Identification of novel immunogenic proteins of Shigella flexneri 2a by proteomic methodologies

Vaccine 22 (2004) 2750–2756

Identification of novel immunogenic proteins of Shigella flexneri 2a by proteomic methodologies Xuanxian Peng∗ , Xintai Ye, Sanying Wang Center for Proteomics, Department of Biology, School of Life Sciences, Xiamen University, Xiamen, Fujian 361005, PR China Received 3 September 2003; accepted 19 January 2004 Available online 13 February 2004

Abstract Shigella spp. are one of the most important etiological factors for people who are living in developing countries and travelers to tropical countries. High priority has been given by the World Health Organization to the development of vaccines to control Shigellosis caused by these bacteria. However, information regarding to profile of immunogenic proteins of Shigella is not available now. In the present study, sub-immunoproteomics was applied to screen novel immunogenic proteins which could be reacted with antisera produced by challenge of a whole bacterium. Our results indicated that 13 immunogens were identified, in which seven proteins and six proteins from outer membrane and soluble proteome, respectively. Of the 13 proteins, 12 showed to be novel immunogens. These results suggest that immunoproteomics can greatly improve the chances of identification and result in discovery of novel immunogenic proteins. © 2004 Elsevier Ltd. All rights reserved. Keywords: Proteomics; Vaccine; Shigella; Immunogen

1. Introduction Shigellosis is a major public health problem in infants and young children in developing countries and is the major etiologic agent of traveler’s diarrhea [1]. In China, Shigella spp. are the second leading cause of intestinal infectious disease [2]. The bacteria are spread via the faecal–oral route. Once ingested, the virulent organisms invade the colonic epithelial cells, multiply intracellularly and spread to adjacent uninfected intestinal cells [3,4]. Invasion of host cells by Shigella flexneri requires expression of virulence genes located on a 230 kb plasmid. The products of these genes include the Ipa invasins, the Mxi and Spa proteins, and the IcsA, IcsB and VirA proteins. The first mediates the invasion of gut epithelia and macrophage apoptosis. The second two form a type III secretion system. And the last three are responsible for intercellular spreading of bacteria in the lower gut [5]. Increasing recognition of the burden of the incidence and mortality rates of infections caused by Shigella spp. has stimulated research to develop vaccines to prevent shigellosis [6]. High priority has been given by the World Health Organization to the development of vaccines that will protect against infections ∗ Corresponding author. Tel.: +86-592-218-1140; fax: +86-592-218-1015. E-mail address: [email protected] (X. Peng).

0264-410X/$ – see front matter © 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2004.01.038

caused by the bacteria [7]. Two approaches for Shigella vaccine are applied including development of attenuated strains without a heterogeneous gene and attenuated hybrid bivalent vaccine strains with a heterogeneous gene which can code to express highly protective antigens [7–9]. Accumulating data indicated that the major protective antigen appeared to be the type specific O-antigen because the presence of antibodies against the serotype-determining lipopolysaccharide (LPS) O-antigen was correlated with protection against disease [7,10]. However, recent studies have shown that more protective antigens, which can screen from immunogenic proteins, may be selected as vaccine candidates [9,10]. Unfortunately, information regarding the profile of immunogenic proteins of Shigella is not available now. Recently, proteomics, a high-efficiency technology with high accuracy, has brought new challenges into this area and it provides an understanding of the expression of a genotype at the phenotypic level in a target cell at a given stage. Much of information about immunogen amounts or activities can be derived from proteomics with Western blotting, namely immunoproteomics [11–13]. Research on vaccinology will be improved by quick high-through screen and identifications of proteins in the science of proteomics [14–16]. Recently, a two-dimensional proteome map of S. flexneri has been characterized, but immunogenic proteins have not been determined [17]. The aims of the present study are

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to identify immunogenic proteins from S. flexneri 2a by a combination of proteomic and Western blotting methods.

