Outer surface proteins, their genes, and their use

Abstract

invention, a series of genes are identified in Group B Streptococcus, the products of which may be located on the outer surface of the organism. The genes, or functional fragments thereof, may be useful in the preparation of therapeutics, e.g. vaccines for the immunization of a patient against microbial infection.

Description

FIELD OF THE INVENTION

[0001]This invention relates to the identification of outer surface proteins, their genes, and their use. More particularly, it relates to their use in therapy, for immunisation and in screening for drugs.

BACKGROUND TO THE INVENTION

[0002]Group B Streptococcus (GBS), also known as Streptococcus aralactiae, is the causative agent of various conditions. In particular, GBS causes:

Early Onset Neonatal Infection.

[0003]This infection usually begins in utero and causes severe septicaemia and pneumonia in infants, which is lethal if untreated and even with treatment is associated with a 10-20% mortality rate.

Late Onset Neonatal Infection.

[0004]This infection occurs in the period shortly after birth until about 3 months of age. It causes a septicaemia, which is complicated by meningitis in 90% of cases. Other focal infections also occur including osteomyelitis, septic arthritis, abscesses and endopthalmitis.

Adult Infections.

[0005]These appear to be increasingly common and occur most frequently in women who have just delivered a baby, the elderly and the immunocompromised. They are characterised by septicaemia and focal infections including osteomyelitis, septic arthritis, abscesses and endopthalmitis.

Urinary Tract Infections.

[0006]GBS is a cause of urinary tract infections and in pregnancy accounts for about 10% of all infections. Veterinary infections.

[0007]GBS causes chronic mastitis in cows. This, in turn, leads to reduced milk production and is therefore of considerable economic importance.

[0008]GBS infections can be treated with antibiotics. However, immunisation is preferable. It is therefore desirable to develop an immunogen that could be used in a therapeutically-effective vaccine.

SUMMARY OF THE INVENTION

[0009]The present invention is based on the identification of a series of genes in GBS, and also related organisms, the products of which may be associated with the outer surface of the organism and may therefore be useful as targets for immunotherapy.

[0010]According to one aspect of the invention, a peptide is encoded by an operon including any of the genes identified herein as MS4, MS10, MS11, MS14 and MS16, obtainable from Group B Streptococcus, or a homologue or functional fragment thereof. Such a peptide is suitable for therapeutic use, e.g. when isolated.

[0011]The term "functional fragments" is used herein to define a part of the gene or peptide which retains the activity of the whole gene or peptide. For example, a functional fragment of the peptide may be used as an antigenic determinant, useful in a vaccine or in the production of antibodies.

[0012]A gene fragment may be used to encode the active peptide. Alternatively, the gene fragment may have utility in gene therapy, targeting the wild-type gene in vivo to exert a therapeutic effect.

[0013]A peptide according to the present invention may comprise any of the amino acid sequences identified herein as SEQ ID NOS. 2, 4, 6, 8, 10 and 12, or a functional fragment thereof.

[0014]Because of the extracellular or cell surface location, the peptides of the present invention may be suitable candidates for the production of therapeutically-effective vaccines against GBS. The term "therapeutically-effective" is intended to include the prophylactic effect of vaccines. For example, a vaccine may comprise a peptide according to the invention, or the means for its expression, for the treatment of infection.

[0015]This vaccine may be administered to females either prior to or during pregnancy to protect mother and neonate against infection by GBS.

[0016]According to another aspect of the invention, the peptides or genes may be used for screening potential antimicrobial drugs or for the detection of virulence.

[0017]A further aspect of this invention is the use of any of the products identified herein, for the treatment or prevention of a condition associated with infection by a Group B Streptococcal strain.

[0018]Although the protein has been described for use in the treatment of patients, veterinary uses of the products of the invention are also considered to be within the scope of the present invention. In particular, the peptides or the vaccines may be used in the treatment of chronic mastitis, especially in cows.

DESCRIPTION OF THE INVENTION

[0019]The present invention is described with reference to Group B Streptococcal strain M732. However, all the GBS strains and many other bacterial strains are likely to include related peptides or proteins having amino acid sequence homology with the peptide of M732. Organisms likely to contain the peptides include, but are not limited to, S. pneumoniae, S. pycgenes, S. suis, S. milleri, Group C and Group G Streptococci and Enterococci. Vaccines to each of these may be developed in the same way as described for GBS.

[0020]Preferably, the peptides that may be useful for the production of vaccines have greater than 40% sequence similarity with the peptides identified herein. More preferably, the peptides have greater than 60% sequence similarity. Most preferably, the peptides have greater than 80% sequence similarity, e.g. 95% similarity.

[0021]Having characterised a gene according to the invention, it is possible to use the gene sequence to establish homologies in other microorganisms. In this way it is possible to determine whether other microorganisms have similar outer surface products. Sequence homologies may be established by searching in existing databases, e.g. EMBL or Genbank.

[0022]Peptides or proteins according to the invention may be purified and isolated by methods known in the art. In particular, having identified the gene sequence, it will be possible to use recombinant techniques to express the genes in a suitable host. Active fragments and homologues can be identified and may be useful in therapy. For example, the peptides or their active fragments may be used as antigenic determinants in a vaccine, to elicit an immune response. They may also be used in the preparation of antibodies, for passive immunisation, or diagnostic applications. Suitable antibodies include monoclonal antibodies, or fragments thereof, including single chain fv fragments. Methods for the preparation of antibodies will be apparent to those skilled in the art.

[0023]The preparation of vaccines based on attenuated microorganisms is known to those skilled in the art. Vaccine compositions can be formulated with suitable carriers or adjuvants, e.g. alum, as necessary or desired, and used in therapy, to provide effective immunisation against Group B Streptococci or other related microorganisms. The preparation of vaccine formulations will be apparent to the skilled person.

[0024]More generally, and as is well known to those skilled in the art, a suitable amount of an active component of the invention can be selected, for therapeutic use, as can suitable carriers or excipients, and routes of administration. These factors will be chosen or determined according to known criteria such as the nature/severity of the condition to be treated, the type or health of the subject etc.

[0025]The products of the present invention were identified as follows:

[0026]Todd-Hewitt broth was inoculated with GBS and allowed to grow overnight at 37° C. The cells were harvested by centrifugation and washed with Phosphate Buffered Saline (PBS). The cells were resuspended in an osmotic buffer (20% (w/v) Sucrose, 20 mM Tris-HCl pH 7.0, 10 mM MgCl₂) containing protease inhibitors (1 mM PMSF, 10 mM Iodoeacetic Acid, 10 mM 1,10-Phenanthroline, 1 μM Pepstatin A) and Mutanolysin at a final concentration of 4 Units per microlitre. This was incubated (shaking) at 37° C. for 2 hours.

[0027]Cells and debris were removed first by high speed centrifugation, then ultra-centrifugation for 1 hour. The resultant supernatant containing cell wall proteins was concentrated under pressure using an ultrafiltration device (10,000 molecular weight cut-off).

[0028]The sample was dialysed against ultra high quality water and lyophilised. After resuspension in loading buffer, the proteins were separated by preparative 2-Dimensional-Gel Electrophoresis. Following electrophoresis an individual spot was chosen for study. The spot was subjected to in-gel tryptic digestion. The resulting peptides were extracted from the gel and purified using microbore RP-HPLC. Fractions were collected every 45 seconds and a portion of these consistent with the regions of UV absorbance were analysed by Delayed Extraction-Matrix Assisted Laser Desorption-Time of Flight Mass Spectrometry (DE-MALDI-TOF-MS). Peptides not observed in a blank preparation were then subjected to sequencing using Nanospray-MS/MS

[0029]Using this peptide sequence information, degenerate oligonucleotides were designed to be used in a polymerase chain reaction (PCR) to amplify the DNA segment lying between the peptide sequences identified.

