WO2001024635A1 - Asue - Google Patents

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Publication number
WO2001024635A1
WO2001024635A1 PCT/US2000/027224 US0027224W WO0124635A1 WO 2001024635 A1 WO2001024635 A1 WO 2001024635A1 US 0027224 W US0027224 W US 0027224W WO 0124635 A1 WO0124635 A1 WO 0124635A1
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Prior art keywords
polypeptide
seq
polynucleotide
sequence
isolated
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PCT/US2000/027224
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English (en)
Inventor
Magdalena Zalacain
Sanjoy Biswas
Thomas B. Mathie
Stephanie Van Horn
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Smithkline Beecham Corporation
Smithkline Beecham Plc
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Publication of WO2001024635A1 publication Critical patent/WO2001024635A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/305—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F)
    • C07K14/31—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F) from Staphylococcus (G)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention relates to newly identified polynucleotides and polypeptides. and their production and uses, as well as their variants, agonists and antagonists, and their uses
  • the mventon relates to polynucleotides and polypeptides of the asuE (tRNA modifying enzymes) family, as well as their variants, herein referred to as "asuE,” “asuE polynucleot ⁇ de(s),” and “asuE 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 toxigemc 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 bacte ⁇ al endocarditis are also relatively common
  • Osteomyelitis, septic arthritis, septic thrombophlebitis and acute bacte ⁇ al endocarditis are also relatively common
  • There are at least three clinical conditions resulting from the toxigemc properties of Staphylococci The manifestation of tliese diseases result from the actions of exotoxins as opposed to tissue invasion and bacteremia These conditions include Staphylococcal food poison
  • 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 that are resistant to some or all of the standard antibiotics This phenomenon has created an unmet medical need and demand for new anti-microbial agents, vaccines, drug screening methods, and diagnostic tests for tins organism
  • the present mvention relates to asuE. m particular asuE polypeptides and asuE polynucleotides, recombinant materials and methods for their production
  • the mvention relates to methods for usmg such polypeptides and polynucleotides, including treatment of microbial diseases, amongst others
  • the invention relates to methods for identifying agonists and antagonists usmg the materials provided by the invention, and for treating microbial infections and conditions associated with such infections with the identified agonist or antagonist compounds
  • the invention relates to diagnostic assays for detecting diseases associated with microbial infections and conditions associated with such infections, such as assays for detecting asuE expression or activity
  • the mvention relates to asuE polypeptides and polynucleotides as descnbed in greater detail below
  • the mvention relates to polypeptides and polynucleotides of an asuE of Staphylococcus aureus, that is related by ammo acid sequence homology to B subtihs trmU polypeptide
  • the invention relates especially to asuE having a nucleotide and ammo acid sequences set out m Table 1 as SEQ ID NO 1 and SEQ ID NO 2 respectively
  • sequences recited in the Sequence Listing below as "DNA” represent an exemplification of the invention, since those of ordinary skill will recognize that such sequences can be usefully employed in polynucleotides in general, including ⁇ bopolynucleotides
  • NCIMB National Collections of Indust ⁇ al and Marine Bacte ⁇ a Ltd
  • the deposit of the deposited stram has been made under the terms of the Budapest Treaty on die International Recognition of the Deposit of Micro-organisms for Purposes of Patent Procedure
  • the deposited stram will be l ⁇ evocably and without rest ⁇ ction or condition released to the public upon the issuance of a patent
  • the deposited stram is provided merely as convemence to those of skill in the art and is not an admission that a deposit is required for enablement, such as that required under 35 U S C ⁇ 112
  • a license may be required to make, use or sell the deposited stram, and compounds denved therefrom, and no such license is hereby granted
  • an isolated nucleic acid molecule encoding a mature polypeptide expressible by the Staphylococcus aureus WCUH 29 strain, which polypeptide is comp ⁇ sed in the deposited stram
  • Further provided by the mvention are asuE polynucleotide sequences m the deposited strain, such as DNA and RNA,
  • this initial ammo acid of a polypeptide of the mvention is leucme
  • AsuE polypeptide of the mvention is substantially phylogenetically related to other proteins of the asuE (tRNA modifying enzymes) family
  • va ⁇ ants of asuE polypeptide encoded by naturally occurring alleles of an asuE gene are particularly prefe ⁇ ed embodiments of the mvention.
  • the present mvention further provides for an isolated polypeptide that (a) comprises or consists of an ammo acid sequence that has at least 95% identity, most preferably at least 97-99% or exact identity, to that of SEQ ID NO 2 over the entire length of SEQ ID NO 2.
