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WO2000068425A1 - ysxC - Google Patents

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Publication number
WO2000068425A1
WO2000068425A1 PCT/US2000/011513 US0011513W WO0068425A1 WO 2000068425 A1 WO2000068425 A1 WO 2000068425A1 US 0011513 W US0011513 W US 0011513W WO 0068425 A1 WO0068425 A1 WO 0068425A1
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WO
WIPO (PCT)
Prior art keywords
polypeptide
seq
polynucleotide
sequence
isolated
Prior art date
Application number
PCT/US2000/011513
Other languages
English (en)
Inventor
Magdalena Zalacain
Sanjoy Biswas
Patrick V. Warren
Martin K. R. Burnham
Karen A. Ingraham
Alison F. Chalker
Chi Young So
David J. Holmes
Richard L. Warren
Christopher M. Traini
Original Assignee
Smithkline Beecham Corporation
Smithkline Beecham Plc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Smithkline Beecham Corporation, Smithkline Beecham Plc filed Critical Smithkline Beecham Corporation
Publication of WO2000068425A1 publication Critical patent/WO2000068425A1/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/315—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Streptococcus (G), e.g. Enterococci

Definitions

  • This invention relates to ne h identified pohnucleotides and polypeptides. and their production and uses, as well as their ⁇ a ⁇ ants, agonists and antagomsts, and their uses hi particular, the invention relates to polynucleotides and pohpeptides of the ys ⁇ C (GTP -binding protem) family, as w ell as their ⁇ a ⁇ ants. herein referred to as " ⁇ sxC.” " ⁇ sxC polynucleot ⁇ de(s).” and “ ⁇ sxC pohpept ⁇ de(s)" as the case may be
  • Streptococci make up a medically important genera of microbes known to cause several types of disease in humans, mcludmg. for example, otitis media, conjunctivitis, pneumonia, bacteremia. meningitis. sinusitis, pleural empyema and endocarditis, and most particularly meningitis, such as for example infection of cerebrospmal fluid Smce its isolation more than 100 years ago. Streptococcus pneumoniae has been one of the more intensively studied microbes For example, much of our earh understanding that DNA is.
  • the present invention relates to ysxC. in particular ysxC polypeptides and ysxC polynucleotides recombinant mate ⁇ als and methods for their production hi another aspect, the 1m ention relates to methods for usmg such polypeptides and polynucleotides, mcludmg treatment of microbial diseases, amongst others In a further aspect, the invention relates to methods for identifying agonists and antagomsts using the materials provided b> the invention, and for treating microbial infections and conditions associated with such infections with the identified agonist or antagonist compounds In a still further aspect, the mvention relates to diagnostic assays for detectmg diseases associated with microbial infections and conditions associated with such infections, such as assays for detecting ysxC expression or activity
  • the mvention relates to ysxC polypeptides and pohnucleotides as desc ⁇ bed in greater detail below
  • the mvention relates to polypeptides and polynucleotides of a of Streptococcus pneumoniae, that is related by ammo acid sequence homology to B subtilis ysxC pohpeptide
  • the invention relates especially to ysxC havmg a nucleotide and ammo acid sequences set out m Table 1 as SEQ ID NO 1 and SEQ ID NO 2 respectively
  • sequences recited in the Sequence Listing below as 'DNA represent an exemplification of the invention, since those of ordinary skill w ill recogmze that such sequences can be usefully employed m polynucleotides m general, including ⁇ bopolynucleotides
  • Streptococcus pneumoniae 0100993 DNA hbrary in E coh was similarly deposited with the NCIMB and assigned deposit number 40800
  • the Streptococcus pneumoniae stram deposit is referred to herem as "the deposited stram” or as "the DNA of the deposited stram "
  • the deposited stram comp ⁇ ses a full length ysxC gene
  • the sequence of the polynucleotides compnsed in the deposited strain, as well as the ammo acid sequence of any polypeptide encoded thereby, are controlling m the event of any conflict with any desc ⁇ ption of sequences herem
  • an isolated nucleic acid molecule encodmg a mature pol>pept ⁇ de expressible by the Streptococcus pneumoniae 0100993 stram which pohpeptide is compnsed in the deposited stram
  • ysxC polynucleotide sequences m the deposited stram. such as DNA and RNA.
  • ammo acid sequences encoded therebv Also provided b the mv ention are ys ⁇ C pohpeptide and polynucleotide sequences isolated from the deposited stram
  • YsxC polypeptide of the mvention is substantially phylogeneticalh related to other protems of the vsxC (GTP -bmdmg protem) famil ⁇
  • polypeptides of Streptococcus pneumoniae referred to herem as "ysxC” and "ysxC pohpeptides” as well as biologicalh. ⁇ iagnosticallv, proph lacticalh . clinically or therapeutically useful va ⁇ ants thereof, and compositions comp ⁇ smg the same
  • the present mvention further provides for an isolated polypeptide that (a) compnses or consists of an ammo acid sequence that has at least 95% identity, most preferabh at least 97-99% or exact identify to that of SEQ ID NO 2 over the entire length of SEQ ID NO 2, (b) a po peptide encoded by an isolated polynucleotide compnsmg or consisting of a polynucleotide sequence that has at least 95 % identity, even more preferably at least 97-99% or exact identity to SEQ ID NO 1 over the entire length of SEQ ID NO 1, (c) a polypeptide encoded by an isolated polynucleotide compnsmg or consistmg of a polynucleotide sequence encodmg a polypeptide that has at least 95% identity even more preferably at least 97-99% or exact identity, to the ammo acid sequence of SEQ ID NO 2 over the entire length of SEQ ID NO 2
  • X-(R 1 ) m -(R 2 )-(R 3 ) n -Y wherem. at the ammo terminus, X is hydrogen, a metal or any other moietv descnbed herem for modified polypeptides, and at the carboxyl terminus, Y is hydrogen, a metal or am other moietv desc ⁇ bed herem for modified polypeptides, Rj 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, covalenth bound to R ] and its carboxy teirninal ammo acid residue is at the right, covalenth' bound to R3 Any stretch of ammo acid residues denoted by either R ⁇ or R3.