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preparation was determined using the Folin–phenal method and BSA was used as standard protein. 2.4. Isolation of soluble proteins

2. Materials and methods 2.1. Bacterial cell culture S. flexneri 2a strain was grown aerobically overnight at 37 ◦ C in a shaker bath as seed (approximately 1012 ). Fresh overnight cultures were diluted into 1:100 in Luria–Bertani Medium and growth was continued at 37 ◦ C for 18 h. The cultures were collected by centrifugation at 6000 × g for 10 min at 4 ◦ C. The gained pellets were resuspended in 50 mM Tris–HCl, pH 7.4 for washing and pelleted again by centrifugation at 6000 × g for 10 min. 2.2. Production of antibodies The antibody production was performed essentially as described by Peng et al. [18]. The bacteria were cultured to high density and harvested by centrifugation at 4000 × g for 10 min and diluted with 0.65% saline. Then, formaldehyde was added at 1% final concentration to inactivate the bacteria for preparation of antigen. The antigen was diluted to 108 /ml of inactivated bacteria with 0.65% saline following three times rinse with sterile saline. Three mice (Mus musculus Km) received three subcutaneous injections with 0.1 ml per animal on nape at internal 1 week in the first and second injections and at internal 10 days in the third injection. We collected and pooled their sera, and then measured the antibody titer using agglutination test. The agglutination test was performed on a slide. Bacteria with concentrations of 108 /ml were used as antigen. The sera were diluted two-fold in PBS at starting dilution of 1:200. 2.3. Isolation and purification of outer membrane proteins (OMPs) OMPs were prepared by the sarkosyl method with modification [19]. Briefly, harvested cells (1012 /ml) were resuspended in 50 mM Tris–HCl, pH 7.4 for washing and pelleted by centrifugation at 6000 × g for 10 min. The supernatant was removed and the pellet was resuspended in three volumes of sonication buffer (50 mM Tris–HCl, 1 mM EDTA, pH 7.4) and subsequently disrupted on ice by intermittent sonic oscillation at 50% power with 30 s and a duty cycle of 5. Unbroken cells and cellular debris were removed by centrifugation at 2500 × g for 10 min where after the supernatant was collected and further centrifuged at 100,000 × g for 40 min at 4 ◦ C. The pellet was resuspended in 2% (w/v) sodium lauryl sarcosinate and incubated at room temperature for 20 min to facilitate inner membrane solubilization. The OMPs were pelleted at 100,000 × g for 40 min at 4 ◦ C, resuspended in the sonication buffer and stored at −70 ◦ C until required. The concentration of the OMPs in the final

After washing harvested cells (1012 /ml) were resuspended in 50 mM Tris–HCl, 1 mM EDTA, pH 7.4 and then sonicated on ice at 50% power with 30 s and a duty cycle of 5. The solution was centrifuged at 12,000 × g for 10 min at 4 ◦ C. The supernatant was collected and stored at −70 ◦ C until required. The concentration of the soluble proteins in the final preparation was determined using the Folin–phenal method and BSA was used as standard protein. 2.5. Two-dimensional electrophoresis and Western blotting The electrophoretic separation of proteins was performed essentially as described by O’Farrell et al. [20] as well as Chen et al. [21]. Isoelectric focusing was made in 12 cm × 3 mm vertical glass tubes with 5% T gel containing 2% ampholine (pH 3–10). The sample was mixed with IEF solution (9.8 M urea, 1.6% ampholine (pH 7–10) and 0.4% ampholine (pH 3–10), 2% Triton X100, 100 mM DTT, 50 mM Tris–HCl). A total amount of 20 ␮g OMPs and 300 ␮g soluble proteins were separately loaded on the first dimensional gel and isoelectric focusing was conducted and run for 400 V for 18 h. For the second dimension, the first dimensional gel was equilibrated for 15 min by incubating/rocking in a solution of 6 ␮M Tris–HCl, pH 6.8, 2% SDS, 5% ␤-mercaptocothanol, 10% Gly, following which the gel was embedded onto a 10% SDS/PAGE gel according to size. Proteins were visualized by staining with Coomassie Brilliant Blue R-250 (Merck, Germany), and then the wet gels were scanned and analyzed in GSC-8000 (Bio-Rad). The tests were repeated five times. Proteins from the other gels were transferred to a NC membrane (Bio-Rad Corporation, USA) for 6 h at 200 mA in transfer buffer (48 mM Tris, 39 mM glycine and 20% methanol) at 4 ◦ C. The NC membrane was blocked for 60 min with 5% skimmed milk in TBS at 37 ◦ C. After rinsing three times with TBS for 5 min each, the NC membrane was incubated with primary antibody, mouse anti-Shigella flexneri 2a (the agglutination titer of the pooled sera was 1:3200), at a dilution of 1:500 in TTBS containing 5% skimmed milk for 1 h at 28 ◦ C on a gentle shaker. The membrane was rinsed three times, 10 min each, and incubated with goat anti-mouse-HRP (CALTAG, CA, USA) at a dilution of 1:1000 in TTBS containing 5% skimmed milk for 1 h at 37 ◦ C. Then the membrane was washed with 50 mM Tris–HCl buffer (pH 7.4) and developed with substrate (DAB) until optimum color developed. 2.6. In situ tryptic digestion of proteins Protein spots were excised from the gels using a clear scalpel as close as the protein spot and subjected to in

