a closely related protem or antibody that bmds the same sites on a bmdmg molecule, such as a bmdmg molecule, without inducing nrdD-rnduced activities, thereby preventmg the action or expression of mdD polypeptides and or polynucleotides by excluding mdD polypeptides and or polynucleotides from bmdmg
Potential antagonists mclude a small molecule that bmds to and occupies the bmdmg site of the polypeptide thereby preventmg bmdmg to cellular bmdmg molecules, such that normal biological activity is prevented Examples of small molecules mclude but are not limited to small orgamc molecules, peptides or peptide-hke molecules Other potential antagonists mclude antisense molecules (see Okano, J Neurochem 56 560 (1991) OLIGODEOXYNUCLEOTIDES AS ANTISENSE INHIBITORS OF GENE EXPRESSION, CRC Press. Boca Raton, FL (1988), for a descnption of these molecules) Preferred potential antagonists mclude compounds related to and vanants of mdD Other examples of potential polypeptide antagonists mclude antibodies or, m some cases, ohgonucleotides or proteins which are closely related to the ligands, substrates, receptors, enzymes, etc , as the case may be. of the polypeptide, e g . a fragment of the ligands, substrates, receptors, enzymes, etc , or small molecules which bmd to the polypeptide of the present mvention but do not elicit a response, so that the activity of the polypeptide is prevented Certam of the polypeptides of the mvention are biomimetics. functional mimetics of the natural mdD polypeptide These functional mimetics may be used for. among other things, antagonizing the activity of mdD polypeptide or as a antigen or lmmunogen m a manner descπbed elsewhere herem Functional mimetics of the polypeptides of the mvention mclude but are not limited to truncated polypeptides For example, preferred functional mimetics mclude. a polypeptide compnsing the polypeptide sequence set forth in SEQ ID NO 2 lacking 20, 30, 40. 50. 60, 70 or 80 ammo- or carboxy-terminal ammo acid residues, mcludmg fusion proteins compπsmg one or more of these truncated sequences Polynucleotides encoding each of these functional mimetics may be used as expression cassettes to express each mimetic polypeptide It is prefeπed that these cassettes compnse 5' and 3' restπction sites to allow for a convement means to hgate the cassettes together when desired It is further prefeπed that these cassettes compnse gene expression signals known in the art or descπbed elsewhere herem
Thus, in another aspect, the present invention relates to a screening kit for identifying agomsts, antagonists, ligands receptors, substrates, enzymes, etc for a polypeptide and/or polynucleotide of the present invention, or compounds which decrease or enhance the production of such polypeptides and/or polynucleotides , which comprises
(a) a polypeptide and/or a polynucleotide of the present mvention,
(b) a recombinant cell expressing a polypeptide and/or polynucleotide of the present invention,
(c) a cell membrane expressing a polypeptide and/or polynucleotide of the present invention, or
(d) antibody to a polypeptide and or polynucleotide of the present invention, which polypeptide is preferably that of SEQ ID NO 2. and which polynucleotide is preferably that of SEQ ID NO 1
It will be appreciated that in any such kit, (a), (b), (c) or (d) may comprise a substantial component
It will be readily appreciated by the skilled artisan that a polypeptide and/or polynucleotide of the present invention may also be used in a method for the structure-based design of an agonist, antagonist or inhibitor 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 sιte(s), bmdmg sιte(s) or motifls) of an agonist, antagonist or inhibitor,
(c) synthesizing candidate compounds that are predicted to bmd to or react with the deduced binding sιte(s) reactive sιte(s). and/or motιf(s), and
(d) testing whether the candidate compounds are indeed agonists, antagonists or inhibitors
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 In a further aspect, the present mvention provides methods of treatmg abnormal conditions such as, for instance, a Disease, related to either an excess of. an under-expression of, an elevated activity of. or a decreased activity of mdD polypeptide and or polynucleotide
If the expression and/or activity of the polypeptide and/or polynucleotide is in excess, several approaches are available One approach compπses administering to an mdividual m need thereof an inhibitor compound (antagonist) as herem descπbed, 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 for example, by blocking the bmdmg of ligands, substrates, receptors, enzymes, etc , or by inhibiting a second signal, and thereby alleviating the abnormal condition In another approach, soluble forms of the polypeptides still capable of binding the hgand. substrate, enzymes, receptors, etc m competition with endogenous polypeptide and or polynucleotide may be administered Typical examples of such competitors include fragments of the mdD polypeptide and/or polypeptide
In a further aspect, the present invention relates to genetically engineered soluble fusion proteins comprising a polypeptide of the present mvention, or a fragment thereof, and vanous portions of the constant regions of heavy or light chains of immunoglobulms of various subclasses (IgG, IgM, IgA, IgE) Preferred as an lmmunoglobulm is the constant part of the heavy chain of human IgG, particularly IgGl, where fusion takes place at the hmge region In a particular embodiment, the Fc part can be removed simply by incorporation of a cleavage sequence which can be cleaved with blood clotting factor Xa Furthermore, this invention relates to processes for the preparation of these fusion protems by genetic engineering, and to the use thereof for drug screenmg. diagnosis and therapy A further aspect of the invention also relates to polynucleotides encoding such fusion proteins Examples of fusion prote technology can be found in International Patent Application Nos W094/29458 and W094/22914
In still another approach, expression of the gene encoding endogenous mdD polypeptide can be inhibited using expression blocking techniques This blocking may be targeted against any step m gene expression, but is preferably targeted against transcription and/or translation An examples of a known technique of this sort mvolve the use of antisense sequences, either internally generated or separately administered (see, for example. O'Connor, J Neurochem (1991) 56 560 in Ohgodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press. Boca Raton, FL (1988)) Alternatively, ohgonucleotides which 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 o gomers can be administered per se or the relevant ohgomers can be expressed in vivo
Each of the 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 Additionally, the polynucleotide sequences encodmg 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 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 physical interaction between a pathogen or pathogens and a eukaryotic, preferably mammalian, host responsible for sequelae of infection In particular, the molecules of the invention may be used in the prevention of adhesion of bacteria, m particular gram positive and/or gram negative bacteria, to eukaryotic, preferably mammalian, extracellular matrix proteins on in-dwelling devices or to extracellular matrix proteins in wounds, to block mdD protem-mediated mammalian cell invasion by. for example, initiating phosphorylation of mammalian tyrosine kmases (Rosenshine et al , Infect Immun 60 2211 (1992), to block bacterial adhesion between eukaryotic, preferably mammalian, extracellular matrix proteins and bacterial mdD proteins that mediate tissue damage and or. to block the normal progression of pathogenesis m infections initiated other than by the implantation of m-dwelhng devices or by other surgical techniques In accordance with yet another aspect of the mvention, there are provided mdD agomsts and antagonists, preferably bacteπstatic or bactencidal agomsts and antagonists
The antagonists and agomsts of the mvention may be employed, for instance, to prevent, inhibit and or treat diseases
