PROTEOLYTIC PATHWAYS OF ACTIVATION AND DEGRADATION OF A BACTERIAL PHOSPHOLIPASE-C DURING INTRACELLULAR INFECTION BY LISTERIA-MONOCYTOGENES

Listeria monocytogenes is a facultative intracellular bacterial pathog en that spreads cell to cell without exposure to the extracellular env ironment. Bacterial cell-to-cell spread is mediated in part by two sec reted bacterial phospholipases C (PLC), a broad spectrum PLC (PC-PLC) and a phosphatidylinositol-specific PLC (PI-PLC). PI-PLC is secreted i n an active state, whereas PC-PLC is secreted as an inactive preenzyme (proPC-PLC) whose activation is mediated in vitro by an L. monocytoge nes metalloprotease (Mpl). Analysis of PI-PLC, PC-PLC, and Mpl single and double mutants revealed that Mpl also plays a role in the spread o f an infection, but suggested that proPC-PLC has an Mpl-independent ac tivation pathway. Using biochemical and microscopic approaches, we des cribe three intracellular proteolytic pathways regulating PC-PLC activ ity. Initially, proPC-PLC secreted in the cytosol of infected cells wa s rapidly degraded in a proteasome-dependent manner. Later during infe ction, PC-PLC colocalized with bacteria in lysosome-associated membran e protein 1-positive vacuoles. Activation of proPC-PLC in vacuoles was mediated by Mpl and an Mpl-independent pathway, the latter being sens itive to inhibitors of cysteine proteases. Lastly, proPC-PLC activatio n by either pathway was sensitive to bafilomycin A(1), a specific inhi bitor of vacuolar ATPase, suggesting that activation was dependent on acidification of the vacuolar compartment. These results are consisten t with a model in which proPC-PLC activation is compartment specific a nd controlled by a combination of bacterial and host factors.