[0030]PCR amplification resulted in the production of several polynucleotide fragments, each of which was cloned into the pCR 2.1-TOPO vector (Invitrogen BV, Netherlands) according to manufacturers protocol.

[0031]The DNA fragment in each plasmid was identified by sequencing and then used to obtain the full-length gene sequence, as follows.

[0032]Using the identified DNA fragment, oligonucleotide primers were designed for genomic DNA sequencing. These primers were designed so as to sequence in an outward direction from the obtained sequence. Once read, the sequence obtained was checked to see if the 5' and 3' termini of the gene had been reached. The presence of these features was identified by checking against homologous sequences, and for the 5' end the presence of an AUG start codon (or accepted alternative) preceded by a Shine-Dalgarno consensus sequence, and for the 3' end, the presence of a translation termination (Stop) codon.

[0033]Upon identification of the full-length gene, primers were designed for amplification of full-length product from GBS genomic DNA. Primers used included restriction enzyme recognition sites (NcoI at the 5' end and EcoO109I at the 3' end) to allow subsequent cloning of the product into the Lactococcal expression system used.

[0034]PCR was carried out using the primers, and the products cloned into a pCR 2.1 cloning vector (In Vitrogen). Following confirmation of the presence of the cloned fragment, the DNA was excised using the restriction enzymes NcoI and EcoO109I.

[0035]The vector into which this fragment was inserted was a modified version of pNZ8048 (Kuipers, O. P. et al. (1998) J. Biotech 64: 15-21). This vector, harbouring a lactococcal origin of replication, a chloramphenicol resistance marker, an inducible nisin promoter and a multicloning site was altered by the replacement of the multicloning site with two 10×His tags, flanked on the 5-most end with an NcoI site, split in the middle with a multicloning site (including an EcoO109I site), and a Stop (termination) codon at the 3' end of the His tags.

[0036]The gene of interest was inserted so that a 10× His tag was in the 3' position relative to the coding region. Following transformation of the recombinant plasmid into L. lactis (strain NZ9000-Kuipers, O. P. et al. (1998) supra), a 400 ml liquid culture was set up and translation of the protein was induced by the addition of nisin to the culture. After a 2 hour incubation, the cells were harvested and lysed by bead beating. The resultant lysate was cleared by centrifugation, then passed over a metal affinity (Talon, Clonetech) column. The column was washed repeatedly before bound proteins were eluted with Imidazole.

[0037]To identify fractions containing the His-tagged recombinant protein, an aliquot from each fraction was analysed by SDS-PAGE, Western blotted and probed with anti-His antibodies.

[0038]The recombinant protein obtained was then used to immunise New Zealand white rabbits, with pre-immune sera being harvested prior to immunisation. Following a boost, the rabbits were sacrificed and sera collected. This sera was used in Western blots, ELISA and animal protection models.

[0039]Using the sera obtained from the animal studies, immunosorption studies were carried out.

[0040]Group B Streptococcus was grown in 20 ml Todd Hewitt broth (THB) for 8 hours, harvested and resuspended in 5 ml PBS. 50 μl aliquots of this were used to coat wells in a 96 well plate (Nunc Immuno-Sorb). This was left at 4° C. overnight to allow for absorbance of the bacteria onto the plate. Plates were washed twice with PBS, then blocked with 3% BSA in PBS for 1 hr at 37° C. Plates were again washed. Serial 10 fold dilutions of the sera were made in PBS and 50 μl of these dilutions were added to the wells of the plate, in duplicate. The plate was covered and incubated for 1 hr at 37° C. The plate was washed, then. 501 anti-rabbit alkaline phosphatase conjugated secondary antibody at a concentration of 1:5000 was added to each well. Following incubation at 37° C. for an hour, the plate was washed again. 50 μl substrate (PNPP) was added to each well, and the reaction allowed to proceed for 30 min before the absorbance was read at 405 nm.

[0041]Animal protection studies were also carried out to test the effectiveness of protection on the immunized rabbits.

[0042]GBS M732 was grown up in THB until mid-log phase was reached--approximately 5 hours. Cells were counted in a counting chamber, and bacteria were diluted to give a concentration of 2×10⁷ bacteria per ml in pre-immune or test sera. 50 μl of this was injected via the intraperitoneal route into 0-1 day old mice. The mice were observed for survival over 48 hours.

[0043]The following Examples illustrate the invention.

EXAMPLE 1

[0044]A first plasmid was termed MS4. The cloned DNA fragment was sequenced and the nucleotide and deduced amino acid sequence (SEQ ID NO. 1 and 2) was used to search protein databases.

[0045]Homologues to the GBS MS4 gene product can be identified in Clostridium perfingens, Haemophilus influenzae, Neisseria flavescens and Thermatoga maritima. In all cases the homologues are the genes for Ornithine Carbamoyltransferase (OCT). In eukaryotic systems this enzyme catalyses the second step in the Urea cycle, the conversion of ornithine to citrulline, a reaction requiring carbomyl phosphate. In prokaryotes, ODC is one of the three enzymes involved in Arginine Deaminase activity--a system which protects bacteria from acid damage. In particular, ODC is responsible for the conversion of citrulline to ornithine and carbamoyl phosphate (the opposite role to that in eukaryotes) (Casiano-Colon, A and Marquis, R. E. 1988. Appl. Environ. Microbiol. 54: 1318-1324, Cunin, R. et al. 1986. Microbiol. Rev. 50: 314-352).

[0046]Animal protection studies were carried out as described above. The results are as follows:

TABLE-US-00001 # pups surviving at time (hrs) Treatment # pups 24 48 PBS 15 6 0 Pre-Immune 41 18 1 Test 41 33 14

EXAMPLE 2

[0047]A second plasmid was termed MS11. The nucleotide and deduced amino acid sequence (SEQ ID NOS. 3 and 4) were used to search protein databases.

[0048]Homologues to the GBS MS11 gene product can be identified in Lactobacillus delbrueckii, Thermotoga maritina, Clostridium acetobulylicum, Bacillus megaterium, Triticum aestivium and Synechocystis PCC6803.

[0049]In all cases the homologues are the genes for the protein Phosphoglycerate Kinase (PGK). PGK is a major enzyme in the glycolytic pathway, being involved in the conversion of Glyceraldehyde-3-phosphate to Phosphoenolpyruvate. In particular, it is involved in the catalysis of the reaction between Glycerate-1,3-diphosphate and 3-Phospho-Glycerate, releasing a phosphate in the forward reaction.

EXAMPLE 3

[0050]A third plasmid was termed pMS16. The 5' and 3' cloned DNA fragments were sequenced and the nucleotide and deduced amino acid sequences for each are shown as SEQ ID NOS. 5 and 6 for the 5' fragment and SEQ ID NOS. 7 and 8 for the 3' fragment.

[0051]Homologues to the GBS MS16 gene product can be identified in Bacillus stearothermophilus, Bacillus subtilis and Mycoplasma genitalium.

[0052]In all cases the homologues are the genes for the protein Glucose-6-Phosphate Isomerase (GPI).

[0053]The enzyme Glucose-6-Phospate Isomerase catalyses the reaction between Glucose-6-phosphate and Fructose-6-Phosphate in both glycolysis (G6P to F6P) and gluconeogenesis (F6P to G6P). Mutations in the gpi gene have been shown to confer purine analogue sensitivity to organisms.

