Mutagenesis of active-site histidines of Listeria monocytogenes phosphatidylinositol-specific phospholipase C: effects on enzyme activity and biological function

Abstract

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.

FIG. 1

Expression of PI-PLC in the J774 murine macrophage-like cell line. J774 cells were infected with each L. monocytogenes strain for 3.5 h, after which the cells were labelled for 1 h with [³⁵S]methionine. PI-PLC was precipitated with rabbit polyclonal anti-PI-PLC bound to protein A-coated beads. For each lane of the resulting autoradiograph the PI-PLC phenotype of the infecting strain is indicated.

FIG. 2

The percentage of bacteria that escaped from the primary vacuole into the host cell cytoplasm, determined as previously described (20). Monolayers of bone marrow-derived macrophages were infected for 90 min. The total number of intracellular bacteria was obtained by staining with fluorescein isothiocyanate-tagged polyclonal anti-Listeria antibody. Cytoplasmic bacteria were inferred by staining with tetramethyl rhodamine isothiocyanate-labelled phalloidin, which stains polymerized F actin.

FIG. 3

Plaque formation by L. monocytogenes derivatives in L2 cell monolayers. Plaquing was done as previously described (35). The size of the plaque formed by each strain was calculated as a percentage of the plaque size of the wild-type strain.