P. aeruginosa is an opportunistic pathogen that causes infections in immuno-compromised individuals and cause infections due to and arsenal of secreted virulence factor. Some virulence factors are translocated to the cell via a type III secretion system, such ExoS. ExoS is a bifunctional toxin with a C-terminal ADP-ribosylation activity, which modify activities of the Ras superfamily. The GAP domain of ExoS abrogates the activation of RhoA, Cdc42 and Rap1. The eukaryotic 14-3-3 proteins are necessary for the activity of ExoS. The interaction between 14-3-3 and ExoS occurs in a novel phosphorylation independent manner.
We will use immunohistochemistry and biochemical analysis, such as pull-down-, live/dead-, GAP-, ADP-ribosylation-assays using specific downstream substrate target proteins and site-specific mutagenesis on cells infected with ExoS. Further, structural crystal analysis is employed, followed by real life analysis to evaluate the pathogenesis in a mouse model of acute pneumonia.
Significance; the overall aim of this application is to increase our understanding of P. aeruginosa infection at a functional molecular level with respect to the critical bacterial toxin ExoS. This knowledge can then form the basis for new strategies of therapy and treatment of P. aeruginosa infections.