Activation of host phospholipases C and D in macrophages after infection with Listeria monocytogenes

Infection of the J774 murine macrophage-derived cell line with Listeria mon ocytogenes results in several elevations of intracellular calcium during th e first 15 min of infection. These appear to result from the actions of sec reted bacterial proteins, including phosphatidylinositol-specific phospholi pase C (PI-PLC), a broad-range phospholipase C, and listeriolysin O (LLO) ( S. J. Wadsworth and H. Goldfine, Infect. Immun, 67:1770-1778, 1999), We hav e measured hydrolysis of host PI and the activation of host polyphosphoinos itide-specific PLC and host phospholipase D (PLD) during infection with wil d-type and mutant L. monocytogenes. Elevated hydrolysis of host PI occurred within the first 10 min of infection and was dependent on both bacterial P I-PLC and LLO, both of which were required for the earliest elevations of i ntracellular calcium in the host cell. A more rapid hydrolysis of host PI w as observed at 30 min after infection, at the time when wild-type bacteria have been internalized. Activation of host PLC, also occurred in the first 10 min of infection but was not dependent on the presence of bacterial PI-P LC, Similar observations were made in murine bone marrow-derived macrophage s. In J774 cells, activation of host PLD was observed after 20 min of infec tion and was dependent on bacterial LLO, Mutants in the bacterial phospholi pases produced levels of PLD activation similar to those produced by the wi ld type. Phorbol myristate acetate (PMA) also activated host PLD, while lon g-term treatment with PMA resulted in loss of the ability of L. monocytogen es to activate host PLD, suggesting an involvement of protein kinase C (PKC ) in the activation of PLD. Rottlerin, an inhibitor of PKC delta in J774 ce lls, also inhibited the activation of PLD, but hispidin, an inhibitor of PK C beta I and beta II, did not. Pretreatment of J774 cells with the PLD inhi bitor, 2,3-diphosphoglycerate partially inhibited escape of the bacteria fr om the primary phagocytic vacuole.