  • m and/or n is greater than 1, mav be either a heteropolymer or a homopolymer. preferably a heteropolymer
  • m is an mteger between 1 and 50, 100 or 500
  • n is an mteger between 1 and 50, 100. or 500
  • a polypeptide of the mvention is denved from Streptococcus pneumoniae. however, it may preferably be obtained from other organisms of the same taxonomic genus A polypeptide of the mvention may also be obtained, for example, from organisms of the same taxonomic family or order
  • a fragment is a vanant pohpeptide 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 ysxC pohpeptides. fragments may be "free-standing,” or compnsed within a larger polypeptide of which they form a part or region, most preferably as a smgle continuous region in a smgle larger polypeptide Preferred fragments mclude, for example, truncation polypeptides having a portion of an ammo acid sequence of Table 1 [SEQ LD NO.2], or of va ⁇ ants thereof, such as a contmuous senes of residues that mcludes an ammo- and/or carboxyl-terminal ammo acid sequence Degradation forms of the pohpeptides of the mvention produced by or m a host cell, particularly a Streptococcus pneumoniae, are also preferred Further preferred are fragments characterized by structural or functional attributes such as fragments
  • 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,
  • Fragments of the polypeptides of the mvention may be employed for producmg the corresponding full-length polypeptide by peptide synthesis, therefore, these va ⁇ ants may be employed as mtermediates for producmg the full-length polypeptides of the mvention
  • polynucleotides that encode a polypeptide herem designated ysxC In a particularly preferred embodiment of the mvention the pohnucleotide comp ⁇ ses a region encodmg ysxC pohpeptides compnsmg a sequence set out m Table 1 [SEQ ID NO 1] that mcludes a full length gene, or a variant thereof The Applicants believe that this full length gene is essential to the growth and/or survival of an organism that possesses it.
  • isolated nucleic acid molecules encodmg and/or expressmg ysxC pohpeptides and pohnucleotides, particularly Streptococcus pneumoniae ysxC polypeptides and polynucleotides.
  • mcludmg for example, unprocessed RNAs. nbozyme RNAs. mRNAs. cDNAs, genomic DNAs, B- and Z-DNAs
  • Further embodiments of the mvention mclude biologicalh . diagnosticalh . prophylactically. clinically or therapeutically useful polynucleotides and pohpeptides. and vanants thereof, and compositions compnsmg the same
  • mcludmg at least one full length gene, that encodes a ysxC polypeptide having a deduced ammo acid sequence of Table 1 [SEQ ID NO 2] and polynucleotides closely related thereto and variants thereof
  • ysxC pohpeptide from Streptococcus pneumoniae compnsmg or consistmg of an ammo acid sequence of Table 1 [SEQ ID NO 2], or a variant thereof
  • a polynucleotide of the mvention encodmg ysxC polypeptide may be obtained usmg standard clonmg and screening methods, such as those for clonmg and sequencmg chromosomal DNA fragments from bacte ⁇ a usmg Streptococcus pneumoniae 0100993 cells as starting matenal, followed by obtammg a full length clone
  • 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 Streptococcus pneumoniae 0100993 in E coh or some other suitable host is probed with a radiolabeled ohgonucleotide, preferably a 17-mer or
  • the present mvention provides for an isolated polynucleotide compnsmg or consistmg of (a) a polynucleotide sequence that has at least 95 % identity, even more preferably at least 97%. still more preferably at least 99%, yet still more preferablv at least 99 5% or exact identity to SEQ ID NO 1 over the entire length of SEQ ID NO 1.
  • a polynucleotide encodmg a polypeptide of the present mvention, mcludmg homologs and orthologs from species other than Streptococcus pneumoniae may be obtained by a process that compnses the steps of screening an appropnate hbrary under stringent hybndization conditions with a labeled or detectable probe consisting 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 comp ⁇ smg said polynucleotide sequence
  • the mvention provides a polyn
  • polynucleotide of the mvention may also compnse 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-mdependent termination signals), nbosome binding sites, Kozak sequences, sequences that stabilize mRNA, mtrons, and polyadenylation signals
  • the polynucleotide sequence may also compnse 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-mdependent termination signals), nbosome binding sites, Kozak sequences, sequences that stabilize mRNA, mtrons, and polyaden
  • a preferred embodiment of the mvention is a polynucleotide of consistmg of or comp ⁇ smg nucleotide 1 to the nucleotide immediately upstream of or mcludmg nucleotide 586 set forth m SEQ ID NO 1 of Table 1. both of that encode a ysxC polypeptide
  • the mvention also mcludes a pohnucleotide consisting of or compnsmg a polynucleotide of the formula
  • Y is hydrogen, a metal, or a modified nucleotide residue, or together with X defines the covalent bond
  • each occurrence of Ri and R3 is independently any nucleic acid residue or modified nucleic acid residue
  • m is an integer betw een 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 of the invention, particularly a nucleic acid sequence selected from Table 1 or a modified nucleic acid sequence thereof In the polynucleotide formula above.
  • R 2 is onented so that its 5' end nucleic acid residue is at the left, bound to Ri and its 3' end nucleic acid residue is at the ⁇ ght, bound to R3
  • Any stretch of nucleic acid residues denoted by either R j and/or R 2 , where m and/or n is greater than 1, may be either a heteropolymer or a homopolymer, preferablv' a heteropolymer
  • the polynucleotide of the above formula is a closed, circular polynucleotide.
  • n is an mteger between 1 and 1000.
  • m is an mteger between 1 and 50. 100 or 500.