  • polypeptides ofthe mvention mclude a polypeptide of Table 1 [SEQ ID NO 2] (in particular a mature polypeptide) as well as polypeptides and fragments, particularly those that has a biological activity of asuE, and also those that have at least 95% identity to a polypeptide of Table 1 [SEQ ID NO 2] and also m
  • the mvention also mcludes a polypeptide consisting of or comp ⁇ smg a polypeptide ofthe formula
  • X-(R 1 ) m -(R 2 )-(R 3 ) n -Y wherein, at the ammo terminus, X is hydrogen, a metal or any other moiety desc ⁇ bed herein for modified polypeptides, and at the carboxyl terminus, Y is hydrogen, a metal or any other moiety 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 mteger between 1 and 1000 or zero, and R 2 is an ammo acid sequence of the mvention, particularly an ammo acid sequence selected from Table 1 or modified forms thereof In the formula above, R 2 is onented so that its ammo terminal ammo acid residue is at the left, covalently bound to Ri and its carboxy termmal ammo acid residue is at the ⁇ ght, covalently bound to R3 Any stretch of ammo acid residues denote
  • a polypeptide of the mvention is denved from Staphylococcus aureus, however, it may preferably be obtained from other organisms ofthe same taxonomic genus
  • a polypeptide of the mvention may also be obtained, for example, from organisms ofthe same taxonomic family or order
  • a fragment is a va ⁇ ant polypeptide having an ammo acid sequence that is entirely the same as part but not all of any ammo acid sequence of any polypeptide of the mvention As with asuE polypeptides. fragments may be "free-standmg,” or comp ⁇ sed within a larger polypeptide of which they form a part or region, most preferably as a smgle continuous region m a smgle larger polypeptide
  • Prefe ⁇ ed fragments mclude, for example, truncation polypeptides having a portion of an ammo acid sequence of Table 1 [SEQ ID NO 2], or of va ⁇ ants thereof, such as a continuous se ⁇ es of residues that mcludes an ammo- and or carboxyl-terminal ammo acid sequence
  • Degradation forms of the polypeptides of the mvention produced by or in a host cell, particularly a Staphylococcus aureus are also prefe ⁇ ed
  • fragments characterized by structural or funcUonal attributes such as fragments that comp ⁇ se alpha-helix and alpha-helix forming regions, beta-sheet and beta-sheet-forming regions, turn and turn-forming regions, coil and coil-forming regions, hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions, substrate binding region, and high antigemc index regions
  • fragments mclude an isolated polypeptide comprising an ammo acid sequence having at least 15, 20, 30, 40, 50 or 100 contiguous ammo acids from the ammo acid sequence of SEQ ID NO.2, or an isolated polypeptide comprising an ammo acid sequence having at least 15, 20, 30, 40, 50 or 100 contiguous ammo acids truncated or deleted from the ammo acid sequence of SEQ ID NO:2
  • Fragments of the polypepUdes of the mvention may be employed for producmg the co ⁇ esponding full-length polypeptide by peptide synthesis, therefore, these va ⁇ ants may be employed as intermediates for producmg the full-length polypeptides ofthe mvention Polynucleotides It is an object ofthe mvention to provide polynucleotides that encode asuE polypeptides, particularly polynucleotides that encode a polypeptide herein designated asuE
  • the polynucleotide comp ⁇ ses a region encoding asuE polypeptides comp ⁇ smg a sequence set out in Table 1 [SEQ ID NO 1] that mcludes a full length gene, or a variant thereof
  • This mvention provides that this full length gene is essential to the growth and or survival of an organism that possesses it, such as Staphylococcus aureus
  • isolated nucleic acid molecules encodmg and/or expressing asuE polypeptides and polynucleotides, particularly Staphylococcus aureus asuE 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 ofthe mvention relates to isolated polynucleotides, including at least one full length gene, that encodes an asuE polypeptide having a deduced ammo acid sequence of Table 1 [SEQ ID NO 2] and polynucleotides closely related thereto and va ⁇ ants thereof
  • a polynucleotide of the mvention encodmg asuE polypeptide may be obtained usmg standard cloning and screening methods, such as those for cloning and sequencmg chromosomal DNA fragments from bacte ⁇ a usmg Staphylococcus aureus WCUH 29 cells as starting mate ⁇ al, followed by obtaining a full length clone
  • a polynucleotide sequence of the invention such as a polynucleotide sequence given in Table 1 [SEQ ID NO 1]
  • a library of clones of chromosomal DNA of Staphylococcus aureus WCUH 29 in E coh or some other suitable host is probed with a radiolabeled ohgonucleotide, 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
  • each polynucleotide set out in Table 1 [SEQ ID NO 1] was discovered m a DNA library de ⁇ ved from Staphylococcus aureus WCUH 29 Moreover, each DNA sequence set out m Table 1 [SEQ ID NO 1] contains an open readmg frame encodmg a protem havmg about the number of ammo acid residues set forth m Table 1 [SEQ ID NO 2] w th a deduced molecular weight that can be calculated usmg ammo acid residue molecular weight values well known to those skilled m the art The polynucleotide of SEQ ID NO 1. between nucleotide number 1 and the stop codon that begins at
  • the present mvention provides for an isolated polynucleotide comp ⁇ smg or consisting of (a) a polynucleotide sequence that has at least 95% identity, even more preferably at least 97-99% or exact identity to SEQ ID NO 1 over the entire length of SEQ ID NO 1, or the entire length of that portion of SEQ ID NO 1 which encodes SEQ ID NO 2, (b) a polynucleotide sequence encodmg a polypeptide that has at least 95% identity, even more preferably at least 97-99% or 100% exact, to the ammo acid sequence of SEQ ID NO 2, over the entire length of SEQ ID NO 2
  • a polynucleotide encodmg a polypeptide of the present mvention may be obtained by a process that comp ⁇ ses the steps of screemng an approp ⁇ ate library under stringent hyb ⁇ dization conditions with a labeled or detectable probe consistmg of or comp ⁇ smg the sequence of SEQ ID NO 1 or a fragment thereof, and isolatmg a full-length gene and/or genomic clones comp ⁇ smg said polynucleotide sequence
  • the mvention provides a polynucleotide sequence identical over its entire length to a codmg sequence (open readmg frame) in Table 1 [SEQ ID NO 1] Also provided by the mvention is a codmg sequence for a mature polypeptide or a fragment thereof, by itself as well as a codmg sequence for a mature polypeptide or a fragment in readmg frame with another codmg sequence, such as a sequence encodmg a leader or secretory sequence, a pre-, or pro- or prepro-protem sequence
  • the polynucleotide ofthe mvention may also comp ⁇ se at least one non-coding sequence, including for example, but not limited to at least one non-coding 5' and 3 " sequence, such as the transc ⁇ bed but non-translated sequences, termination signals (such as rho-dependent and rho-mdependent termination signals), ⁇ bosome binding sites, Kozak sequences, sequences that stabilize mRNA, mtrons.
  • the polynucleotide sequence may also compnse additional codmg sequence encodmg additional ammo acids
  • a marker sequence that facilitates purification of a fused polypep de can be encoded
  • the marker sequence is a hexa-histidine pepttde, as provided m the pQE vector (Qiagen, hie ) and desc ⁇ bed 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).
