Auto‐ADP‐ribosylation inhibits the catalytic activity of ExoS, a type‐III effector of Pseudomonas aeruginosa

Abstract

Pseudomonas aeruginosa, an opportunistic organism responsible for infections in immunocompromised hosts, produces a number of virulence factors, e.g., Exoenzyme S (ExoS), which are delivered into host cells through a type III secretion system. ExoS is a bifunctional type III effector which has ADP-ribosyltranferase (ART) activity localized in the carboxyl-terminal domain and GTPase-Activating Protein (GAP) activity for Rho GTPases in an amino-terminal domain. The ART domain is dependent on Factor-Activating ExoS (FAS), a 14-3-3 protein, for expression of ART activity. ExoS was previously shown to auto-ADP-ribosylate arginine 146 in the GAP domain, inhibiting GAP activity. We report here that the 49-kDa full length ExoS and the ART domain of ExoS auto-ADP-ribosylate both GAP- and ART- domains in a FAS-dependent manner. Increasing [NAD] and the time of incubation increased the amount of ADP-ribose bound to ExoS and the ART domain. ART activity of ExoS (ADP-ribosyl-agmatine formation) was reduced dramatically by auto-ADP-ribosylation. The ADP-ribose amino acid linkage was hydroxylamine sensitive, consistent with modification of arginine. FAS, ADP-ribosylated by ExoS, still stimulated ExoS activity. Auto-ADP-ribosylation thus down regulates the activities of both the GAP- and ART- domains and thereby, may limit the intracellular activity and toxicity of ExoS. Intramural Research Program, NIH/NHLBI