EXAMPLE 4

[0054]A fourth plasmid was termed pMS14. The cloned DNA fragment was sequenced and the nucleotide and deduced amino acid sequence (SEQ ID NOS. 9 and 10) was used to search protein databases.

[0055]Homologues to the GBS MS14 gene product can be identified in Bacillus subtilis, Bacillus stearothermophilus, Mus musculus, Bos taurus and Zea mays. In all cases the homologues are the genes for the protein Purine Nucleoside Phosphatase (PNP). The function of this enzyme is to cleave the nucleosides guanosine or inosine to their respective basis and sugar-1-phosphate molecules in the presence of orthophosphate.

EXAMPLE 5

[0056]A fifth plasmid was termed pMS10. The cloned DNA fragment was sequenced. The nucleotide and deduced amino acid sequence (SEQ ID NOS. 11 and 12) was used to search protein databases.

[0057]Homologues to the GBS MS10 gene product can be identified in Streptococcus mutans, Nicotiana plumb, Pisum sativum and, Zea mays. In all cases the homologues are the genes for the protein Nonphosphorylating, NADP-Dependent Glyceraldehyde-3-Phosphate Dehydrogenase (NPGAP-3-DH). NPGAP-3-DH has been reported as being an important means of generating NADPH for biosynthetic reactions in S. mutans (as opposed to NAD-specific GAP-3-DH which satisfies the requirements of the glycolytic pathway) (Boyd, D. A., Cvitkovitch, D. G. and Hamilton, I. R 1995 J. Bacteriol. 177: 2622-2727).

