Involvement of Listeria monocytogenes Phosphatidylinositol-Specific Phospholipase C and Host Protein Kinase C in Permeabilization of the Mac
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感染与免疫杂志 2005年第7期
Department of Microbiology, School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6076
ABSTRACT
We have previously shown that phosphatidylinositol-specific phospholipase C (PI-PLC) produced by Listeria monocytogenes activates a host protein kinase C (PKC) cascade which promotes escape of the bacterium from a macrophage-like cell phagosome. Here, we provide evidence linking bacterial PI-PLC and host PKC to phagosome permeabilization, which precedes escape.
TEXT
Listeria monocytogenes, a facultative intracellular bacterium, is internalized by the host cell before escaping from the phagosome into the cytoplasm, where it multiplies. Listeriolysin O (LLO) forms pores in both the cell and the vacuolar membrane (18). These pores are too small to permit bacteria to cross the membrane (16), but they allow the bidirectional diffusion of electrolytes between the cytoplasm and the phagosome (11, 18). Aside from the need for LLO (2), the factors regulating permeabilization of the phagosome are poorly understood.
L. monocytogenes secretes a phosphatidylinositol-specific phospholipase C (PI-PLC) which catalyzes the cleavage of the membrane lipid PI into inositol phosphate and diacylglycerol (DAG) (9, 17). DAG is an important activator of host protein kinases C (PKC). In the murine macrophage cell line J774, the four main isoforms of PKC are PKC , I, II, and . PKC , I, and II are activated by intracellular Ca2+ and/or DAG. The activation of PKC is Ca2+ independent. Activation of host PKC is observed prior to entry of L. monocytogenes (22).
To examine the involvement of host PKC I and PKC II, we used specific inhibitors. The inhibitors and final concentrations were as follows: SK&F 96365, 25 μM; thapsigargin, 1 μM; hispidin, 5 μM; G 6983, 10 μM; and RO-31-8425, 10 μM. RO-31-8425 exhibits higher inhibitory activity with PKC I than with PKC II (23). G 6983 and hispidin (10, 22) inhibit PKC I and PKC II to the same extent. We checked the potential effects of the inhibitors on bacterial growth and infectivity. For as long as 8 h, there was no effect of hispidin, G 6983, or RO-31-8425 on L. monocytogenes multiplication in brain heart infusion, as determined by measuring the optical density at 620 nm (data not shown). We also tested for potential inhibition of bacterial entry into J774 cells and did not observe any effect of the PKC inhibitors used in this study on L. monocytogenes entry at 35 min postinfection (data not shown).
Since LLO and PI-PLC are needed for efficient escape from the macrophage phagosome, we determined the effects of inhibitors on the expression of LLO and PI-PLC. We analyzed hemolytic activities of culture supernatants obtained from the wild-type strain 10403S, grown with or without inhibitors, on sheep red blood cells at pH 5.5. Hispidin suppressed L. monocytogenes hemolytic activity in a time-dependent manner (Fig. 1A), but RO-31-8425 and G 6983 had no significant effect (Fig. 1B). Hispidin was therefore excluded from the subsequent experiments.
PI-PLC enzymatic activity of L. monocytogenes supernatants from cultures grown in the presence or absence of PKC inhibitors was analyzed as previously described (6). Supernatants from wild-type L. monocytogenes cultured for 8 h with PKC inhibitors did not exhibit any significant reduction compared to untreated bacterial supernatant (P, 0.614 for RO-31-8425 and 0.803 for G 6983).