Hehcobacter pylori (herein "H pylori") bactena infect the stomachs of over one-third of the world's population causing stomach cancer, ulcers, and gastritis (International Agency for Research on Cancer (1994) Schistosomes, Liver Flukes and Hehcobacter Pylori (International Agency for Research on Cancer. L\ on. France, http //www uicc ch ecp/ecp2904 htm) Moreover, the International Agency for Research on Cancer recently recognized a cause-and-effect relationship between H pylori and gastric adenocarcinoma. classifying the bacterium as a Group I (definite) carcinogen Preferred antimicrobial compounds of the invention (agonists and antagonists of nrdD 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 carcinoma Such treatment should also prevent, inhibit and/or cure gastric ulcers and gastritis Vaccines
There are provided by the mvention, products, compositions and methods for assessmg mdD expression, treatmg disease, assaying genetic vanation, and administering a nrdD polypeptide and/or polynucleotide to an orgamsm to raise an lmmunological response against a bactena. especially a Staphylococcus aureus bactena Another aspect of the mvention relates to a method for inducing an immunological response in an individual, particularly a mammal which comprises inoculating the individual with mdD polynucleotide and or polypeptide. or a fragment or variant thereof, adequate to produce antibody and/ or T cell immune response to protect said individual from infection, particularly bacterial infection and most particularly Staphylococcus aureus infection Also provided are methods whereby such immunological response slows bacterial replication Yet another aspect of the invention relates to a method of mducmg immunological response in an individual which comprises delivering to such mdividual a nucleic acid vector, sequence or πbozyme to direct expression of mdD polynucleotide and/or polypeptide, or a fragment or a vanant thereof, for expressing mdD polynucleotide and/or polypeptide, or a fragment or a variant thereof in vivo m order to induce an immunological response, such as, to produce antibody and/ or T cell immune response, including, for example, cytokine-producing T cells or cytotoxic T cells, to protect said individual, preferably a human, from disease, whether that disease is already established withm the mdividual or not One example of administering the gene is by accelerating it mto the desired cells as a coating on particles or otherwise Such nucleic acid vector may comprise DNA, RNA, a πbozyme, a modified nucleic acid, a DNA/RNA hybrid, a DNA-protein complex or an RNA-protem complex
A further aspect of the invention relates to an immunological composition that when introduced into an individual, preferably a human, capable of having induced within it an immunological response, mduces an immunological response in such individual to a mdD polynucleotide and or polypeptide encoded therefrom, wherein the composition comprises a recombinant mdD polynucleotide and/or polypeptide encoded therefrom and/or comprises DNA and/or RNA which encodes and expresses an antigen of said mdD polynucleotide. polypeptide encoded therefrom, or other polypeptide of the invention The immunological response may be used therapeutically or prophylactically and may take the form of antibody immunity and or cellular immunity, such as cellular immunity arising from CTL or CD4+ T cells A mdD polypeptide or a fragment thereof may be fused with co-protem or chemical moiety which may or may not by itself produce antibodies, but which is capable of stabilizing the first protem and producing a fused or modified protein which will have antigenic and/or rmmunogenic properties, and preferably protective properties Thus fused recombinant protein, preferably further comprises an antigenic co-protein, such as hpoprotein D from Hemophύus influenzae, Glutathione-S-transferase (GST) or beta-galactosidase or any other relatively large co-protem which solubihzes the protem and facilitates production and purification thereof Moreover, the co-protein may act as an adjuvant in the sense of providing a generalized stimulation of the immune system of the organism receiving the protem The co-protein may be attached to either the ammo- or carboxy-terminus of the first protein Provided by this invention are compositions, particularly vaccine compositions, and methods comprising the polypeptides and/or polynucleotides of the invention and lmmunostimulatory DNA sequences, such as those described in Sato. Y et al Science 273 352 (1996)
Also, provided by this invention are methods usmg the described polynucleotide or particular fragments thereof, which have been shown to encode non-vaπable regions of bacterial cell surface proteins, in polynucleotide constructs used in such genetic immunization experiments in animal models of infection with Staphylococcus aureus Such experiments will be particularly useful for identifying protem epitopes able to provoke a prophylactic or therapeutic immune response It is believed that this approach will allow for the subsequent preparation of monoclonal antibodies of particular value, deπved from the requisite organ of the animal successfully resisting or clearmg infection, for the development of prophylactic agents or therapeutic treatments of bacterial mfection, particularly Staphylococcus aureus infection, in mammals, particularly humans
A polypeptide of the invention may be used as an antigen for vaccmation of a host to produce specific antibodies which protect against invasion of bactena. for example by blocking adherence of bacteria to damaged tissue Examples of tissue damage mclude wounds in skm or connective tissue caused, for example, by mechanical, chemical, thermal or radiation damage or by implantation of indwelling devices, or wounds in the mucous membranes, such as the mouth, throat, mammary glands, urethra or vagina
The invention also includes a vaccine formulation which comprises an lmmunogenic recombinant polypeptide and/or polynucleotide of the invention together with a suitable carrier, such as a pharmaceutically acceptable earner Since the polypeptides and polynucleotides may be broken down in the stomach, each is preferably administered parenterally, mcludmg, for example, administration that is subcutaneous, intramuscular, intravenous, or intradermal Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti- oxidants. buffers, bacteristatic compounds and solutes which render the formulation isotomc with the bodily fluid, preferably the blood, of the individual, and aqueous and non-aqueous sterile suspensions which may include suspending agents or thickening agents The formulations may be presented in unit- dose or multi-dose containers, for example, sealed ampoules and vials and may be stored m a freeze-dπed condition requiring only the addition of the sterile liquid carrier immediately prior to use The vaccine formulation may also include adjuvant systems for enhancing the lmmunoge city of the formulation, such as oil-m water systems and other systems known m the art The dosage will depend on the specific activity of the vaccine and can be readily determined by routine experimentation
While the invention has been described with reference to certain mdD polypeptides and polynucleotides, it is to be understood that this covers fragments of the naturally occurring polypeptides and polynucleotides, and similar polypeptides and polynucleotides with additions, deletions or substitutions which do not substantially affect the lmmunogemc properties of the recombinant polypeptides or polynucleotides
Compositions, kits and administration
In a further aspect of the mvention there are provided compositions compπsmg a mdD polynucleotide and/or a mdD polypeptide for administration to a cell or to a multicellular orgamsm
