The arginine finger domain of ExoT contributes to actin cytoskeleton disruption and inhibition of internalization of Pseudomonas aeruginosa by epithelial cells and macrophages

Pseudomonas aeruginosa, an important nosocomial pathogen of humans, express es a type III secretion system that is required for virulence. Previous stu dies demonstrated that the lung-virulent strain PA103 has the capacity to b e either cytotoxic or invasive. Analyses of mutants suggest that PA103 deli vers a negative regulator of invasion, or anti-internalization factor, to h ost cells via a type III secretion system. In this work we show that the ty pe III secreted protein ExoT inhibits the internalization of PA103 by polar ized epithelial cells (Madin-Darby canine kidney cells) and J774.1 macropha ge-like cells. ExoS, which is closely related to ExoT but has additional AD P-ribosylating activity, can substitute for ExoT as an anti-internalization factor. ExoT contains a signature arginine finger domain found in GTPase-a ctivating proteins. Mutation of the conserved arginine in ExoT diminished i ts anti-internalization activity and altered its ability to disrupt the act in cytoskeleton. Cell fractionation experiments showed that ExoT is translo cated into host cells and that mutation of the arginine finger did not disr upt translocation. In a mouse model of acute pneumonia, PA103 DeltaU DeltaT reached the lungs as efficiently as PA103 DeltaU but showed reduced coloni zation of the liver. This finding suggests that the ability to resist inter nalization may be important for virulence in vivo.