title: The Rickettsia prowazekii ExoU homologue possesses phospholipase A1 (PLA1), PLA2, and lyso-PLA2 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.
authors.affiliation: Laboratory of Molecular Biology, Department of Microbiology and Immunology, University of South Alabama College of Medicine, Mobile, AL 36688, USA.
authors.forename: Nicole A
authors.initials: NA
authors.lastname: Housley
authors.forename: Herbert H
authors.initials: HH
authors.lastname: Winkler
authors.forename: Jonathon P
authors.initials: JP
authors.lastname: Audia
mesh_terms: D006868:Hydrolysis; D008245:Lysophospholipase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010713:Phosphatidylcholines / Q000378:metabolism; D054466:Phospholipases A1 / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D054467:Phospholipases A2 / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012283:Rickettsia prowazekii / Q000201:enzymology* / Q000235:genetics; D013482:Superoxide Dismutase / Q000378:metabolism; D000072105:Superoxide Dismutase-1
chemical_list: D010713:Phosphatidylcholines; D011994:Recombinant Proteins; D013482:Superoxide Dismutase; D000072105:Superoxide Dismutase-1; D054466:Phospholipases A1; D054467:Phospholipases A2; D008245:Lysophospholipase