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situ digestion with trypsin according to previously described procedures [22], with modifications reported in our laboratory [23]. The gel particles were desdained with water/acetonitrile 1:1 (v/v). Solvent volumes used in the washing steps should roughly equal two times the gel volume. Then all liquid was removed and enough acetonitrile was added to cover the particles. After the gel pieces become white and shrunk, the acetonitrile was removed and gel pieces were rehydrodrated in 0.1 M NH4 HCO3 for 5 min. An equal volume of acetonitrile was added and incubated for 15 min. All liquid was removed and gel particles dried down in a vacuum. Then the particles were swelled in 10 nM DTT/0.1 M NH4 HCO3 at 56 ◦ C for 45 min to reduce the protein. Excess liquid was removed and replaced quickly with roughly the same volume of freshly prepared 55 mM iodoacetamide/0.1 M NH4 HCO3 , incubated for 30 min at room temperature in the dark. After iodoacetamide solution was removed, the gel particles were washed with 0.1 M NH4 HCO3 /50% acetonitrile. Gel particles were completely dried down in a vacuum followed by rehydration with a digestion buffer containing 50 mM NH4 HCO3 , 5 mM CaCl2 and 12.5 ng/␮l of trypsin at 4 ◦ C for 45 min. Then remaining enzyme supernatant was removed and replaced with 5–20 ␮l of the same buffer without enzyme to keep the gel pieces wet during enzymatic cleavage (37 ◦ C, overnight). Peptides were extracted first by adding a sufficient volume of 25 mM NH4 HCO3 to cover the pieces for 15 min then adding the same volume of acetonitrile for another 15 min, and recovering the supernatant. Extraction with 5% TFA/50% acetonitrile was repeated two times and all extracts were pooled together for drying in a vacuum. 2.7. Matrix-assisted laser desorption-ionization time of flight mass spectrometry (MALDI-TOF-MS) analysis The sample solution (30–100 ppm) with equivalent matrix solution was applied onto the MALDI-TOF-target and

prepared for MALDI-TOF-MS analysis according to a previously described procedure [24–26]. A-cyano-4-hydroxycinnamic acid (HCCA) was used as the matrix. MALDI-TOF spectra were calibrated using trypsin auto-digestion peptide signals and matrix ion signals. All MALDI analyses were performed by a fuzzy logic feedback control system (Reflex III MALDI-TOF system, Bruker) equipped with delayed ion extraction. Peptide masses were searched against the SWISS-PROT database using the Peptident program (http://www.expasy.org/tools/peptident.html) or NCBI database using the Mascot program (http://www. matrixscience.com).

