Investigation of genes involved in interactions and grazing defence of Vibrio cholerae with protozoa

Vibrio cholerae is exposed to predation by heterotrophic protists in the environment. Here we investigate whether the quorum sensing regulator, HapR or a hypothetical adhesin (VC1804), play roles in the interaction of V. cholerae with the amoeba, Acanthamoeba castellanii. V. cholerae rapidly colonises the surface of A. castellanii during co-incubation. Results presented here show that the hapR mutant was less efficient in colonisation of A. castellanii, and was deficient in intracellular survival in once ingested compared to the wild type. Furthermore, a transposon insertion in VC1804 resulted in a significant decrease in attachment to and uptake by A. castellanii, as well as a significant decrease in attachment to human epithelial cells (HepG2). Furthermore epi-fluorescent microscopy showed that V. cholerae was taken up in food vacuoles by both A. castellanii and Tetrahymena pyriformis but was subsequently expelled from the protozoa within egested food vacuoles, where viability was maintained. In addition, the roles of the outer membrane proteins OmpA and OmpV in antiprotozoal activities of V. cholerae were examined. Although the ompV mutant did not show any significant difference in susceptibility to grazing by A. castellanii and T. pyriformis when grown in biofilms, it was significantly less grazing resistant in the planktonic phase when grazed by T. pyriformis in comparison to the wild type. Interestingly, this decrease in grazing resistance was not observed when a suspension feeder, Cafeteria roenbergensis was the predator. The ompA mutant was not significantly different compared to the wild type in defence against grazing by A. castellanii, T. pyriformis and C. roenbergensis. Co-cultivation of the ompA and ompV mutants with A. castellanii showed that neither of these outer membrane proteins plays a role in colonisation of A. castellanii. Overall this project aimed to further identify factors involved in predation resistance of V. cholerae. Protozoan predation on bacteria acts as an evolutionary force, leading to the evolution of antiprotozoal mechanisms which may also function as virulence factors enabling bacteria not only to survive in the environment but to successfully cause disease in their hosts