Localization of protein kinase C ε to macrophage vacuoles perforated by Listeria monocytogenes cytolysin

Three proteins secreted by Listeria monocytogenes facilitate escape from macrophage vacuoles: the cholesterol-dependent cytolysin listeriolysin O (LLO), a phosphoinositide-specific phospholipase C (PI-PLC) and a broad-range phospholipase C (PC-PLC). LLO and PI-PLC can activate several members of the protein kinase C (PKC) family during infection. PKC epsilon is a novel PKC that contributes to macrophage activation, defence against bacterial infection, and phagocytosis; however, a role for PKC epsilon in Lm infections has not been described. To study PKC epsilon dynamics, PKC epsilon-YFP chimeras were visualized in macrophages during Lm infection. PKC epsilon-YFP was recruited to forming vacuoles during macrophage phagocytosis of Lm and again later to fully formed Lm vacuoles. The PKC epsilon-YFP localization to the fully formed Lm vacuole was LLO-dependent but independent of PI-PLC or PC-PLC. PKC epsilon-YFP recruitment often followed LLO perforation of the membrane, as indicated by localization of PKC epsilon-YFP to Lm vacuoles after they released small fluorescent dyes into the cytoplasm. PKC epsilon-YFP recruitment to vesicles also followed phagocytosis of LLO-containing liposomes or osmotic lysis of endocytic vesicles, indicating that vacuole perforation by LLO was the chief cause of the PKC epsilon response. These studies implicate PKC epsilon in a cellular mechanism for recognizing damaged membranous organelles, including the disrupted vacuoles created when Lm escapes into cytoplasm.