  • n is an mteger between 1 and 50, 100, or 500 It is most preferred that a pohnucleotide of the mvention is de ⁇ ved from Streptococcus pneumoniae, 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
  • polynucleotides encodmg ysxC vanants that have the ammo acid sequence of ysxC pohpeptide of Table 1 [SEQ ID NO 2] m which several, a few. 5 to 10. 1 to 5 1 to 3, 2, 1 or no ammo acid residues are substituted, modified, deleted and/or added, m any combmation Especially preferred among these are silent substitutions, additions and deletions, that do not alter the properties and activities of ysxC pohpeptide
  • Preferred isolated polynucleotide embodiments also mclude polynucleotide fragments, such as a polynucleotide comprising a nuclic acid sequence having at least 15, 20, 30, 40, 50 or 100 contiguous nucleic acids from the polynucleotide sequence of SEQ ID NO. l, or an polynucleotide comprising a nucleic acid sequence havmg at least 15, 20, 30, 40, 50 or 100 contiguous nucleic acids truncated or deleted from the 5' and/or 3' end of the polynucleotide sequence of SEQ ID NO: l
  • polynucleotides that are at least 95% or 97% identical over their entire length to a pohnucleotide encodmg ysxC polypeptide having an ammo acid sequence set out m Table 1 [SEQ ID NO 2], and polynucleotides that are complementarv to such polynucleotides
  • polynucleotides that compnse a region that is at least 95% are especially preferred
  • those with at least 97% are highly preferred 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 preferred Preferred embodiments are polynucleotides encodmg pohpeptides that retain substantially the same biological function or activity as a mature polypeptide encoded by a DNA of Table 1 [SEQ ID NO 1]
  • polynucleotides that hybndize, particularly under stringent conditions, to ysxC 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 pohnucleotides that hybndize under stringent conditions to the polynucleotides desc ⁇ bed herem
  • the terms "stringent conditions” and “stringent hybndization conditions” mean hybndization occurring only if there is at least 95% and preferably at least 97% identity between the sequences
  • a specific example of stringent hybndization conditions is overnight mcubation at 42°C m a solution comp ⁇ smg 50% formamide.
  • 5x SSC 150mM NaCl. 15mM tnsodium citrate), 50 mM sodium phosphate (pH7 6), 5x Denhardt's solution. 10% dextran sulfate, and 20 micrograms/ml of denatured, sheared salmon sperm DNA. follow ed by w ashmg 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 Sprmg Harbor, N Y , (1989), particularh Chapter 1 1 therein Solution hybridization may also be used with the polynucleotide sequences provided by the invention
  • the invention also provides a polynucleotide consisting of or compnsmg a polynucleotide sequence obtained by screemng an appropnate library compnsmg a complete gene for a poly nucleotide sequence set forth m SEQ ID NO 1 under stringent hybridization conditions with a probe having 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 obtammg such a polynucleotide include, for example, probes and primers fully described elsewhere herem
  • the polynucleotides of the mvention may be used as a hybndization probe for RNN cD ⁇ A and genomic D ⁇ A to isolate full-length cD ⁇ As and genomic clones encodmg ysxC and to isolate cD ⁇ A and genomic clones of other genes that have a high identity, particularly high sequence identity, to a ysxC gene
  • Such probes generally will compnse at least 15 nucleotide residues or base pairs
  • such probes will have at least 30 nucleotide residues or base pairs and may have at least 50 nucleotide residues or base pairs
  • Particularly preferred probes will have at least 20 nucleotide residues or base pairs and will have lee than 30 nucleotide residues or base pairs
  • a codmg region of a ysxC gene may be isolated by screening usmg a D ⁇ A sequence provided m Table 1 [SEQ ID NO 1] to synthesize an ohgonucleotide probe
  • a labeled ohgonucleotide having a sequence complementary to that of a gene of the mvention is then used to screen a library of cDNA. genomic DNA or niRNA to determine which members of the hbrary the probe hyb ⁇ dizes to
  • polynucleotides and polypeptides of the mvention may be employ ed, for example, as research reagents and matenals for discovery of treatments of and diagnostics for diseases, particularh human diseases, as further discussed herem relating to polynucleotide assays
  • the mvention also provides polynucleotides that encode a polypeptide that is a mature protem plus additional ammo or carboxyl-terminal ammo acids, or ammo acids mtenor to a mature polypeptide (when a mature form has more than one polypeptide chain, for instance)
  • Such sequences may play a role m processmg of a protem from precursor to a mature form, may allow protem transport, may lengthen or shorten protem half-life or may facilitate manipulation of a protem for assay or production, among other things
  • the additional ammo acids may be processed away from a mature protem by cellular enzymes
  • a precursor protem. having a mature form of the polypeptide fused to one or more prosequences may be an inactive form of the polypeptide When prosequences are removed such inactive precursors generally are activated Some or all of the prosequences may be removed before activation Generally, such precursors are called proproteins
  • the mvention also relates to vectors that compnse a polynucleotide or polynucleotides of the mvention, host cells that are genetically engmeered with vectors of the mvention and the production of polypeptides of the mvention by recombinant techmques Cell-free translation systems can also be employed to produce such proteins usmg RNAs denved 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 compnsmg expression systems
  • the present mvention relates to expression systems that compnse a polynucleotide or polynucleotides of the present mvention, to host cells that are genetically engmeered with such expression systems, and to the production of polypeptides of the mvention by recombinant techmques For recombinant production of the polypeptide
  • 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 Sprmg Harbor Laboratory Press, Cold Sprmg Harbor, N Y (1989). such as, calcium phosphate transfection, DEAE-dextian mediated transfection, transvection, micromjection. cationic hpid-mediated transfection, electroporation. transduction, scrape loading, ballistic mtroduction and infection
  • appropnate hosts include bactenal cells, such as cells of streptococci, staphylococci, enterococci E coh, streptomyces, cyanobacte ⁇ a, Bacillus subtilis, and Streptococcus pneumoniae, fungal cells, such as cells of a yeast, Kluveromyces, Saccharomyces. a basidiomycete.