Sequence CWU 1

1211014DNAStreptococcus agalactiaeCDS(1)..(1014) 1atg aca caa gta ttt caa gga cgt agt ttc tta gca gaa aaa gat ttt 48Met Thr Gln Val Phe Gln Gly Arg Ser Phe Leu Ala Glu Lys Asp Phe1 5 10 15tct cgt gag gaa ttt gaa tat ctt att gat ttt tca gct cat tta aaa 96Ser Arg Glu Glu Phe Glu Tyr Leu Ile Asp Phe Ser Ala His Leu Lys 20 25 30gac ctt aaa aaa cgt ggt gtt cct cat cat tat ctt gaa ggt aaa aat 144Asp Leu Lys Lys Arg Gly Val Pro His His Tyr Leu Glu Gly Lys Asn 35 40 45att gct ctc tta ttt gaa aaa aca tct act cgt act cgc gca gcc ttt 192Ile Ala Leu Leu Phe Glu Lys Thr Ser Thr Arg Thr Arg Ala Ala Phe 50 55 60aca act gca gca att gac cta ggc gct cat ccg gaa tac ctt ggt gca 240Thr Thr Ala Ala Ile Asp Leu Gly Ala His Pro Glu Tyr Leu Gly Ala65 70 75 80aat gat att caa ctt ggt aaa aaa gaa tca aca gaa gat act gct aag 288Asn Asp Ile Gln Leu Gly Lys Lys Glu Ser Thr Glu Asp Thr Ala Lys 85 90 95gtt tta gga cgt atg ttt gat ggt att gaa ttc cgt ggt ttt agc caa 336Val Leu Gly Arg Met Phe Asp Gly Ile Glu Phe Arg Gly Phe Ser Gln 100 105 110aga atg gtt gaa gag ctt gct gaa ttt tct gga gta cct gtc tgg aat 384Arg Met Val Glu Glu Leu Ala Glu Phe Ser Gly Val Pro Val Trp Asn 115 120 125ggt tta aca gat gaa tgg cat cca aca caa atg cta gct gac tac ctt 432Gly Leu Thr Asp Glu Trp His Pro Thr Gln Met Leu Ala Asp Tyr Leu 130 135 140act atc aaa gaa aac ttc ggt aaa ctt gaa ggt att act ctt gtt tac 480Thr Ile Lys Glu Asn Phe Gly Lys Leu Glu Gly Ile Thr Leu Val Tyr145 150 155 160tgt ggt gac gga cgt aac aat gtt gcc aac tcg ctt tta gtg gct ggg 528Cys Gly Asp Gly Arg Asn Asn Val Ala Asn Ser Leu Leu Val Ala Gly 165 170 175act ttg atg ggg gtc aat gta cac atc ttt tct cca aaa gaa ctt tty 576Thr Leu Met Gly Val Asn Val His Ile Phe Ser Pro Lys Glu Leu Phe 180 185 190ccw gct gaa gag att gtt aaa ttg gct gaa gga tat gcc aaa gaa tct 624Pro Ala Glu Glu Ile Val Lys Leu Ala Glu Gly Tyr Ala Lys Glu Ser 195 200 205ggg gct cac gtt ctc gtt act gat aat gta gac gaa gct gta aag gga 672Gly Ala His Val Leu Val Thr Asp Asn Val Asp Glu Ala Val Lys Gly 210 215 220gca gac gtc ttt tac act gat gtc tgg gta tcg atg gga gaa gaa gat 720Ala Asp Val Phe Tyr Thr Asp Val Trp Val Ser Met Gly Glu Glu Asp225 230 235 240aag ttc aaa gaa cgc gtt gaa ctt ctt caa cca tat caa gta aac atg 768Lys Phe Lys Glu Arg Val Glu Leu Leu Gln Pro Tyr Gln Val Asn Met 245 250 255gaa ctg att aaa aaa gct aat aat gat aat ctt atc ttc tta cac tgc 816Glu Leu Ile Lys Lys Ala Asn Asn Asp Asn Leu Ile Phe Leu His Cys 260 265 270tta cct gca ttc cat gat aca aat acc gtt tat ggc aaa gac gtc gct 864Leu Pro Ala Phe His Asp Thr Asn Thr Val Tyr Gly Lys Asp Val Ala 275 280 285gaa aaa ttt ggg gtc aag gaa atg gaa gtt act gat gaa gtc ttc cgt 912Glu Lys Phe Gly Val Lys Glu Met Glu Val Thr Asp Glu Val Phe Arg 290 295 300agc aaa tat gct cgt cat ttc gac caa gct gaa aat cgt atg cac act 960Ser Lys Tyr Ala Arg His Phe Asp Gln Ala Glu Asn Arg Met His Thr305 310 315 320att aaa gct gta atg gct gca acc ctt gga aat ctt ttc att cca aaa 1008Ile Lys Ala Val Met Ala Ala Thr Leu Gly Asn Leu Phe Ile Pro Lys 325 330 335gtt taa 1014Val2337PRTStreptococcus agalactiae 2Met Thr Gln Val Phe Gln Gly Arg Ser Phe Leu Ala Glu Lys Asp Phe1 5 10 15Ser Arg Glu Glu Phe Glu Tyr Leu Ile Asp Phe Ser Ala His Leu Lys 20 25 30Asp Leu Lys Lys Arg Gly Val Pro His His Tyr Leu Glu Gly Lys Asn 35 40 45Ile Ala Leu Leu Phe Glu Lys Thr Ser Thr Arg Thr Arg Ala Ala Phe 50 55 60Thr Thr Ala Ala Ile Asp Leu Gly Ala His Pro Glu Tyr Leu Gly Ala65 70 75 80Asn Asp Ile Gln Leu Gly Lys Lys Glu Ser Thr Glu Asp Thr Ala Lys 85 90 95Val Leu Gly Arg Met Phe Asp Gly Ile Glu Phe Arg Gly Phe Ser Gln 100 105 110Arg Met Val Glu Glu Leu Ala Glu Phe Ser Gly Val Pro Val Trp Asn 115 120 125Gly Leu Thr Asp Glu Trp His Pro Thr Gln Met Leu Ala Asp Tyr Leu 130 135 140Thr Ile Lys Glu Asn Phe Gly Lys Leu Glu Gly Ile Thr Leu Val Tyr145 150 155 160Cys Gly Asp Gly Arg Asn Asn Val Ala Asn Ser Leu Leu Val Ala Gly 165 170 175Thr Leu Met Gly Val Asn Val His Ile Phe Ser Pro Lys Glu Leu Phe 180 185 190Pro Ala Glu Glu Ile Val Lys Leu Ala Glu Gly Tyr Ala Lys Glu Ser 195 200 205Gly Ala His Val Leu Val Thr Asp Asn Val Asp Glu Ala Val Lys Gly 210 215 220Ala Asp Val Phe Tyr Thr Asp Val Trp Val Ser Met Gly Glu Glu Asp225 230 235 240Lys Phe Lys Glu Arg Val Glu Leu Leu Gln Pro Tyr Gln Val Asn Met 245 250 255Glu Leu Ile Lys Lys Ala Asn Asn Asp Asn Leu Ile Phe Leu His Cys 260 265 270Leu Pro Ala Phe His Asp Thr Asn Thr Val Tyr Gly Lys Asp Val Ala 275 280 285Glu Lys Phe Gly Val Lys Glu Met Glu Val Thr Asp Glu Val Phe Arg 290 295 300Ser Lys Tyr Ala Arg His Phe Asp Gln Ala Glu Asn Arg Met His Thr305 310 315 320Ile Lys Ala Val Met Ala Ala Thr Leu Gly Asn Leu Phe Ile Pro Lys 325 330 335Val31197DNAStreptococcus agalactiaeCDS(1)..(1197) 3atg gct aaa ttg act gtt aaa gac gtt gat ttg aag gta aaa aaa gtc 48Met Ala Lys Leu Thr Val Lys Asp Val Asp Leu Lys Val Lys Lys Val1 5 10 15ctc gtt cgt gtt gac ttt aat gtg cct ttg aaa gac ggc gtt atc act 96Leu Val Arg Val Asp Phe Asn Val Pro Leu Lys Asp Gly Val Ile Thr 20 25 30aac gac aac cgt atc act gcg gct ctt cca aca atc aag tat atc atc 144Asn Asp Asn Arg Ile Thr Ala Ala Leu Pro Thr Ile Lys Tyr Ile Ile 35 40 45gaa caa ggt ggt cgt gct atc ctc ttc tct cac ctt gga cgt gtt aaa 192Glu Gln Gly Gly Arg Ala Ile Leu Phe Ser His Leu Gly Arg Val Lys 50 55 60gaa gaa gct gac aaa gaa gga aaa tca ctt gca ccg gta gct gct gat 240Glu Glu Ala Asp Lys Glu Gly Lys Ser Leu Ala Pro Val Ala Ala Asp65 70 75 80tta gct gct aaa ctt ggt caa gat gtt gta ttc cca ggt gtt act cgt 288Leu Ala Ala Lys Leu Gly Gln Asp Val Val Phe Pro Gly Val Thr Arg 85 90 95ggt gca aaa tta gaa gaa gca atc aat gct ttg gaa gat gga caa gtt 336Gly Ala Lys Leu Glu Glu Ala Ile Asn Ala Leu Glu Asp Gly Gln Val 100 105 110ctt ttg gtt gaa aac act cgt ttt gaa gat gtt gac ggt aag aaa gaa 384Leu Leu Val Glu Asn Thr Arg Phe Glu Asp Val Asp Gly Lys Lys Glu 115 120 125tct aag aat gac gaa gaa ctt ggt aaa tac tgg gct tca ctt gga gat 432Ser Lys Asn Asp Glu Glu Leu Gly Lys Tyr Trp Ala Ser Leu Gly Asp 130 135 140gga atc ttc gtt aac gat gca ttt ggt aca gca cac cgt gct cat gca 480Gly Ile Phe Val Asn Asp Ala Phe Gly Thr Ala His Arg Ala His Ala145 150 155 160tca aac gta ggt att tca gca aac gtt gaa aaa gct gta gct ggt ttc 528Ser Asn Val Gly Ile Ser Ala Asn Val Glu Lys Ala Val Ala Gly Phe 165 170 175ctt ctt gaa aac gaa att gct tac atc caa gaa gca gtt gaa act cca 576Leu Leu Glu Asn Glu Ile Ala Tyr Ile Gln Glu Ala Val Glu Thr Pro 180 185 190gaa cgc cca ttc gta gct att ctt ggt ggc tca aaa gtt tct