Mutagenesis of active-site histidines of Listeria monocytogenes phosphatidylinositol-specific phospholipase C: effects on enzyme activity and biological function.
Listeria monocytogenes, a gram-positive facultative intracellular pathogen, produces two distinct phospholipases C. PC-PLC, encoded by plcB, is a broad-range phospholipase, whereas PI-PLC, encoded by plcA, is specific for phosphatidylinositol. It was previously shown that PI-PLC plays a role in efficient escape of L. monocytogenes from the primary phagosome. To further understand the function of PI-PLC in intracellular growth, site-directed mutagenesis of plcA was performed. Two potential active-site histidine residues were mutated independently to alanine, serine, and phenylalanine. With the exception of the activity of the enzyme containing H38F, which was unstable, the PI-PLC enzyme activities of culture supernatants containing each mutant enzyme were <1% of wild-type activity. In addition, the levels of expression of the mutant PI-PLC proteins were equivalent to wild-type expression. Derivatives of L. monocytogenes containing these specific plcA mutations were found to have phenotypes similar to that of the plcA deletion strain in an assay for escape from the primary vacuole, in intracellular growth in a murine macrophage cell line, and in a plaquing assay for cell-to-cell spread. Thus, catalytic activity of PI-PLC is required for all its intracellular functions.
Bannam
T
T
Department of Microbiology, University of Pennsylvania School of Medicine, Philadelphia 19104-6076, USA.
Goldfine
H
H
D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002384:Catalysis; D002460:Cell Line; D004789:Enzyme Activation / Q000235:genetics; D005347:Fibroblasts / Q000382:microbiology; D006639:Histidine / Q000235:genetics* / Q000378:metabolism; D007424:Intracellular Fluid / Q000382:microbiology; D007739:L Cells; D008089:Listeria monocytogenes / Q000201:enzymology* / Q000235:genetics* / Q000254:growth & development; D008264:Macrophages / Q000382:microbiology; D051379:Mice; D016297:Mutagenesis, Site-Directed; D010649:Phenylalanine / Q000235:genetics; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D054801:Phosphoinositide Phospholipase C; D010738:Type C Phospholipases / Q000235:genetics* / Q000378:metabolism*; D014617:Vacuoles / Q000382:microbiology
D010649:Phenylalanine; D006639:Histidine; D010738:Type C Phospholipases; D054801:Phosphoinositide Phospholipase C; D043265:Phosphatidylinositol Diacylglycerol-Lyase