The mvention also relates to compositions compnsrng a polynucleotide and/or a polypeptides discussed herem or their agomsts or antagonists The polypeptides and polynucleotides of the mvention may be employed m combination with a non-steπle or stenle earner or earners for use with cells, tissues or orgamsms, such as a pharmaceutical earner suitable for administration to an mdividual Such compositions compnse. for instance, a media additive or a therapeutically effective amount of a polypeptide and/or polynucleotide of the mvention and a pharmaceutically acceptable earner or excipient Such earners may mclude, but are not limited to, salme, buffered salme. dextrose, water, glycerol. ethanol and combmations thereof The formulation should suit the mode of administration The mvention further relates to diagnostic and pharmaceutical packs and kits compnsrng one or more containers filled with one or more of the ingredients of the aforementioned compositions of the mvention
Polypeptides, polynucleotides and other compounds of the mvention may be employed alone or m conjunction with other compounds, such as therapeutic compounds
The pharmaceutical compositions may be admmistered in any effective, convement manner mcludmg, for instance, admmistration by topical, oral, anal, vaginal, mtravenous, mtrapeπtoneal. intramuscular, subcutaneous, mtranasal or lntradermal routes among others
In therapy or as a prophylactic, the active agent may be administered to an mdividual as an mjectable composition, for example as a sterile aqueous dispersion, preferably isotomc
Alternatively the composition may be formulated for topical application for example m the form of ointments, creams, lotions, eye ointments, eye drops, ear drops, mouthwash, impregnated dressings and sutures and aerosols, and may contain appropriate conventional additives, including, for example, preservatives, solvents to assist drug penetration, and emollients in omtments and creams Such topical formulations may also contain compatible conventional carriers, for example cream or ointment bases, and ethanol or oleyl alcohol for lotions Such carriers may constitute from about 1% to about 98% by weight of the formulation, more usually they will constitute up to about 80% by weight of the formulation
In a further aspect, the present mvention provides for pharmaceutical compositions compnsrng a therapeutically effective amount of a polypeptide and/or polynucleotide, such as the soluble form of a polypeptide and or polynucleotide of the present mvention, agonist or antagonist peptide or small molecule compound, in combination with a pharmaceutically acceptable earner or excipient Such earners mclude, but are not limited to, salme. buffered salme. dextrose, water, glycerol, ethanol, and combmations thereof The mvention further relates to pharmaceutical packs and kits compnsrng one or more containers filled with one or more of the ingredients of the aforementioned compositions of the mvention Polypeptides, polynucleotides and other compounds of the present mvention may be employed alone or in conjunction with other compounds, such as therapeutic compounds The composition will be adapted to the route of admmistration, for instance by a systemic or an oral route Prefeπed forms of systemic admmistration mclude injection, typically by mtravenous injection Other injection routes, such as subcutaneous, intramuscular, or mtrapeπtoneal, can be used Alternative means for systemic admmistration mclude transmucosal and transdermal admmistration usmg penetrants such as bile salts or fusidic acids or other detergents In addition, if a polypeptide or other compounds of the present mvention can be formulated m an entenc or an encapsulated formulation, oral administration may also be possible Admimstration of these compounds may also be topical and or localized, in the form of salves, pastes, gels, and
For administration to mammals, and particularly humans, it is expected that the daily dosage level of the active agent will be from 0 01 mg/kg to 10 mg/kg, typically around 1 mg/kg The physician in any event will determine the actual dosage which will be most suitable for an mdividual and will vary with the age, weight and response of the particular individual The above dosages are exemplary of the average case There can. of course, be individual instances where higher or lower dosage ranges are merited, and such are within the scope of this mvention
In-dwellmg devices include surgical implants, prosthetic devices and catheters, l e , devices that are introduced to the body of an individual and remain in position for an extended time Such devices mclude. for example, artificial jomts, heart valves, pacemakers, vascular grafts, vascular catheters, cerebrospmal fluid shunts, urinary catheters, continuous ambulatory pentoneal dialysis (CAPD) catheters
The composition of the mvention may be administered by injection to achieve a systemic effect agamst relevant bacteria shortly before insertion of an in-dwelling device Treatment may be continued after surgery during the m-body time of the device In addition, the composition could also be used to broaden peπoperative cover for any surgical technique to prevent bacterial wound infections, especially Staphylococcus aureus wound infections
Many orthopedic surgeons consider that humans with prosthetic joints should be considered for antibiotic prophylaxis before dental treatment that could produce a bacteremia Late deep mfection is a serious complication sometimes leading to loss of the prosthetic joint and is accompanied by significant morbidity and mortality It may therefore be possible to extend the use of the active agent as a replacement for prophylactic antibiotics in this situation
In addition to the therapy described above, the compositions of this invention may be used generally as a wound treatment agent to prevent adhesion of bacteria to matrix protems exposed in wound tissue and for prophylactic use m dental treatment as an alternative to, or in conjunction with, antibiotic prophylaxis
Alternatively, the composition of the invention may be used to bathe an indwelling device immediately before insertion The active agent will preferably be present at a concentration of 1 μg/ml to lOmg/ml for bathing of wounds or indwelling devices
A vaccine composition is conveniently in mjectable form Conventional adjuvants may be employed to enhance the immune response A suitable unit dose for vaccination is 0 5-5 microgram kg of antigen, and such dose is preferably administered 1-3 times and with an interval of 1-3 weeks With the mdicated dose range, no adverse toxicological effects will be observed with the compounds of the invention which would preclude their administration to suitable individuals
Sequence Databases, Sequences in a Tangible Medium, and Algorithms Polynucleotide and polypeptide sequences form a valuable information resource with which to determme their 2- and 3-dιmensιonal structures as well as to identify further sequences of similar homology These approaches are most easily facilitated by storing the sequence in a computer readable medium and then usmg the stored data m a known macromolecular structure program or to search a sequence database usmg well known searching tools, such as GCC
The polynucleotide and polypeptide sequences of the invention are particularly useful as components in databases useful for search analyses as well as in sequence analysis algonthms As used in this section entitled "Sequence Databases, Sequences in a Tangible Medium, and Algorithms," and m claims related to this section, the terms "polynucleotide of the invention" and "polynucleotide sequence of the invention" mean any detectable chemical or physical characteristic of a polynucleotide of the invention that is or may be reduced to or stored in a tangible medium, preferably a computer readable form For example, chromatographic scan data or peak data, photographic data or scan data therefrom, called bases, and mass spectrographic data As used m this section entitled Databases and Algonthms and m claims related thereto, the terms "polypeptide of the invention" and "polypeptide sequence of the invention" mean any detectable chemical or physical characteristic of a polypeptide of the mvention that is or may be reduced to or stored in a tangible medium, preferably a computer readable form For example, chromatographic scan data or peak data, photographic data or scan data therefrom, and mass spectrographic data The mvention provides a computer readable medium having stored thereon polypeptide sequences of the invention and/or polynucleotide sequences of