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J. Biol. Chem., Vol. 278, Issue 42, 41326-41332, October 17, 2003

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

Rebecca M. Phillips, David A. Six, Edward A. Dennis and Partho Ghosh 

From the Department of Chemistry and Biochemistry, University of California at San Diego, La Jolla, California 92093-0314

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.

Received for publication, March 11, 2003 , and in revised form, June 12, 2003.

Note Added in Proof—Similar conclusions regarding ExoU activation and function on heterologous expression of the protein in yeast have been reported recently (Sato, H., Frank, D. W., Hillard, C. J., Feix, J. B., Pankhaniya, R. R., Moriyama, K., Finck-Barbancon, V., Buchaklian, A., Lei, M., Long, R. M., Wiener-Kronish, J., and Sawa, T. (2003) Embo J. 22, 2959–2969).

^* This work was supported by the Cystic Fibrosis Foundation (to P. G.), Keck Distinguished Young Scholar in Medicine Award (to P. G.), National Institutes of Health Grants GM 20,501 and GM 64,611 (to E. A. D.), National Institutes of Health/NCI National Research Service Award T32 CA09523 (to R. M. P.), and National Institutes of Health Training Grant DK07202 (to D. A. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Chemistry and Biochemistry, Mail Code 0314, University of California at San Diego, 9500 Gilman Dr., La Jolla, CA 92093-0314. Tel.: 858-822-1139; Fax: 858-534-7042; E-mail pghosh{at}ucsd.edu.

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