gat aag 624Glu Arg Pro Phe Val Ala Ile Leu Gly Gly Ser Lys Val Ser Asp Lys 195 200 205att ggt gtt atc gaa aac ctt ctt gaa aaa gct gat aaa gtt ctt atc 672Ile Gly Val Ile Glu Asn Leu Leu Glu Lys Ala Asp Lys Val Leu Ile 210 215 220ggt ggt ggt atg act tac aca ttc tac aaa gct caa ggt atc gaa atc 720Gly Gly Gly Met Thr Tyr Thr Phe Tyr Lys Ala Gln Gly Ile Glu Ile225 230 235 240ggt aac tca ctt gta gaa gaa gac aaa ttg gat gtt gct aaa gac ctc 768Gly Asn Ser Leu Val Glu Glu Asp Lys Leu Asp Val Ala Lys Asp Leu 245 250 255ctt gaa aaa tca aac ggt aaa ttg atc ttg cca gtt gac tca aaa gaa 816Leu Glu Lys Ser Asn Gly Lys Leu Ile Leu Pro Val Asp Ser Lys Glu 260 265 270gca aac gca ttt gct ggt tat act gaa gtt cgc gac act gaa ggt gaa 864Ala Asn Ala Phe Ala Gly Tyr Thr Glu Val Arg Asp Thr Glu Gly Glu 275 280 285gca gtt tca gaa ggg ttc ctt ggt ctt gac atc ggt cct aaa tca atc 912Ala Val Ser Glu Gly Phe Leu Gly Leu Asp Ile Gly Pro Lys Ser Ile 290 295 300gct aaa ttt gat gaa gca ctt act ggt gct aaa aca gtt gta tgg aac 960Ala Lys Phe Asp Glu Ala Leu Thr Gly Ala Lys Thr Val Val Trp Asn305 310 315 320gga cct atg ggt gtc ttt gaa aac cct gac ttc caa gct ggt aca atc 1008Gly Pro Met Gly Val Phe Glu Asn Pro Asp Phe Gln Ala Gly Thr Ile 325 330 335ggt gta atg gac gct atc gtt aaa caa cca ggc gtt aaa tca atc atc 1056Gly Val Met Asp Ala Ile Val Lys Gln Pro Gly Val Lys Ser Ile Ile 340 345 350ggt ggt ggt gat tca gca gca gct gct atc aac ctt ggt cgt gct gac 1104Gly Gly Gly Asp Ser Ala Ala Ala Ala Ile Asn Leu Gly Arg Ala Asp 355 360 365aaa ttc tca tgg atc tct act ggt ggt gga gca agc atg gaa ttg ctc 1152Lys Phe Ser Trp Ile Ser Thr Gly Gly Gly Ala Ser Met Glu Leu Leu 370 375 380gaa ggt aaa gta tta cca ggt ttg gca gca ttg act gaa aaa taa 1197Glu Gly Lys Val Leu Pro Gly Leu Ala Ala Leu Thr Glu Lys385 390 3954398PRTStreptococcus agalactiae 4Met Ala Lys Leu Thr Val Lys Asp Val Asp Leu Lys Val Lys Lys Val1 5 10 15Leu Val Arg Val Asp Phe Asn Val Pro Leu Lys Asp Gly Val Ile Thr 20 25 30Asn Asp Asn Arg Ile Thr Ala Ala Leu Pro Thr Ile Lys Tyr Ile Ile 35 40 45Glu Gln Gly Gly Arg Ala Ile Leu Phe Ser His Leu Gly Arg Val Lys 50 55 60Glu Glu Ala Asp Lys Glu Gly Lys Ser Leu Ala Pro Val Ala Ala Asp65 70 75 80Leu Ala Ala Lys Leu Gly Gln Asp Val Val Phe Pro Gly Val Thr Arg 85 90 95Gly Ala Lys Leu Glu Glu Ala Ile Asn Ala Leu Glu Asp Gly Gln Val 100 105 110Leu Leu Val Glu Asn Thr Arg Phe Glu Asp Val Asp Gly Lys Lys Glu 115 120 125Ser Lys Asn Asp Glu Glu Leu Gly Lys Tyr Trp Ala Ser Leu Gly Asp 130 135 140Gly Ile Phe Val Asn Asp Ala Phe Gly Thr Ala His Arg Ala His Ala145 150 155 160Ser Asn Val Gly Ile Ser Ala Asn Val Glu Lys Ala Val Ala Gly Phe 165 170 175Leu Leu Glu Asn Glu Ile Ala Tyr Ile Gln Glu Ala Val Glu Thr Pro 180 185 190Glu Arg Pro Phe Val Ala Ile Leu Gly Gly Ser Lys Val Ser Asp Lys 195 200 205Ile Gly Val Ile Glu Asn Leu Leu Glu Lys Ala Asp Lys Val Leu Ile 210 215 220Gly Gly Gly Met Thr Tyr Thr Phe Tyr Lys Ala Gln Gly Ile Glu Ile225 230 235 240Gly Asn Ser Leu Val Glu Glu Asp Lys Leu Asp Val Ala Lys Asp Leu 245 250 255Leu Glu Lys Ser Asn Gly Lys Leu Ile Leu Pro Val Asp Ser Lys Glu 260 265 270Ala Asn Ala Phe Ala Gly Tyr Thr Glu Val Arg Asp Thr Glu Gly Glu 275 280 285Ala Val Ser Glu Gly Phe Leu Gly Leu Asp Ile Gly Pro Lys Ser Ile 290 295 300Ala Lys Phe Asp Glu Ala Leu Thr Gly Ala Lys Thr Val Val Trp Asn305 310 315 320Gly Pro Met Gly Val Phe Glu Asn Pro Asp Phe Gln Ala Gly Thr Ile 325 330 335Gly Val Met Asp Ala Ile Val Lys Gln Pro Gly Val Lys Ser Ile Ile 340 345 350Gly Gly Gly Asp Ser Ala Ala Ala Ala Ile Asn Leu Gly Arg Ala Asp 355 360 365Lys Phe Ser Trp Ile Ser Thr Gly Gly Gly Ala Ser Met Glu Leu Leu 370 375 380Glu Gly Lys Val Leu Pro Gly Leu Ala Ala Leu Thr Glu Lys385 390 3955516DNAStreptococcus agalactiaeCDS(1)..(516) 5atg aca cat att aca ttt gac tta ttc aaa gtc ttg ggt caa ttt gta 48Met Thr His Ile Thr Phe Asp Leu Phe Lys Val Leu Gly Gln Phe Val1 5 10 15ggc gaa cac gag tta gac tac cta cca cca caa gta agt gca gca gat 96Gly Glu His Glu Leu Asp Tyr Leu Pro Pro Gln Val Ser Ala Ala Asp 20 25 30gct ttc ctt cgt caa ggg act ggt cct gga tca gat ttt ctc gga tgg 144Ala Phe Leu Arg Gln Gly Thr Gly Pro Gly Ser Asp Phe Leu Gly Trp 35 40 45atg gaa cct cca gaa aac tat gac aaa gaa gaa ttt tct cgc att caa 192Met Glu Pro Pro Glu Asn Tyr Asp Lys Glu Glu Phe Ser Arg Ile Gln 50 55 60aaa gcc gct gaa aag att aaa tca gat agc gaa gta ctc gtg gtt att 240Lys Ala Ala Glu Lys Ile Lys Ser Asp Ser Glu Val Leu Val Val Ile65 70 75 80ggt att ggt ggt tcg tac ctt ggt gca aaa gca gca att gac ttt ttg 288Gly Ile Gly Gly Ser Tyr Leu Gly Ala Lys Ala Ala Ile Asp Phe Leu 85 90 95aat aat cat ttt gct aat ttg caa acc gca gaa gaa cgt aaa gcg cct 336Asn Asn His Phe Ala Asn Leu Gln Thr Ala Glu Glu Arg Lys Ala Pro 100 105 110cag att ctt tat gct gga aat tct att tca tct act tac ctt gcc gat 384Gln Ile Leu Tyr Ala Gly Asn Ser Ile Ser Ser Thr Tyr Leu Ala Asp 115 120 125tta gtt gaa tac gtc caa gat aaa gaa ttc tca gta aat gtc att tca 432Leu Val Glu Tyr Val Gln Asp Lys Glu Phe Ser Val Asn Val Ile Ser 130 135 140aaa tca ggt aca aca act gaa cca gcg att gct ttc cgt gta ttt aaa 480Lys Ser Gly Thr Thr Thr Glu Pro Ala Ile Ala Phe Arg Val Phe Lys145 150 155 160gaa ctt cta gtt aaa aag tac cgg tca aga aga agc 516Glu Leu Leu Val Lys Lys Tyr Arg Ser Arg Arg Ser 165 1706172PRTStreptococcus agalactiae 6Met Thr His Ile Thr Phe Asp Leu Phe Lys Val Leu Gly Gln Phe Val1 5 10 15Gly Glu His Glu Leu Asp Tyr Leu Pro Pro Gln Val Ser Ala Ala Asp 20 25 30Ala Phe Leu Arg Gln Gly Thr Gly Pro Gly Ser Asp Phe Leu Gly Trp 35 40 45Met Glu Pro Pro Glu Asn Tyr Asp Lys Glu Glu Phe Ser Arg Ile Gln 50 55 60Lys Ala Ala Glu Lys Ile Lys Ser Asp Ser Glu Val Leu Val Val Ile65 70 75 80Gly Ile Gly Gly Ser Tyr Leu Gly Ala Lys Ala Ala Ile Asp Phe Leu 85 90 95Asn Asn His Phe Ala Asn Leu Gln Thr Ala Glu Glu Arg Lys Ala Pro 100 105 110Gln Ile Leu Tyr Ala Gly Asn Ser Ile Ser Ser Thr Tyr Leu Ala Asp 115 120 125Leu Val Glu Tyr Val Gln Asp Lys Glu Phe Ser Val Asn Val Ile Ser 130 135 140Lys Ser Gly Thr Thr Thr Glu Pro Ala Ile Ala Phe Arg Val Phe Lys145 150 155 160Glu Leu Leu Val Lys Lys Tyr Arg Ser Arg Arg Ser 165 1707318DNAStreptococcus agalactiaeCDS(1)..(318) 7att aac cga aga ttt aga tgg tct tgg tta tct tca aga aaa gat gta 48Ile Asn Arg Arg Phe Arg Trp Ser Trp Leu Ser Ser Arg Lys Asp Val1 5 10 15gat ttt gtt aat aaa aaa gca aca gat ggt gtg ctt ctt gct cat aca 96Asp Phe Val Asn Lys Lys Ala Thr Asp Gly Val Leu Leu Ala His Thr 20 25 30gat ggt ggg gtt cca aat atg ttt gta acg ctt cct aca caa gac gct 144Asp

