Legionella pneumophila, an intracellular pathogen causing a severe pneumonia, possesses distinct lipolytic activities which have not been completely assigned to specific enzymes so far. We cloned and characterized a gene, plaC, encoding a protein with high homology to PlaA, the major secreted lysophospholipase A of L. pneumophila and to other hydrolytic enzymes belonging to the GDSL family. Here we show that L. pneumophila plaC mutants possessed reduced phospholipase A and lysophospholipase A activities and lacked glycerophospholipid:cholesterol acyltransferase activity in their culture supernatants. The mutants' reduced phospholipase A and acyltransferase activities were complemented by reintroduction of an intact copy of plaC. Additionally, plaC conferred increased lysophospholipase A and glycerophospholipid:cholesterol acytransferase activities to recombinant Escherichia coli. Furthermore, PlaC was shown to be another candidate exported by the L. pneumophila type II secretion system and was activated by a factor present in the bacterial culture supernatant dependent on the zinc metalloprotease. Finally, the role of plaC in intracellular infection of Acanthamoeba castellanii and U937 macrophages with L. pneumophila was assessed, and plaC was found to be dispensable. Thus, L. pneumophila possesses another secreted lipolytic enzyme, a protein with acyltransferase, phospholipase A, and lysophospholipase A activities. This enzyme is distinguished from the previously characterized phospholipases A and lysophospholipases A by its capacity not only to cleave fatty acids from lipids but to transfer them to cholesterol. Cholesterol is an important compound of eukaryotic membranes, and an acyltransferase might be a tool for host cell modification to fit the needs of the bacterium.Legionella pneumophila is a gram-negative bacterium which is found in freshwater environments, where it associates with amoebae. The inhalation of Legionella-containing aerosols can lead to an acute pneumonia, Legionnaires' disease. In the human lung the bacteria are able to replicate within alveolar macrophages and epithelial cells, causing tissue damage and eventually leading to lung failure. Some of the factors which promote intracellular replication are exported by or depend on other factors exported by the type II secretion system Lsp or the type IVB secretion system Dot/Icm (6,22,32,39,40). The type II secretion system is responsible for translocation of a variety of hydrolytic activities, because L. pneumophila type II secretion mutants show considerably reduced protease, acid phosphatase, lipase, lysophospholipase A (LPLA), phospholipase A (PLA), p-nitrophenylphosphorylcholine (p-NPPC) hydrolase, and nuclease activities in their culture supernatants (22,39). Accordingly, substrates of the type II secretion system include the major zinc metalloprotease ProA or Msp, the major acid phosphatase Map, the lipase LipA, and the major secreted LPLA PlaA (3,4,17,22). Studies with knockout mutants indicate that none of these proteins are essential...

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