The Study Of Interaction Mechanism Between Bile Acid Molecules And DsbA Of Vibrio Cholerae

The disease cholera is caused by Vibrio cholerae, a member of Gram-negative bacterium. This causative agent can cause host severe vomiting, diarrhea, dehydration and even death. After entering and colonizing in intestine, tcp and ct, cholera toxin gene, can be expressed in host. After analyzing the small intestine extracts, we found that bile acid molecules help TcpP form more functional dimer, which induces expression of a large amount of toxin factors. The expression of toxin gene doesn’t be induced by bile acid molecules. TcpP fails to form dimer when knocking out vcdsbA. We speculated that the interaction between bile acid and DsbA could regulate downstream toxin gene network, which affects a large amount of toxin factors when Vibrio cholerae enters human intestinal tract.To investigate the interaction between bile acid molecules and DsbA, we cloned vc0034(vcdsbA) into the prokaryotic expression vector pET28 a, expressed by transforming the recombinant plasmid into E.coli Rosetta. The expressed DsbA was purified by nickel ions resin. Then we analyzed the interaction between bile acid molecules and DsbA by an iothermal titration calorimetry(ITC) assay. The results showed that equilibrium dissociation constant(KD) was 0.93×10-4 M, while the stoichiometric ratio(n) was 1.17 and the enthalpy(ΔH) was-3647 cal/mol. The entropy(ΔS) was 6.22 cal/mol. In order to further exploit the biological function of DsbA, we measured the reductase activity of DsbA by insulin reductase assay, and found that there was a interaction between DsbA and TC.In order to define key amino acid sites of the interaction between DsbA and bile acid molecules, we constructed a mutant gene library by error prone PCR. This library was further screened by detecting the luminescence of candidate strains. The vcdsbA mutant was selected after sequencing. Three mutants(dsbAH113L, dsbAD135 V, dsbAE188A) were screened from nearly ten thousand strains. To further characterize roles of these three amino acid sites, the mutant protein DsbAH113 L, DsbAD135 V, DsbAE188 A were expressed in E.coli. The purified mutant proteins were not sensitive to TC in EcdsbA deleted E.coli strain, although these mutant protein were sensitive in the dsbA deletion strain of V.cholerae. This experiment indicates that bile acid affect reductase activity of DsbA and its derivatives, suggesting that H113, D135, E188 are not the binding sites of DsbA towards TC. Thus, the interaction mechanism still needs to be further investigated. This study lays an experimental foundation for screening of small molecular inhibitors against DsbA.