Gly Gly Val Pro Asn Met Phe Val Thr Leu Pro Thr Gln Asp Ala 35 40 45tac act ctt ggt tac act att tac ttc ttt gag tta gca att ggc ctt 192Tyr Thr Leu Gly Tyr Thr Ile Tyr Phe Phe Glu Leu Ala Ile Gly Leu 50 55 60tca ggt tat ctt aac tca gta aat cca ttt gat caa ccg ggg gta gaa 240Ser Gly Tyr Leu Asn Ser Val Asn Pro Phe Asp Gln Pro Gly Val Glu65 70 75 80gca tat aaa cgt aat atg ttc gca ttt ggt aaa cct gga ttc gaa gag 288Ala Tyr Lys Arg Asn Met Phe Ala Phe Gly Lys Pro Gly Phe Glu Glu 85 90 95ctt agc gct gaa ttg aat gca cgt ctt taa 318Leu Ser Ala Glu Leu Asn Ala Arg Leu 100 1058105PRTStreptococcus agalactiae 8Ile Asn Arg Arg Phe Arg Trp Ser Trp Leu Ser Ser Arg Lys Asp Val1 5 10 15Asp Phe Val Asn Lys Lys Ala Thr Asp Gly Val Leu Leu Ala His Thr 20 25 30Asp Gly Gly Val Pro Asn Met Phe Val Thr Leu Pro Thr Gln Asp Ala 35 40 45Tyr Thr Leu Gly Tyr Thr Ile Tyr Phe Phe Glu Leu Ala Ile Gly Leu 50 55 60Ser Gly Tyr Leu Asn Ser Val Asn Pro Phe Asp Gln Pro Gly Val Glu65 70 75 80Ala Tyr Lys Arg Asn Met Phe Ala Phe Gly Lys Pro Gly Phe Glu Glu 85 90 95Leu Ser Ala Glu Leu Asn Ala Arg Leu 100 1059804DNAStreptococcus agalactiaeCDS(1)..(804) 9atg aca tta tta gaa aaa att aat gag act aga gac ttt ttg caa gca 48Met Thr Leu Leu Glu Lys Ile Asn Glu Thr Arg Asp Phe Leu Gln Ala1 5 10 15aaa ggc gtc aca gca cca gaa ttt ggy ctt att tta ggc tct ggt tta 96Lys Gly Val Thr Ala Pro Glu Phe Gly Leu Ile Leu Gly Ser Gly Leu 20 25 30gga gaa ttg gct gaa gaa atc gaa aat cct att gtt gtg gat tat gca 144Gly Glu Leu Ala Glu Glu Ile Glu Asn Pro Ile Val Val Asp Tyr Ala 35 40 45gac atc ccm aat tgg gga cag tca aca gta gtt ggt cat gct gga aaa 192Asp Ile Pro Asn Trp Gly Gln Ser Thr Val Val Gly His Ala Gly Lys 50 55 60ttt agt gta tgg gat tta tca ggc cgt aag gta tta gcg ctt caa ggt 240Phe Ser Val Trp Asp Leu Ser Gly Arg Lys Val Leu Ala Leu Gln Gly65 70 75 80cgt ttt cat ttt tay gaa ggw aat aca atg gaa gtc gtt act ttc cca 288Arg Phe His Phe Tyr Glu Gly Asn Thr Met Glu Val Val Thr Phe Pro 85 90 95gta cgt atc atg aga gca ttg gct tgc cac agt gtg ctt gtg act aat 336Val Arg Ile Met Arg Ala Leu Ala Cys His Ser Val Leu Val Thr Asn 100 105 110gca gcg ggt ggg att gga tac gga cca gga act tta atg ctg atc aaa 384Ala Ala Gly Gly Ile Gly Tyr Gly Pro Gly Thr Leu Met Leu Ile Lys 115 120 125gac cac atc aat atg att ggg act aac cct ctc ata ggt gag aac ctt 432Asp His Ile Asn Met Ile Gly Thr Asn Pro Leu Ile Gly Glu Asn Leu 130 135 140gaa gaa ttt gga cca cgt ttc cca gac atg tcg gat gct tay aca gca 480Glu Glu Phe Gly Pro Arg Phe Pro Asp Met Ser Asp Ala Tyr Thr Ala145 150 155 160aca tat cga caa aaa gct cac caa att gct gaa aac gat atc aaa ctc 528Thr Tyr Arg Gln Lys Ala His Gln Ile Ala Glu Asn Asp Ile Lys Leu 165 170 175gaa gaa ggt gtg tac ttg ggt gta tca gga ccc act tat gaa aca cct 576Glu Glu Gly Val Tyr Leu Gly Val Ser Gly Pro Thr Tyr Glu Thr Pro 180 185 190gca gaa att cgt gca ttc caa aca atg ggc gca caa gcg gta ggt atg 624Ala Glu Ile Arg Ala Phe Gln Thr Met Gly Ala Gln Ala Val Gly Met 195 200 205tcc acg gtt cca gag gtg atc gtt gca gct cac tca ggg ctt aaa gtg 672Ser Thr Val Pro Glu Val Ile Val Ala Ala His Ser Gly Leu Lys Val 210 215 220tta gga att tca gca att act aac ctt gcc gct ggc ttc caa tca gag 720Leu Gly Ile Ser Ala Ile Thr Asn Leu Ala Ala Gly Phe Gln Ser Glu225 230 235 240ctc aat cat gag gag gtc gtt gaa gtt act cag cgt att aaa gaa gat 768Leu Asn His Glu Glu Val Val Glu Val Thr Gln Arg Ile Lys Glu Asp 245 250 255ttc aag gga tta ggt aaa tca tta gtt gct gaa ctc 804Phe Lys Gly Leu Gly Lys Ser Leu Val Ala Glu Leu 260 26510268PRTStreptococcus agalactiae 10Met Thr Leu Leu Glu Lys Ile Asn Glu Thr Arg Asp Phe Leu Gln Ala1 5 10 15Lys Gly Val Thr Ala Pro Glu Phe Gly Leu Ile Leu Gly Ser Gly Leu 20 25 30Gly Glu Leu Ala Glu Glu Ile Glu Asn Pro Ile Val Val Asp Tyr Ala 35 40 45Asp Ile Pro Asn Trp Gly Gln Ser Thr Val Val Gly His Ala Gly Lys 50 55 60Phe Ser Val Trp Asp Leu Ser Gly Arg Lys Val Leu Ala Leu Gln Gly65 70 75 80Arg Phe His Phe Tyr Glu Gly Asn Thr Met Glu Val Val Thr Phe Pro 85 90 95Val Arg Ile Met Arg Ala Leu Ala Cys His Ser Val Leu Val Thr Asn 100 105 110Ala Ala Gly Gly Ile Gly Tyr Gly Pro Gly Thr Leu Met Leu Ile Lys 115 120 125Asp His Ile Asn Met Ile Gly Thr Asn Pro Leu Ile Gly Glu Asn Leu 130 135 140Glu Glu Phe Gly Pro Arg Phe Pro Asp Met Ser Asp Ala Tyr Thr Ala145 150 155 160Thr Tyr Arg Gln Lys Ala His Gln Ile Ala Glu Asn Asp Ile Lys Leu 165 170 175Glu Glu Gly Val Tyr Leu Gly Val Ser Gly Pro Thr Tyr Glu Thr Pro 180 185 190Ala Glu Ile Arg Ala Phe Gln Thr Met Gly Ala Gln Ala Val Gly Met 195 200 205Ser Thr Val Pro Glu Val Ile Val Ala Ala His Ser Gly Leu Lys Val 210 215 220Leu Gly Ile Ser Ala Ile Thr Asn Leu Ala Ala Gly Phe Gln Ser Glu225 230 235 240Leu Asn His Glu Glu Val Val Glu Val Thr Gln Arg Ile Lys Glu Asp 245 250 255Phe Lys Gly Leu Gly Lys Ser Leu Val Ala Glu Leu 260 265111428DNAStreptococcus agalactiaeCDS(1)..