  • Candida albicans and Aspergillus insect cells such as cells of Drosophila S2 and Spodoptera Sf9. animal cells such as CHO, COS, HeLa, C127, 3T3.
  • Such vectors mclude. among others, chromosomal-, episomal- and virus-denved vectors, for example, vectors denved from bactenal plasmids, from bactenophage, from transposons. from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses such as baculovrruses.
  • papova viruses such as SV40, vaccinia viruses, adenovrruses, fowl pox viruses, pseudorabies viruses, picornaviruses and retroviruses. and vectors de ⁇ ved from combinations thereof, such as those de ⁇ ved from plasmid and bactenophage genetic elements, such as cosmids and phagemids
  • the expression system constructs may compnse control regions that regulate as well as engender expression
  • any system or vector suitable to maintain, propagate or express polynucleotides and/or to express a polypeptide m a host may be used for expression m this regard
  • the appropnate DNA sequence may be inserted mto the expression system by any of a vanety of well-known and routme techmques, such as, for example, those set forth m Sambrook et al , MOLECULAR CLONING, A LABORATORY MANUAL, (supra)
  • appropnate secretion signals may be incorporated mto the expressed polypeptide These signals may be endogenous to the polypeptide or they may be heterologous signals
  • This mvention is also related to the use of ysxC polynucleotides and polypeptides of the mvention for use as diagnostic reagents Detection of ysxC polynucleotides and/or polypeptides in a eukaryote, particularly a mammal, and especially a human, will provide a diagnostic method for diagnosis of disease, staging of disease or response of an infectious orgamsm to drugs Eukaryotes, particularly mammals, and especially humans, particularly those infected or suspected to be infected with an orgamsm compnsmg the ysxC gene or protein, may be detected at the nucleic acid or ammo acid level by a vanety of well known techmques as well as by methods provided herem
  • Polypeptides and polynucleotides for prognosis, diagnosis or other analysis may be obtained from a putattvely infected and/or infected individual's bodily mate ⁇ als
  • Polynucleotides from any of these sources, particularly DNA or RNN may be used directly for detection or may be amplified enzymatically b ⁇ usmg PCR or any other amplification technique pnor to analysis
  • RNN particularly mR ⁇ N cD ⁇ A and genomic D ⁇ A may also be used m the same ways Usmg amplification, characterization of the species and stram of infectious or resident orgamsm present m an mdividual, may be made by an analy sis of the genotype of a selected polynucleotide of the orgamsm
  • Deletions and insertions can be detected by a change m size of the amplified product m compa ⁇ son to a genotype of a reference sequence selected from a related orgamsm
  • Po t mutations can be identified by hybndizmg amplified D ⁇ A to labeled ysxC polynucleotide sequences Perfectly or significantly matched sequences can be distinguished from imperfectly or more significantly mismatched duplexes by D ⁇ ase or R ⁇ ase digestion, for D ⁇ A or R ⁇ A respectively, or by detecting differences m melting temperatures or renaturation kmetics 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 D ⁇ A or R ⁇ A sequencmg See, for example, Myers et al , Science, 230 1242 (1985) Sequence changes at specific locations also may be revealed by nuclease protection assays
  • an array of ohgonucleotides probes comp ⁇ smg ysxC 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, for example, Chee et al , Science, 274 610 (1996))
  • the present mvention relates to a diagnostic kit that comp ⁇ ses (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 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 comprise 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 of the present mvention as diagnostic reagents Detection of a mutated form of a polynucleotide of the mvention, preferable, SEQ ID NO 1. that is associated with a disease or pathogenicity will provide a diagnostic tool that can add to. or define, a diagnosis of a disease, a prognosis of a course of disease, a determination of a stage of disease, or a susceptibility to a disease, that results from under-expression, over-expression or altered expression of the pohnucleotide Orgamsms, particularly infectious orgamsms. carrying mutations m such polynucleotide may be detected at the polynucleotide level by a vanety of techniques, such as those descnbed elsewhere herem
  • a polynucleotide and/or polypeptide sequence between orgamsms possessing a first phenotype and orgamsms possessmg a different, second different phenotype can also be determmed If a mutation is observed m some or all orgamsms possessmg the first phenotype but not m any orgamsms possessmg the second phenotype, then the mutation is likely to be the causative agent of the first phenotype
  • a polynucleotide and/or polypeptide of the mvention may also be detected at the polynucleotide or polypeptide level by a vanety of techniques, to allow for serotypmg, for example
  • RT-PCR can be used to detect mutations in the RNA It is particularly preferred to use RT-PCR m conjunction with automated detection systems, such as, for example, GeneScan RNN cD ⁇ A or genomic D ⁇ A may also be used for the same purpose, PCR
  • PCR primers complementary to a polynucleotide encodmg ysxC polypeptide can be used to identify and analyze mutations The mvention further provides these primers with 1.
  • primers may be used for, among other thmgs, amplifying ysxC D ⁇ A and/or R ⁇ A isolated from a sample denved from an mdividual, 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 va ⁇ ous techniques for elucidation of the polynucleotide sequence In this way, mutations m 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 Streptococcus pneumoniae, comprising determining from a sample de ⁇ ved from an mdividual, such as a bodily material, an increased level of expression of polynucleotide havmg a sequence of Table 1 [SEQ ID NO 1] Increased or decreased expression of a ysxC polynucleotide can be measured usmg any on of the methods well known m the art for the quantitation of polynucleotides, such as, for example, amplification, PCR, RT-PCR, RNase protection, Northern blotting, spectrometry and other hybndization methods
  • a diagnostic assay m accordance with the mvention for detecting over-expression of ysxC 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 ysxC polypeptide.
  • Assay techmques that can be used to determine levels of a ysxC polypeptide.
