In Vivo Phospholipase Activity of the Pseudomonas aeruginosa Cytotoxin ExoU and Protection of Mammalian Cells with Phospholipase A2 Inhibitors

Abstract

A number of clinical isolates of Pseudomonas aeruginosa are cytotoxic to mammalian cells due to the action of the 74-kDa protein ExoU, which is secreted into host cells by the type III secretion system and whose function is unknown. Here we report that the swift and profound cytotoxicity induced by purified ExoU or by an ExoUexpressing strain of P. aeruginosa is blocked by various inhibitors of cytosolic (cPLA 2 ) and Ca 2؉ -independent (iPLA 2 ) phospholipase A 2 enzymes. In contrast, no cytoprotection is offered by inhibitors of secreted phospholipase A 2 enzymes or by a number of inhibitors of signal transduction pathways. This suggests that phospholipase A 2 inhibitors may represent a novel mode of treatment for acute P. aeruginosa infections. We find that 300 -600 molecules of ExoU/cell are required to achieve half-maximal cell killing and that ExoU localizes to the host cell plasma membrane in punctate fashion. We also show that ExoU interacts in vitro with an inhibitor of cPLA 2 and iPLA 2 enzymes and contains a putative serine-aspartate catalytic dyad homologous to those found in cPLA 2 and iPLA 2 enzymes. Mutation of either the serine or the aspartate renders ExoU non-cytotoxic. Although no phospholipase or esterase activity is detected in vitro, significant phospholipase activity is detected in vivo, suggesting that ExoU requires one or more host cell factors for activation as a membrane-lytic and cytotoxic phospholipase.

Cloning and Characterization of the Gene Encoding the Major Cell-Associated Phospholipase A of Legionella pneumophila , plaB , Exhibiting Hemolytic Activity

“…However, it is noteworthy that the L. pneumophila plaB mutants constructed in this study were defective in cell-associated phospholipase A activity although the Km r cassette disrupting the plaB gene was placed after the putative catalytic domains, suggesting that the C terminus might be important for activation, stability, or proper transport of PlaB. Indeed, the cytotoxic activity of the type III secreted P. aeruginosa cytotoxin ExoU, recently found to be a phospholipase A (45,50), has been suggested to depend on the C-terminal region adjacent to the catalytic domain, since a mutant containing a transposon insertion 88 nucleotides from the exoU stop codon secretes a stable protein but is defective in cell killing (29). Furthermore, several type I secreted bacterial proteins, such as the E. coli hemolysin HlyA or the Erwinia chrysanthemi metalloprotease PrtG, contain their signal for transport via an ABC transporter in their C termini (8).…”

Bacterial clearance of Pseudomonas aeruginosa is enhanced by the inhibition of COX‐2

“…of enzymes possessing phospholipase A2 activity [33,34]. A recent study by Saliba et al [35] showed that ExoU modulates the production of eicosonoid mediators by endothelial cells, favoring the inflammatory response associated with P. aeruginosa infection.…”

Mutation of the phospholipase catalytic domain of the Pseudomonas aeruginosa cytotoxin ExoU abolishes colonization promoting activity and reduces corneal disease severity

“…The N-terminal domain of the protein encodes the phospholipase activity. Mutations of the catalytic serine (S142) or aspartate (D344) residues abolishes both the phospholipase activity and cytotoxicity towards mammalian and yeast cells (Phillips et al, 2003;Sato et al, 2003;Rabin and Hauser 2005). Mutation of the N-terminal region of ExoU also reduces P. aeruginosa virulence of in a murine model of acute pneumonia (Pankhaniya et al, 2004).…”