(1428) 11ttg aca aaa gaa tat caa aat tat gtc aat ggc gaa tgg aaa tca tct 48Leu Thr Lys Glu Tyr Gln Asn Tyr Val Asn Gly Glu Trp Lys Ser Ser1 5 10 15gtt aat cag att gag att ttg tca cca att gat gat tct tca ttg gga 96Val Asn Gln Ile Glu Ile Leu Ser Pro Ile Asp Asp Ser Ser Leu Gly 20 25 30ttc gtg cca gcg atg act cga gaa gaa gtt gat cat gct atg aaa gcg 144Phe Val Pro Ala Met Thr Arg Glu Glu Val Asp His Ala Met Lys Ala 35 40 45ggt cgt gag gct tta cca gct tgg gct gct tta aca gta tat gaa cgt 192Gly Arg Glu Ala Leu Pro Ala Trp Ala Ala Leu Thr Val Tyr Glu Arg 50 55 60gca caa tac ctt cat aaa gcc gca gac att att gaa cgt gat aaa gaa 240Ala Gln Tyr Leu His Lys Ala Ala Asp Ile Ile Glu Arg Asp Lys Glu65 70 75 80gaa att gct act gtt tta gca aaa gaa att tct aaa gct tac aat gct 288Glu Ile Ala Thr Val Leu Ala Lys Glu Ile Ser Lys Ala Tyr Asn Ala 85 90 95tca gta act gag gtt gta agg aca gct gat ctt att cgt tat gca gca 336Ser Val Thr Glu Val Val Arg Thr Ala Asp Leu Ile Arg Tyr Ala Ala 100 105 110gaa gaa gga att cgt tta tca act tca gct gac gaa ggt gga aaa atg 384Glu Glu Gly Ile Arg Leu Ser Thr Ser Ala Asp Glu Gly Gly Lys Met 115 120 125gat gct tca aca ggt cat aag ttg gct gtt att cgt cgt caa cca gta 432Asp Ala Ser Thr Gly His Lys Leu Ala Val Ile Arg Arg Gln Pro Val 130 135 140ggt atc gtt tta gca atc gca cct tat aat tac cct gtt aac ctc tca 480Gly Ile Val Leu Ala Ile Ala Pro Tyr Asn Tyr Pro Val Asn Leu Ser145 150 155 160gga tca aaa att gcg cca gct cta att ggt gga aac gtt gtg atg ttt 528Gly Ser Lys Ile Ala Pro Ala Leu Ile Gly Gly Asn Val Val Met Phe 165 170 175aaa cca cca aca caa ggt tca gtc tca gga ctt gtt tta gca aaa gct 576Lys Pro Pro Thr Gln Gly Ser Val Ser Gly Leu Val Leu Ala Lys Ala 180 185 190ttt gca gaa gca ggt ctt cca gca ggt gtc ttt aat act att aca gga 624Phe Ala Glu Ala Gly Leu Pro Ala Gly Val Phe Asn Thr Ile Thr Gly 195 200 205cgc ggt tct gag att gga gat tac att gtt gag cat gaa gaa gtt aat 672Arg Gly Ser Glu Ile Gly Asp Tyr Ile Val Glu His Glu Glu Val Asn 210 215 220ttt att aac ttt aca gga tca acg cca gtt gga caa cgt att ggt aag 720Phe Ile Asn Phe Thr Gly Ser Thr Pro Val Gly Gln Arg Ile Gly Lys225 230 235 240ttg gca gga atg cgt cca att atg ctt gag ttg ggc ggt aag gat gca 768Leu Ala Gly Met Arg Pro Ile Met Leu Glu Leu Gly Gly Lys Asp Ala 245 250 255ggt atc gtc tta gct gat gct gac ctt gat aac gct gct aaa caa atc 816Gly Ile Val Leu Ala Asp Ala Asp Leu Asp Asn Ala Ala Lys Gln Ile 260 265 270gtt gca ggt gct tat gat tac tct gga caa cgc tgt acg gca att aag 864Val Ala Gly Ala Tyr Asp Tyr Ser Gly Gln Arg Cys Thr Ala Ile Lys 275 280 285cgt gtg ctt gtc gtt gaa gaa gtt gcw gat gaa ttg gca gaa aaa ata 912Arg Val Leu Val Val Glu Glu Val Ala Asp Glu Leu Ala Glu Lys Ile 290 295 300tct gaa aat gta gca aaa tta tca gta ggt gat cca ttt gat aat gca 960Ser Glu Asn Val Ala Lys Leu Ser Val Gly Asp Pro Phe Asp Asn Ala305 310 315 320acg gtg aca ccg gtt att gat gat aat tca gct gac ttt att gaa agc 1008Thr Val Thr Pro Val Ile Asp Asp Asn Ser Ala Asp Phe Ile Glu Ser 325 330 335tta gta gta gat gca cgt caa aaa ggt gcg aaa gaa ttg aat gaa ttt 1056Leu Val Val Asp Ala Arg Gln Lys Gly Ala Lys Glu Leu Asn Glu Phe 340 345 350aaa cgt gat ggt cgt cta tta act cca gga ttg ttt gat cat gtt act 1104Lys Arg Asp Gly Arg Leu Leu Thr Pro Gly Leu Phe Asp His Val Thr 355 360 365tta gat atg aaa cta gct tgg gaa gag cct ttt gga cca att ctc cca 1152Leu Asp Met Lys Leu Ala Trp Glu Glu Pro Phe Gly Pro Ile Leu Pro 370 375 380att att cgt gtc aag gat gca gaa gaa gct gtt gct att gcc aac aaa 1200Ile Ile Arg Val Lys Asp Ala Glu Glu Ala Val Ala Ile Ala Asn Lys385 390 395 400tct gat ttt gga tta caa tca tca gtc ttt aca cgt gat ttc caa aaa 1248Ser Asp Phe Gly Leu Gln Ser Ser Val Phe Thr Arg Asp Phe Gln Lys 405 410 415gca ttt gat ata gca aat aaa ctt gaa gtt ggt aca gtt cac att aac 1296Ala Phe Asp Ile Ala Asn Lys Leu Glu Val Gly Thr Val His Ile Asn 420 425 430aat aag act gga cgt ggt ccw gat aat ttc cca ttc tta gga ctc aaa 1344Asn Lys Thr Gly Arg Gly Pro Asp Asn Phe Pro Phe Leu Gly Leu Lys 435 440 445gga tct ggt gca ggt gtt caa ggt atc aga tat tca att gaa gca atg 1392Gly Ser Gly Ala Gly Val Gln Gly Ile Arg Tyr Ser Ile Glu Ala Met 450 455 460aca aat gta aaa tcg att gtt ctc gat atg aaa tag 1428Thr Asn Val Lys Ser Ile Val Leu Asp Met Lys465 470 47512475PRTStreptococcus agalactiae 12Leu Thr Lys Glu Tyr Gln Asn Tyr Val Asn Gly Glu Trp Lys Ser Ser1 5 10 15Val Asn Gln Ile Glu Ile Leu Ser Pro Ile Asp Asp Ser Ser Leu Gly 20 25 30Phe Val Pro Ala Met Thr Arg Glu Glu Val Asp His Ala Met Lys Ala 35 40 45Gly Arg Glu Ala Leu Pro Ala Trp Ala Ala Leu Thr Val Tyr Glu Arg 50 55 60Ala Gln Tyr Leu His Lys Ala Ala Asp Ile Ile Glu Arg Asp Lys Glu65 70 75 80Glu Ile Ala Thr Val Leu Ala Lys Glu Ile Ser Lys Ala Tyr Asn Ala 85 90 95Ser Val Thr Glu Val Val Arg Thr Ala Asp Leu Ile Arg Tyr Ala Ala 100 105 110Glu Glu Gly Ile Arg Leu Ser Thr Ser Ala Asp Glu Gly Gly Lys Met 115 120 125Asp Ala Ser Thr Gly His Lys Leu Ala Val Ile Arg Arg Gln Pro Val 130 135 140Gly Ile Val Leu Ala Ile Ala Pro Tyr Asn Tyr Pro Val Asn Leu Ser145 150 155 160Gly Ser Lys Ile Ala Pro Ala Leu Ile Gly Gly Asn Val Val Met Phe 165 170 175Lys Pro Pro Thr Gln Gly Ser Val Ser Gly Leu Val Leu Ala Lys Ala 180 185 190Phe Ala Glu Ala Gly Leu Pro Ala Gly Val Phe Asn Thr Ile Thr Gly 195 200 205Arg Gly Ser Glu Ile Gly Asp Tyr Ile Val Glu His Glu Glu Val Asn 210 215 220Phe Ile Asn Phe Thr Gly Ser Thr Pro Val Gly Gln Arg Ile Gly Lys225 230 235 240Leu Ala Gly Met Arg Pro Ile Met Leu Glu Leu Gly Gly Lys Asp Ala 245 250 255Gly Ile Val Leu Ala Asp Ala Asp Leu Asp Asn Ala Ala Lys Gln Ile 260 265 270Val Ala Gly Ala Tyr Asp Tyr Ser Gly Gln Arg Cys Thr Ala Ile Lys 275 280 285Arg Val Leu Val Val Glu Glu Val Ala Asp Glu Leu Ala Glu Lys Ile 290 295 300Ser Glu Asn Val Ala Lys Leu Ser Val Gly Asp Pro Phe Asp Asn Ala305 310 315 320Thr Val Thr Pro Val Ile Asp Asp Asn Ser Ala Asp Phe Ile Glu Ser 325 330 335Leu Val Val Asp Ala Arg Gln Lys Gly Ala Lys Glu Leu Asn Glu Phe 340 345 350Lys Arg Asp Gly Arg Leu Leu Thr Pro Gly Leu Phe Asp His Val Thr 355 360 365Leu Asp Met Lys Leu Ala Trp Glu Glu Pro Phe Gly Pro Ile Leu Pro 370 375 380Ile Ile Arg Val Lys Asp Ala Glu Glu Ala Val Ala Ile Ala Asn Lys385 390 395 400Ser Asp Phe Gly Leu Gln Ser Ser Val Phe Thr Arg Asp Phe Gln Lys 405 410 415Ala Phe Asp Ile Ala Asn Lys Leu Glu Val Gly Thr Val His Ile Asn 420 425 430Asn Lys Thr Gly Arg Gly Pro Asp Asn Phe Pro Phe Leu Gly Leu Lys 435 440 445Gly Ser Gly Ala Gly Val Gln Gly Ile Arg Tyr Ser Ile Glu Ala Met 450 455 460Thr Asn Val Lys Ser Ile Val Leu Asp Met Lys465 470 475