the invention For example, a computer readable medium is provided comprising and having stored thereon a member selected from the group consisting of a polynucleotide comprising the sequence of a polynucleotide of the mvention, a polypeptide comprising the sequence of a polypeptide sequence of the invention, a set of polynucleotide sequences wherein at least one of the sequences comprises the sequence of a polynucleotide sequence of the invention, a set of polypeptide sequences wherein at least one of the sequences comprises the sequence of a polypeptide sequence of the invention, a data set representing a polynucleotide sequence comprising the sequence of polynucleotide sequence of the invention, a data set representing a polynucleotide sequence encoding a polypeptide sequence comprising the sequence of a polypeptide sequence of the invention, a polynucleotide comprising the sequence of a polynucleotide sequence of the invention, a polypeptide comprising the sequence of a polypeptide sequence of the mvention, a set of polynucleotide sequences wherein at least one of the sequences comprises the sequence of a polynucleotide sequence of the invention, a set of polypeptide sequences wherein at least one of said sequences comprises the sequence of a polypeptide sequence of the invention, a data set representing a polynucleotide sequence compnsrng the sequence of a polynucleotide sequence of the invention, a data set representing a polynucleotide sequence encoding a polypeptide sequence comprising the sequence of a polypeptide sequence of the invention The computer readable medium can be any composition of matter used to store information or data, including for example, commercially available floppy disks, tapes, chips, hard drives, compact disks, and video disks Also provided by the invention are methods for the analysis of character sequences or strings, particularly genetic sequences or encoded genetic sequences Preferred methods of sequence analysis mclude, for example, methods of sequence homology analysis, such as identity and similanty analysis, RNA structure analysis, sequence assembly, cladistic analysis, sequence motif analysis, open reading frame determination, nucleic acid base calling, nucleic acid base trimming, and sequencing chromatogram peak analysis
A computer based method is provided for performing homology identification This method comprises the steps of providing a polynucleotide sequence comprising the sequence a polynucleotide of the invention in a computer readable medium, and comparing said polynucleotide sequence to at least one polynucleotide or polypeptide sequence to identify homology A computer based method is also provided for performing homology identification, said method comprising the steps of providing a polypeptide sequence comprising the sequence of a polypeptide of the mvention in a computer readable medium, and comparing said polypeptide sequence to at least one polynucleotide or polypeptide sequence to identify homology
A computer based method is still further provided for polynucleotide assembly, said method comprising the steps of providing a first polynucleotide sequence comprising the sequence of a polynucleotide of the invention in a computer readable medium, and screening for at least one overlapping region between said first polynucleotide sequence and a second polynucleotide sequence
A further embodiment of the invention provides a computer based method for performing homology identification, said method comprising the steps of providing a polynucleotide sequence comprising the sequence of a polynucleotide of the invention in a computer readable medium, and comparing said polynucleotide sequence to at least one polynucleotide or polypeptide sequence to identify homology
A further embodiment of the invention provides a computer based method for performing homology identification, said method comprising the steps of providing a polypeptide sequence compnsrng the sequence of a polypeptide of the mvention in a computer readable medium, and comparing said polypeptide sequence to at least one polynucleotide or polypeptide sequence to identify homology
A further embodiment of the invention provides a computer based method for polynucleotide assembly, said method comprising the steps of providing a first polynucleotide sequence comprising the sequence of a polynucleotide of the invention in a computer readable medium, and screening for at least one overlapping region between said first polynucleotide sequence and a second polynucleotide sequence
In another prefeπed embodiment of the mvention there is provided a computer readable medium having stored thereon a member selected from the group consisting of a polynucleotide comprising the sequence of SEQ ID NO 1 , a polypeptide comprising the sequence of SEQ ID NO 2, a set of polynucleotide sequences wherein at least one of said sequences compnses the sequence of SEQ ID NO 1 , a set of polypeptide sequences wherein at least one of said sequences comprises the sequence of SEQ ID NO 2. a data set representing a polynucleotide sequence comprising the sequence of SEQ ID NO 1, a data set representing a polynucleotide sequence encoding a polypeptide sequence compnsrng the sequence of SEQ ID NO 2, a polynucleotide comprising the sequence of SEQ ID NO 1 , a polypeptide compnsrng the sequence of SEQ ID NO 2, a set of polynucleotide sequences wherein at least one of said sequences comprises the sequence of SEQ ID NO 1, a set of polypeptide sequences wherein at least one of said sequences comprises the sequence of SEQ ID NO 2. a data set representing a polynucleotide sequence comprising the sequence of SEQ ID NO 1. a data set representing a polynucleotide sequence encodmg a polypeptide sequence comprising the sequence of SEQ ID NO 2 A further preferred embodiment of the invention provides a computer based method for performing homology identification, said method comprising the steps of providing a polynucleotide sequence comprising the sequence of SEQ ID NO 1 m a computer readable medium, and comparing said polynucleotide sequence to at least one polynucleotide or polypeptide sequence to identify homology
A still further preferred embodiment of the invention provides a computer based method for performing identification, said method comprising the steps of providmg a polypeptide sequence comprising the sequence of SEQ ID NO 2 in a computer readable medium, and comparing said polypeptide sequence to at least one polynucleotide or polypeptide sequence to identify homology
A further embodiment of the invention provides a computer based method for polynucleotide assembly, said method comprising the steps of providing a first polynucleotide sequence comprising the sequence of SEQ ID NO 1 in a computer readable medium, and screening for at least one overlappmg region between said first polynucleotide sequence and a second polynucleotide sequence A further embodiment of the invention provides a computer based method for performing homology identification, said method comprising the steps of providing a polynucleotide sequence comprising the sequence of SEQ ID NO 1 in a computer readable medium, and comparing said polynucleotide sequence to at least one polynucleotide or polypeptide sequence to identify homology A further embodiment of the mvention provides a computer based method for performing homology identification, said method comprising the steps of providing a polypeptide sequence compnsrng the sequence of SEQ ID NO 2 in a computer readable medium, and comparing said polypeptide sequence to at least one polynucleotide or polypeptide sequence to identify homology
A further embodiment of the invention provides a computer based method for polynucleotide assembly, said method comprising the steps of providing a first polynucleotide sequence comprising the sequence of SEQ ID NO 1 in a computer readable medium, and screemng for at least one overlappmg region between said first polynucleotide sequence and a second polynucleotide sequence