  • a sample denved from a host such as a bodily matenal. 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
  • Polypeptides and polynucleotides of the mvention may also be used to assess the bmdmg of small molecule substrates and ligands in, for example, cells, cell-free preparations, chemical libraries, and natural product mixtures
  • substrates and ligands may be natural substrates and ligands or may be structural or functional mimetics See, e g , Cohgan et al , Current Protocols in Immunology 1 (2) Chapter 5 (1991)
  • Polypeptides and polynucleotides of the present mvention are responsible for many biological functions, mcludmg many disease states, m particular the Diseases herem mentioned It is therefore desirable to devise screemng methods to identify compounds that agonize (e g , stimulate) or that antagonize (e g .inhibit) the function of the polypeptide or polynucleotide Accordingly, m a further aspect, the present m
  • agomsts and antagomsts so-identified may be natural or modified substrates, ligands. receptors, enzymes, etc . as the case may be, of ysxC polypeptides and polynucleotides, or may be structural or functional mimetics thereof (see Cohgan et al , Current Protocols in Immunology 1(2) Chapter 5 (1991))
  • the screemng methods may simply measure the bmdmg of a candidate compound to the polypeptide or polynucleotide, or to cells or membranes bearing the polypeptide or polynucleotide, or a fusion protein of the polypeptide by means of a label directly or indirectly associated with the candidate compound Alternatively, the screemng method may mvolve competition with a labeled competitor
  • these screemng methods may test whether the candidate compound results m a signal generated by activation or inhibition of the polypeptide or polynucleotide.
  • detection systems appropnate to the cells compnsmg the polypeptide or polynucleotide Inhibitors of activation are generally assayed m the presence of a known agomst 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 m screemng methods for mverse agomsts.
  • the screemng methods may simply compnse the steps of mixing a candidate compound with a solution compnsmg a polypeptide or polynucleotide of the present mvention, to form a mixture, measunng ysxC polypeptide and/or polynucleotide activity m the mixture, and comparing the ysxC polypeptide and/or polynucleotide activity of the mixture to a standard Fusion protems, such as those made from Fc portion and ysxC polypeptide, as herem 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
  • the polynucleotides, polypeptides and antibodies that bmd to and/or interact with a polypeptide of the present mvention may also be used to configure screemng methods for detectmg the effect of added compounds on the production of mRNA and/or polypeptide m cells
  • an ELISA assay may be constructed for measuring secreted or cell associated levels of polypeptide using monoclonal and polyclonal antibodies by standard methods known m the art This can be used to discover agents that may inhibit or enhance the production of polypeptide (also called antagonist or agomst, respectively) from suitably manipulated cells or tissues
  • the mvention also provides a method of screening compounds to identify those that enhance (agomst) or block (antagonist) the action of ysxC polypeptides or polynucleotides, particularly those compounds that are bactenstatic and/or bacte ⁇ cidal
  • the method of screemng may mvolve high-throughput techniques For example, to screen for ago
  • ysxC polypeptides that bmd ell 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 in this regard mclude but are not limited to colonmetnc, labeled substrate converted mto product, a reporter gene that is responsive to changes m ysxC polynucleotide or polypeptide activity, and bmdmg assays known in the art
  • Polypeptides of the mvention may be used to identify membrane bound or soluble receptors, if any, for such polypeptide, through standard receptor bmdmg techniques known m the art
  • techmques mclude, but are not limited to, ligand bmdmg and crosshnking assays in which the polypeptide is labeled with a radioactive isotope (for instance, ⁇ 1), chemically modified (for instance, biotinylated), or fused to a peptide sequence suitable for detection or purification, and incubated with a source of the putative receptor (e g , cells, cell membranes, cell supernatants, tissue extracts, bodily matenals)
  • a source of the putative receptor e g , cells, cell membranes, cell supernatants, tissue extracts, bodily matenals
  • Other methods mclude biophysical techmques such as surface plasmon resonance and spectroscopy
  • These screemng methods may also be used to identify
  • the fluorescence polanzation value for a fluorescently-tagged molecule depends on the rotational correlation time or tumblmg rate Protein complexes, such as formed by y sxC polypeptide associating with another ysxC polypeptide or other polypeptide labeled to compnse a fluorescently - labeled molecule will have higher polarization values than a fluorescently labeled monome ⁇ c protem It is preferred that this method be used to characterize small molecules that disrupt pohpeptide complexes
  • Fluorescence energy transfer may also be used characterize small molecules that interfere with the formation of ysxC polypeptide d mers. trrmers. tetramers or higher order structures, or structures formed by ysxC polypeptide bound to another polypeptide YsxC 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 ill inhibit fluorescence energy transfer
  • YsxC 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 ysxC polypeptide -coated surface and specific bmdmg can be detected m real-time by momtonng the change in resonance angle caused by a change m local refractive mdex
  • This technique can be used to characterize the effect of small molecules on kinetic rates and equihbnum bmdmg constants for ysxC polypeptide self-association as well as an association of ysxC polypeptide and another polypeptide or small molecule
  • a scintillation proximity assay may be used to charactenze the mteraction betw een an association of ysxC polypeptide with
  • identifying compounds that bmd to or otherwise interact with and inhibit or activate an activity or expression of a polypeptide and/or polynucleotide of the mvention comp ⁇ smg 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 preferablv bemg associated with a second component capable of providing a detectable signal m response to the bmdmg or mteraction of the polypeptide and/or polynucleotide w th the compound, and determinmg whether the compound bmds to or otherwise mteracts with and activates or inhibits an activity or expression of the polypeptide and/
  • an assay for ysxC agomsts is a competitiv e assay that combmes vsxC and a potential agomst with ysxC -bmdmg molecules recombmant ysxC bmdmg molecules, natural substrates or ligands, or substrate or hgand mimetics, under appropnate conditions for a competitive inhibition assay YsxC can be labeled, such as by radioactivity or a colonmetnc compound such that the number of ysxC molecules bound to a bmdmg molecule or converted to product can be determmed accurately to assess the effectiveness of the potential antagomst
  • a polypeptide and/or polynucleotide of the present mvention may also be used in a method for the structure-based design of an agonist or antagomst of the polypeptide and/or polynucleotide, by
  • the present mvention provides methods of treating abnormal conditions such as for instance, a Disease, related to either an excess of, an under-expression of, an elevated activity of, or a decreased activity of ysxC polypeptide and/or polynucleotide
  • expression of the gene encodmg endogenous ysxC polypeptide can be inhibited usmg expression blocking techniques
  • This blocking may be targeted agamst any step in gene expression, but is preferably targeted agamst transcnption and/or translation
  • An examples of a known techmque of this sort mvolve the use of antisense sequences, either internally generated or separately administered (see, for example, O'Connor. J Neurochem (1991) 56 560 in Ohgodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, FL (1988))
  • ohgonucleotides that form triple helices with the gene can be supplied (see.