3. Results 3.1. OMP profile in 2-DE and 2-DE immunoblotting Fig. 1A shows a representative example of the OMPs separated on a 2-DE gel, where 20 ␮g of total protein was applied. Approximately 80 protein spots were detected on the Coomassie stained gel. Eight protein spots could be identified to be positive on 2-DE maps. The eight proteins on 2-DE were named from O1 to O8. Fig. 1B shows representative results from 2-DE Western blotting. 3.2. Soluble protein profile in 2-DE and 2-DE immunoblotting The profile of the soluble proteins, separated by 2-DE and stained by Coomassie R-250, is shown in Fig. 2A with approximately 300 spots. The most spots distributed on the map from pI 5.0 to 8.0 and molecular weight from 10 to 140 kD. Of the 300 proteins on 2-DE gel, six spots were detected to be positive reactivity with sera of mouse

Fig. 1. 2-DE and 2-DE Western blotting of OMPs of S. flexneri 2a. Totally 20 ␮g proteins were loaded onto a gel, and approximately 80 spots could be detected after proteins were stained with Coomassie R-250. Of these proteins, eight could be reacted with antibody against S. flexneri 2a. (A) 2-DE; (B) 2-DE Western blotting.

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Fig. 2. 2DE and 2-DE Western blotting of soluble proteins of S. flexneri 2a. Totally 300 ␮g proteins were loaded onto a gel, and approximately 300 spots could be detected after proteins were stained with Coomassie R-250. Of these proteins, six could be reacted with antibody against S. flexneri 2a. (A) SDS-PAGE; (B) 2-DE Western blotting.

anti-Shigella flexneri 2a (Fig. 2B). They were named from S1 to S6. 3.3. Protein identification The 14 proteins, eight OMPS and six soluble proteins, were identified by MALDI-TOF-MS on the basis of peptide mass matching, following in-gel digestion with trypsin.

The peptide masses were matched with the theoretical peptide masses of all proteins from the other proteobacteria of the SWISS-PROT database except for O2. Therefore, 13 proteins were successfully identified by MALDI-TOF-MS. Fig. 3 shows S6 map from MALDI-TOF mass spectrum of the analysis of the peptides after in situ tryptic digestion. Tables 1 and 2 list the identified proteins from OMPs and soluble proteins, respectively.

Fig. 3. A representative map from MALDI-TOF mass spectrum of the analysis of the peptides after in situ tryptic digestion (S6).

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Table 1 The search results for the identification of immunogenic OMPs Spot

Accession number

Description

Species

Mw (kD)

pI

Coverage (%)

Peptides matched

1 3 4 5

Gi/16767473 Gi/22126369 Gi/603576 Gi/28901046

Salmoella typhimurium Yersinia pestis Neisseria meningitidis Vibrio parahaemolyticus

24828 31167 35704 36162

6.74 9.42 7.14 5.57

45 42 46 39

13 9 13 11

6 7

Gi/16123748 Gi/17547695

Yersinia pestis Ralstonia solanacearum

87168 88003

9.60 9.46

41 37

16 9

8

Gi/21230836

Putative outer membrane lipoprotein Flagellar biosynthesis protein Serotype 15 outer membrane protein Putative multidrug efflux membrane fusion protein Putative Rhs accessory genetic element Probable penicillin-binding (peptidoglycan synthetase) transmembrane protein Outer membrane usher protein FasD

Xanthomonas campestris

88005

6.31

26

9

Table 2 The search results for the identification of immunogenic soluble proteins Spot

Accession

Description

Species

Mw (kD)

pI

Coverage (%)

Peptides matched

1 2 3 4 5 6

Gi/15429330 Gi/16264235 Gi/15964616 Gi/15965841 Gi/23013417 Gi/22966457

Major antigenic protein MAP1 Putative trifolitoxin immunity protein Hypothetical signal peptide protein Hypothetical protein Hypothetical protein Conserved hypothetical protein

Cowdria rumiuautium Sinorhizobium meliloti Sinorhizobium meliloti Sinorhizobium meliloti Magnetospirillum magnetotacticum Brucella suis