All publications and references, including but not limited to patents and patent applications, cited in this specification are herein incorporated by reference in their entirety as if each mdividual publication or reference were specifically and individually indicated to be incorporated by reference herem as being fully set forth Any patent application to which this application claims pnonty is also incorporated by reference herem in its entirety in the manner described above for publications and references
GLOSSARY The following definitions are provided to facilitate understanding of certam terms used frequently herem
"Antιbody(ιes)" as used herein includes polyclonal and monoclonal antibodies, chimeπc. single chain, and humanized antibodies, as well as Fab fragments, including the products of an Fab or other lmmunoglobulin expression library "Antigemcally equivalent deπvatιve(s)" as used herem encompasses a pohpeptide, polynucleotide. or the equivalent of either which will be specifically recognized b\ certain antibodies which, when raised to the protem, polypeptide or polynucleotide according to the mvention. interferes with the immediate physical interaction between pathogen and mammalian host
"Bispecific antibody(ιes)" means an antibody comprising at least two antigen bmdmg domains, each domain directed against a different epitope
"Bodily mateπal(s) means any matenal denved from an mdividual or from an orgamsm infecting, infesting oi inhabiting an mdividual. mcludmg 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 autopsy mateπals "Dιsease(s)" means any disease caused by or related to infection by a bactena. mcludmg , for example, disease, such as. infections of the upper respiratory tract (e g , otitis media, bacteπal tracheitis, acute epiglottitis, thyroiditis). lower respiratory (e g , empyema, lung abscess), cardiac (e g , infective endocarditis), gastrointestinal (e g , secretory diaπhoea. splemc absces, retropeπtoneal abscess), CNS (e g , cerebral abscess), eye (e g . blephantis. conjunctivitis, keratitis, endophthalmitis, preseptal and orbital celluhtis, darcryocystitis), kidney and urinary tract (e g . epididymitis, rntrarenal and pennephnc absces, toxic shock syndrome), skin (e g , impetigo, follicu tis, cutaneous abscesses, celluhtis, wound infection, bacteπal myositis) bone and joint (e.g , septic arthntis, osteomyelitis)
"Fusion protem(s)" refers to a protein encoded by two, often unrelated, fused genes or fragments thereof In one example. EP-A-0464 discloses fusion proteins comprising vanous portions of constant region of lmmunoglobuhn molecules together with another human protem or part thereof In many cases, employing an lmmunoglobuhn Fc region as a part of a fusion protein is advantageous for use m therapy and diagnosis resulting in. for example, improved pharmacokinetic properties [see, e g . EP-A 0232262] On the other hand, for some uses it would be desirable to be able to delete the Fc part after the fusion protein has been expressed, detected and puπfied
"Host cell(s)" is a cell which has been transformed or transfected, or is capable of transformation or transfection by an exogenous polynucleotide sequence
"Identity," as known in the art, is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as the case may be, as determined by comparing the sequences In the art, "identity" also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences "Identity" can be readily calculated by known methods, including but not limited to those described m (Computational Molecular Biology Lesk. A M . ed . Oxford University Press. New York, 1988, Biocomputtng Informatics and Genome Projects Smith, D W ed Academic Press, New York. 1993. Computer Analysis of Sequence Data. Part I. Griffm, A M . and Griffin. H G , eds , Humana Press. New Jersey, 1994. Sequence Analysis in Molecular Biology, von Heinje, G , Academic Press, 1987, and Sequence Analysis Primer, Gπbskov. M and Devereux, J . eds , M Stockton Press, New York. 1991, and Caπllo, H . and Lrpman. D . SIAM J Applied Math , 48 1073 (1988) Methods to determine identity are designed to give the largest match between the sequences tested Moreover, methods to determine identity are codified m publicly available computer programs Computer program methods to determine identity between two sequences include, but are not limited to, the GCG program package (Devereux, J , et al , Nucleic Acids Research 12(1) 387 (1984)), BLASTP, BLASTN. and FASTA (Altschul, S F et al , J Molec Bwl 215 403-410 (1990) The BLAST X program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S , et al , NCBI NLM NIH Bethesda, MD 20894. Altschul, S , et al , J Mol Bwl 215 403-410 (1990) The well known Smith Waterman algorithm may also be used to determine identity
Parameters for polypeptide sequence companson include the following
1) Algorithm Needleman and Wunsch, J Mol Biol 48 443-453 (1970) Comparison matrix BLOSSUM62 from Hentikoff and Hentikoff. Proc Natl Acad Sci USA
89 10915-10919 (1992)
Gap Penalty 12
Gap Length Penalty 4
A program useful with these parameters is publicly available as the "gap" program from Genetics Computer Group, Madison WI The aforementioned parameters are the default parameters for peptide comparisons (along with no penalty for end gaps)
Parameters for polynucleotide companson include the following
1) Algorithm Needleman and Wunsch, J Mol Biol 48 443-453 (1970)
Comparison matrix matches = +10. mismatch = 0 Gap Penalty 50
Gap Length Penalty 3
Available as The "gap" program from Genetics Computer Group, Madison WI These are the default parameters for nucleic acid comparisons
A preferred meaning for "identity" for polynucleotides and polypeptides, as the case may be, are provided in (1) and (2) below
(1) Polynucleotide embodiments further mclude an isolated polynucleotide comprising a polynucleotide sequence having at least a 50, 60, 70. 80. 85. 90. 95, 97 or 100% identity to the reference sequence of SEQ ID NO 1. wherein said polynucleotide sequence may be identical to the reference sequence of SEQ ID NO 1 or ma\ mclude up to a certain integer number of nucleotide alterations as compared to the reference sequence, wherein said alterations are selected from the group consisting of at least one nucleotide deletion, substitution, including transition and transversion. or insertion, and wherein said alterations may occur at the 5' or 3' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among the nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence, and wherein said number of nucleotide alterations is determined by multiplying the total number of nucleotides SEQ ID
NO 1 by the integer defining the percent identity divided by 100 and then subtracting that product from said total number of nucleotides in SEQ ID NO 1, or
nn ≤ *n " (xn * y), wherein nn is the number of nucleotide alterations. xn is the total number of nucleotides in SEQ ID NO 1, y is 0 50 for 50%, 0 60 for 60%, 0 70 for 70%, 0 80 for 80%, 0 85 for 85%, 0 90 for 90%, 0 95 for 95%, 0 97 for 97% or 1 00 for 100%, and • is the symbol for the multiplication operator, and wherein any non-integer product of xn and y is rounded down to the nearest integer prior to subtractmg it from xn Alterations of a polynucleotide sequence encoding the polypeptide of SEQ ID NO 2 may create nonsense, missense or frameshift mutations in this coding sequence and
Figure imgf000040_0001
alter the polypeptide encoded by the polynucleotide following such alterations
By way of example, a polynucleotide sequence of the present invention may be identical to the reference sequence of SEQ ID NO 1. that is it may be 100% identical, or it may include up to a certain mteger number of nucleic acid alterations as compared to the reference sequence such that the percent identity is less than 100% identity Such alterations are selected from the group consisting of at least one nucleic acid deletion, substitution, including transition and transversion, or insertion, and w herem said alterations may occur at the 5' or 3' terminal positions of the reference polynucleotide sequence or anywhere between those terminal positions, mterspersed either individually among the nucleic acids in the reference sequence or m one or more contiguous groups withm the reference sequence The number of nucleic acid alterations for a given percent identity is determined by multiplying the total number of nucleic acids in SEQ ID NO 1 by the integer defining the percent identity divided by 100 and then subtracting that product from said total number of nucleic acids m SEQ ID NO 1. or