Since the effects of RO-31-8425 and G 6983 on escape were not previously determined (22), we measured their effects on the escape of L. monocytogenes from the primary phagocytic vacuole as described previously (13, 21). As shown in Fig. 2A, PI-PLC deficiency in both the PI-PLC– and PI-PLC–/phosphatidylcholine-PLC (PC-PLC)– strains (Table 1) leads to a reduction in the level of escape compared to that of the wild-type strain (P, <10–5 and 0.001, respectively). In contrast, the PC-PLC– strain showed no significant reduction (P = 0.053). This confirms previous observations indicating a role of PI-PLC in escape from the macrophage phagosome (1, 4, 20), even though this defect did not influence escape in the human epithelial cell line Henle 407 (14). Since PI-PLC activity leads to activation of host PKC , J774 cells were infected with wild-type L. monocytogenes, with or without PKC inhibitors, which were added 10 min before infection. As shown in Fig. 2B, both inhibitors reduced the level of escape (P, 0.008 for RO-31-8425 and 0.0006 for G 6983). The inhibition of escape by G 6983 was significantly greater than that by RO-31-8425 (P < 10–4).
Perforation of the phagosome membrane has been observed prior to escape. It was measured according to the method of Beauregard et al. (2), with slight modifications. If inhibitors were used, treatment started 15 min before infection. At time point 0, the supernatant was replaced by a solution of 5mM HPTS (8-hydroxypyrene-1,3,6-trisulfonic acid, trisodium salt) in Ringer buffer (RB), and cells were infected at a multiplicity of infection of 25 to 30 bacteria per cell. After 10 min of incubation, the coverslips were washed and subsequently kept in warm RB (with inhibitors when indicated). Permeabilization was observed with a fluorescence microscope. The HPTS spectrum is pH dependent: excitation at 405 nm induces fluorescence of acidic to neutral compartments, but around neutral pH there is also fluorescence after excitation at 440 nm. This fluorescence at 440 nm, which is indicative of vacuolar permeabilization, appears first in vacuoles and then diffuses into the cytosol. The number of cells/field acquiring fluorescence at an excitation of 440 nm between 30 and 60 min postinfection was used to compare the different strains and conditions. Both of the PI-PLC-deficient strains displayed significant reduction in permeabilization (Fig. 3A) (P, <10–5 for both strains), which paralleled their reduced level of escape (Fig. 2A). As observed by Beauregard et al. (2), LLO was absolutely required for permeabilization (data not shown). The effects of PKC inhibitors are illustrated in Fig. 3B. As we observed for escape from the primary phagosome, G 6983 (inhibiting both PKC I and PKC II) leads to greater reduction in permeabilization than RO-31-8425 (with a higher inhibitory activity toward PKC I than PKC II) (P = 0.005). Taken together, these data indicate that PI-PLC in addition to LLO plays a significant role in permeabilization of the primary phagosome and that a PKC pathway is involved. Both PKC isoforms appear to play a role in mediating permeabilization of the phagocytic vacuole.
It was previously shown that inhibitors of the calcium fluxes needed for PKC mobilization strongly inhibit escape from the phagosome (21). SK&F 96365, which inhibits Ca2+ entry, and thapsigargin, which affects release of Ca2+ from intracellular stores, also strongly inhibited vacuolar permeabilization (Fig. 3C) (P, <10–5 for both inhibitors).
The reduction of permeabilization induced by PKC inhibitors did not parallel a reduction in LLO activity, confirming that the effect of bacterial PI-PLC on permeabilization is independent of LLO modulation. Another possible explanation is that loss of PI-PLC activity results in decreased acidification of the phagosome, which would decrease LLO activity. This question has been addressed by Lee Shaughnessy and Joel Swanson, who have found no defect in phagosome acidification in the PI-PLC– strain (personal communication).
