Aeromonas species are ubiquitous water-borne bacteria that are able to cause a variety of diseases in poikilothermics and humans. Aeromonas hydrophila is one of the most pathogenic species, responsible for aeromonad septicaemia in fish, gastroenteritis and wound infections in humans. The T3SS is utilized to inject protein effectors directly into host cells. One of the major genetic regulators of the T3SS in several Gram-negative bacterial species is the AraC-like protein ExsA. Lateral flagella are expressed by bacteria upon contact with host cells or a surface and are required for host cell adherence and biofilm formation. However, no direct link between the T3SS and the lateral flagella system has yet been found in A. hydrophila. Moreover, the genetic regulation of the T3SS that involves the master regulator ExsA has not been determined in A. hydrophila AH-3.
The aim of this project is to determine the genetic regulation of the T3SS and the potential interaction between the T3SS and the lateral flagella system in A. hydrophila AH-3.
The genes encoding the T3SS regulatory components including exsA, exsD, exsC and exsE were mutated and the activities of the T3SS promoters were measured in exs mutant backgrounds. The interactions between each of the Exs proteins were investigated using BACTH assay and Far-Western Blot. Together, the findings suggested a regulatory cascade that the effector protein ExsE was bound to the chaperone protein ExsC, which sequestered the anti-activator ExsD from inhibiting the T3SS master regulator ExsA via direct protein-protein interactions.
The T3SS regulatory components were also shown to affect the expression of the lateral flagella system using swarming assays. The activities of the lateral flagella promoters were shown to be repressed by the absence of ExsD and ExsE, suggesting that the T3SS master regulator ExsA was a negative regulator of the lateral flagella system.