nn ≤ xn " (xn * y),
wherein nn is the number of nucleic acid alterations, xn is the total number of nucleic acids m SEQ ID NO 1, y is, for instance 0 70 for 70%, 0 80 for 80%, 0 85 for 85% etc , • is the symbol for the multiplication operator, and wherein any non-mteger product of xn and y is rounded down to the nearest mteger prior to subtracting it from xn
(2) Polypeptide embodiments further include an isolated polypeptide compnsrng a polypeptide having at least a 50,60, 70, 80, 85, 90, 95, 97 or 100% identity to a polypeptide reference sequence of SEQ ID NO 2, wherein said polypeptide sequence may be identical to the reference sequence of SEQ ID NO 2 or may include up to a certain integer number of ammo acid alterations as compared to the reference sequence, wherein said alterations are selected from the group consisting of at least one amino acid deletion, substitution, including conservative and non-conservative substitution, or insertion, and wherein said alterations may occur at the amino- or carboxy-termmal positions of the reference polypeptide sequence or anywhere between those terminal positions, interspersed either individually among the amino acids m the reference sequence or in one or more contiguous groups within the reference sequence, and wherein said number of amino acid alterations is determined by multiplying the total number of ammo acids in SEQ ID NO 2 by the mteger defining the percent identity divided by 100 and then subtracting that product from said total number of ammo acids m SEQ ID NO 2, or
na < xa - (xa • y),
wherein na is the number of ammo acid alterations, xa is the total number of amino acids in SEQ ID NO 2. y is 0 50 for 50%, 0 60 for 60%, 0 70 for 70%, 0 80 for 80%, 0 85 for 85%, 0 90 for 90%, 0 95 for 95%, 0 97 for 97% or 1 00 for 100%, and • is the symbol for the multiplication operator, and wherein any non-integer product of xa and y is rounded down to the nearest integer prior to subtractmg it from xa
By way of example, a polypeptide sequence of the present invention may be identical to the reference sequence of SEQ ID NO 2, that is it may be 100% identical, or it may mclude up to a certain integer number of amino acid alterations as compared to the reference sequence such that the percent identity is less than 100% identity Such alterations are selected from the group consistmg 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-terminal positions of the reference polypeptide sequence or anywhere between those terminal positions, interspersed either individually among the amino acids in the reference sequence or in one or more contiguous groups within the reference sequence The number of ammo acid alterations for a given % identity is determined by multiplying the total number of ammo acids m SEQ ID NO 2 by the integer defining the percent identity divided by 100 and then subtracting that product from said total number of amino acids in SEQ ID NO 2, or
na < xa - (xa • y),
wherein na is the number of amino acid alterations, xa is the total number of amino acids in SEQ ID NO 2, y is. for instance 0 70 for 70%. 0 80 for 80%, 0 85 for 85% etc , and • is the symbol for the multiplication operator, and wherem any non-mteger product of xa and y is rounded down to the nearest mteger prior to subtracting it from xa
"Immunologically equivalent deπvatιve(s)" as used herein encompasses a polypeptide, poh nucleotide or the equivalent of either which when used in a suitable formulation to raise antibodies in a \ ertebrate. the antibodies act to interfere with the immediate physical interaction between pathogen and mammalian host "Immunospecific" means that charactenstic of an antibody whereby it possesses substantially greater affinity for the polypeptides of the mvention or the polynucleotides of the mvention than its affimty for other related polypeptides or polynucleotides respectively, particularly those polypeptides and polynucleotides in the pπor art "Indιvιdual(s)" means a multicellular eukaryote. mcludmg. but not limited to a metazoan, a mammal, an ovid, a bovid. a simian, a primate, and a human
"Isolated" means altered "by the hand of man" from its natural state, / , if it occurs m nature, it has been changed or removed from its onginal environment, or both For example, a polynucleotide or a polypeptide naturally present m a Irving orgamsm is not "isolated," but the same polynucleotide or polypeptide separated from the coexistmg matenals of its natural state is "isolated", as the term is employed herem Moreover, a polynucleotide or polypeptide that is mtroduced mto an orgamsm by transformation, genetic manipulation or by any other recombinant method is "isolated" even if it is still present in said orgamsm, which orgamsm may be living or non-living
"Organιsm(s)" means a (I) prokaryote, mcludmg but not limited to, a member of the genus Streptococcus Staphylococcus, Bordetella, Corynebactenum, Mycobacterium, Neissena, Haemophilus, Actinomycetes Streptomycetes, Nocardia, Enterobacter, Yersinia, Fancisella, Pasturella, Moraxella, Acmetobacter Erysipelothnx, Branhamella, Actinobacillus, Streptobacillus, Listena, Calymmatobactenum, Brucella, Bacillus, Clostndium, Treponema, Eschench a, Salmonella, Kleibsiella, Vibrio, Proteus, Erwinia, Borrelia, Leptospira, Spirillum, Campylobacter, Shigella, Legionella, Pseudomonas, Aeromonas, Rickettsia, Chlamydia, Borrelia and Mycoplasma, and further mcludmg, but not limited to, a member of the species or group, Group A Streptococcus, Group B Streptococcus, Group C Streptococcus, Group D Streptococcus, Group G Streptococcus, Streptococcus pneumon ae, Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus faecahs, Streptococcus faec um, Streptococcus durans, Neissena gonorrheae, Neissena meningitidis, Staphylococcus aureus, Staphylococcus epidermidis, Corynebactenum dipthenae, Gardnerella vaginahs, Mycobacterium tuberculosis, Mycobacterium bovis, Mycobactenum ulcerans, Mycobactenum leprae, Actinomyctes israelu, Listena monocytogenes, Bordetella pertusis, Bordatella parapertusis, Bordetella bronchi septica, Eschenchia colt, Shigella dysentenae, Haemophilus influenzae, Haemophilus aegyptius, Haemophilus parainjluenzae, Haemophilus ducreyi, Bordetella, Salmonella typhi, Citrobacter freundn, Proteus mirabihs, Proteus vulgans, Yersinia pestis, Kleibsiella pneumoniae, Serratia marcessens, Serratia hquefaciens Vibrio cholera, Shigella dysentern, Shigella flexnen, Pseudomonas aemginosa, Franscisella tularensis, Brucella abortis, Bacillus anthracis, Bacillus cereus, Clostndium perfnngens Clostndium tetani, Clostndium botulinum, Treponema palhdum, Rickettsia rickettsn and Chlamydia trachomitis, (n) an archaeon, mcludmg but not limited to Archaebacter, and (m) a unicellular or filamentous eukaryote. mclud g but not limited to, a protozoan, a fungus, a member of the genus Saccharomyces, Kluveromyces, or Candida, and a member of the species Saccharomyces cenv seae, Kluveromyces lactis, or Candida albicans
"Polynucleotιde(s)" generally refers to any polynbonucleoti.de or polydeoxynbonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA "Polynucleotide(s)" mclude, without limitation, smgle- and double-stranded DNA. DNA that is a mixture of single- and double-stranded regions or single-, double- and tπple-stranded regions, smgle- and double-stranded RNA, and RNA that is mixture of s gle- and double-stranded regions, hybnd molecules compπsmg DNA and RNA that may be single-stranded or, more typically, double-stranded, or tπple-stranded regions, or a mixture of smgle- and double-stranded regions In addition, "polynucleotide" as used herem refers to tnple-stranded regions compπsmg RNA or DNA or both RNA and DNA The strands m such regions may be from the same molecule or from different molecules The regions may mclude all of one or more of the molecules, but more typically mvolve only a region of some of the molecules One of the molecules of a tnple-he cal region often is an oligonucleotide As used herem, the term "polynucleotide(s)" also mcludes DNAs or RNAs as descnbed above that contam one or more modified bases Thus, DNAs or RNAs with backbones modified for stability or for other reasons are "polynucleotide(s)" as that term is mtended herem Moreover, DNAs or RNAs compπsmg unusual bases, such as mosme, or modified bases, such as tπtylated bases, to name just two examples, are polynucleotides as the 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 the art The term "polynucleotιde(s)" as it is employed herem 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, mcludmg, for example, simple and complex cells "Polynucleotide(s)" also embraces short polynucleotides often refenedto as olιgonucleotide(s)