  • Polynucleotides of the mvention also mclude, but are not limited to, polynucleotides comp ⁇ smg a structural gene and its naturally associated sequences that control gene expression
  • a prefe ⁇ ed embodiment ofthe mvention is a polynucleotide of consistmg of or comp ⁇ smg nucleotide 1 to the nucleotide immediately upstream of or mcludmg nucleotide 1117 set forth in SEQ ID NO 1 of Table 1, both of that encode an asuE polypeptide
  • the mvention also mcludes a polynucleotide consistmg of or comp ⁇ smg a polynucleotide of the formula
  • R ⁇ and R3 are independently any nucleic acid residue or modified nucleic acid residue
  • m is an integer between 1 and 3000 or zero
  • n is an mteger between 1 and 3000 or zero
  • R 2 is a nucleic acid sequence or modified nucleic acid sequence ofthe invention, particularly a nucleic acid sequence selected from Table 1 or a modified nucleic acid sequence thereof In the polynucleotide formula above, R 2 is oriented so that its 5' end nucleic acid residue is at the left, bound to R ⁇ and its 3' end
  • polynucleotide of the mvention is de ⁇ ved from Staphylococcus aureus, however, it may preferably be obtained from other organisms ofthe same taxonomic genus
  • a polynucleotide ofthe mvention may also be obtained, for example, from organisms ofthe same taxonomic family or order
  • polynucleotide encodmg a polypeptide encompasses polynucleotides that mclude a sequence encoding a polypepfide of the mvention, particularly a bacte ⁇ al polypeptide and more particularly a polypeptide ofthe Staphylococcus aureus asuE havmg an ammo acid sequence set out in Table 1 [SEQ ID NO 2]
  • the term also encompasses polynucleotides that mclude a smgle continuous region or discontinuous regions encodmg the polypeptide (for example,
  • the mvention further relates to va ⁇ ants ofthe polynucleotides desc ⁇ bed herem that encode va ⁇ ants of a polypeptide havmg a deduced ammo acid sequence of Table 1 [SEQ ID NO 2] Fragments of polynucleotides of the mvention may be used, for example, to synthesize flill-lengtli polynucleotides of the mvention
  • prefe ⁇ ed embodiments are polynucleotides encodmg asuE va ⁇ ants that have the ammo acid sequence of asuE 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 combmation
  • Prefe ⁇ ed isolated polynucleotide embodiments also mclude polynucleotide fragments, such as a polynucleotide comprising a nuchc acid sequence having at least 15, 20, 30, 40, 50 or 100 contiguous nucleic acids from the polynucleotide sequence of SEQ ID NO 1 , or an polynucleotide comprising a nucleic acid sequence having at least 15, 20, 30, 40, 50 or 100 contiguous nucleic acids truncated or deleted from the 5' and/or 3' end of the polynucleotide sequence of SEQ ID NO. l
  • prefe ⁇ ed embodiments of the invention are polynucleotides that are at least 95% or 97% identical over their entire length to a polynucleotide encodmg asuE polypeptide havmg an ammo acid sequence set out in Table 1 [SEQ ID NO 2], and polynucleotides that are complementary to such polynucleotides
  • Most highly prefe ⁇ ed are polynucleotides that comp ⁇ se a region that is at least 95% are especially prefe ⁇ ed
  • 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 prefe ⁇ ed. with at least 99% bemg the more prefe ⁇ ed
  • Prefe ⁇ ed embodiments are polynucleotides encodmg polypeptides that retain substantially the same biological function or activity as a mature polypeptide encoded by a DNA of Table 1 [SEQ ID NO 1]
  • the mvention further relates to polynucleotides that hyb ⁇ dize to the polynucleotide sequences provided herem
  • the mvention especially relates to polynucleotides that hyb ⁇ dize under stringent conditions to the polynucleotides desc ⁇ bed herem
  • a specific example of stringent hybridization conditions is overnight incubation at 42°C in a solution comprismg 50% formamide.
  • 5x SSC 150mM NaCl, 15mM t ⁇ sodium citrate), 50 mM sodium phosphate (pH7 6), 5x Denhardt's solution, 10% dextran sulfate, and 20 rrucrograms/ml of denatured, sheared salmon sperm DNA, followed by washmg the hybridization support in 0 lx SSC at about 65°C Hybridization and wash conditions are well known and exemplified m Sambrook, et al , Molecular Cloning A Laboratory Manual. Second Edition. Cold Spring Harbor, N Y , (1989), particularly Chapter 11 therein Solution hybndization may also be used with the polynucleotide sequences provided by the invention
  • the invention also provides a polynucleotide consisting of or comprismg a polynucleotide sequence obtained by screening an appropriate library comprismg a 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 m SEQ ID NO 1 or a fragment thereof, and isolatmg said polynucleotide sequence Fragments useful for obtainmg such a polynucleotide mclude, 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.
  • RNA, cDNA and genomic DNA may be used as a hybndization probe for RNA, cDNA and genomic DNA to isolate full-length cDNAs and genomic clones encodmg asuE and to isolate cDNA and genomic clones of other genes that have a high identity, particularly high sequence identity, to an asuE 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 an asuE gene may be isolated by screening usmg a DNA sequence provided m Table 1 [SEQ ID NO 1] to synthesize an ohgonucleotide probe
  • a labeled oligonucleotide havmg a sequence complementary to that of a gene ofthe mvention is then used to screen a library of cDNA, genomic DNA or mRNA to determine which members ofthe library the probe hybndizes to
  • polynucleotides and polypeptides of the mvention may be employed, for example, as research reagents and mate ⁇ als for discovery of treatments of and diagnostics for diseases, particularly human diseases, as further discussed herem relatmg to polynucleotide assays
  • the polynucleotides ofthe invention that are ohgonucleotides derived from a sequence of Table
  • SEQ ID NOS 1 or 2 may be used in the processes herein as described, but preferably for PCR, to determine whether or not the polynucleotides identified herein m whole or in part are transcribed m bacteria in infected tissue It is recogmzed that such sequences will also have utility in diagnosis of the stage of infection and type of infection the pathogen has attained
  • the mvention also provides polynucleotides that encode a polypeptide that is a mature protein plus additional ammo or carboxyl-terminal ammo acids, or am o acids inte ⁇ or to a mature polypeptide (when a mature form has more than one polypeptide chain, for instance) Such sequences may play a role in processmg of a protem from precursor to a mature form, may allow protem transport, may lengthen or shorten protem half-life or may facilitate manipulation of a protem for assay or production, among other things As generally is the case in vivo, the additional ammo acids may be processed away from
  • a precursor protein, havmg a mature form ofthe polypeptide fused to one or more prosequences may be an inactive form ofthe polypeptide
  • prosequences When prosequences are removed such mactive precursors generally are activated
  • Some or all ofthe prosequences may be removed before activation Generally, such precursors are called proprotems
  • the entire polypeptide encoded by an open readmg frame is often not required for activity Accordmgly, it has become routine m molecular biology to map die boundanes of the primary structure required for activity with N-terminal and C-termnal deletion experiments
  • anv contiguous fragment of SEQ ID NO 2 which retains at least 20%, preferably at least 50%, of an activity ofthe polypeptide encoded by the gene for SEQ ID NO 2 is withm the invention, as are corresponding fragment which are 70%, 80%, 90%, 95%,97%, 98% or 99% identical to such contiguous fragments
  • the contiguous fragment comprises at least 70% ofthe ammo acid residues of SEQ ID NO 2, preferably at least 80%. 90% or 95% ofthe residues
  • a polynucleotide ofthe mvention may encode a mature protem, a mature protem plus a leader sequence (that may be refe ⁇ ed to as a preprotein), a precursor of a mature protem havmg one or more prosequences that are not the leader sequences of a preprotein. or a preproprotein. that is a precursor to a proprotein. havmg a leader sequence and one or more prosequences, that generally are removed during processmg steps that produce active and mature forms ofthe polypeptide
  • 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 ofthe mvention by recombinant techniques
  • Cell-free translation systems can also be employed to produce such proteins usmg RNAs denved from the DNA constructs ofthe mvention
  • Recombinant polypeptides ofthe present mvention may be prepared by processes ell known m those skilled in the art from genetically engineered host cells compnsing expression systems Accordingh .
