The mechanism of action of the Pseudomonas aeruginosa-encoded type III cytotoxin, ExoU

Abstract

Pseudomonas aeruginosa delivers the toxin ExoU to eukaryotic cells via a type III secretion system. Intoxication with ExoU is associated with lung injury, bacterial dissemination and sepsis in animal model and human infections. To search for ExoU targets in a genetically tractable system, we used controlled expression of the toxin in Saccharomyces cerevisiae. ExoU was cytotoxic for yeast and caused a vacuolar fragmentation phenotype. Inhibitors of human calcium-independent (iPLA 2 ) and cytosolic phospholipase A 2 (cPLA 2 ) lipase activity reduce the cytotoxicity of ExoU. The catalytic domains of patatin, iPLA 2 and cPLA 2 align or are similar to ExoU sequences. Sitespeci®c mutagenesis of predicted catalytic residues (ExoUS142A or ExoUD344A) eliminated toxicity. ExoU expression in yeast resulted in an accumulation of free palmitic acid, changes in the phospholipid pro®les and reduction of radiolabeled neutral lipids. ExoUS142A and ExoUD344A expressed in yeast failed to release palmitic acid. Recombinant ExoU demonstrated lipase activity in vitro, but only in the presence of a yeast extract. From these data we conclude that ExoU is a lipase that requires activation or modi®cation by eukaryotic factors.

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).…”

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

“…This mutant lacks both ExoU and ExoT, the two effectors of the type III secretion system known to be produced by this strain (Vallis et al, 1999). The PA103ΔUT mutant was complemented with plasmid pUCP18 containing either the exoU gene (pUCPexoU) which fully restores cytotoxic activity towards eukaryotic cells, or the exoU gene in which the aspartate catalytic site of the N-terminal phospholipase domain is mutated (pUCPexoUD344A), and thus it lacks phospholipase and in vitro cytotoxic activity, or an empty vector control (pUCP18) (Sato et al, 2003). Plasmid complemented mutants were grown on trypticase soy agar (TSA) supplemented with carbenicillin 300 μg/ml overnight (~18 h) at 37 °C.…”

“…It has been shown that ExoU exhibits a patatin-like phospholipase activity (Sato et al, 2003;Sato and Frank 2004;Sato et al, 2005) in combination with host factors, e.g. superoxide dismutase (Sato et al, 2006).…”

“…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).…”

Phospholipases during membrane dynamics in malaria parasites

“…Pseudomonas aeruginosa and Legionella pneumophila, respectively, have been described as crucial secreted virulence factors which are injected by the bacteria into the HC (Phillips et al, 2003;Sato et al, 2003;Shohdy et al, 2005;VanRheenen et al, 2006; reviewed in Banerji and Flieger, 2004;Flores-Díaz et al, 2016). The four plasmodial PLPs exhibit the characteristic lipase motif GXSXG containing the catalytic S. Three of the four PLPs further clearly comprised the second member D of the catalytic S-D dyad, while the fourth enzyme (PF3D7_0924000) possesses several candidates for catalytic Ds (Fig.…”