"Polypeptιde(s)" refers to any peptide or protem compπsmg two or more amino acids jomed to each other b} peptide bonds or modified peptide bonds "Polypeptide(s)" refers to both short chains, commonly refeπed to as peptides o gopeptides and ohgomers and to longer chains generally refeπed to as proteins Polypeptides may contam ammo acids other than the 20 gene encoded ammo acids "Polypeptide(s)" mclude those modified either by natural processes, such as processmg and other post-translational modifications, but also by chemical modification techmques Such modifications are well descπbed m 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 varying degree at several sites m a given polypeptide Also, a given polypeptide may contam many types of modifications Modifications can occur anywhere in a polypeptide. mcludmg the peptide backbone, the ammo acid side-chains, and the ammo or carboxyl termini Modifications mclude, for example, acetylation, acylation. ADP- nbosylation. amidation. covalent attachment of flavrn, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide deπvative, covalent attachment of a pid or lipid denvative, covalent attachment of phosphotidy nositol cross-linking, cvchzation, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteme. formation of pyroglutamate. formylation. gamma- carboxylation GPI anchor formation, hydroxylation, lodination, methylation, mynstoylation, oxidation, proteolytic processmg, phosphorylation, prenylation, racemization, glycosylation, hpid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP-nbosylation. selenoylation, sulfation. transfer-RNA mediated addition of ammo acids to protems, such as arginylation, and ubiquitination See, for instance, PROTEINS - STRUCTURE AND MOLECULAR PROPERTIES, 2nd E . T E Creighton, W H Freeman and Company, New York (1993) and Wold, F , Posttranslational Protem Modifications Perspectives and Prospects, pgs 1-12 m POSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B C Johnson, Ed . Academic Press, New York (1983), Seifter et al , Meth Enzymol 182 626-646 (1990) and Rattan et al , Protein Synthesis Posttranslational Modifications and Aging, Ann N Y Acad Sci 663 48-62 (1992) Polypeptides may be branched or cyclic, with or without branching Cyclic, branched and branched circular polypeptides may result from post-translational natural processes and may be made by entirely synthetic methods, as well
"Recombinant expression system(s)" refers to expression systems or portions thereof or polynucleotides of the mvention mtroduced or transformed mto a host cell or host cell lysate for the production of the polynucleotides and polypeptides of the mvention
"Subtraction set" is one or more, but preferably less than 100, polynucleotides compnsrng at least one polynucleotide of the invention
"Vaπant(s)" as the term is used herein, is a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide respectively, but retains essential properties A typical vanant of a polynucleotide differs in nucleotide sequence from another, reference polynucleotide Changes m the nucleotide sequence of the variant may or may not alter the ammo acid sequence of a 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 m amino acid sequence from another, reference Generally, differences are limited so that the sequences of the reference polypeptide and the vanant 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 m 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 mclude vaπants of each of the polypeptides of the mvention. that is polypeptides that vary from the referents by conservative ammo acid substitutions, whereby a residue is substituted b\ 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 or aromatic residues Phe and Tyr Particularly prefeπed are vaπants m which several. 5-10, 1-5, 1-3 1-2 or 1 ammo acids are substituted, deleted, or added in any combination A vanant of a polynucleotide or polypeptide may be a naturally occurring such as an allelic variant, or it ma% be a variant that is not known to occur naturally Non-naturally occurring vanants of polynucleotides and polypeptides may be made by mutagenesis techniques, by direct synthesis, and by other recombinant methods known to skilled artisans EXAMPLES
The examples below are earned out usmg standard techniques, which are well known and routme to those of skill m the art, except where otherwise descπbed m detail The examples are illustrative, but do not limit the mvention Example 1 Strain selection, Library Production and Sequencing The polynucleotide having a DNA sequence given m Table 1 [SEQ ID NO 1] was obtained from a library of clones of chromosomal DNA of Staphylococcus aureus in E cob The sequencing data from two or more clones containmg overlapping Staphylococcus aureus DNAs was used to construct the contiguous DNA sequence in SEQ ID NO 1 Libraries may be prepared by routine methods, for example Methods 1 and 2 below
Total cellular DNA is isolated from Staphylococcus aureus WCUH 29 according to standard procedures and size-fractionated by either of two methods Method 1 Total cellular DNA is mechanically sheared by passage through a needle in order to size- fractionate according to standard procedures DNA fragments of up to l lkbp in size are rendered blunt by treatment with exonuclease and DNA polymerase. and EcoRI linkers added Fragments are hgated mto 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 mto library vectors (e g . Rsal, Pall, Alul, Bshl235I), and such fragments are size-fractionated according to standard procedures EcoRI linkers are hgated to the DNA and the fragments then hgated into the vector Lambda ZapII that have been cut with EcoRI, the library packaged by standard procedures, and E coli infected with the packaged library The library is amplified by standard procedures

Claims

What is claimed is:
1 An isolated polypeptide selected from the group consisting of
(1) an isolated polypeptide comprising an amino acid having at least
(a) 70% identity,
(b) 80% identity,
(c) 90% identity, or
(d) 95% identity to the ammo acid sequence of SEQ ID NO 2 over the entire length of SEQ ID NO 2, (n) an isolated polypeptide comprising the ammo acid sequence of SEQ ID NO 2, (in) an isolated polypeptide which is the amino acid sequence of SEQ ID NO 2, and (iv) a polypeptide which is encoded by a recombinant polynucleotide comprising the po micleotide sequence of SEQ ID NO 1
2 An isolated polynucleotide selected from the group consistmg of
(I) an isolated polynucleotide compnsrng a polynucleotide sequence encodmg a polypeptide that has at least
(a) 70% identity,
(b) 80% identity,
(c) 90% identity, or
(d) 95% identity. to the ammo acid sequence of SEQ ID NO 2, over the entire length of SEQ ID NO 2, (n) an isolated polynucleotide compπsmg a polynucleotide sequence that has at least
(a) 70% identity
(b) 80% identity.
(c) 90% identity, or
(d) 95% identity. over its entire length to a polynucleotide sequence encoding the polypeptide of SEQ ID NO 2,
(m) an isolated polynucleotide compπsmg a nucleotide sequence which has at least
(a) 70%o identity.
(b) 80% identify
(c) 90% identity, or
(d) 95% identify to that of SEQ ID NO 1 over the entire length of SEQ ID NO 1 , (iv) an isolated polynucleotide compnsrng a nucleotide sequence encodmg the polypeptide of SEQ ID
NO 2,
(v) an isolated polynucleotide which is the polynucleotide of SEQ ID NO 1 ,
(vi) an isolated polynucleotide obtainable by screening an appropnate library under stringent hybndization conditions with a probe havmg the sequence of SEQ ID NO 1 or a fragment thereof.
