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Infection and Immunity, November 2002, p. 6094-6106, Vol. 70, No. 11
0019-9567/02/$04.00+0     DOI: 10.1128/IAI.70.11.6094-6106.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Characterization of the Gene Encoding the Major Secreted Lysophospholipase A of Legionella pneumophila and Its Role in Detoxification of Lysophosphatidylcholine

Department of Microbiology-Immunology, Northwestern University Medical School, Chicago, Illinois 60611,¹ Abteilung für Transfusionsmedizin Universitätslinikum Tübingen, D-72076 Tübingen, Germany²

Received 20 June 2002/ Returned for modification 25 July 2002/ Accepted 13 August 2002

We previously showed that Legionella pneumophila secretes, via its type II secretion system, phospholipase A activities that are distinguished by their specificity for certain phospholipids. In this study, we identified and characterized plaA, a gene encoding a phospholipase A that cleaves fatty acids from lysophospholipids. The plaA gene encoded a 309-amino-acid protein (PlaA) which had homology to a group of lipolytic enzymes containing the catalytic signature GDSL. In Escherichia coli, the cloned gene conferred trypsin-resistant hydrolysis of lysophosphatidylcholine and lysophosphatidylglycerol. An L. pneumophila plaA mutant was generated by allelic exchange. Although the mutant grew normally in standard buffered yeast extract broth, its culture supernatants lost greater than 80% of their ability to release fatty acids from lysophosphatidylcholine and lysophosphatidylglycerol, implying that PlaA is the major secreted lysophospholipase A of L. pneumophila. The mutant's reduced lipolytic activity was confirmed by growth on egg yolk agar and thin layer chromatography and was complemented by reintroduction of an intact copy of plaA. Overexpression of plaA completely protected L. pneumophila from the toxic effects of lysophosphatidylcholine, suggesting a role for PlaA in bacterial detoxification of lysophospholipids. The plaA mutant grew like the wild type in U937 cell macrophages and Hartmannella vermiformis amoebae, indicating that PlaA is not essential for intracellular infection of L. pneumophila. In the course of characterizing plaA, we discovered that wild-type legionellae secrete a phospholipid cholesterol acyltransferase activity, highlighting the spectrum of lipolytic enzymes produced by L. pneumophila.

Editor: D. L. Burns

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