We hypothesize that PI-PLC enters the host cell's cytosol via pores formed by LLO. This initially occurs from outside the cell (19) but may continue once the bacteria are inside a phagosome (7). PI-PLC cleaves PI in host cell membranes, producing DAG. DAG production also results from activation of host PLC by an LLO-dependent signaling pathway (8). DAG activates Ca2+-independent PKC , leading to the opening of a Ca2+ channel and elevation of intracellular Ca2+ levels, which continues via release of Ca2+ from intracellular stores (22). The data presented in this paper show that preceding escape from the phagosome, permeabilization of the phagosomal membrane has the same requirements as escape: LLO and PI-PLC activities and activation of PKC isoforms. At this time, we can only speculate on the involvement of PKC in these processes. Early endosomes are known to traffic through sorting endosomes with other organelles including lysosomes and phagosomes (5, 15). It has been shown that PKC I and PKC II mobilize to early endosomes within the first 5 min of infection (22). It is possible that phosphorylation of proteins on early endosomes modifies the program of phagosomal maturation, resulting in permeabilization and lysis. We believe that the search for PKC targets involved in phagosomal maturation will be rewarding.
ACKNOWLEDGMENTS
This study was supported by NIH grant AI-45153 to H.G.
REFERENCES
1. Bannam, T., and H. Goldfine. 1999. Mutagenesis of active-site histidines of Listeria monocytogenes phosphatidylinositol-specific phospholipase C: effects on enzyme activity and biological function. Infect. Immun. 67:182-186.
2. Beauregard, K. E., K. D. Lee, R. J. Collier, and J. A. Swanson. 1997. pH-dependent perforation of macrophage phagosomes by listeriolysin O from Listeria monocytogenes. J. Exp. Med. 186:1159-1163.
3. Bishop, D. K., and D. J. Hinrichs. 1987. Adoptive transfer of immunity to Listeria monocytogenes—the influence of in vitro stimulation on lymphocyte subset requirements. J. Immunol. 139:2005-2009.
4. Camilli, A., L. G. Tilney, and D. A. Portnoy. 1993. Dual roles of plcA in Listeria monocytogenes pathogenesis. Mol. Microbiol. 8:143-157.
5. Desjardins, M. 1995. Biogenesis of phagolysosomes—the kiss and run hypothesis. Trends Cell Biol. 5:183-186.
6. Goldfine, H., and C. Knob. 1992. Purification and characterization of Listeria monocytogenes phosphatidylinositol-specific phospholipase C. Infect. Immun. 60:4059-4067.
7. Goldfine, H., and S. J. Wadsworth. 2002. Macrophage intracellular signaling induced by Listeria monocytogenes. Microbes Infect. 4:1335-1343.
8. Goldfine, H., S. J. Wadsworth, and N. C. Johnston. 2000. Activation of host phospholipases C and D in macrophages after infection with Listeria monocytogenes. Infect. Immun. 68:5735-5741.
9. Griffith, O. H., and M. Ryan. 1999. Bacterial phosphatidylinositol-specific phospholipase C: structure, function, and interaction with lipids. Biochim. Biophys. Acta 1441:237-254.
10. Gschwendt, M., S. Dieterich, J. Rennecke, W. Kittstein, H. J. Mueller, and F. J. Johannes. 1996. Inhibition of protein kinase C mu by various inhibitors. Differentiation from protein kinase c isoenzymes. FEBS Lett. 392:77-80.
11. Higgins, D. E., N. Shastri, and D. A. Portnoy. 1999. Delivery of protein to the cytosol of macrophages using Escherichia coli K-12. Mol. Microbiol. 31:1631-1641.
12. Jones, S., and D. A. Portnoy. 1994. Characterization of Listeria monocytogenes pathogenesis in a strain expressing perfringolysin O in place of listeriolysin O. Infect. Immun. 62:5608-5613.
13. Jones, S., and D. A. Portnoy. 1994. Intracellular growth of bacteria. Methods Enzymol. 236:463-467.
14. Marquis, H., V. Doshi, and D. A. Portnoy. 1995. The broad-range phospholipase C and a metalloprotease mediate listeriolysin O-independent escape of Listeria monocytogenes from a primary vacuole in human epithelial cells. Infect. Immun. 63:4531-4534.
