Acanthamoeba castellanii as a host and model to study bacterial virulence

Abstract: Vibrio cholerae is the causative agent of cholera, a waterborne diarrheal disease and it is believed to be strictly extracellular bacterium. Cholera remains as a global public health threat affecting most of the developing world. In endemic areas, women and children are among the high risk. Recently outbreaks are reported from many countries. V. cholerae O1 and O139 have been associated with epidemic and pandemic outbreaks. Shigellosis is a global human health problem, especially in developing countries, with substandard hygiene and unsafe water supplies. The morbidity and mortality due to shigellosis are especially high among children in developing countries. Acanthamoebae are ubiquitous free-living amoebae that are distributed worldwide, living in diverse environments. Acanthamoeba has the ability to act as a host for bacterial pathogens. The aim of this thesis is to study the interaction of free-living amoebae A. castellanii and waterborne bacteria of V. cholerae and Shigella species, and the possibility to use the amoebae as model to study bacterial virulence. V. cholerae and Shigella were co-cultivated with A. castellanii and the interaction was studied by cell count, viable count, fluorescence microscopy, electron microscopy and flow cytometry analysis. The results showed that V. cholerae grew and survived intracellularly in the cytoplasm of trophozoites, and that the bacteria were found in the cysts of A. castellanii. V. cholerae O139 MO10 did not inhibit growth of the amoeba instead enhanced growth and survival of V. cholerae O139 MO10 occurred. The wild type V. cholerae O139 MO10 and its capsule mutant or capsule and LPS double mutant grew inside A. castellanii indicting no special role of those molecules in the interaction. The co-cultivation of A. castellanii with Shigella showed that S. dysenteriae or S. sonnei grew and survived in the presence of amoebae for more than three weeks. Gentamicin assay showed that Shigella were viable inside the A. castellanii which confirmed by electron microscopy which disclosed the Shigella localized in the cytoplasm of the A. castellanii. S. flexneri (wild type) and mutant resulted in the cell death at 37°C and inhibition at 30°C. IpaB was found to play an important role in killing A. castellanii. In conclusion the interaction showed a facultative intracellular behavior of V. cholerae and a possible role of A. castellanii as an environmental reservoir of V. cholerae species. Growth and survival of both amoebae and bacteria in recultivation of A. castellanii harboring intracellular V. cholerae indicate endosymbiontshost relation between these microorganisms. Neither the capsule nor the LPS O side chain of V. cholerae O139 was found to play any important role in the interaction with A. castellanii disclosing the ability of V. cholerae to multiply and survive inside A. castellanii as well as the role of A. castellanii as environmental hosts for V. cholerae. The relationship between, S. dysenteriae, S. sonnei with A. castellanii is symbiotic, and amoeba may act as a reservoir for Shigella in environmental water. The interaction showed that wild type S. flexneri, IpaB mutant and virulence plasmid-cured kill A. castellanii by inducing necrosis. This thesis shows that A. castellanii may act as reservoir for waterborne bacteria and the characteristics of Acanthamoeba makes it suitable non mammalian cell to be used in the investigation of host bacterial interaction.