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...

The hemolytic phospholipase C (PlcHR) expressed by Pseudomonas aeruginosa is the original member of a Phosphoesterase Superfamily, which includes phosphorylcholine-specific phospholipases C (PC-PLC) produced by frank and opportunistic pathogens. PlcHR, but not all its family members, is also a potent sphingomyelinase (SMase). Data presented herein indicate that picomolar (pM) concentrations of PlcHR are selectively lethal to endothelial cells (EC). An RGD motif of PlcHR contributes to this selectivity. Peptides containing an RGD motif (i.e., GRGDS), but not control peptides (i.e., GDGRS), block the effects of PlcHR on calcium signaling and cytotoxicity to EC. Moreover, RGD variants of PlcHR (e.g., RGE, KGD) are significantly reduced in their binding and toxicity, but retain the enzymatic activity of the wild type PlcHR. PlcHR also inhibits several EC-dependent in vitro assays (i.e., EC migration, EC invasion, and EC tubule formation), which represent key processes involved in angiogenesis (i.e., formation of new blood vessels from existing vasculature). Finally, the impact of PlcHR in an in vivo model of angiogenesis in transgenic zebrafish, and ones treated with an antisense morpholino to knock down a key blood cell regulator, were evaluated because in vitro assays cannot fully represent the complex processes of angiogenesis. As little as 2 ng/embryo of PlcHR was lethal to ∼50% of EGFP-labeled EC at 6 h after injection of embryos at 48 hpf (hours post-fertilization). An active site mutant of PlcHR (Thr178Ala) exhibited 120-fold reduced inhibitory activity in the EC invasion assay, and 20 ng/embryo elicited no detectable inhibitory activity in the zebrafish model. Taken together, these observations are pertinent to the distinctive vasculitis and poor wound healing associated with P. aeruginosa sepsis and suggest that the potent antiangiogenic properties of PlcHR are worthy of further investigation for the treatment of diseases where angiogenesis contributes pathological conditions (e.g., vascularization of tumors, diabetic retinopathy).

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Listeria monocytogenes secretes several proteins that have been shown to contribute to virulence. Among these is listeriolysin O (LLO), a pore-forming hemolysin that is absolutely required for virulence. Two other virulence factors are phospholipases: a phosphatidylinositol-specific phospholipase C (PI-PLC [plcA]) and a broad-range PLC (plcB). Although mutations in plcA or plcB resulted in small increases in mouse 50% lethal dose (LD 50 ), deletions in both genes resulted in a 500-fold increase in LD 50 . We have examined the role of these secreted proteins in host intracellular signaling in the J774 macrophage-like cell line. Measurements of cytosolic free calcium ([Ca 2؉ ] i ) have revealed a rapid spike upon exposure of these cells to wild-type L. monocytogenes. This is followed by a second peak at 5 min and a third prolonged peak with a maximal [Ca 2؉ ] i of 800 to 1,000 nM. The pattern of calcium changes was greatly altered by deletion of any of the three virulence factors. An LLO mutant produced none of these elevations in [Ca 2؉ ] i ; however, a transient elevation was observed whenever these bacteria entered the cell. A PI-PLC mutant produced a diminished single elevation in [Ca 2؉ ] i at 15 to 30 min. A broad-range PLC mutant produced only the first calcium spike. Studies with inhibitors suggested that the first elevation arises from influx of calcium from the extracellular medium through plasma membrane channels and that the second and third elevations come from release of Ca 2؉ from intracellular stores. We observed that internalization of wild-type bacteria and the broad-range PLC mutant was delayed for 5 to 10 min, but the LLO and PI-PLC mutants were internalized rapidly upon infection. Inhibitors that affected calcium signaling changed the kinetics of association of wild-type bacteria with J774 cells, the kinetics of entry, and the efficiency of escape from the primary phagosome.

The earliest events in the interaction of Listeria monocytogenes with mammalian cells appear to involve the activities of bacterial secreted proteins before internalization of these bacteria. On infection of the J774 murine macrophage cell line, these activities delay uptake of wild-type bacteria into the phagosome (36). Subsequent growth in the cytoplasm and cellto-cell spread are completely dependent on the ability of the bacterium to mediate escape from a vacuole (12,26,35). Two genes, hly and plcA, in a cluster of six genes on the bacterial chromosome have been implicated in escape from the primary vacuole of a macrophage. They encode listeriolysin O (LLO) and a phosphatidylinositol-specific phospholipase C (PI-PLC), respectively. A third gene, prfA, which is adjacent to plcA, encodes a positive regulatory protein, PrfA, which is required for the induction of all genes in this virulence cluster (25,29). LLO has been shown to be absolutely required for escape from the primary vacuole of a macrophage (12, 26) and for mouse virulence (9,13,18,26,29). Assays for escape from the primary vacuole show that mutants in PI-PLC are between 30 and 65% less likely to be found in the cytoplasm of a bone marrowderived macrophage than a wild-type strain at 1.5 h postinfection (7, 33). These mutants show reduced growth compared to the wild type in mouse liver; however, the mouse 50% lethal dose is only slightly increased upon intravenous infection (7).PI-PLC of L. monocytogenes is a member of a family of homologous enzymes secreted by gram-positive bacteria. Like other bacterial PI-PLCs, the enzyme from L. monocytogenes has high specificity for PI with no detectable activity on PI-4-P or PI-4,5-P 2 , eukaryotic lipids involved in intracellular signaling. It has relatively low activity on glycosyl-PI-anchored eukaryotic membrane proteins, which are actively cleaved by other bacterial PI-PLCs (14, 16).The ability of L. monocytogenes to escape from a phagosome, grow in the cytoplasm, and spread from cell to cell is essential for the pathogenesis of this food-borne, human and animal pathogen. In humans, infections with L. monocytogenes tend to occur in immunocompromised adults, pregnant women, and the elderly. They can produce septic abortions of the fetus and meningoencephalitis and are often fatal (11,28).Since bacterial LLO and PI-PLC activities appear to be important for elevation of intracellular Ca 2ϩ in host cells (36), it seemed possible that there is a connection between escape from the vacuole and activation of certain host cell functions that are dependent on elevated intracellular Ca 2ϩ . Among these is the activation of host PLC isoforms, which hydrolyze PI-4-P and PI-4,5-P 2 (27, 31). The hydrolysis of host phosphoinositides by bacterial and host PLCs also results in the formation of diacylglycerol (DAG), which is an activator of eukaryotic protein kinase C (PKC) isoforms (24). Activation of the classical isoforms of PKC also requires elevated intracellular Ca 2ϩ . Since PKC has been implicated in activation of pho...