In vivo phospholipase activity of the Pseudomonas aeruginosa cytotoxin ExoU and protection of mammalian cells with phospholipase A2 inhibitors.

TitleIn vivo phospholipase activity of the Pseudomonas aeruginosa cytotoxin ExoU and protection of mammalian cells with phospholipase A2 inhibitors.
Publication TypeJournal Article
Year of Publication2003
AuthorsPhillips, Rebecca M., Six David A., Dennis Edward A., and Ghosh Partho
JournalJ Biol Chem
Volume278
Issue42
Pagination41326-32
Date Published2003 Oct 17
ISSN0021-9258
KeywordsAmino Acid Sequence, Animals, Bacterial Proteins, CHO Cells, Carboxylic Ester Hydrolases, Chromatography, Thin Layer, Cricetinae, Cytosol, Enzyme Activation, Enzyme Inhibitors, Microscopy, Fluorescence, Molecular Sequence Data, Mutation, Phospholipases A, Phospholipases A2, Pseudomonas aeruginosa, Sequence Homology, Amino Acid, Serine, Signal Transduction
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 ExoU-expressing strain of P. aeruginosa is blocked by various inhibitors of cytosolic (cPLA2) and Ca2+ -independent (iPLA2) phospholipase A2 enzymes. In contrast, no cytoprotection is offered by inhibitors of secreted phospholipase A2 enzymes or by a number of inhibitors of signal transduction pathways. This suggests that phospholipase A2 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 cPLA2 and iPLA2 enzymes and contains a putative serine-aspartate catalytic dyad homologous to those found in cPLA2 and iPLA2 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.

DOI10.1074/jbc.M302472200
Alternate JournalJ. Biol. Chem.
PubMed ID12915403
Grant ListDK07202 / DK / NIDDK NIH HHS / United States
GM20501 / GM / NIGMS NIH HHS / United States
GM64611 / GM / NIGMS NIH HHS / United States
T32 CA09523 / CA / NCI NIH HHS / United States