The Rickettsia prowazekii ExoU Homologue Possesses Phospholipase A ₁ (PLA ₁ ), PLA ₂ , and Lyso-PLA ₂ Activities and Can Function in the Absence of Any Eukaryotic Cofactors In Vitro

Abstract

Here we have characterized the Rickettsia prowazekii RP534 protein, a homologue of the Pseudomonas aeruginosa ExoU phospholipase A (PLA) secreted cytotoxin. Our studies showed that purified recombinant RP534 PLA possessed the predicted PLA 2 and lyso-PLA 2 activities based on what has been published for P. aeruginosa ExoU. RP534 also displayed PLA 1 activity under the conditions tested, whereas ExoU did not. In addition, recombinant RP534 displayed a basal PLA activity that could hydrolyze phosphatidylcholine in the absence of any eukaryotic cofactors. Interestingly, the addition of bovine liver superoxide dismutase 1 (SOD1), a known activator of P. aeruginosa ExoU, resulted in an increased rate of RP534-catalyzed phospholipid hydrolysis, indicating that mechanisms of activation of the ExoU family of PLAs may be evolutionarily conserved. The mechanism of SOD1-dependent stimulation of RP534 was further examined using active site mutants and a fluorogenic phospholipid substrate whose hydrolysis by RP534 over a short time course is measureable only in the presence of SOD1. These studies suggest a mechanism by which SOD1 stimulates RP534 activity once it has bound to the substrate. We also show that antibody raised against RP534 was useful for immunoprecipitating active RP534 from R. prowazekii lysed cell extracts, thus verifying that this protein is expressed and active in rickettsiae isolated from embryonated hen egg yolk sacs.

Secretome of obligate intracellularRickettsia

“…A subsequent report confirmed the PLA 2 activity of R . prowazekii Pat2 (Housley et al ., 2011) . Recently, we determined that Pat1 of R .…”

Ubiquitin Activates Patatin-Like Phospholipases from Multiple Bacterial Species

“…A growing number of reports demonstrate that bacterial pathogens, particularly Gram-negative species, use patatin-like PLA 2 enzymes as effector molecules to target host cellular membranes (23)(24)(25)(26)(27). Previous work has shown that exoenzyme U (ExoU), a patatin-like phospholipase encoded by the opportunistic pathogen Pseudomonas aeruginosa, is injected by a type III secretion system (T3SS) into host cells as an effector (27)(28)(29).…”

“…ϩ Gram-negative bacteria considering that bioinformatic analyses revealed several close orthologs to ExoU (23)(24)(25)(26)(27). We queried three additional enzymes from bacterial species representing different ecological niches and pathogenic potentials to determine if ubiquitin activation was a common property of these proteins (24,27,31,32).…”

RalF-Mediated Activation of Arf6 Controls Rickettsia typhi Invasion by Co-Opting Phosphoinositol Metabolism

“…Quickly after inducing phagocytosis, bacteria escape into the host cell's cytoplasm and actively replicate. Several adhesion and cytoskeleton-binding proteins (Sca5, Adr1, Adr2, Sca4, Sca0, Sca1, Sca2, and RickA) have been identified for their role in rickettsial entry (33–39), as have several enzymes putatively involved in phagosomal escape (TlyC, PLD, Pat1, and Pat2) (40–43). Importantly, these pathogenicity factors are variably encoded across diverse Rickettsia species, indicating the likelihood of multiple strategies for rickettsial invasion of host cells (44).…”