  • die present mvention relates to expression systems that comp ⁇ se a polynucleotide or polynucleotides ofthe present mvention, to host cells that are genetically engmeered with such expression systems, and to the production of polypeptides ofthe mvention by recombinant techniques
  • 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 desc ⁇ bed in many standard laboratory manuals, such as Davis, et al , BASIC METHODS IN MOLECULAR BIOLOGY. (1986) and Sambrook, et al , MOLECULAR CLONING A LABORATORY MANUAL, 2nd Ed , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N Y (1989), such as. calcium phosphate transfection. DEAE-dextran mediated transfection, transvection, micromjection, catiomc hpid-mediated transfection, electroporation, transduction, scrape loading, ballistic mtroduction and infection
  • bacte ⁇ al cells such as cells of streptococci, staphylococci, enterococci E coh, streptomyces, cyanobacte ⁇ a, Bacillus subtihs, and Staphylococcus aureus
  • fungal cells such as cells of a yeast, Kluveromyces , Saccharomyces, a basidiomycete.
  • Candida albicans and Aspergillus insect cells such as cells of Drosoph a S2 and Spodoptera Sf .
  • animal cells such as CHO, COS, HeLa, C 127, 3T3, BHK, 293.
  • CV-1 and Bowes melanoma cells and plant cells, such as cells of a gymnosperm or angiosperm
  • vectors include, among others, chromosomal-, episomal- and virus-de ⁇ ved vectors, for example, vectors denved from bactenal plasmids, from bactenophage, from transposons, from yeast episomes.
  • the expression system constructs may compnse control regions that regulate as well as engender expression Generally, any system or vector suitable to maintain, propagate or express polynucleotides and or to express a polypeptide in a host may be used for expression m this regard
  • the appropnate DNA sequence may be inserted mto the expression system by any of a vanety of well-known and routine techniques, such as, for example, those set forth m Sambrook et al , MOLECULAR CLONING, A LABORATORY MANUAL, (
  • Polypeptides ofthe mvention can be recovered and pu ⁇ fied from recombinant cell cultures by well-known methods mcludmg ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic mteraction chromatography, affinity chromatography, hydroxylapatite chromatography, and lectin chromatography Most preferably, high performance liquid chromatography is employed for punfication
  • Well known techniques for refolding protem may be employed to regenerate active conformation when the polypeptide is denatured during isolation and or punfication
  • This mvention is also related to the use of asuE polynucleotides and polypeptides ofthe mvention for use as diagnostic reagents Detection of asuE 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 asuE gene or protein, may be detected at the nucleic acid or ammo acid level by a vanety of well known techniques as well as by methods provided herem
  • Polypeptides and polynucleotides for prognosis, diagnosis or other analysis may be obtained from a putatively infected and or infected individual's bodily mate ⁇ als
  • Polynucleotides from any of these sources may be used directly for detection or may be amplified enzymatically by usmg PCR or any other amplification technique pnor to analysis RNA, particularly mRNA, cDNA and genomic DNA may also be used m the same ways Usmg amplification, characte ⁇ zation ofthe species and stram of infectious or resident organism present m an individual, may be made by an analysis of the genotype of a selected polynucleotide ofthe organism Deletions and insertions can be detected by a change m size ofthe amplified product m companson to a genotype of a reference sequence selected from a related orgamsm, preferably a different species ofthe same genus or a different stram ofthe same species Pom
  • an array of oligonucleotides probes comp ⁇ smg asuE nucleotide sequence or fragments thereof can be constructed to conduct efficient screemng of. for example, genetic mutations, serotype, taxonomic classification or identification Array technology methods are well known and have general applicability and can be used to address a vanety of questions m molecular genetics mcludmg gene expression, genetic linkage, and genetic vanabihty (see.
  • the present invention relates to a diagnostic kit that comprises (a) a polynucleotide ofthe 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 mvention, preferably the polypeptide of SEQ ID NO 2 or a fragment thereof, or (d) an antibody to a polypeptide of the present mvention, preferably to the polypeptide of SEQ ID NO 2
  • any such kit, (a), (b), (c) or (d) may comp ⁇ se a substantial component
  • Such a kit will be of use in diagnosing a disease or susceptibility to a Disease, among others
  • This mvention also relates to the use of polynucleotides ofthe present mvention as diagnostic reagents Detection of a mutated form of a polynucleotide ofthe mvention, preferable.
  • SEQ ID NO 1. that is associated with a disease or pathogenicity will provide a diagnostic tool that can add to, or define, a diagnosis of a disease, a prognosis of a course of disease, a determination of a stage of disease, or a susceptibility to a disease, that results from under-expression, over-expression or altered expression ofthe polynucleotide
  • Organisms, particularly infectious organisms, carrying mutations m such polynucleotide may be detected at the polynucleotide level by a vanety of techmques, such as those descnbed elsewhere herem
  • the differences in a polynucleotide and/or polypeptide sequence between organisms possessing a first phenotype and organisms possessing a different, second different phenotype can also be determined If a mutation is observed in some or all organisms possessing the first phenotype but not in any organisms possessing the second phenotype, then the mutation is likely to be the causative agent of the first phenotype
  • a polynucleotide and/or polypepttde ofthe mvention may also be detected at the polynucleotide or polypeptide level by a vanety of techmques, to allow for serotypmg, for example
  • RT-PCR can be used to detect mutations m the RNA It is particularly prefe ⁇ ed 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 primers complementary to a polynucleotide encodmg asuE polypeptide can be used to identify and analyze mutations
  • the mvention further provides these primers with 1, 2, 3 or 4 nucleotides removed from the 5' and or the 3' end
  • These primers may be used for. among other things, amplifying asuE DNA and/or RNA isolated from a sample denved from an individual, such as a bodily matenal
  • the p ⁇ mers may be used to amplify a polynucleotide isolated from an infected individual, such that the polynucleotide may then be subject to vanous techmques for elucidation of the polynucleotide sequence In this way. mutations in the polynucleotide sequence may be detected and used to diagnose and/or prognose the infection or its stage or course, or to serotype and/or classify the infectious agent
  • the mvention further provides a process for diagnosmg, disease, preferably bacterial infections, more preferably infections caused by Staphylococcus aureus, comprising determining from a sample derived from an individual, such as a bodily material, an increased level of expression of polynucleotide havmg a sequence of Table 1 [SEQ ID NO 1] Increased or decreased expression of an asuE 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.
