Screen And Characterization Of Vibrio Cholerae Genes Expressed In Vivo And Responsible For Biofilm Formation In Seawater

Vibrio cholerae is both an inhabitant of natural aquatic environments and the etiologic agent of the human diarrheal disease cholera. In the natural aquatic settings, V.cholerae usually persists in biotic or abiotic surfaces in forms of biofilm, a structural community of bacteria adherent to a surface and enclosed in a self-produced exopolysaccharide matrix, and this is proposed to play critical roles in V.cholerae persistence in the natural environment and might account for seasonal cholera epidemics. When entering the host environment, V.cholerae employs a set of new genes that significantly differ from those expressed in vitro to fulfill successful infection.Antibiotics-based in vivo expression technology (IVET) is a method to identify in vivo induced (ivi) genes during host-pathogen interactions. In this study, a novel ICR adult mouse model for selection of V.cholerae El Tor C6706 genes that are specifically induced in vivo was established. This IVET system employed a kan-rpsL gene fusion as a dual selection marker both in vivo and in vitro. The library was pooled by transconjugants with promoterless kan-rpsL on the chromosome and then subjected to two rounds of in vivo and in vitro selection. Clones harboring an in vivo activated promoter upstream the reporter genes exhibit kanamycin resistance in vivo while kanamycin sensitivity and streptomycin resistance in vitro. Using this strategy, five positive clones, encoding four ivi genes of V.cholerae El Tor C6706, have been identified. One of the ivi genes identified in this study, VC0303, encodes a sensor histidine kinase; another three genes identified involve in amino acid and nucleotide metabolism, which is consistent with the previous IVET selection results. Our findings support the hypothesis that to survive and proliferate in the host, V.cholerae must regulate various uniquely in vivo expressed genes, like genes of sensing and metabolism. By monitoring the activities of lacZ reporter gene to VC0303 in different in vitro conditions, we concluded that VC0303 could not be induced in LB and AKI media. To determine genes responsible for V.choleare enhanced biofilm formation in seawater, a random transposon mutagenesis strategy was used. Through a polystyrene microtiter plate assay 10 strains exhibiting enhanced biofilm formation in artificial seawater (ASW) were screened and 9 genes responsible for V.cholerae biofilm formation in ASW were identified. Among them, rfbU encodes a mannosaltransferase and had been shown to be essential for V.cholerae O antigen production. The rfbU mutant formed an increased biofilm in ASW, and expression of the downstream rjbV gene in C6706 cannot recover this phenotype, suggesting that rfbU plays a negative role in V.cholerae biofilm formation in ASW. Meanwhile, the lipopolysaccharide (LPS) assay showed that rfbU mutant could not produce LPS in both LB and ASW, suggesting that LPS played an inhibitory role in V.cholerae biofilm formation in ASW but this effect was not observed in LB medium. Moreover, gene disruption and chromosomal constitutive expression of a phosphate peptone pathway (PPP) operon showed that it was required for V.cholerae enhanced biofilm in ASW. All of our findings will provide basis for better understanding of the underlying of V.cholerae biofilm formation and persistence in seawater.