  • polynucleotide sequences provided herein may be used in the discovery and development of antibacterial compounds
  • the encoded protem upon expression, can be used as a target for the screemng of antibacterial drugs
  • polynucleotide sequences encodmg the ammo terminal 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 mterest
  • the mvention also provides the use of the polypeptide, polynucleotide, agomst or antagomst of the mvention to mterfere with the initial physical mteraction between a pathogen or pathogens and a eukaryotic, preferably mammalian, host responsible for sequelae of infection
  • the molecules of the mvention may be used in the prevention of adhesion of bacteria, in particular gram positive and/or gram negative bacteria, to eukaryotic, preferably mammalian, extracellular matrix protems on in-dwelling devices or to extracellular matrix protems in wounds, to block bacte ⁇ al adhesion between eukaryotic, preferably mammalian, extracellular matrix protems and bacte ⁇ al ysxC protems that mediate tissue damage and/or, to block the normal progression of pathogenesis in infections initiated other than by the implantation of m-dwelling devices or by other surgical techniques
  • ysxC agonists and antagomsts preferably bacte ⁇ static or bacte ⁇ cidal agomsts and antagomsts
  • the antagomsts and agomsts of the mvention may be employed, for instance, to prevent inhibit and/or treat diseases
  • Hehcobacter pylori (herein "H pylori”) bacteria infect the stomachs of over one-third of the world's population causmg stomach cancer, ulcers, and gastntis (International Agency for Research on Cancer (1994) Schistosomes, Liver Flukes and Hehcobacter Pylori (International Agency for Research on Cancer. Lyon.
  • 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 aste, such as, bone, blood, serum, cerebrospinal fluid, semen, sahva, muscle, cartilage, organ tissue, skin, urine, stool or autopsy matenals
  • D ⁇ sease(s) means any disease caused by or related to infection by a bacte ⁇ a. mcludmg . for example, otitis media, conjunctivitis, pneumonia, bacteremia, meningitis, sinusitis, pleural empyema and endocarditis, and most particularly meningitis, such as for example infection of cerebrospinal fluid
  • “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
  • identity also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strmgs of such sequences
  • Identity can be readily calculated by known methods, mcludmg but not limited to those descnbed m (Computational Molecular Biology, Lesk, A M , ed .
  • Parameters for polypeptide sequence companson include the following Algonthm 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)
  • Polynucleotide embodiments further include an isolated polynucleotide compnsmg a polynucleotide sequence havmg at least a 95, 97 or 100% identity to the reference sequence of SEQ ID NO 1, wherein said polynucleotide sequence may be identical to the reference sequence of SEQ ID NO 1 or may mclude up to a certam mteger 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, mcludmg transition and transversion.
  • alterations may occur at the 5' or 3' terminal positions of the reference nucleotide sequence or anywhere bet een those terminal positions, interspersed either individually among the nucleotides in the reference sequence or in one or more contiguous groups vvithin the reference sequence, and wherein said number of nucleotide alterations is determmed by multiplying the total number of nucleotides m SEQ ID NO 1 by the integer defimng the percent identity divided by 100 and then subtracting that product from said total number of nucleotides in SEQ ID NO 1, or
  • n n is the number of nucleotide alterations
  • x n is the total number of nucleotides SEQ ID NO 1
  • y is 0 95 for 95%. 0 97 for 97%. 0 99 for 99%, 0 995 for 99 5% or 1 00 for 100%.
  • any non-mteger product of x n and y is rounded down to the nearest mteger pnor to subtractmg it from x n
  • Alterations of a polynucleotide sequence encodmg the polypeptide of SEQ ID NO 2 may create nonsense, missense or frameshift mutations in this codmg sequence and thereby alter the polypeptide encoded by the polynucleotide following such alterations
  • Polypeptide embodiments further include an isolated polypeptide comprising a polypeptide having at least a 95, 97 or 100% identity to a polypeptide reference sequence of SEQ ID NO 2, wherein said polypeptide sequence may be identical to the reference sequence of SEQ ID NO 2 or may include up to a certain mteger number of ammo acid alterations as compared to the reference sequence, wherem said alterations are selected from the group consistmg of at least one ammo acid deletion, substitution, including conservative and non-conservative substitution, or insertion, and wherem said alterations may occur at the ammo- or carboxy-terrmnal positions of the reference polypeptide sequence or anywhere between those terminal positions, interspersed either individually among the ammo acids in the reference sequence or m one or more contiguous groups within the reference sequence, and wherein said number of ammo acid alterations is determmed by multiplying the total number of ammo acids in SEQ ID NO 2 by the integer defimng the percent identity divided by 100 and then subtracting that product from
  • n a is the number of amino acid alterations
  • x a is the total number of amino acids m SEQ ID NO:2
  • y is 0.95 for 95%, 0.97 for 97% or 1.00 for 100%.
  • • is the symbol for the multiplication operator, and wherein any non-integer product of x a and y is rounded down to the nearest mteger prior to subtracting it from x a .
  • “Individual(s)” means a multicellular eukaryote. mcludmg. but not limited to a metazoan. a mammal, an ovid, a bov d. a simian, a primate, and a human.