Claims

1. A composition of matter comprising:a) an isolated peptide selected from the group consisting of:1) an amino acid sequence encoded by a polynucleotide obtainable from a Group B Streptococcus, wherein said polynucleotide comprises a gene selected from the group consisting of MS4, MS10, MS11, MS14 and MS16;2) an amino acid sequence selected from the group consisting of SEQ ID NO: 2, 4, 6, 8, 10 and 12;3) a homologue of 1) or 2); and 4) a functional fragment of 1) or 2); orb) an isolated polynucleotide:1) encoding a polypeptide of any one of a1) to a4); or2) comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1, 3, 5, 7, 9, and 11; orc) a host cell transformed to express a polynucleotide of b1) or b2); ord) a vaccine comprising any one of a1), a2), a3), a4), b1), or b2); ore) an antibody raised against a peptide of any of a1) to a4).

2. An isolated peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 2, 4, 6, 8, 10 and 12, or a homologue or furictional fragment of any of the foregoing.

3. A method for screening for potential drugs, wherein said method comprises the use of a peptide encoded by a polynucleotide, wherein said polynucleotide sequence comprises a gene obtainable from a Group B Streptococcus, wherein said gene is selected from the group consisting of MS4, MS10, MS11, MS14 and MS16; or wherein said polynucleotide sequence comprises a homologue or a functional fragment of one of said Group B Streptococcus genes.

4. The method according to claim 3, wherein said peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 2, 4, 6, 8, 10 and 12.

5. A method for the detection of virulence; comprising the use of a peptide encoded by a polynucleotide sequence, wherein said polynucleotide sequence comprises a gene obtainable from a Group B Streptococcus, wherein said gene is selected from the group consisting of MS4, MS 10, MS11, MS14 and MS16; or wherein said polynucleotide sequence comprises a homologue or a functional fragment of one of said Group B Streptococcus genes.

6. The method according to claim 5, wherein said peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 2, 4, 6, 8, 10 and 12.

7. A method for the treatment or prevention of a condition associated with bacterial infection, wherein said method comprises administering to a patient in need of such treatment or prevention, an effective amount of a peptide encoded by a polynucleotide sequence, wherein said polynucleotide sequence comprises a gene obtainable from a Group B Streptococcus, wherein said gene is selected from the group consisting of MS4, MS10, MS11, MS14 and MS16; or wherein said polynucleotide sequence comprises a homologue or a functional fragment of one of said Group B Streptococcus genes.

8. The method according to claim 7, wherein the infection is a Group B Streptococcal infection.

9. The method according to claim 7, wherein the infection is a local infection.

10. The method according to claim 7, wherein the infection is a urinary tract infection.

11. The method according to claim 7, wherein the peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 2, 4, 6, 8, 10 and 12, or a homologue or functional fragment of any of the foregoing.

12. The method according to claim 7, wherein the peptide comprises an amino acid sequence selected from the group consisting SEQ ID NO: 2, 4, 6, 8, 10 and 12.

13. The method according to claim 7, wherein the peptide comprises a non-phosphorylating NADP-dependent glyceraldehyde-3-phosphate dehydrogenase.

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