  • a diagnostic assay in accordance with the mvention for detectmg over-expression of asuE 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 an asuE polypeptide. in a sample denved from a host, such as a bodily matenal, are well-known to those of skill in the art Such assay methods mclude radioimmunoassays. competitive-binding assays. Western Blot analysis, antibody sandwich assays, antibody detection and ELISA assays
  • Polypeptides and polynucleotides of the mvention may also be used to assess the binding of small molecule substrates and ligands m, for example, cells, cell-free preparations, chemical libranes, and natural product mixtures
  • substrates and ligands may be natural substrates and ligands or may be structural or functional mimetics See, e g , Coligan et al , Current Protocols in Immunology 1 (2) Chapter 5 (1991)
  • Polypeptides and polynucleotides ofthe present mvention are responsible for many biological functions, mcludmg many disease states, m particular the Diseases herem mentioned It is therefore desirable to devise screemng methods to identify compounds that agonize (e g , stimulate) or that antagonize (e g , ⁇ nh ⁇ b ⁇ t) the function ofthe polypeptide or polynucleotide Accordmgly, m a further aspect, the present mvention provides for a method of screemng compounds to identify those that agomze or that antagonize the function of a polypeptide or polynucleotide ofthe mvention, as well as related polypeptides and polynucleotides In general, agonists or antagonists (e g , inhibitors) may be employed for therapeutic and prophylactic purposes for such Diseases as herem mentioned Compounds may be identified from a vanety of sources, for example, cells, cell-free preparations, chemical libran
  • agonists and antagonists so-identified may be natural or modified substrates, ligands, receptors, enzymes etc . as the case may be, of asuE polypeptides and polynucleotides, or may be structural or functional mimetics thereof (see Coligan et al .
  • the screemng methods may simply measure the binding of a candidate compound to the polypeptide or polynucleotide, or to cells or membranes bearmg the polypeptide or polynucleotide, or a fusion protem ofthe polypeptide by means of a label directly or indirectly associated with the candidate compound Alternatively, the screemng method may mvolve competition with a labeled competitor Further, these screemng methods may test whether the candidate compound results m a signal generated by activation or inhibition ofthe 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 ofthe candidate compound is observed Constitutively active polypeptide and/or constitutively expressed polypeptides and polynucleotides may be employed m screening methods for inverse
  • polypeptides, polypeptides and antibodies that bind to and/or interact with a polypeptide ofthe present invention may also be used to configure screening methods for detectmg the effect of added compounds on the production of mRNA and/or polypeptide in cells
  • an ELISA assay may be constructed for measuring secreted or cell associated levels of polypeptide using monoclonal and polyclonal antibodies by standard methods known in the art This can be used to discover agents that may inhibit or enhance the production of polypeptide (also called antagomst or agomst, respectively) from suitably manipulated cells or tissues
  • the mvention also provides a method of screening compounds to identify those that enhance (agomst) or block (antagomst) the action of asuE polypeptides or polynucleotides, particularly those compounds that are bactenstatic and/or bactencidal
  • the method of screemng may mvolve high-throughput teclmiques
  • a synthetic reaction mix for example, a synthetic reaction mix, a cellular compartment, such as a membrane, cell envelope or cell wall, or a preparation of any thereof, compnsmg asuE polypeptide and a labeled substrate or hgand of such polypeptide is mcubated in the absence or the presence of a candidate molecule that may be an asuE agomst or antagomst
  • the ability of the candidate molecule to agonize or antagonize the asuE polypeptide is reflected m decreased binding ofthe labeled gand or decreased production of product from such substrate Molecules that
  • a reporter system Reporter systems that may be useful m this regard mclude but are not limited to colo ⁇ met ⁇ c, labeled substrate converted mto product, a reporter gene that is responsive to changes in asuE polynucleotide or polypeptide activity, and binding assays known m the art Polypeptides ofthe 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 techmques include, but are not limited to, hgand bmding and crosslinking assays in which the polypeptide is labeled with a radioactive isotope (for instance.