30420 29303 28711 40126 40864 67104

4.93 5.79 8.86 6.72 6.77 9.83

52 49 41 34 29 38

14 15 10 10 6 12

4. Discussion In this study, an attempt is made to screen immunogenrelated proteins from S. flexneri 2a by proteomic techniques. Fourteen proteins were found by immunoproteomic methodologies and then identified by means of MALDI-TOF-MS. These proteins were identified through matching the above information with SWISS-PROT database except for O2. O1, O3–O8 and S1–S6 were successfully identified as putative outer membrane lipoprotein, flagellar biosynthesis protein, serotype 15 outer membrane protein, putative multidrug efflux membrane fusion protein, putative Rhs accessory genetic element, probable penicillin-binding (peptidoglycan synthetase) transmembrane protein, outer membrane usher protein FasD, major antigenic protein MAP1, putative trifolitoxin immunity protein, hypothetical signal peptide protein, hypothetical protein, hypothetical protein, conserved hypothetical protein, respectively. Of the 13 immunogenic proteins, one was known immunogenic protein and 12 were first reported here to be immungenic proteins which could be reacted with antisera produced by challenge of a whole bacterium. These results suggest that immunoproteomics can greatly improve the chances of identification and result in discovery of novel immunogenic proteins. Our approach is carried out by sub-proteomics: OMP and soluble protein sub-proteomics. Sub-cellular proteomics may decrease the complexity that lies in analysis of the whole cell in the identification of the resolved proteins, in which proteins within cell are pre-fractionated/sub-fractionated depending on its function and compartment specialty prior to 2-D [27–29].

Most of the 13 proteins have been characterized by bioimformatics. Putative outer membrane lipoprotein (O1) was obtained by the analysis of the sequence of the chromosome and virulence plasmid of S. typhimurium strain LT2. Function study from its gene homologues showed that they might be exported to the periplasm or outer membrane [30]. Flagellar biosynthesis protein (O3), serotype 15 outer membrane protein (O4), putative multidrug efflux membrane fusion protein (O5) are related to protein secretion, porin activity and transporter activity, protein transporter activity and protein secretion, respectively [31–33]. Putative Rhs accessory genetic element (O6) functions in ATP binding, ATPase activity, coupled to transmembrane movement of ions, phosphorylative mechanism [31]. Probable penicillin-binding (peptidoglycan synthetase) transmembrane protein (O7) possesses catalytic activity, penicillin binding and peptidase activity, and outer membrane usher protein FasD (O8) shows transporter activity [34,35]. Major antigenic protein MAP1 (S1) is coded by the major antigenic protein genes (map1 genes), showing immunogenic function [36]. S2, S3, S4 and S5 were obtained by sequences analysis of Sinorhizobium meliloti, in which S4 and S5 were identified as a same protein [37,38]. Conserved hypothetical protein (S6) was obtained by the analysis of the sequence of the Brucella suis from different organisms [39]. In summary, our results suggest that sub-immunoproteomics, a specific, quick and high-through screen method, can be better applied in analysis of immunogenic proteins from microorganisms. The sub2-D gel that was viewed as complementary approaches to 2-D PAGE exhibits

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a unique library of S. flexneri 2a. Fourteen proteins were identified to be immunogenic by the employment of the library, in which one is known as immunogenic protein and 12 are first reported here to be novel immunogenic proteins which could be reacted with antisera produced by challenge from a whole bacterium. The usefulness of the identified proteins to develop a Shigella-specific vaccine is desirable. Our approach has thus provided information on novel candidates for vaccine designation.

