THE 2 DISTINCT PHOSPHOLIPASES-C OF LISTERIA-MONOCYTOGENES HAVE OVERLAPPING ROLES IN ESCAPE FROM A VACUOLE AND CELL-TO-CELL SPREAD

Listeria monocytogenes secretes two distinct phospholipases C, a phosp hatidylinositol-specific phospholipase C (PI-PLC) and a broad-range ph ospholipase C (PC-PLC). In this study, single in-frame deletion mutant s with mutations in each PLC and a double mutant lacking both PLCs wer e characterized with regard to virulence in mice, escape from a primar y vacuole, and cell-to-cell spread in cell culture. The mutant lacking PI-PLC, previously shown to be twofold less virulent than the wild ty pe in mice, had a minor defect in escape from a primary vacuole but wa s not notably affected in cell-to-cell spread. The mutant lacking PC-P LC was 20-fold less virulent in mice and was defective in cell-to-cell spread but had no measurable defect in escape from a primary vacuole. The mutant lacking both PLCs was 500-fold less virulent in mice and w as severely diminished in its ability to escape from the primary vacuo le and to spread cell to cell. Cellular levels of diacylglycerol and c eramide, products of PLC activity, accumulated beginning 3 to 4 h afte r infection of cells with wild-type bacteria. The bacterial PLCs were partially responsible for this activity, since cells infected with the mutant lacking both PLCs had a reduced increase in diacylglycerol and no increase in ceramide. Elevation of diacylglycerol in the absence o f bacterial PLCs indicated that host cell phospholipase(s) was activat ed during infection. The results of this study were consistent with th e two bacterial PLCs having overlapping functions throughout the cours e of intracellular infection. Furthermore, the PC-PLC, and possibly PI -PLC, appeared to be enzymatically active intracellularly.