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Chapter 3 : Analysis of Acid Phosphatase and Esterase/Lipase Mutants of Legionella pneumophila
The identification of a pilD gene in Legionella pneumophila offered new insight into the molecular pathogenesis of legionellosis. Three mutants, NU254, NU255, and NU256, were selected from the screening and further studied. Supernatants from these mutants contained minimal acid phosphatase activity, while possessing normal levels of other pilD-dependent exoproteins. To determine if the map gene was necessary for intracellular replication of Legionella, macrophage-like U937 cells and Hartmannella vermiformis amoebae were infected at multiplicity of infection of 0.1 and 1, respectively. The authors have demonstrated that L. pneumophila has two acid phosphatases that can be differentiated by their different sensitivity to tartrate, and while the tartrate-sensitive Map seems not to be essential for intracellular replication, the role of the tartrate-resistant phosphatase has yet to be determined. The gene that was interrupted by the transposon was determined, and a basic local alignment search tool (BLAST) analysis of the sequence showed that the putative protein shared some homology to lipase enzymes. Supernatants from the mutant presented a defect in its ability to hydrolyze p-nitrophenyl (PNP) palmitate (indication of esterase/lipase) and in the liberation of free fatty acid from phosphatidylcholine (indication of phospholipase A).