In Vivo Rho GTPase-Activating Protein Activity of Pseudomonas aeruginosa Cytotoxin ExoS

Abstract

ExoS is a bifunctional type III cytotoxin secreted by Pseudomonas aeruginosa, which comprises a C-terminal ADP ribosyltransferase domain and an N-terminal Rho GTPase-activating protein (GAP) domain. In vitro, ExoS is a Rho GAP for Rho, Rac, and Cdc42; however, the in vivo modulation of Rho GTPases has not been addressed. Using a transient transfection system and delivery by P. aeruginosa, interactions were examined between the Rho GAP domain of ExoS and Rho GTPases in CHO cells. Rho GTPases were expressed as green fluorescent protein (GFP) fusion proteins to facilitate quantitation. GFP fusions of wild-type and dominant active Rho, Rac, and Cdc42 localized to discrete regions of CHO cells and appeared functional based upon their modulation of the actin cytoskeleton. Coexpression of the Rho GAP domain of ExoS changed the intracellular distribution of GFP-Rac and GFP-Cdc42 from a predominately membrane location to a cytosolic location. Coexpression of the Rho GAP domain of ExoS did not change the distribution of GFP-Rho, which was primarily in the cytosol. Coexpression of dominant active Rac (DARac) and DACdc42 inhibited actin reorganization by the Rho GAP domain but did not maintain the formation of actin stress fibers, which indicated that Rho had been inactivated. Similar results were observed when ExoS was delivered into CHO cells by P. aeruginosa. These data indicate that in vivo the Rho GAP activity of ExoS stimulates the reorganization of the actin cytoskeleton by inhibition of Rac and Cdc42 and stimulates actin stress fiber formation by inhibition of Rho.

Interactions between effector proteins of the Pseudomonas aeruginosa type III secretion system do not significantly affect several measures of disease severity in mammals

“…ExoS is a bifunctional toxin containing a GTPase-activating protein (GAP) domain and an ADPribosyltransferase (ADPRT) domain. The amino-terminal GAP activity acts on Rho family GTPases while the carboxylterminal ADPRT activity is directed towards Ras and other host-cell proteins (Fraylick et al, 2001;Goehring et al, 1999;Henriksson et al, 2000Henriksson et al, , 2002Krall et al, 2002;Olson et al, 1999;Rocha et al, 2003;Vincent et al, 1999). As a result of these enzymic activities, intoxication with ExoS is associated with several observable phenotypes, including cytotoxicity and inhibition of bacterial internalization by both phagocytic and non-phagocytic mammalian cells (Cowell et al, 2000;Fleiszig et al, 1997;Frithz-Lindsten et al, 1997;Henriksson et al, 2000;Kaufman et al, 2000;Olson et al, 1997Olson et al, , 1999Pederson & Barbieri, 1998;Vincent et al, 1999).…”

The galU Gene of Pseudomonas aeruginosa Is Required for Corneal Infection and Efficient Systemic Spread following Pneumonia but Not for Infection Confined to the Lung

“…The P. aeruginosa TTSS has in recent years been shown to be linked to increased severity of nosocomial pneumonia in humans (19,42) and to be important for pathogenesis in murine models of lung (2,49) and eye (29) infections. The effector proteins of the P. aeruginosa TTSS include ExoS and ExoT, which function as both ADP-ribosyltransferases and GTPase-activating proteins (15,26,27); ExoY, which is an adenylate cyclase (58); and ExoU, which has recently been shown to be a lipase (13,44). Interestingly, the exoS and exoU genes appear to be mutually exclusive: P. aeruginosa strains have either one or the other (14,42,57).…”

Activation of ExoU Phospholipase Activity Requires Specific C-Terminal Regions

“…The activity of each effector is dependent upon interaction with a cofactor present in eukaryotic but not prokaryotic cells. ExoS and ExoT are bifunctional enzymes that possess both Rho GTPase-activating protein and ADP-ribosyltransferase activities (23,25,51). The ADP ribosylation of eukaryotic proteins by ExoS and ExoT requires activation by members of the 14-3-3 family of scaffolding proteins (13).…”