  • ⁇ • 'I chemically modified (for instance, biotinylated). or fused to a peptide sequence suitable for detection or purification, and mcubated with a source ofthe putative receptor (e g , cells, cell membranes, cell supematants, tissue extracts, bodily materials)
  • a source ofthe putative receptor e g , cells, cell membranes, cell supematants, tissue extracts, bodily materials
  • Other methods include biophysical techniques such as surface plasmon resonance and spectroscopy These screening methods may also be used to identify agomsts and antagomsts ofthe polypeptide that compete with the binding ofthe 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 by asuE polypeptide associatmg with another asuE polypeptid
  • labeled to comprise a fluorescenfly- labeled molecule will have higher polarization values than a fluorescently labeled monome ⁇ c protem It is preferred that this method be used to characte ⁇ ze small molecules that disrupt polypeptide complexes
  • Fluorescence energy transfer may also be used characterize small molecules that interfere with the formation of asuE polypeptide dimers, t ⁇ mers. tetramers or higher order structures, or structures formed by asuE polypeptide bound to another polypeptide AsuE polypeptide can be labeled with both a donor and acceptor fluorophore Upon mixmg ofthe two labeled species and excitation ofthe donor fluorophore. fluorescence energy transfer can be detected by observing fluorescence ofthe acceptor Compounds that block drme ⁇ zation will inhibit fluorescence energy transfer
  • AsuE 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 asuE polypeptide -coated surface and specific binding can be detected in real-time by monitoring the change in resonance angle caused by a change in local refractive mdex This technique can be used to characterize the effect of small molecules on kinetic rates and equilibrium bmdmg constants for asuE polypeptide self-association as well as an association of asuE polypeptide and another polypeptide or small molecule
  • a scintillation proximity assay may be used to characterize the interaction between an association of asuE polypeptide with another asuE polypeptide or a different polypeptide
  • AsuE polypeptide can be coupled to a scmtillation-filled bead Addition of radio-labeled asuE polypeptide results bmdmg where the radioactive source molecule is m close proximity to the scintillation fluid
  • signal is emitted upon asuE polypeptide binding and compounds that prevent asuE polypeptide self-association or an association of asuE polypeptide and another polypeptide or small molecule will dimmish signal
  • identifying compounds that bmd to or otherwise mteract with and inhibit or activate an activity or expression of a polypeptide and/or polynucleotide of the mvention compnsing contacting a polypeptide and/or polynucleotide of the mvention with a compound to be screened under conditions to permit bmdmg to or other mteraction between the compound and the polypeptide and/or polynucleotide to assess the bmdmg to or other mteraction with the compound, such bmdmg or mteraction preferably bemg associated with a second component capable of providing a detectable signal m response to the bmdmg or mteraction ofthe polypeptide and/or polynucleotide with the compound, and determining whether die compound bmds to or otherwise mteracts with and activates or inhibits an activity or expression of the polypeptide and/or polynu
  • an assay for asuE agomsts is a competitive assay that combmes asuE and a potential agomst with asuE-binding molecules, recombinant asuE bmdmg molecules, natural substrates or ligands, or substrate or gand mimetics, under appropnate conditions for a competitive inhibition assay AsuE can be labeled, such as by radioactivity or a colonmet ⁇ c compound, such that the number of asuE molecules bound to a bmdmg molecule or converted to product can be determined accurately to assess the effectiveness ofthe potential antagomst
  • a polypeptide and or polynucleotide of the present mvention may also be used m a method for the structure-based design of an agomst or antagomst ofthe polypeptide and/or polynucleotide, by (a) determining in the first instance the three- dimensional structure ofthe polypeptide
  • 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 asuE polypeptide and/or polynucleotide
  • the expression and/or activity ofthe polypeptide and/or polynucleotide is m excess, several approaches are available
  • One approach comp ⁇ ses admimstermg to an mdividual m need thereof an inhibitor compound (antagomst) as herem descnbed, optionally m combination with a pharmaceutically acceptable earner, in an amount effective to inhibit the function and/or expression ofthe polypeptide and/or polynucleotide, such as, for example, by blockmg 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 ofthe 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 ofthe asuE polypeptide and/or polypeptide
  • expression ofthe gene encoding endogenous asuE polypeptide can be inhibited using expression blockmg teclmiques
  • This blocking may be targeted against any step in gene expression, but is preferably targeted agamst transcription and or translation
  • An examples of a knowm 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.
  • ohgonucleotides that form triple helices with the gene can be supplied (see, for example, Lee et al Nucleic Acids Res (1979) 6 3073, Cooney et al , Science (1988) 241 456, Dervan et al , Science (1991) 251 1360) These ohgomers can be administered />er se or the relevant o gomers can be expressed in vivo
  • Each of the polynucleotide sequences provided herein may be used m the discovery and development of antibacterial compounds
  • the encoded protem upon expression, can be used as a target for the screening of antibacterial drugs
  • the polynucleotide sequences encodmg the ammo termmal regions of the encoded protem 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 m the prevention of adhesion of bacteria, in particular gram positive and/or gram negative bacteria, to eukaryotic, preferably mammalian, extracellular matrix proteins on in-dwelling devices or to extracellular matrix proteins m wounds, to block bacterial adhesion between eukaryotic, preferably mammalian, extracellular matrix proteins and bacterial asuE protems that mediate tissue damage and/or, to block the normal progression of pathogenesis in infections initiated other than by the implantation of in-dwelling devices or by other surgical techniques
  • agomsts and antagomsts preferably bactenstatic or bactencidal agomsts and antagomsts
  • the antagomsts and agomsts of the mvention may be employed, for instance, to prevent, inhibit and/or treat diseases
  • Antagomsts ofthe mvention mclude, among others, small organic molecules, peptides, polypeptides and antibodies that bmd to a polynucleotide and/or polypeptide of the mvention and thereby inhibit or extinguish its activity or expression
  • Antagomsts also may be small organic molecules, a peptide, a polypeptide such as a closely related protem or antibody that bmds the same sites on a bmdmg molecule, such as a bmd g molecule, without mducmg asuE-mduced activities, thereby preventmg the action or expression of asuE polypeptides and/or polynucleotides by excludmg asuE polypeptides and/or polynucleotides from bmdmg
  • Antagomsts of die mvention also mclude a small molecule tiiat bmds to and occupies the bmdmg site of the polypeptide thereby preventmg bmdmg to cellular binding molecules, such that normal biological activity is prevented
  • small molecules include but are not limited to small organic molecules, peptides or peptide-like molecules
  • Other antagomsts mclude antisense molecules (see Okano.
  • antagomsts mclude compounds related to and va ⁇ ants of asuE
  • polypeptide antagomsts mclude antibodies or, in some cases, oligonucleotides or proteins that are closely related to the ligands, substrates, receptors, enzymes, etc , as the case may be, ofthe polypeptide, e g , a fragment ofthe ligands. substrates, receptors, enzymes, etc . or small molecules that bmd to the polypeptide of die present mvention but do not ehcit a response, so that the activity ofthe polypeptide is prevented
  • Small molecules ofthe invention preferably have a molecular weight below 2,000 daltons, more preferably between 300 and 1,000 daltons, and most preferably between 400 and 700 daltons It is preferred that these small molecules are organic molecules
  • Hehcobacter pylori (herein "H pylori”) bacteria 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, Lyon, France, http //www uicc ch/ecp/ecp2904 htm)
  • the International Agency for Research on Cancer recently recognized a cause-and-effect relationship between H pylori and gastric adenocarcmoma.
  • Prefe ⁇ ed antimicrobial compounds of the mvention should be useful m the treatment of H pylori infection Such treatment should decrease the advent of H /? /o ⁇ - ⁇ nduced cancers, such as gastromtestinal carcinoma Such treatment should also prevent, inhibit and or cure gastric ulcers and gastritis
  • Bodily mate ⁇ al(s) means any matenal denved from an mdividual or from an orgamsm infecting, infesting or inhabiting an mdividual, mcludmg but not limited to. cells, tissues and waste, such as, bone, blood, serum, 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 bacte ⁇ a, mcludmg , foi example, disease, such as, infections ofthe upper respiratory tract (e g , otitis media, bacte ⁇ al tracheitis.
