Effect of virulence factors on survival strategies of vibrio cholerae and vibrio mimicus

Vibrio is a genus of gram-negative bacteria comprising nearly 70 species and they are clinically human pathogens that can cause many infections. This study is mainly based on two species such as Vibrio cholerae O1 El Tor, which cause cholera disease and Vibrio mimicus, that cause gastroenteritis, open wounds infection, and septicemia. Therefore, this thesis aimed to study the effect of their virulence factors; melanin pigment, LuxO protein, ToxR protein, outer membrane proteins and vesicles on survival strategies; biofilm, rugose morphology and intracellular survival of V. cholerae and V. mimicus in Acanthamoeba castellanii using cell culture, viable cell count, gentamicin assay, vital staining, light-, fluorescent- and electron microscopy, microphotography, spectrophotometry, protein expression and isolation analysis, constructing internal in-frame gene deletion mutants and statistical analysis. The results showed that V. cholerae mutant strain produced more melanin, showed increased UV resistance, expressed more toxin-coregulated pilus and cholera toxin and also increased colonization of infant mouse model compared to the wild-type V. cholerae. These findings suggest a possible role of melanin pigment formation in V. cholerae virulence factor expression. V. mimicus had an enhanced growth in presence of A. castellanii and showed an intracellular behaviour in the amoeba. The bacteria were localized in the cytoplasm of amoeba trophozoites and the intracellular bacteria were viable for more than 2 weeks. Surprisingly, the toxR mutant of V. cholerae produced outer membrane protein T, significant biofilm and rugose colonies compared to the wild- type that produced OmpU, which showed decreased biofilm and did not form rugose colonies at 30°C. However, during the association with the amoebae it was observed that A. castellanii enhanced the survival of V. cholerae wild type compared to toxR mutant strain at 37°C. Interestingly, neither the wild- type nor toxR mutant strain could form rugose colonies in association with the amoebae. Therefore it shows that ToxR does seem to play some regulatory role in the OmpT/OmpU expression shift, the changes in biofilm, rugosity and survival with A. castellanii, suggesting a new role for this regulatory protein in the environments. Interestingly, outer membrane protein A (Omp A) suppressed the survival of alone cultivated wild-type V. cholerae, whereas the ompA mutant released more outer membrane vesicles and inhibited the viability of the amoebae. Co-cultivation of bacterial strains with A. castellanii enhanced the survival of both wild-type and ompA mutant but OmpA protein has no effect on attachment, engulfment and intracellular growth of V. cholerae. In conclusion, this study based on the role of virulence factors on environmental survival strategies of V. cholerae and V. mimcus has demonstrated that melanin pigment plays an enhanced role in virulence and virulence factor expression of the bacteria such as resistance for UV- light, expression of toxin-coregulated pilus, cholera toxin and colonization ability. V. mimicus shows an intracellular growth and survival in A. castellanii and neither LuxO regulator nor protease have role on the intracellular behaviour, which is reported for the first time. The regulatory protein ToxR of V. cholerae suggests a new role in the expression of OmpT/OmpU and in the formation of biofilm, switching morphotypes from smooth to rugose colony and in association with the free living amoeba A. castellanii. The outer membrane protein A of V. cholerae suppresses the survival of alone cultivated wild-type V. cholerae and has no effect on attachment, engulfment and intracellular growth of V. cholerae interacted with the amoebae, whereas ompA mutant released more outer membrane vesicles, which inhibits the viability of amoebae.