Mobilization of Protein Kinase C in Macrophages Induced byListeria monocytogenesAffects Its Internalization and Escape from the Phagosome

Abstract

Listeriolysin O (LLO) and a phosphatidylinositol-specific phospholipase C (PI-PLC) are known virulence factors of Listeria monocytogenes in both tissue cultures and the murine model of infection. LLO is a member of a family of pore-forming cholesterol-dependent cytotoxins and is known to play an essential role in escape from the primary phagocytic vacuole of macrophages. PI-PLC plays an accessory role, in that PI-PLC mutants are partially defective in escape. We have shown that both of these molecules are essential for initiating rapid increases in the calcium level in the J774 murine macrophage cell line (S. J. Wadsworth and H. Goldfine, Infect. Immun. 67:1770-1778, 1999). Here we show that both LLO and PI-PLC are required for translocation of protein kinase C ␦ (PKC ␦) to the periphery of J774 cells and for translocation of PKC ␤ II to early endosomes beginning within the first minute after addition of bacteria to the culture medium. Treatment with the calcium channel blocker SK&F 96365 inhibited translocation of PKC ␤ II but not PKC ␦. Our findings lead us to propose a host signaling pathway requiring LLO and the formation of diacylglycerol by PI-PLC in which calcium-independent PKC ␦ is responsible for the initial calcium signal and the subsequent PKC ␤ II translocation. LLO-dependent translocation of PKC ␤ I to early endosomes also occurs between 1 and 4 min after infection, but this occurs in the absence of PI-PLC. All of these signals were observed in cells that had not internalized bacteria. Blocking PKC ␤ translocation with hispidin resulted in more rapid uptake of wild-type bacteria and greatly reduced escape from the primary phagocytic vacuoles of J774 cells.The capacity to survive and grow within macrophages is a hallmark of infections with Listeria monocytogenes. Our recent studies have focused on the earliest stages of the encounter between L. monocytogenes and a macrophage, as represented by the J774 murine macrophage cell line, a well-studied tissue culture model of infection (5,30,33). We have observed that the cytosolic calcium level is elevated at 1, 5, and 10 min after infection with wild-type L. monocytogenes but not after infection with a strain with a listeriolysin O (LLO) mutation. Strains with deletions in the genes encoding two secreted phospholipases C did not produce some or all of these signals (35).Of specific interest to workers in our laboratory are signal transduction pathways activated by the two phospholipases that contribute significantly to virulence in the mouse model of infection (5, 30). One of these, a phosphatidylinositol (PI)-specific phospholipase C (PI-PLC), encoded by plcA, hydrolyzes host PI, leading to production of the eukaryotic signaling molecule diacylglycerol (DAG) (4,12,23,30). The other, a broad-range phospholipase C (BR-PLC), encoded by plcB, acts on many host polar lipids, including sphingomyelin, leading to production of DAG and ceramide (10, 11). The elevated calcium levels produced by the combined actions of LLO and PI-PLC appear to be part of a signali...

Listeria monocytogenes phosphatidylinositol-specific phospholipase C has evolved for virulence by greatly reduced activity on GPI anchors

“…Studies of L. monocytogenes infections in J774 cells have shown that diacylglycerol generated by hydrolysis of PI mobilizes PKC ␤-isoforms, which seem to play a role in escape from the phagosomes (24,25). In this study, we demonstrated that the replacement of LmPI-PLC with BcPI-PLC inhibits L. monocy- (14).…”

Harnessing Listeria monocytogenes to target tumors

“…It has been shown by others that LLO is involved in the rapid increase in intracellular Ca 2+ levels in a macrophage cell line, J774. 79 We found that Listeria increased intracellular Ca 2+ levels resulting in the production of high levels of mROS. 70 These results imply that NADPH oxidase and excessive intracellular calcium contribute to tumor cell death upon LM-LLO infection causing mitochondrial failure.…”

The arsenal of virulence factors deployed byListeria monocytogenesto promote its cell infection cycle

“…122 It was proposed that CFTR transiently increases phagosomal chloride concentration after infection, potentiating LLO-mediated pore formation and vacuole lysis, which suggests that Listeria exploits mechanisms of cellular ion homeostasis to escape the phagosome. As other CDCs, LLO is a strong signaling protein that can induce several events in host cells, such as apoptosis, NFκB activation, upregulation of adhesion molecules and cytokines, 123 activation of MAP kinase pathways 124 and protein kinase C. 125 In addition, LLO influences the levels of intracellular calcium 108,126,127 and stimulates phosphoinositide metabolism 128 to favor bacterial entry. It is also a potent aggregator of lipid rafts 129 and recently, LLO was shown to suppress the host immune response by decreasing protein SUMOylation (see evasion of host defense section).…”