  • “Host cell(s)” is a cell that has been introduced (e g . transformed or transfected) or is capable of mtroduction (e g , transformation or transfection) by an exogenous polynucleotide sequence
  • Identity is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as the case may be. as determined by comparing the sequences In die 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, mcludmg but not limited to those described m
  • Methods to determine identity are designed to give the largest match between the sequences tested Moreover, methods to determine identity are codified in 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.
  • Polynucleotide embodiments further include an isolated polynucleotide comprising a polynucleotide sequence having at least a 95, 97 or 100% identity to the reference sequence of SEQ ID NO 1, wherem said polynucleotide sequence may be identical to the reference sequence of SEQ ID NO 1 or may include up to a certain integer number of nucleotide alterations as compared to the reference sequence, wherem said alterations are selected from the group consistmg of at least one nucleotide deletion, substitution, including transition and transversion, or insertion, and wherem said alterations may occur at the 5' or 3' terminal positions ofthe 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 in SEQ ID NO 1 by the integer defining the percent identity divided by 100 and then subtracting that product from
  • n n is the number of nucleotide alterations.
  • x n is the total number of nucleotides m SEQ ID NO 1
  • y is 0 95 for 95%, 0 97 for 97% or 1 00 for 100%.
  • • is the symbol for the multiplication operator, and wherein any non-integer product of x n and y is rounded down to the nearest integer prior to subtracting it from x n
  • Alterations of a polynucleotide sequence encoding the polypeptide of SEQ ID NO 2 may create nonsense, missense or frameshift mutations in this coding sequence and thereby alter the polypeptide encoded by the polynucleotide following such alterations
  • Polypeptide embodiments further mclude an isolated polypeptide comprismg a polypeptide having at least a 95, 97 or 100% identity to a polypeptide reference sequence of SEQ ID NO 2, wherein said polypeptide sequence may be identical to the reference sequence of SEQ ID NO 2 or may include up
  • n a is the number of ammo acid alterations
  • x a is the total number of ammo acids in SEQ ID NO.2
  • y is 0 95 for 95%, 0 97 for 97% or 1 00 for 100%
  • • is the symbol for the multiplication operator, and wherein any non-integer product of x a and y is rounded down to the nearest integer prior to subtracting it from x a
  • 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, i e . if it occurs in nature, it has been changed or removed from its onginal environment, or both
  • a polynucleotide or a polypeptide naturally present in a living orgamsm is not “isolated,” but the same polynucleotide or polypeptide separated from the coexistmg mate ⁇ als 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 metiiod is "isolated” even if it is still present in said orgamsm. which orgamsm may be living or non-living
  • Organ ⁇ sm(s) means a (l) prokaryote.
  • mcludmg but not limited to, a member of the genus Streptococcus, Staphylococcus, Bordetella, Corynebactenum Mycobacterium, Neissena, Haemophilus Act nomycetes, Streptomycetes, Nocardia, Enterobacter, Yersinia, Fancisella Pasturella, Moraxella Acmetobacter, Ery ⁇ elothnx, Branhamella, Actinobacillus, Streptobacillus, Listeria, Calymmatobactenum, Brucella, Bacillus, Clostndium, Treponema, Eschenchia, Salmonella, Kleibsiella Vbno, Proteus, Erwinia, Borreha Leptospira, Spirillum, Campylobacter Shigella Legwnella, Pseudomonas, Aeromon
  • mcludmg but not limited to Archaebacter
  • mcludmg but not limited to Archaebacter
  • mcludmg but not limited to Archaebacter
  • mcludmg but not limited to Archaebacter
  • mcludmg but not limited to Archaebacter
  • mcludmg but not limited to Archaebacter
  • mcludmg but not limited to. a protozoan, a fungus, a member of the genus Saccharomyces, Kluveromyces, or Candida
  • Saccharomyces cenviseae Kluveromyces lactis
  • Polynucleot ⁇ de(s) generally refers to any polynbonucleotide or polydeoxynbonucleotide, that may be unmodified RNA or DNA or modified RNA or DNA "Polynucleot ⁇ de(s)” mclude. without limitation. smgle- and double-stranded DNA, DNA that is a mixture of smgle- and double-stranded regions or smgle-. double- and t ⁇ ple-stranded regions, smgle- and double-stranded RNA. and RNA that is mixture of smgle- and double-stranded regions, hybnd molecules comp ⁇ smg DNA and RNA that may be single-stranded or.
  • polynucleotide refers to tnple-stranded regions compnsing RNA or DNA or botii 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 ofthe molecules, but more typically mvolve only a region of some ofthe molecules
  • One of die molecules of a tiiple-he cal region often is an oligonucleotide As used herem.
  • polynucleotide(s) also mcludes DNAs or RNAs as desc ⁇ bed above that comp ⁇ se one or more modified bases
  • DNAs or RNAs with backbones modified for stability or for other reasons are "polynucleot ⁇ de(s)” as that term is intended herem
  • polynucleotides as the term is used herem
  • t ⁇ tylated bases to name ust two examples
  • polynucleotides as the term is used herem
  • Polynucleot ⁇ de(s) also embraces short polynucleotides often refe ⁇ ed to as ol ⁇ gonucleotide(s)
  • Polypeptide(s) refers to any peptide or protem comp ⁇ smg two or more ammo acids joined to each other by peptide bonds or modified peptide bonds
  • Polypept ⁇ de(s) refers to both short chains, commonly refe ⁇ ed to as peptides, ohgopeptides and ohgomers and to longer chams generally refe ⁇ ed to as proteins
  • Polypeptides may comp ⁇ se ammo acids othe 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 techniques Such modifications are well desc ⁇ bed in basic texts and m more detailed monographs, as well as in a voluminous research literature, and they are well known to uiose of skill in the art It will be appreciated diat the same type of modification may be present m the same or varying degree at several sites m a given polypeptide Also,
  • acetylation, acylation, ADP-nbosylation, amidation covalent attachment of flavm, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide de ⁇ vative, covalent attachment of a hpid or pid de ⁇ vative.
