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Legionella pneumophila genes that encode lipase and phospholipase C activities^a

Department of Microbiology-Immunology, Northwestern University Medical School, 320 East Superior Street, Chicago, IL 60611-3010, USA¹

Author for correspondence: Nick Cianciotto. Tel: +1 312 503 0385. Fax: +1 312 503 1339. e-mail: n-cianciotto{at}northwestern.edu

Legionella pneumophila, the agent of Legionnaires’ disease, is an intracellular parasite of aquatic protozoans and human macrophages. The type II protein secretion system of the Gram-negative Legionella organism promotes intracellular infection. A lipase activity and a p-nitrophenylphosphorylcholine (pNPPC) hydrolytic activity are two of the factors that are diminished in L. pneumophila type II secretion mutants. The Legionella lipase activity was found to include free fatty acid release from di- and triacylglycerol substrates, in addition to the previously reported cleavage of monoacylglycerol. In a number of other bacterial systems, the release of p-nitrophenol from pNPPC is due to a phospholipase C. In an attempt to identify exoproteins that potentiate intracellular infection, three genes were identified and mutated in L. pneumophila strain 130b that were predicted to encode either a secreted lipase or a phospholipase C. The first two genes, which were designated lipA and lipB, encoded proteins containing the lipase consensus sequence [LIV]-X-[LIVFY]-[LIVMST]-G-[HYWV]-S-X-G-[GSTAC]. Mutations in lipA in particular reduced supernatant activity against mono- and triacylglycerols. However, loss of lipA and/or lipB did not impair the ability of L. pneumophila to infect Hartmannella amoebae or U937 cell macrophages. The third L. pneumophila gene, which was denoted plcA, encoded a protein that was highly homologous with a phospholipase C from Pseudomonas fluorescens. Inactivation of plcA diminished secreted pNPPC hydrolase activity but did not influence Legionella infection of host cells. Taken together, these data indicate that L. pneumophila has multiple lipases and possibly several phospholipase C enzymes but that LipA, LipB and PlcA are not among those exoproteins required for optimal intracellular infection.

Keywords: Legionaires’ disease, type II secretion, lipolytic enzymes, macrophages, intracellular bacteria

Abbreviations: 1,2-DG, 1,2-dipalmitoylglycerol; FFA, free fatty acid; 1-MG, 1-monopalmitoylglycerol; PLC, phospholipase C; PNP, p-nitrophenol; pNPPC, p-nitrophenylphosphorylcholine

^a The GenBank accession numbers for the L. pneumophila lipA, lipB and plcA sequences are AF454863, AF454864 and AF454865, respectively.

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