Mechanisms of intracellular resistance and the role of the escape of vibrio cholerae from protist hosts

Vibrio cholerae is the cause of the acute diarrhoeal disease cholera, and is a natural inhabitant of marine and fresh water systems. Reports have shown that V. cholerae survives intracellularly in and escapes from protist hosts. However, the mechanisms of this interaction are poorly understood and the biological roles of the escape remain unknown. V. cholerae and two protozoan models were used here to investigate the mechanisms of intracellular survival and the impact of the escape from protozoal digestion on in vivo colonisation in a mouse model of infection. The release of V. cholerae in expelled food vacuoles (EFVs) from T. pyriformis was investigated. Co-incubation experiments revealed that the release of EFVs from T. pyriformis is largely mediated by the bacterium. In addition, several mutants of V. cholerae were tested and it was established that the virulence factor, OmpU, positively regulated by the master of virulence, ToxR, plays a key role in the release of V. cholerae-EFVs from T. pyriformis. Furthermore, the biological role of EFVs during infection represent a major gap in our understanding for the transmission of infectious diseases. Here, purified V. cholerae-EFVs were used to study their impact on the viability and intestinal colonisation of V. cholerae. Results showed that EFVs confer stress protection and a growth and colonisation advantage to V. cholerae. The intracellular survival and trafficking of V. cholerae in A. castellanii was also studied. It was established that the uptake of V. cholerae was mediated by an actin-dependant mechanism and that ammonium increases the intracellular viability of V. cholerae, possibly through phagosome-lysosome inhibition. Intracellular trafficking experiments revealed that V. cholerae remain viable within the normal intracellular pathway, suggesting that factors required for the resistance to host defences such as antimicrobial peptides, hydrolases and low pH are involved in the process. Transposon sequencing analysis showed that novel genes and virulence factors are involved in intracellular survival and escape and in intestinal colonisation of V. cholerae-EFVs. In summary, this project provided strong evidence that V. cholerae factors used to survive intracellularly in the environment play an important role in the pathogenicity of this prevalent pathogen