Utilizzo dell'ameba come ospite per studiare le interazioni batterio-ospite: un sistema modello con Vibrio cholerae

Vibrio cholerae, the causative agent of cholera, is a versatile bacterium with multiple strains exhibiting varying survival mechanisms. Understanding these mechanisms is crucial for grasping how V. cholerae persists in diverse environments. This study leverages Acanthamoeba castellanii as a host to investigate the interactions between V. cholerae and its environmental hosts, aiming to uncover the strategies employed by the bacterium to thrive. The methodology involved infecting A. castellanii with different V. cholerae strains, including wild-type and mutants (ΔhapR, ΔmakA, ΔmakB, and ΔmakE), through a series of experiments. Techniques such as passaging amoeba cells, bacterial plate streaking, optical density measurements, seeding amoeba plates, and infection assays were employed. Advanced imaging methods, including holography and cell staining with Propidium Iodide and Acridine Orange, were utilized alongside fluorescence detection and CFU counting for intracellular bacteria analysis. Key findings highlighted that nutrient deprivation in DASW (a seawater mimic) triggered encystment in A. castellanii, a protective response to osmotic stress. In contrast, nutrient-rich PYG medium fostered higher bacterial growth and intracellular persistence. The ΔhapR strain uniquely formed biofilms in PYG, suggesting defense, colonization, or symbiotic mechanisms. CFU counts revealed significant bacterial loads, with ΔmakA, ΔmakB, and ΔmakE mutants showing zero intracellular counts, indicating their necessity for intracellular survival. Wild-type V. cholerae efficiently invaded and survived within A. castellanii, especially in PYG medium. Conclusions drawn from these experiments emphasize the critical role of nutrient availability in bacterial growth and persistence. Higher encystation rates in DASW and increased bacterial survival in PYG medium were observed. The ΔhapR strain demonstrated significant amoeba killing through biofilm formation. Gentamicin treatment was more effective in nutrient-deprived DASW, highlighting the impact of nutritional stress on antibiotic susceptibility. This research provides valuable insights into V. cholerae's survival strategies and interactions with environmental hosts, contributing to a deeper understanding of bacterial-host mechanisms.