The ADP Ribosyltransferase Domain of Pseudomonas aeruginosa ExoT Contributes to Its Biological Activities

Abstract

ExoT is a type III secreted effector protein found in almost all strains of Pseudomonas aeruginosa and is required for full virulence in an animal model of acute pneumonia. It is comprised of an N-terminal domain with GTPase activating protein (GAP) activity towards Rho family GTPases and a C-terminal ADP ribosyltransferase (ADPRT) domain with minimal activity towards a synthetic substrate in vitro. Consistent with its activity as a Rho family GTPase, ExoT has been shown to inhibit P. aeruginosa internalization into epithelial cells and macrophages, disrupt the actin cytoskeleton through a Rho-dependent pathway, and inhibit wound repair in a scrape model of injured epithelium. We have previously shown that mutation of the invariant arginine of the GAP domain to lysine (R149K) results in complete loss of GAP activity in vitro but only partially inhibits ExoT anti-internalization and cell rounding activity. We have constructed in-frame deletions and point mutations within the ADPRT domain in order to test whether this domain might account for the residual activity observed in ExoT GAP mutants. Deletion of a majority of the ADPRT domain (residues 234 to 438) or point mutations of the ADPRT catalytic site (residues 383 to 385) led to distinct changes in host cell morphology and substantially reduced the ability of ExoT to inhibit in vitro epithelial wound healing over a 24-h period. In contrast, only subtle effects on the efficiency of ExoT-induced bacterial internalization were observed in the ADPRT mutant forms. Expression of each domain individually in Saccharomyces cerevisiae was toxic, whereas expression of each of the catalytically inactive mutant domains was not. Collectively, these data demonstrate that the ADPRT domain of ExoT is active in vivo and contributes to the pathogenesis of P. aeruginosa infections.

Pseudomonas aeruginosa type III-secreted toxin ExoT inhibits host-cell division by targeting cytokinesis at multiple steps

“…Crucial to its success as a human opportunistic pathogen is its ability to inhibit epithelial wound repair. Previously published studies have demonstrated that the GAP and ADPRT domains of ExoT work together to inhibit cell migration, a key step in epithelial wound repair (17,18). We now show that these domain activities also combine to effectively block cell division, another important cellular process in wound repair, by targeting cytokinesis at multiple steps.…”

“…We have previously demonstrated that P. aeruginosa inhibits the repair of wounded epithelium in an ExoT-dependent manner (17,18). In this article, we report that P. aeruginosa inhibition of wound repair is in part due to its inhibition of host-cell division by targeting cytokinesis at multiple steps.…”

“…We have previously shown that P. aeruginosa inhibits the repair of wounded epithelium through ExoU-dependent and ExoU-independent mechanisms (17,18). During studies designed to examine whether ExoU-independent epithelial cell injury is reversible, we observed that a portion of A549 human lung epithelial cells failed to complete cytokinesis, the final stage of cell division in which the daughter cells separate (Fig.…”

A novel human antimicrobial factor targets Pseudomonas aeruginosa through its type III secretion system

“…To distinguish between these possibilities, we complemented PA103 DUDT with a pUCP20 expression vector harbouring either the wild-type ExoT or its inactive form ExoT(R149K, AAA) that has been shown to be secreted through the T3SS (Garrity-Ryan et al, 2004). PA103 DUDT+pExoT and PA103 DUDT+pExoT (R149K, AAA) strains were both highly sensitive to CA-AMF antimicrobial activity in the CM (Fig.…”

“…The pUCP20 : : pscJ was transformed into PA103 pscJ : : Tn5 bacteria which were made competent as described by Chuanchuen et al (2002). pUCP20 : : exoT and pUCP20 : : exoT(R149K, AAA) have been described previously described (Garrity-Ryan et al, 2004).…”