15. Maxfield, F. R., and T. E. Mcgraw. 2004. Endocytic recycling. Nat. Rev. Mol. Cell Biol. 5:121-132.
16. Palmer, M. 2001. The family of thiol-activated, cholesterol-binding cytolysins. Toxicon 39:1681-1689.
17. Portnoy, D. A., T. Chakraborty, W. Goebel, and P. Cossart. 1992. Molecular determinants of Listeria monocytogenes pathogenesis. Infect. Immun. 60:1263-1267.
18. Repp, H., Z. Pamukci, A. Koschinski, E. Domann, A. Darji, J. Birringer, D. Brockmeier, T. Chakraborty, and F. Dreyer. 2002. Listeriolysin of Listeria monocytogenes forms Ca2+-permeable pores leading to intracellular Ca2+ oscillations. Cell. Microbiol. 4:483-491.
19. Sibelius, U., T. Chakraborty, B. Krogel, J. Wolf, F. Rose, R. Schmidt, J. Wehland, W. Seeger, and F. Grimminger. 1996. The listerial exotoxins listeriolysin and phosphatidylinositol-specific phospholipase C synergize to elicit endothelial cell phosphoinositide metabolism. J. Immunol. 157:4055-4060.
20. Smith, G. A., H. Marquis, S. Jones, N. C. Johnston, D. A. Portnoy, and H. Goldfine. 1995. The two distinct phospholipases C of Listeria monocytogenes have overlapping roles in escape from a vacuole and cell-to-cell spread. Infect. Immun. 63:4231-4237.
21. Wadsworth, S. J., and H. Goldfine. 1999. Listeria monocytogenes phospholipase C-dependent calcium signaling modulates bacterial entry into J774 macrophage-like cells. Infect. Immun. 67:1770-1778.
22. Wadsworth, S. J., and H. Goldfine. 2002. Mobilization of protein kinase C in macrophages induced by Listeria monocytogenes affects its internalization and escape from the phagosome. Infect. Immun. 70:4650-4660.
23. Wilkinson, S. E., P. J. Parker, and J. S. Nixon. 1993. Isoenzyme specificity of bisindolylmaleimides, selective inhibitors of protein kinase C. Biochem. J. 294:335-337.(Mathilde A. Poussin and H)
ABSTRACT
We have previously shown that phosphatidylinositol-specific phospholipase C (PI-PLC) produced by Listeria monocytogenes activates a host protein kinase C (PKC) cascade which promotes escape of the bacterium from a macrophage-like cell phagosome. Here, we provide evidence linking bacterial PI-PLC and host PKC to phagosome permeabilization, which precedes escape.
TEXT
Listeria monocytogenes, a facultative intracellular bacterium, is internalized by the host cell before escaping from the phagosome into the cytoplasm, where it multiplies. Listeriolysin O (LLO) forms pores in both the cell and the vacuolar membrane (18). These pores are too small to permit bacteria to cross the membrane (16), but they allow the bidirectional diffusion of electrolytes between the cytoplasm and the phagosome (11, 18). Aside from the need for LLO (2), the factors regulating permeabilization of the phagosome are poorly understood.
L. monocytogenes secretes a phosphatidylinositol-specific phospholipase C (PI-PLC) which catalyzes the cleavage of the membrane lipid PI into inositol phosphate and diacylglycerol (DAG) (9, 17). DAG is an important activator of host protein kinases C (PKC). In the murine macrophage cell line J774, the four main isoforms of PKC are PKC , I, II, and . PKC , I, and II are activated by intracellular Ca2+ and/or DAG. The activation of PKC is Ca2+ independent. Activation of host PKC is observed prior to entry of L. monocytogenes (22).