  • Isolated means altered “by the hand of man” from its natural state, i. e.. if it occurs m nature, it has been changed or removed from its original 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 coexisting materials of its natural state is "isolated", as the term is employed herem
  • a polynucleotide or polypeptide that is introduced into an organism by transformation, genetic manipulation or by any other recombinant method is "isolated” even if it is still present m said orgamsm. which organism may be living or non-living.
  • Organism(s) means a (i) prokaryote, includmg but not limited to, a member of the genus Streptococcus, Staphylococcus, Bordetella, Corynebactenum, Mycobactenum, Neissena, Haemophilus, Actinomycetes, Streptomycetes, Nocardia, Enterobacter, Yersinia, Fancisella, Pasturella. Moraxella, Acmetobacter, Eryspelothrix, Branhamella, Actinobacillus, Streptobac ⁇ lus.
  • Neissena gonorrheae Neissena memngitidis, Staphylococcus aureus, Staphylococcus epidermidis, Corynebactenum dipthenae, Gardnerella vaginahs, Mycobactenum tuberculosis, Mycobactenum bovis, Mycobacterium ulcerans, Mycobactenum leprae, Actinomyctes israelii, Listena monocytogenes, Bordetella pertusis, Bordatella parapertusis, Bordetella bronchiseptica, Eschenchia coh, Shigella dysenteriae, Haemophilus mfluenzae, Haemophilus aegyptius, Haemophilus paramfluenzae, Haemophilus ducreyi, Bordetella, Salmonella typhi, Citrobacler freundu, Proteus mirabihs, Proteus vulgaris, Yersinia
  • a unicellular or filamentous eukaryote including but not limited to, a protozoan, a fungus, a member of the genus Saccharomyces Kluveromyces, or Candida, and a member of the species Saccharomyces cenviseae Kluveromyces lactis or Candida albicans
  • Polynucleot ⁇ de(s) generally refers to any poly ⁇ bonucleotide or poly deoxynbonucleotide. that may be uimiodified RNA or DNA or modified RNA or DNA "Polynucleot ⁇ de(s)” mclude. without limitation.
  • smgle- and double-stranded DNN DNA that is a mixture of smgle- and double-stranded regions or smgle-, double- and t ⁇ ple-stranded regions, smgle- and double-stranded RNN and R ⁇ A that is mixture of smgle- and double-stranded regions, hyb ⁇ d molecules compnsmg D ⁇ A and R ⁇ A that mav be single-stranded or.
  • polynucleotide refers to t ⁇ ple-stranded regions comp ⁇ smg R ⁇ A or D ⁇ A or both R ⁇ A and D ⁇ A
  • 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 t ⁇ ple-hehcal region often is an ohgonucleotide
  • polynucleotide(s) also mcludes D ⁇ As or R ⁇ As as desc ⁇ bed above that compnse one or more modified bases
  • D ⁇ As or R ⁇ As w th backbones modified for stability or for other reasons are "polynucleotide(s)" as that
  • Polypeptide(s) refers to any peptide or protem comp ⁇ smg two or more ammo acids jomed to each other by peptide bonds or modified peptide bonds "Polypeptide(s)” refers to both short chams.
  • Polypeptides may compnse ammo acids other than the 20 gene encoded ammo acids "Polypept ⁇ de(s)" mclude those modified either by natural processes, such as processmg and other post-translational modifications, but also by chemical modification techmques Such modifications are well descnbed m basic texts and m more detailed monographs, as well as m 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 compnse many types of modifications Modifications can occur anywhere m a polypeptide, mcludmg the peptide backbone, the ammo acid side-chains, and the ammo or carboxyl termini Modifications mclude,
  • cross-hnkmg cychzation. disulfide bond formation, demethylation. formation of covalent cross-hnks formation of cysteine. formation of pyroglutamate, formylation. gamma-carboxylation, GPI anchor formation, hydroxy lation, lodmation methylation. my ⁇ stoylation. oxidation, proteolytic processmg. phosphorylation. preny lation. racemization glycosylation. hpid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP-nbosylation, selenovlation. sulfation.
  • 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
  • 'Na ⁇ ant(s) is a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide respectively, but retams essential properties
  • a ty ⁇ ical variant of a polynucleotide differs in nucleotide sequence from another, reference polynucleotide Changes in the nucleotide sequence of the variant may or may not alter the ammo acid sequence of a polypeptide encoded by the reference polynucleotide Nucleotide changes may result in amino acid substitutions additions, deletions, fusion proteins and truncations in the polypeptide encoded by the reference sequence, as discussed below
  • a typical variant of a polypeptide differs m ammo acid sequence from another, reference polypeptide Generally, differences are limited so that the sequences of the reference polypeptide and the vanant are closely similar overall and, m many regions, identical
  • a variant and reference polypeptide may differ m am o acid
  • a vanant of a polynucleotide or polypeptide may be a naturally occurring such as an allehc vanant, or it may be a variant that is not known to occur naturally
  • Non-naturally occurrmg variants of polynucleotides and polypeptides may be made by mutagenesis techniques, by direct synthesis, and by other recombmant methods known to skilled artisans
  • the polynucleotide having a DNA sequence given in Table 1 [SEQ ID NO 1] was obtained from a library of clones of chromosomal DNA of Streptococcus pneumoniae in E coh
  • the sequencing data from two or more clones comprising overlapping Streptococcus pneumoniae DNAs was used to construct the contiguous DNA sequence in SEQ ID NO 1 Libranes 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 into the vector Lambda ZapII that has been cut with EcoRI, the library packaged by standard procedures and E coh infected with the packaged library
  • the library is amplified by standard procedures
  • Total cellular DNA is partially hydrolyzed with a one or a combmation of restriction enzymes appropnate to generate a series of fragments for cloning into library vectors (e g . Rsal. Pall. Alul Bshl235I). and such fragments are size-fractionated according to standard procedures EcoRI linkers are hgated to the DNA and the fragments then hgated mto the vector Lambda ZapII that have been cut with EcoRI, the library packaged by standard procedures, and E coh mfected with the packaged library The library is amplified by standard procedures Example 2 ysxC Characterization The S. pneumoniae ysxC gene is expressed during infection in a respiratory tract infection model
