Shigella flexneri 2a is an important pathogen causing bacillary dysentery in humans. In order to investigate any potential vaccine candidate proteins present in outer membrane proteins (OMPs) and extracellular proteins of S. flexneri 2a 2457T, we use the proteome mapping and database analyzing techniques. A subproteome map and database of OMPs were established first. One hundred and nine of the total 126 marked spots were cut out and processed to MALDI-TOF-MS and PMF. Eighty-seven spots were identified and they represented 55 OMP entries. Furthermore, immunoproteomics analysis of OMPs and extracellular proteins were performed. Total of 34 immunoreactive spots were identified, in which 22 and 12 were from OMPs and extracellular proteins, respectively. Eight novel antigens were found and some of these antigens may be potential vaccine candidate proteins. These results are useful for future studying of pathogenicity, vaccine, and novel antibacterial drugs. Maps and tables of all identified proteins are available on the Internet at www.proteomics.com.cn.

Bacillus anthracis has always been an important pathogen because it can cause lethal inhalational anthrax, and may be used as a bioweapon or by bioterrorists. In this study, a 2-DE reference map and database of B. anthracis A16R was constructed. In total, 534 spots were processed, and 406 spots representing 299 proteins were identified. Gel-estimated pIs and molecular masses mostly matched well with their theoretical predictions, but some discrepancies also existed. Spot and protein corresponding analysis revealed that post-translational modifications might be common in B. anthracis. Through the MASCOT search, the similarity of B. anthracis, B. cereus and B. thuringiensis was further verified by protein level and a possible annotation error in B. anthracis strain Ames 0581 genome was found. Proteins of energy metabolism, fatty acid and phospholipid metabolism, protein synthesis, and cellular processes represented a large part of the most abundant proteins. At the same time, 27 hypothetical proteins were experimentally proved. There were 28 proteins also identified as spore composition in recently spore-related research, which indicated that they might play some roles in different phases such as growth, sporulation and outgrowth. Maps and information about all identified proteins are available on the Internet at http://www.mpiib-berlin.mpg.de/2D-PAGE and http://www.proteomics.com.cn.

The complete Shiga toxin B subunit and two N-terminal segments of the B subunit have been inserted into a cell surface exposed loop of the LamB protein, and expression of the hybrid proteins from three different promoter systems, i.e., (i) an in vitro-inducible tac promoter that provides high-level expression, (ii) the iron-regulated aerobactin promoter presumably induced in vivo under the iron-limiting conditions of the intestinal mucosal environment, and (iii) a synthetic, modified 13-lactamase promoter providing moderate level constitutive expression, has been analyzed in Escherichia coli, SalmoneUla typhimurium, and attenuated antigen carrier strains of S. typhimurium (aroA mutants). The hybrid vaccine strains were used to immunize mice by the oral and intraperitoneal routes. S. typhimurium aroA mutants apparently have a membrane export defect which prevents the transport of LamB and its derivatives across the cytoplasmic membrane. High-level expression of hybrid proteins through use of the tac promoter proved deleterious to the vaccine strains and prevented the production of viable cells at reasonable cell densities. The lower levels of gene expression observed with the tl-lactamase and aerobactin promoters did not have this effect. Immunization of mice with S. typhimurium aroA strains carrying the hybrid genes expressed from these two promoters resulted in significant B subunit-specific mucosal and serum antibody responses. This suggests that such expression systems may be useful when incorporated into candidate antidysentery live oral vaccines for inducing protection against the effect of Shiga toxin in infections caused by Shigella dysenteriae 1 and other Shiga toxinor Shiga-like toxin-producing pathogens. Bacillary dysentery is an invasive disease of the colon in humans and higher primates and is transmitted via the fecal-oral route. The disease is associated with poor hygiene, overcrowding, and stress, and hence, the majority of cases occur in regions of developing countries that have suboptimal sanitation. However, outbreaks have also been associated with war zones and mental institutions in developed countries. The causative agents are various species of Shigella and enteroinvasive strains of Escherichia coli. The current pandemic of bacillary dysentery caused by Shigella dysenteriae serotype 1 is characterized by high fatality rates * Corresponding author. t This paper is dedicated to the memory of J.-P. Lecocq, acclaimed scientist and friend, who is sorely missed.

Shigella flexneri is a Gram-negative facultatively intracellular pathogen responsible for bacillary dysentery in humans. In this study, extracellular proteins from the culture medium and whole cell proteins in cellular extracts of S. flexneri 2a strain 2457T were examined by two-dimensional (2-D) gel electrophoresis using immobilized pH gradient (IPG) technology. Proteins were identified by matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) in combination with Mascot search program. In total, among the 488 proteins spots processed, 388 proteins were identified. The identified proteins represented 169 genes. By comparing results of Mascot search against databases of Escherichia coli and genomes of S. flexneri 2a, one S. flexneri-specific protein was identified and one possible gap was found in 2457T genome sequences. Although this proteome map is still incomplete, it is already a useful reference for future studies involving pathogenicity, vaccine development, design of novel antibacterial drugs, etc. Proteome maps and a table of all identified proteins are available on the internet at www.proteomics.com.cn.

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