Modulation of Rho signal transduction by bacterial Rho GAPs

ExoS is a bifunctional cytotoxin that encodes a FAS dependent ADP-ribosyltransferase domain in the C terminus and a Rho GTPase-Activating Protein (Rho GAP) domain in the N terminus.; A mutated form of P. aeruginosa (PA103), which expresses ExoT but not ExoU, stimulates the rounding of CHO cells. Initial studies focused on characterizing this enzymatic activity in the amino-terminus of ExoT. Transient transfection of ExoT stimulated a non-cytotoxic rounding of CHO cells and reorganization of the actin cytoskeleton. In vitro studies identified ExoT 78--237 as a GAP for RhoA, Rac, and Cdc42. Mutation of the conserved arginine, R149K, abolished GAP activity and inhibited ExoT induced actin disruption, which is consistent with X-ray crystallography studies of RhoGAPs. These studies suggest that ExoT possesses a biologically relevant activity as a RhoGAP and imply that there are at least two types of P. aeruginosa with respect to ExoS/ExoT, represented by strain 388, which expresses both ExoS and ExoT and strain PA103, which expresses only ExoT.; The in vivo activity of the RhoGAP domain of ExoS was addressed by examining the effects of intracellular expression of dominant active Rho GTPases on ExoS induced actin reorganization. Co-expression of dominant active Rac and Cdc42 reversed the reorganization of the actin cytoskeleton elicited by the RhoGAP domain of ExoS. These cells lacked actin stress fibers, which could be stimulated by expression of DARho. Taken together, these data indicate that Rac, Cdc42, and Rho are in vivo targets of the RhoGAP domain of ExoS. The inactivation of Rac and Cdc42 is responsible for the actin reorganization elicited by ExoS while inactivation of Rho is responsible for the loss of actin stress fibers.; YopE is a type-III cytotoxin of Yersinia that is also a Rho GAP. While considerable structural homology and similarities in in vitro catalytic activity exists for the Rho GAP domains of ExoS and YopE, the intracellular localization of YopE and modification of Rho GTPases within mammalian cells are not clear. A fusion protein containing the N-terminal 84 amino acids of YopE localized to a punctate perinuclear region in CHO cells, which co-localized with a fusion protein containing the membrane localization domain (MLD) of ExoS. Residues 54--75 of YopE (YopE-MLD) were found to be necessary and sufficient for this intracellular localization. Substitution of the ExoS-MLD with the YopE-MLD in ExoS localized ExoS to intracellular membranes in mammalian cells and allowed ExoS to ADP-ribosylate Ras and other membrane proteins. This indicates that the ExoS-MLD and YopE-MLD are functionally homologous. While sharing limited primary amino acid homology, the ExoS-MLD and YopE-MLD possess common hydrophobic moments, which may represent the intracellular targeting determinant of the respective cytotoxin. Using dominant active forms of the Rho GTPases, both Rac and Rho were inhibited by YopE and a chimeric protein that comprised the MLD of ExoS and the RhoGAP domain of YopE. This showed that in vivo modulation of Rho GTPases by ExoS and YopE were unique and that Rho GTPase specificity is defined by their catalytic domains. (Abstract shortened by UMI.)...