To examine the involvement of host PKC I and PKC II, we used specific inhibitors. The inhibitors and final concentrations were as follows: SK&F 96365, 25 μM; thapsigargin, 1 μM; hispidin, 5 μM; G 6983, 10 μM; and RO-31-8425, 10 μM. RO-31-8425 exhibits higher inhibitory activity with PKC I than with PKC II (23). G 6983 and hispidin (10, 22) inhibit PKC I and PKC II to the same extent. We checked the potential effects of the inhibitors on bacterial growth and infectivity. For as long as 8 h, there was no effect of hispidin, G 6983, or RO-31-8425 on L. monocytogenes multiplication in brain heart infusion, as determined by measuring the optical density at 620 nm (data not shown). We also tested for potential inhibition of bacterial entry into J774 cells and did not observe any effect of the PKC inhibitors used in this study on L. monocytogenes entry at 35 min postinfection (data not shown).
Since LLO and PI-PLC are needed for efficient escape from the macrophage phagosome, we determined the effects of inhibitors on the expression of LLO and PI-PLC. We analyzed hemolytic activities of culture supernatants obtained from the wild-type strain 10403S, grown with or without inhibitors, on sheep red blood cells at pH 5.5. Hispidin suppressed L. monocytogenes hemolytic activity in a time-dependent manner (Fig. 1A), but RO-31-8425 and G 6983 had no significant effect (Fig. 1B). Hispidin was therefore excluded from the subsequent experiments.
PI-PLC enzymatic activity of L. monocytogenes supernatants from cultures grown in the presence or absence of PKC inhibitors was analyzed as previously described (6). Supernatants from wild-type L. monocytogenes cultured for 8 h with PKC inhibitors did not exhibit any significant reduction compared to untreated bacterial supernatant (P, 0.614 for RO-31-8425 and 0.803 for G 6983).
Since the effects of RO-31-8425 and G 6983 on escape were not previously determined (22), we measured their effects on the escape of L. monocytogenes from the primary phagocytic vacuole as described previously (13, 21). As shown in Fig. 2A, PI-PLC deficiency in both the PI-PLC– and PI-PLC–/phosphatidylcholine-PLC (PC-PLC)– strains (Table 1) leads to a reduction in the level of escape compared to that of the wild-type strain (P, <10–5 and 0.001, respectively). In contrast, the PC-PLC– strain showed no significant reduction (P = 0.053). This confirms previous observations indicating a role of PI-PLC in escape from the macrophage phagosome (1, 4, 20), even though this defect did not influence escape in the human epithelial cell line Henle 407 (14). Since PI-PLC activity leads to activation of host PKC , J774 cells were infected with wild-type L. monocytogenes, with or without PKC inhibitors, which were added 10 min before infection. As shown in Fig. 2B, both inhibitors reduced the level of escape (P, 0.008 for RO-31-8425 and 0.0006 for G 6983). The inhibition of escape by G 6983 was significantly greater than that by RO-31-8425 (P < 10–4).
Perforation of the phagosome membrane has been observed prior to escape. It was measured according to the method of Beauregard et al. (2), with slight modifications. If inhibitors were used, treatment started 15 min before infection. At time point 0, the supernatant was replaced by a solution of 5mM HPTS (8-hydroxypyrene-1,3,6-trisulfonic acid, trisodium salt) in Ringer buffer (RB), and cells were infected at a multiplicity of infection of 25 to 30 bacteria per cell. After 10 min of incubation, the coverslips were washed and subsequently kept in warm RB (with inhibitors when indicated). Permeabilization was observed with a fluorescence microscope. The HPTS spectrum is pH dependent: excitation at 405 nm induces fluorescence of acidic to neutral compartments, but around neutral pH there is also fluorescence after excitation at 440 nm. This fluorescence at 440 nm, which is indicative of vacuolar permeabilization, appears first in vacuoles and then diffuses into the cytosol. The number of cells/field acquiring fluorescence at an excitation of 440 nm between 30 and 60 min postinfection was used to compare the different strains and conditions. Both of the PI-PLC-deficient strains displayed significant reduction in permeabilization (Fig. 3A) (P, <10–5 for both strains), which paralleled their reduced level of escape (Fig. 2A). As observed by Beauregard et al. (2), LLO was absolutely required for permeabilization (data not shown). The effects of PKC inhibitors are illustrated in Fig. 3B. As we observed for escape from the primary phagosome, G 6983 (inhibiting both PKC I and PKC II) leads to greater reduction in permeabilization than RO-31-8425 (with a higher inhibitory activity toward PKC I than PKC II) (P = 0.005). Taken together, these data indicate that PI-PLC in addition to LLO plays a significant role in permeabilization of the primary phagosome and that a PKC pathway is involved. Both PKC isoforms appear to play a role in mediating permeabilization of the phagocytic vacuole.
