Actin cytoskeleton disruption by ExoY and its effects on Pseudomonas aeruginosa invasion

Three of the Type III-secreted effectors of Pseudomonas aeruginosa (ExoS, ExoT, and ExoY) each alter mammalian cell morphology in culture without causing a loss of cell viability. For ExoS and ExoT this property involves RhoGAP activity, and leads to actin cytoskeleton disruption and a reduced capacity for internalizing bacteria. ExoY does not possess RhoGAP activity. Instead, cell rounding depends upon its adenylate cyclase catalytic region. Since anti-phagocytic activities of ExoS and ExoT are associated with cell rounding and cytoskeleton disruption, we hypothesized that ExoY would also inhibit P. aeruginosa invasion of epithelial cells coinciding with adenylate cyclase-mediated cytoskeleton disruption. The results showed actin disruption of epithelial cells at 2 h post-infection associated with both adenylate cyclase-active ExoY and its catalytic mutant form ExoYK81M, and which coincided with inhibition of bacterial invasion (76% inhibition by ExoY, and 37% by ExoYK81M). Surprisingly, at 4h post-infection, neither form of ExoY inhibited invasion despite extensive actin disruption. These data suggest that ExoY, like ExoS and ExoT, contains more than one active domain affecting mammalian cell function. The data also suggest that cytoskeleton disruption does not necessarily predict invasion inhibitory activity, supporting the recently proposed model that P. aeruginosa internalization can proceed through more than one pathway.