(vn) an isolated polynucleotide encodmg a mature polypeptide expressed by the mdD gene contamed in the Staphylococcus aureus, and
(vm) a polynucleotide sequence complementary to said isolated polynucleotide of (I), (n), (m), (iv), (v),
3 An antibody antigenic to or immunospecific for the polypeptide of claim 1
4 A method for the treatment of an individual
(I) m need of enhanced activity or expression of the polypeptide of claim 1 comprising the step of
(a) administering to the individual a therapeutically effective amount of an agonist to said polypeptide, or
(b) providing to the individual an isolated polynucleotide compπsmg a polynucleotide sequence encodmg said polypeptide m a form so as to effect production of said polypeptide activity in vivo, or
(n) having need to inhibit activity or expression of the polypeptide of claim 1 compnsrng
(a) administering to the individual a therapeutically effective amount of an antagonist to said polypeptide. or
(b) administering to the individual a nucleic acid molecule that inhibits the expression of a polynucleotide sequence encoding said polypeptide, or
(c) administering to the individual a therapeutically effective amount of a polypeptide that competes with said polypeptide for its hgand. substrate , or receptor
5 A process for diagnosing or prognosing a disease or a susceptibility to a disease in an individual related to expression or activity of the polypeptide of claim 1 m an mdividual comprising the step of
(a) determining the presence or absence of a mutation in the nucleotide sequence encoding said polypeptide in the genome of said individual, or
(b) analyzing for the presence or amount of said polypeptide expression m a sample derived from said individual
6 A method for screening to identify compounds that activate or that inhibit the function of the polypeptide of claim 1 which compπses a method selected from the group consistmg of
(a) measuring the bmdmg of a candidate compound to the polypeptide or to the cells or membranes bearing the polypeptide or a fusion protein thereof by means of a label directly or indirectly associated with the candidate compound,
(b) measuring the bmdmg of a candidate compound to the polypeptide or to the cells or membranes bearing the polypeptide or a fusion protem thereof m the presence of a labeled competitor,
(c) testing whether the candidate compound results in a signal generated
Figure imgf000048_0001
activation or inhibition of the polypeptide, usmg detection systems appropriate to the cells or cell membranes bearing the polypeptide,
(d) mixing a candidate compound with a solution containing a polypeptide of claim 1, to form a mixture, measuring activity of the polypeptide in the mixture, and comparing the activity of the mixture to a standard,
(e) detecting the effect of a candidate compound on the production of mRNA encoding said polypeptide and said polypeptide in cells, using for instance, an ELISA assay, or
(f) (1) contacting a composition compπsmg the polypeptide with the compound to be screened under conditions to permit mteraction between the compound and the polypeptide to assess the mteraction of a compound, such mteraction bemg associated with a second component capable of providing a detectable signal in response to the mteraction of the polypeptide with the compound, and
(2) determirung whether the compound interacts with and activates or inhibits an activity of the polypeptide by detectmg the presence or absence of a signal generated from the mteraction of the compound with the polypeptide
7 An agonist or an antagonist of the activity or expression polypeptide of claim 1
8 An expression system compnsrng a polynucleotide capable of producing a pohpeptide of claim 1 when said expression system is present in a compatible host cell
9 A host cell comprising the expression system of claim 8 or a membrane thereof expressing a polypeptide selected from the group consisting of
(I) an isolated polypeptide comprising an amino acid sequence selected from the group having at least
(a) 70% identity,
(b) 80% identity, (c) 90% identity, or
(d) 95% identity to the ammo acid sequence of SEQ ID NO 2 over the entire length of SEQ ID NO 2, (n) an isolated po peptide comprising the ammo acid sequence of SEQ ID NO 2, (in) an isolated polypeptide which is the amino acid sequence of SEQ ID NO 2, and (iv) a polypeptide which is encoded by a recombinant polynucleotide compnsrng the polynucleotide sequence of SEQ ID NO 1
10 A process for producing a polypeptide selected from the group consisting of
(I) an isolated polypeptide comprising an amino acid sequence selected from the group having at least
(a) 70% identity,
(b) 80% identity,
(c) 90% identity, or
(d) 95% identity to the ammo acid sequence of SEQ ID NO 2 over the entire length of SEQ ID NO 2, (n) an isolated polypeptide comprising the amino acid sequence of SEQ ID NO 2, (in) an isolated polypeptide which is the ammo acid sequence of SEQ ID NO 2, and
(iv) a polypeptide which is encoded by a recombinant polynucleotide comprising the polynucleotide sequence of SEQ ID NO 1, comprising the step of culturmg a host cell of claim 9 under conditions sufficient for the production of said polypeptide
11 A process for producing a host cell comprising the expression system of claim 8 or a membrane thereof expressing a polypeptide selected from the group consisting of
(1) an isolated polypeptide comprising an ammo acid sequence selected from the group having at least
(a) 70% identity,
(b) 80% identity,
(c) 90% identity, or
(d) 95% identity to the amino acid sequence of SEQ ID NO 2 over the entire length of SEQ ID NO 2, (n) an isolated polypeptide comprising the amino acid sequence of SEQ ID NO 2, (in) an isolated polypeptide which is the amino acid sequence of SEQ ID NO 2. and (iv) a polypeptide which is encoded by a recombinant polynucleotide comprising the polynucleotide sequence of SEQ ID NO 1, said process comprising the step of transforming or transfectmg a cell with an expression system compnsrng a polynucleotide capable of producing said polypeptide of (1), (n), ( ) or (iv) when said expression system is present in a compatible host cell such the host cell, under appropriate culture conditions, produces said polypeptide of (1), (u), (in) or (iv)
12 A host cell produced by the process of claim 11 or a membrane thereof expressmg a polypeptide selected from the group consistmg of
(l) an isolated polypeptide comprising an amino acid sequence selected from the group having at least
(a) 70% identity,
(b) 80% identity,
(c) 90% identity, or
(d) 95% identity to the amino acid sequence of SEQ ID NO 2 over the entire length of SEQ ID NO 2, (u) an isolated polypeptide comprising the amino acid sequence of SEQ ID NO 2, (in) an isolated polypeptide which is the ammo acid sequence of SEQ ID NO 2, and (iv) a polypeptide which is encoded by a recombinant polynucleotide comprising the polynucleotide sequence of SEQ ID NO 1
13 A computer readable medium having stored thereon a member selected from the group consisting of a polynucleotide comprising the sequence of SEQ ID NO 1. a polypeptide comprising the sequence of SEQ ID NO 2, a set of polynucleotide sequences wherein at least one of said sequences comprises the sequence of SEQ ID NO 1, a set of polypeptide sequences wherein at least one of said sequences comprises the sequence of SEQ ID NO 2, a data set representing a polynucleotide sequence compnsrng the sequence of SEQ ID NO 1. a data set representing a polynucleotide sequence encoding a polypeptide sequence comprising the sequence of SEQ ID NO 2 a polynucleotide comprising the sequence of SEQ ID NO 1. a polypeptide comprising the sequence of SEQ ID NO 2, a set of polynucleotide sequences wherein at least one of said sequences comprises the sequence of SEQ ID NO 1, a set of polypeptide sequences wherein at least one of said sequences comprises the sequence of SEQ ID NO 2, a data set representing a polynucleotide sequence comprising the sequence of SEQ ID NO 1. a data set representing a polynucleotide sequence encoding a polypeptide sequence comprising the sequence of SEQ ID NO 2
14. A computer based method for performing homology identification, said method comprising the steps of providing a polynucleotide sequence comprising the sequence of SEQ ID NO: 1 in a computer readable medium; and comparing said polynucleotide sequence to at least one polynucleotide or polypeptide sequence to identify homology.
15. A further embodiment of the invention provides a computer based method for polynucleotide assembly, said method comprising the steps of: providing a first polynucleotide sequence comprising the sequence of SEQ ID NO: 1 in a computer readable medium; and screening for at least one overlapping region between said first polynucleotide sequence and a second polynucleotide sequence.
PCT/US1999/012975 1998-06-17 1999-06-09 NrdD WO1999065530A1 (en)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0786519A2 (en) * 1996-01-05 1997-07-30 Human Genome Sciences, Inc. Staphylococcus aureus polynucleotides and sequences
EP0841394A2 (en) * 1996-09-24 1998-05-13 Smithkline Beecham Corporation Staphylococcus aureus polynucleotides, polypeptides and their uses

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0786519A2 (en) * 1996-01-05 1997-07-30 Human Genome Sciences, Inc. Staphylococcus aureus polynucleotides and sequences
EP0841394A2 (en) * 1996-09-24 1998-05-13 Smithkline Beecham Corporation Staphylococcus aureus polynucleotides, polypeptides and their uses

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