Acknowledgements This work was sponsored by grants from the “863” Project of China (No. 2002AA629050), the Foundation of Chinese Education Ministry for Ph.D. Program. References [1] Barry EM, Altboum Z, Losonsky G, Levine MM. Immune responses elicited against multiple enterotoxigenic Escherichia coli fimbriae and mutant LT expressed in attenuated Shigella vaccine strains. Vaccine 2003;21:333–40. [2] Peng XX, Luo W, Zhang JY, Wang SY, Lin SC. Rapid detection of Shigella species in environmental sewage by an immuno-capture PCR with universal primers. Appl Environ Microbiol 2002;68:2580–3. [3] Walker JC, Verma NK. Identification of a putative pathogenicity island in Shigella flexneri using subtractive hybridisation of the S. flexneri and Escherichia coli genomes. FEMS Microbiol Lett 2002;213:257–64. [4] Morona R, Van DBL. Multicopy icsA is able to suppress the virulence defect caused by the wzzSF mutation in Shigella flexneri. FEMS Microbiol Lett 2003;221:213–9. [5] McKenna S, Beloin C, Dorman CJ. In vitro DNA-binding properties of VirB, the Shigella flexneri virulence regulatory protein. FEBS lett 2003;545:183–7. [6] Anderson RJ, Pasetti MF, Sztein MB, Levine MM, Noriega FR. DguaBA attenuated Shigella flexneri 2a strain CVD 1204 as a Shigella vaccine and as a live mucosal delivery system for fragment C of tetanus toxin. Vaccine 2000;18:2193–202. [7] Falt IC, Mills D, Schweda EKH, Timmis KN, Lindberg AA. Construction of recombinant aroA salmonellae stably producing the Shigella dysenteriae serotype 1O-antigen and structural characterization of the Salmonella/Shigella hybrid LPS. Microb Pathog 1996;20:11–30. [8] Tzschaschel BD, Klee SR, de Lorenzo V, Timmis KN, Guzmán CA. Towards a vaccine candidate against Shigella dysenteriae 1: expression of the Shiga toxin B-subunit in an attenuated Shigella flexneri aroD carrier strain. Microb Pathog 1996;21:277–88. [9] Mukhopadhaya A, Mahalanabis D, Khanam J, Chakrabarti MK. Protective efficacy of oral immunization with heat-killed Shigella flexneri 2a in animal model: study of cross protection, immune response and antigenic recognition. Vaccine 2003;21:3043–50. [10] Biswas T. Role of porin of Shigella dysenteriae type 1 in modulation of lipopolysaccharide mediated nitric oxide and interleukin-1 release by murine peritoneal macrophages. FEMS Immunol Med Microbiol 2000;29:129–36. [11] Klade CS. Proteomics approaches towards antigen discovery and vaccine development. Curr Opin Mol Ther 2002;4:216–23. [12] Stanislavsky ES, Makarenko TA, Kholodkova EV, Lugowski C. R-form lipopolysaccharides (LPS) of Gram-negative bacteria as possible vaccine antigens. FEMS Immunol Med Microbiol 1997;18:139–45.

