From Fish To Human Beings: Pathogenesis Investigation Of Several Important Vibrio Species

Vibrios are common free-living aquatic bacteria that are classified as Gram-negative facultative anaerobes. Although Vibrios always exist as normal flora in environment, several Vibrio species cause diseases in human and fish. In this work, we indentified the virulence factors and their regulation by quorum sensing system in fish pathogen Vibrio alginolyticus, and examined the mechanism of reactogenicity of cholera live-attentuated vaccine, and finally tried to understand the pathogenesis of Vibrio parahaemolyticus by using novel animal model (infant rabbits).Vibrio alginolyticus, a moderately halophilic gram-negative bacterium, is considered as an opportunistic pathogen of aquatic animals. However, in the South China Sea, V. alginolyticus has been reported to be the dominant causative agent of high-mortality vibriosis in the large yellow croaker, sea bream, grouper, kuruma prawn, as well as shellfish species. In marine Vibrio species, the Vibrio harveyi-type LuxR protein, a key player in a quorum sensing system, was found to control the expression of some virulence-relative genes. In this study, the luxR homologue in Vibrio alginolyticus was identified and named luxRval, whose expression was greatly induced at high cell density. The luxRval in-frame deletion mutant showed a significant down-regulation of total extracellular protease activity, and also resulted in changes of colony morphology, extracellular polysaccharide (EPS) production, and mobility. The extracellulat alkaline serine protease asp, which was shown to be a virulence factor of Vibrio alginolyticus as a purified protein, was cloned from V. alginolyticus. The regulation of asp gene was analyzed by using RT-PCR and quantitative real-time PCR methods, we proved that its transcription was greatly induced at the late stage of growth and was regulated by luxO-luxRval regulatory system. The asp null mutant was also constructed by homologous recombination using suicide plasmid pNQ705-1. Compared to the wild-type strain, the asp null mutant exhibited a significant decrease of total extracellular protease activity, causing a 15-fold decrease in virulence of V. alginolyticus and decrease of cytotoxicity to EPC cells.Cholera is a severe diarrheal disease caused by the motile gram-negative rod Vibrio cholerae. Live-attenuated V. cholerae vaccines harboring deletions of the genes encoding cholera toxin have great promise for reducing the global burden of cholera. However, development of live vaccines has been hampered by the tendency of such strains to induce non-choleric'reactogenic'diarrhea in human subjects. The molecular bases of reactogenicity are unknown, but it has been speculated that reactogenic diarrhea is a response to V. cholerae's flagellum and/or the motility that it enables. Here, we used an infant rabbit model of reactogenicity to determine what V. cholerae factors trigger this response. We found that V. cholerae ctx mutants that produced flagellins induced diarrhea, regardless of whether the proteins were assembled into a flagellum or whether the flagellum was functional. In contrast,～90% of rabbits infected with V. cholerae lacking all 5 flagellin-encoding genes did not develop diarrhea. Thus, flagellin production, independent of flagellum assembly or motility, is sufficient for reactogenicity. The intestinal colonization and intra-intestinal localization of the non-reactogenic flagellin-deficient strain were indistinguishable from those of a flagellated motile strain, while the flagellin-deficient strain stimulated less mRNA transcripts coding for pro-inflammatory cytokines in the intestine. Thus, reactogenic diarrhea may be a consequence of an innate host inflammatory response to V. cholerae flagellins. Our results suggest a simple genetic blueprint for engineering of defined non-reactogenic live-attenuated V. cholerae vaccine strains.Vibrio parahaemolyticus, a gram-negative marine bacterium, is a worldwide cause of food-borne gastroenteritis. However, the pathogenesis of V. parahaemolyticus was not understood very well due to lack of animal model. Recently, genome sequencing of the clinical V. parahaemolyticus strain RIMD2210633 suggested that hymolysin gene tdh and two sets of typeⅢsecretion system (TTSS), TTSS1 and TTSS2, existed in genome. In this study, a new animal model (infant rabbit) was developed to study pathogenesis of V. parahaemolyticus. We found that oral infection of V. parahaemolyticus could cause diarrhea to～90% infant rabbits and fluid accumulation in intestine. Diarrhea was normally developed around 30 hours after inovulation but varied in different rabbits. We also infected rabbits with putative virulence factor mutants, and found that intestinal fluid accumulation in rabbits was diminished by infection of mutants which contained the deletion of the TTSS2-related genes, but fluid accumulation was not diminished in rabbits when infected by tdh and T3SS1 mutant. Compared to those infected by T3SS2 mutant, the intestinal colonization and pro-inflammatory cytokines in rabbits were much higher when infected by WT, tdh and T3SS1 mutant. Histologic analysis showed that there was much more disease of cell sloughing, heterophil assemble, cell proliferation, congestion, villus tip edema, and villus shorting in rabbits infected by WT tdh and T3SS1 mutant. The rabbits that infected by T3SS2 mutants were completely normal. Finally, we used the confocal microscopy to examine the localization of V. parahaemolyticus in intestion, and found V. parahaemolyticus formed microcolonies and discontinuously distributed in diatal small intestine.