  • Excised lungs from a 48 hour respiratory tract infection of Streptococcus pneumoniae 0100993 in the mouse is efficiently disrupted and processed m the presence of chaotropic agents and RNAase inhibitor to provide a mixture of animal and bacterial RNA
  • the optimal conditions for disruption and processing to give stable preparations and high yields of bacterial RNA are followed by the use of hybridisation to a radiolabelled ohgonucleotide specific to Streptococcus pneumomae 16S RNA on
  • DNA and protem free preparations of RNA obtamed are suitable for Reverse Transcnption PCR (RT-PCR) usmg unique pnmer pairs designed from the sequence of each gene of Streptococcus pneumoniae 0100993 a) Isolation of tissue infected with Streptococcus pneumoniae 0100993 from a mouse animal model of infection (lungs)
  • Streptococcus pneumomae 0100993 is seeded onto TSA (Tryptic Soy Agar. BBL) plates containing 5% horse blood and allowed to grow overnight at 37°C in a C02 incubator Bacterial growth is scraped into 5 ml of phosphate-buffered saline (PBS) and adjusted to an A600 ⁇ 0 6 (4 x 106/ml) Mice (male CBA/J-1 mice, approximately 20g) were anaesthetized with isoflurane and 50 microhters of the prepared bacterial moculum is delivered by mtranasal instillation Animals are allowed to recover and observed twice daily for signs of moribundancy Forty-eight hours after infection the animals are euthanized by carbon dioxide overdose and their torsos swabbed with ethanol and then RNAZap The torso is then opened, and the lungs are aseptically removed Half of each pair of lungs is placed m a cryovial
  • 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
  • RNA preparations are stored at -80 oC for up to one month
  • the RNA precipitate can be stored at the wash stage of the protocol in 75% ethanol for at least one year at -20 oC
  • RNA isolation Quality of the RNA isolated is assessed by running samples on 1% agarose gels 1 x TBE gels stamed with ethidium bromide are used to visualise total RNA y lelds To demonstrate the isolation of bacterial RNA from the infected tissue 1 x MOPS.
  • DNA was removed from 50 microgram samples of RNA by a 30 mmute treatment at 37°C with 20 units of RNAase-free DNAasel (GenHunter) in the buffer supplied m a final volume of 57 microhters
  • the DNAase was inactivated and remov ed by treatment with TRIzol LS Reagent (Gibco BRL. Life Technologies) according to the manufacturers protocol
  • DNAase treated RNA was resuspended in 100 microhtres of DEPC treated water with the addition of Rnasm as described before d)
  • RT/PCR controls may include +/- reverse transcnptase reactions. 16S rRNA primers or DNA specific primer pairs designed to produce PCR products from non-transc ⁇ bed Streptococcus pneumoniae 0100993 genomic sequences To test the efficiency of the primer pairs they are used in DNA PCR with Streptococcus pneumomae 0100993 total DNA PCR reactions are set up and run as described above using approx 1 microgram of DNA in place of the cDNA
  • Primer pairs which fail to give the predicted sized product in either DNA PCR or RT/PCR are PCR failures and as such are urunformative Of those which give the correct size product with DNA PCR two classes are distmguished in RT/PCR 1 Genes which are not transcnbed m vivo reproducibh fail to give a product in RT/PCR, and 2 Genes which are transcnbed m vivo reproducibly give the correct size product in RT/PCR and show a stronger signal m the +RT samples than the signal (if at all present) in -RT controls
  • Example 3 The ysxC gene is essential for S. pneumoniae in vitro growth. Demonstration of gene essentiality to bacterial viability
  • allehc replacement cassette was generated using PCR technology
  • the cassette consisted of a pair of 500bp chromosomal DNA fragments flanking an erythromycin resistance gene
  • the chromosomal DNA sequences are the 500bp preceding and following the DNA sequence encoding the ysxC gene contained in Seq ID NO 1
  • the allehc replacement cassette was introduced mto S pneumoniae R6 by transformation
  • Competent cells were prepared according to published protocols DNA w as introduced into the cells by mcubation of ng quantities of allehc replacement cassette with 10" cells at 30°C for 30 minutes The cells were transferred to 37°C for 90 mmutes to allow expression of the erythromycin resistance gene Cells were plated m agar containing lug erythromycin per ml Following incubation at 37°C for 36 hours, colonies are picked and grown overnight in Todd-Hewitt broth supplemented with 0 5% yeast extract Typically 1000 transformants contaimng the appropriate allehc replacement are obtained If no transformants are obtamed in three separate transformation experiments as was the case for this gene ysxC, then the gene is considered as being essential m vitro

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Abstract

L'invention concerne des polypeptides ysxC et des polynucléotides codant pour les polypeptides ysxC, ainsi que des procédés pour la production desdits polypeptides par des techniques de recombinaison. L'invention porte également sur des procédés d'utilisation de polypeptides ysxC pour le criblage de composés antibactériens.
PCT/US2000/011513 1999-05-05 2000-04-28 ysxC WO2000068425A1 (fr)

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Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
GASC ET AL.: "Organization around the dnaA gene of streptococcus pneumoniae", MICROBIOLOGY, vol. 144, no. PT2, February 1998 (1998-02-01), pages 433 - 439, XP002931232 *
PRIEBE ET AL.: "Nucleotide sequence of the hexA gene for DNA mismatch repair in streptococcus pneumoniae and homology of hexA to mutS of escherichia coli and salmonella typhimurium", JOURNAL OF BACTERIOLOGY, vol. 170, no. 1, January 1988 (1988-01-01), pages 190 - 196, XP002931231 *

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