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[13] Utt M, Nilsson I, Ljungh A, Wadstrom T. Identification of novel immunogenic proteins of Helicobacter pylori by proteome technology. J Immunol Methods 2002;259:1–10. [14] Chakravarti DN, Fiske MJ, Fletcher LD, Zagursky RJ. Application of genomics and proteomics for identification of bacterial gene products as potential vaccine candidates. Vaccine 2001;19:601–12. [15] André FE. Vaccinology: past achievements, present roadblocks and future promises. Vaccine 2003;21:593–5. [16] Poland GA, Ovsyannikova IG, Johnson KL, Naylor S. The role of mass spectrometry in vaccine development. Vaccine 2002;19:2692– 700. [17] Liao X, Ying T, Wang H, Wang J, Shi ZX, Feng EL, et al. A two-dimensional proteome map of Shigella flexneri. Electrophoresis 2003;24:2864–82. [18] Peng XX, Zhang JY, Wang SY, Lin ZL, Zhang WY. Immuno-capture PCR for detection of Aeromonas hydrophila. J Microbiol Methods 2002;49:335–8. [19] Molloy MP, Herbert BR, Slade MB, Rabilloud T, Nouwens AS, Williams KL, et al. Proteomic analysis of the Escherichia coli outer membrane. Eur J Biochem 2000;267:2871–81. [20] O’Farrell PZ, Goodman HG, O’Farrel PH. High resolution two-dimensional electrophoresis of basic as well as acidic proteins. J Cell 1997;12:1133–42. [21] Chen ZJ, Wang SY, Chen JA, Peng XX. The influence of temperature on the protein expression of human lung cancer cell line A549. ACTA Biol Exp Sin 2002;35:179–83. [22] Kussmann M, Nordhoff E, RahbekNielsen H, Haebel S, RosselLarsen M, Jakobsen L, et al. Matrix-assisted laser desorption/ionization mass spectrometry sample preparation techniques designed for various peptide and protein analytes. J Mass Spectrom 1997;32:593–601. [23] Wang SY, Wu MS, Chen JA, Chen ZJ, Peng XX. Primany establishment of proteome map to Aeromonas hydrophia. J Xiamen Univ (Nat Sci) 2003;42:143–6. [24] Zhang YL, Wang SY, Peng XX. Identification of a type of human IgG-like protein in shrimp Penaeus vannamei by mass spectrometry. J Exp Mar Biol Ecol 2004, available online. [25] Jensen ON, Podtelejnikov A, Mann M. Delayed extraction improves speciaficity in database searches by MALDI peptide maps. Rapid Commun Mass Spectrom 1996;10:1371–8. [26] Jensen O, Podtelejnikov A, Mann M. Identification of the components of simple protein mixtures by high accuracy peptide mass mapping. Anal Chem 1997;69:4741–50. [27] Pattern WF. Proteome analysis II. Protein subcellular redistribution: linking physiology to genomics via the proteome and separation technologies involved. J Chromatogr B 1999;722:203–23. [28] Oosthuizen MC, Steyn B, Theron J, Cosette P, Lindsay D, von Holy A, et al. Proteomic analysis reveals differential protein expression by Bacillus cereus during biofilm formation. Appl Environ Microbiol 2002;68:2770–80. [29] Thompson DK, Beliaev AS, Giometti CS, Tollaksen SL, Khare T, Lies DP, et al. Transcriptional and proteomic analysis of a ferric uptake regulator (Fur) mutant of Shewanella oneidensis: possible involvement of fur in energy metabolism, transcriptional regulation, and oxidative stress. Appl Environ Microbiol 2001;67:3396–405. [30] McClelland M, Sanderson KE, Spieth J, Clifton SW, Latreille P, Cowtrey L, et al. Complete genome sequence of Salmonella enterica serovar typhimurium LT2. Nature 2001;413:852–6. [31] Parkhill J, Wren BW, Thomson NR, Boutin A, Mayhew GF, Liss P, et al. Genome sequence of Yersinia pestis KIM. J Bacteriol 2002;184:4601–11. [32] Butcher S, Sarvas M, Runeberg-Nyman K. Class-3 porin protein of Neisseria meningitidis: cloning and structure of the gene. Gene 1991;105:125–8. [33] Makino K, Oshima K, Kurokawa K, Yokoyama K, Uda T, Tagomori K, et al. Genome sequence of Vibrio parahaemolyticus: a pathogenic mechanism distinct from that of V. cholerae. Lancet 2003;361:743–9.

2756

X. Peng et al. / Vaccine 22 (2004) 2750–2756

[34] Salanoubat M, Genin S, Artiguenave F, Gouzy J, Mangenot S, Arlat M, et al. Genome sequence of the plant pathogen Ralstonia solanacearum. Nature 2002;415:497–502. [35] da Silva ACR, Ferro JA, Reinach FC, Farah CS, Furlan LR, Quaggio RB, et al. Comparison of the genomes of two Xanthomonas pathogens with differing host specificities. Nature 2002;417:459– 63. [36] Allsopp MTEP, Dorfling CM, Maillard JC, Bensaid A, Haydon DT, van Heerden H, et al. Ehrlichia ruminantium major antigenic protein gene (map1) variants are not geographically constrained and show no evidence of having evolved under positive selection pressure. J Clin Microbiol 2001;39:4200–3.

[37] Finan TM, Weidner S, Wong K, Buhrmester J, Chain P, Vorholter FJ, et al. The complete sequence of the 1,683-kb pSymB megaplasmid from the N2-fixing endosymbiont Sinorhizobium meliloti. Proc Natl Acad Sci USA 2001;98:9889–94. [38] Capela D, Barloy-Hubler F, Gouzy J, Bothe G, Ampe F, Batut J, et al. Analysis of the chromosome sequence of the legume symbiont Sinorhizobium meliloti strain 1021. Proc Natl Acad Sci USA 2001;98:9877–82. [39] Paulsen IT, Seshadri R, Nelson KE, Eisen JA, Heidelberg JF, Read TD, et al. The Brucella suis genome reveals fundamental similarities between animal and plant pathogens and symbionts. Proc Natl Acad Sci USA 2002;99:13148–53.