It was previously shown that inhibitors of the calcium fluxes needed for PKC mobilization strongly inhibit escape from the phagosome (21). SK&F 96365, which inhibits Ca2+ entry, and thapsigargin, which affects release of Ca2+ from intracellular stores, also strongly inhibited vacuolar permeabilization (Fig. 3C) (P, <10–5 for both inhibitors).
The reduction of permeabilization induced by PKC inhibitors did not parallel a reduction in LLO activity, confirming that the effect of bacterial PI-PLC on permeabilization is independent of LLO modulation. Another possible explanation is that loss of PI-PLC activity results in decreased acidification of the phagosome, which would decrease LLO activity. This question has been addressed by Lee Shaughnessy and Joel Swanson, who have found no defect in phagosome acidification in the PI-PLC– strain (personal communication).
We hypothesize that PI-PLC enters the host cell's cytosol via pores formed by LLO. This initially occurs from outside the cell (19) but may continue once the bacteria are inside a phagosome (7). PI-PLC cleaves PI in host cell membranes, producing DAG. DAG production also results from activation of host PLC by an LLO-dependent signaling pathway (8). DAG activates Ca2+-independent PKC , leading to the opening of a Ca2+ channel and elevation of intracellular Ca2+ levels, which continues via release of Ca2+ from intracellular stores (22). The data presented in this paper show that preceding escape from the phagosome, permeabilization of the phagosomal membrane has the same requirements as escape: LLO and PI-PLC activities and activation of PKC isoforms. At this time, we can only speculate on the involvement of PKC in these processes. Early endosomes are known to traffic through sorting endosomes with other organelles including lysosomes and phagosomes (5, 15). It has been shown that PKC I and PKC II mobilize to early endosomes within the first 5 min of infection (22). It is possible that phosphorylation of proteins on early endosomes modifies the program of phagosomal maturation, resulting in permeabilization and lysis. We believe that the search for PKC targets involved in phagosomal maturation will be rewarding.
ACKNOWLEDGMENTS
This study was supported by NIH grant AI-45153 to H.G.
REFERENCES
1. Bannam, T., and H. Goldfine. 1999. Mutagenesis of active-site histidines of Listeria monocytogenes phosphatidylinositol-specific phospholipase C: effects on enzyme activity and biological function. Infect. Immun. 67:182-186.
2. Beauregard, K. E., K. D. Lee, R. J. Collier, and J. A. Swanson. 1997. pH-dependent perforation of macrophage phagosomes by listeriolysin O from Listeria monocytogenes. J. Exp. Med. 186:1159-1163.
3. Bishop, D. K., and D. J. Hinrichs. 1987. Adoptive transfer of immunity to Listeria monocytogenes—the influence of in vitro stimulation on lymphocyte subset requirements. J. Immunol. 139:2005-2009.
4. Camilli, A., L. G. Tilney, and D. A. Portnoy. 1993. Dual roles of plcA in Listeria monocytogenes pathogenesis. Mol. Microbiol. 8:143-157.
5. Desjardins, M. 1995. Biogenesis of phagolysosomes—the kiss and run hypothesis. Trends Cell Biol. 5:183-186.
6. Goldfine, H., and C. Knob. 1992. Purification and characterization of Listeria monocytogenes phosphatidylinositol-specific phospholipase C. Infect. Immun. 60:4059-4067.