  • 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 mediods, as well
  • Recombinant expression system(s) refers to expression systems or portions thereof or polynucleotides ofthe mvention introduced or transformed mto a host cell or host cell lysate for the production of die polynucleotides and polypeptides ofthe mvention "Var ⁇ ant(s)' " as the term is used herein, is a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide respectively, but retains essential properties
  • a typical variant of a polynucleotide differs in nucleotide sequence from another, reference polynucleotide Changes m the nucleotide sequence of the variant may or may not alter the ammo acid sequence of a polypeptide encoded by the reference polynucleotide Nucleotide changes may result m ammo acid substitutions, additions, deletions, fusion proteins and truncations in the polypeptide encoded by the reference sequence, as discussed below A typical van
  • a variant of a polynucleotide or polypeptide may be a naturally occurring such as an allehc variant, or it may be a variant that is not known to occur naturally
  • Non-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 polynucleotide havmg a DNA sequence given in Table 1 [SEQ ID NO 1] was obtained from a library of clones of chromosomal DNA of Staphylococcus aureus in E coll
  • the sequencing data from two or more clones comprising overlappmg Staphylococcus aureus DNAs was used to construct the contiguous DNA sequence SEQ ID NO 1 Libraries may be prepared by routine methods, for example Methods 1 and 2 below
  • Total cellular DNA is mechanically sheared by passage through a needle in order to size- fractionate according to standard procedures
  • DNA fragments of up to 1 lkbp in size are rendered blunt by treatment with exonuclease and DNA polymerase, and EcoRI linkers added Fragments are hgated mto the vector Lambda ZapII that has been cut with EcoRI, the library packaged by standard procedures and E coh infected with the packaged library
  • the library is amplified by standard procedures
  • Total cellular DNA is partially hydrolyzed with a one or a combination of restriction 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 accordmg 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 coh infected with the packaged library
  • the library is amplified by standard procedures
  • the asuE gene is expressed durine infection of Staphylococcus aureus WCUH29 in a pyelonephritis infection model
  • Necrotic fatty tissue from a kidney from a seven day pyelonephritis infection of Staphylococcus aureus WCUH29 in the mouse is efficiently disrupted and processed in the presence of acid phenol and detergent to provide a mixture of animal and bacte ⁇ al RNA
  • the resultant total RNA is free of DNA and protem (mcludmg RNAases and DNAases)
  • the optimal conditions for disruption and processing to give high yields of bacterial mRNA with transc ⁇ pts of long length are followed by reverse transcribing the resultmg mRNA to cDNA and amplified with ORF-specific primers for a bacterial gene known to be expressed constitutively and at low copy number in Staphylococcus aureus WCUH29
  • Infected tissue samples in 2-ml cyro-strorage tubes, are removed from -80°C storage into a dry ice ethanol bath In a microbiological safety cabinet the samples are disrupted up to eight at a time while the remaining samples are kept frozen in the dry ice ethanol bath To disrupt the bacteria within the tissue sample. 50-100 mg of the tissue is transfered to a FastRNA tube containing a silica/ceramic matrix (BIO 101) Innnediately . 1 ml of extraction reagents (FastRNA reagents. BIO 101) are added to give a sample to reagent volume ratio of approximately 1 to 20 The tubes are shaken in a reciprocating shaker (FastPrep FP120.
  • FastPrep FP120 reciprocating shaker
  • RNA preparations are stored in this isopropanol solution at -80°C if necessary
  • the RNA is pelleted (12,000g for 10 mm ), washed with 75% ethanol (v/v m DEPC-treated water), air-dried for 5-10 mm, and resuspended in 0 1 ml of DEPC-treated water
  • DNA was removed from 50 microgram samples of RNA by a 30 minute treatment at 37°C with 10 units of RNAase-free DNAasel (GeneHunter) in the buffer supplied in a final volume of 57 microhters
  • RNA was precipitated with 5 microhters of 3 M NaOAc and 200 microhters 100% EtOH, and pelleted by cent ⁇ fugation at 12,000g for 10 minutes
  • the RNA is pelleted (12,000g for 10 mm ), washed with 75% ethanol (v/v in DEPC-treated water), air-dried for 5-10 mm. and resuspended in 10-20 microhters of DEPC-treated water RNA yield is quantitated by OD260 after 1 1000 dilution of the cleaned RNA sample RNA is stored at -80°C if necessary and reverse-transcribed within one week
  • PCR reactions are set up on ice in 0 2ml tubes by adding the following components 43 microhtres PCR Master Mix (Advanced Biotechnologies Ltd ), 1 microhtre PCR primers (optimally 18-25 basepairs in length and designed to possess similar annealing temperatures), each primer at 1 OmM initial concentration, and 5 microhtres cDNA
  • PCR reactions are run on a Perkm Elmer GeneAmp PCR System 9600 as follows 2 minutes at 94 oC, then 50 cycles of 30 seconds each at 94 oC. 50 oC and 72 oC followed by 7 minutes at 72 oC and then a hold temperature of 20 oC (the number of cycles is optimally 30-50 to determine the appearance or lack of a PCR product and optimally 8-30 cycles if an estimation ofthe starting quantity of cDNA from the RT reaction is to be made), 10 microhtre ahquots are then run out on 1 % 1 x TBE gels stained with ethidium bromide, with PCR product, if present, sizes estimated by comparison to a 100 bp DNA Ladder (Gibco BRL, Life Technologies) Alternatively if the PCR products are conveniently labelled by the use of a labelled PCR primer (e g labelled at the 5'end with a dye) a suitable aliquot ofthe PCR product is run out on a polyacrylamide sequencing gel
  • Primer pairs which fail to give the predicted sized product in either DNA PCR or RT/PCR are PCR failures and as such are uninformative Of those which give the correct size product with DNA PCR two classes are distinguished in RT/PCR 1 Genes which are not transcribed m vivo reproducibly fail to give a product in RT/PCR, and 2 Genes which are transcribed in vivo reproducibly give the correct size product in RT/PCR and show a stronger signal in the +RT samples than the signal (if at all present) in -RT controls

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Abstract

L'invention concerne des polypeptides asuE, des polynucléotides codant pour ces polypeptides asuE et des procédés pour produire ces polypeptides par des techniques recombinantes. Elle concerne aussi des procédés pour utiliser les polypeptides asuE pour cribler les composés antibactériens.
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WO2003083099A2 (fr) * 2002-04-02 2003-10-09 Affinium Pharmaceuticals, Inc. Nouveaux polypeptides purifies impliques dans la synthese et la modification des proteines

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