7. Goldfine, H., and S. J. Wadsworth. 2002. Macrophage intracellular signaling induced by Listeria monocytogenes. Microbes Infect. 4:1335-1343.
8. Goldfine, H., S. J. Wadsworth, and N. C. Johnston. 2000. Activation of host phospholipases C and D in macrophages after infection with Listeria monocytogenes. Infect. Immun. 68:5735-5741.
9. Griffith, O. H., and M. Ryan. 1999. Bacterial phosphatidylinositol-specific phospholipase C: structure, function, and interaction with lipids. Biochim. Biophys. Acta 1441:237-254.
10. Gschwendt, M., S. Dieterich, J. Rennecke, W. Kittstein, H. J. Mueller, and F. J. Johannes. 1996. Inhibition of protein kinase C mu by various inhibitors. Differentiation from protein kinase c isoenzymes. FEBS Lett. 392:77-80.
11. Higgins, D. E., N. Shastri, and D. A. Portnoy. 1999. Delivery of protein to the cytosol of macrophages using Escherichia coli K-12. Mol. Microbiol. 31:1631-1641.
12. Jones, S., and D. A. Portnoy. 1994. Characterization of Listeria monocytogenes pathogenesis in a strain expressing perfringolysin O in place of listeriolysin O. Infect. Immun. 62:5608-5613.
13. Jones, S., and D. A. Portnoy. 1994. Intracellular growth of bacteria. Methods Enzymol. 236:463-467.
14. Marquis, H., V. Doshi, and D. A. Portnoy. 1995. The broad-range phospholipase C and a metalloprotease mediate listeriolysin O-independent escape of Listeria monocytogenes from a primary vacuole in human epithelial cells. Infect. Immun. 63:4531-4534.
15. Maxfield, F. R., and T. E. Mcgraw. 2004. Endocytic recycling. Nat. Rev. Mol. Cell Biol. 5:121-132.
16. Palmer, M. 2001. The family of thiol-activated, cholesterol-binding cytolysins. Toxicon 39:1681-1689.
17. Portnoy, D. A., T. Chakraborty, W. Goebel, and P. Cossart. 1992. Molecular determinants of Listeria monocytogenes pathogenesis. Infect. Immun. 60:1263-1267.
18. Repp, H., Z. Pamukci, A. Koschinski, E. Domann, A. Darji, J. Birringer, D. Brockmeier, T. Chakraborty, and F. Dreyer. 2002. Listeriolysin of Listeria monocytogenes forms Ca2+-permeable pores leading to intracellular Ca2+ oscillations. Cell. Microbiol. 4:483-491.
19. Sibelius, U., T. Chakraborty, B. Krogel, J. Wolf, F. Rose, R. Schmidt, J. Wehland, W. Seeger, and F. Grimminger. 1996. The listerial exotoxins listeriolysin and phosphatidylinositol-specific phospholipase C synergize to elicit endothelial cell phosphoinositide metabolism. J. Immunol. 157:4055-4060.
20. Smith, G. A., H. Marquis, S. Jones, N. C. Johnston, D. A. Portnoy, and H. Goldfine. 1995. The two distinct phospholipases C of Listeria monocytogenes have overlapping roles in escape from a vacuole and cell-to-cell spread. Infect. Immun. 63:4231-4237.
21. Wadsworth, S. J., and H. Goldfine. 1999. Listeria monocytogenes phospholipase C-dependent calcium signaling modulates bacterial entry into J774 macrophage-like cells. Infect. Immun. 67:1770-1778.
22. Wadsworth, S. J., and H. Goldfine. 2002. Mobilization of protein kinase C in macrophages induced by Listeria monocytogenes affects its internalization and escape from the phagosome. Infect. Immun. 70:4650-4660.
23. Wilkinson, S. E., P. J. Parker, and J. S. Nixon. 1993. Isoenzyme specificity of bisindolylmaleimides, selective inhibitors of protein kinase C. Biochem. J. 294:335-337.(Mathilde A. Poussin and H)