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Infection and Immunity, July 2005, p. 4410-4413, Vol. 73, No. 7
0019-9567/05/$08.00+0     doi:10.1128/IAI.73.7.4410-4413.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Involvement of Listeria monocytogenes Phosphatidylinositol-Specific Phospholipase C and Host Protein Kinase C in Permeabilization of the Macrophage Phagosome

Mathilde A. Poussin and Howard Goldfine^*

Department of Microbiology, School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6076

Received 11 June 2004/ Returned for modification 20 July 2004/ Accepted 22 February 2005

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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 ß to phagosome permeabilization, which precedes escape.

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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 , ßI, ßII, and . PKC , ßI, and ßII are activated by intracellular Ca²⁺ and/or DAG. The activation of PKC is Ca²⁺ independent. Activation of host PKC ß is observed prior to entry of L. monocytogenes (22).

To examine the involvement of host PKC ßI and PKC ßII, we used specific inhibitors. The inhibitors and final concentrations were as follows: SK&F 96365, 25 µM; thapsigargin, 1 µM; hispidin, 5 µM; Gö 6983, 10 µM; and RO-31-8425, 10 µM. RO-31-8425 exhibits higher inhibitory activity with PKC ßI than with PKC ßII (23). Gö 6983 and hispidin (10, 22) inhibit PKC ßI and PKC ßII 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, Gö 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 ß 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 Gö 6983 had no significant effect (Fig. 1B). Hispidin was therefore excluded from the subsequent experiments.

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FIG. 1. Determination of hemolytic activity. Supernatants were collected after culture of L. monocytogenes in liquid medium for 8 h with or without inhibitors, and the lytic effects of the supernatants on sheep red blood cells were evaluated by measuring the optical density at 405 nm resulting from hemolysis. (A) Hispidin was added during growth at 6 h (), 4 h (+), 2 h (*), or 0 h (x), or cells were left untreated for 8 h (). Dilutions are indicated on the x axis. (B) Bacteria were grown for 8 h in the presence of hispidin, Gö 6983, or RO-31-8425 or left untreated (NT) for 8 h. Results are expressed as the dilution giving 50% hemolysis. Data represent averages + standard deviations of results from three independent experiments. The asterisk indicates a P of <0.05 as determined using a two-tailed Student t test.

PI-PLC enzymatic activity of L. monocytogenes supernatants from cultures grown in the presence or absence of PKC ß inhibitors was analyzed as previously described (6). Supernatants from wild-type L. monocytogenes cultured for 8 h with PKC ß inhibitors did not exhibit any significant reduction compared to untreated bacterial supernatant (P, 0.614 for RO-31-8425 and 0.803 for Gö 6983).

Since the effects of RO-31-8425 and Gö 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 ß, J774 cells were infected with wild-type L. monocytogenes, with or without PKC ß 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 Gö 6983). The inhibition of escape by Gö 6983 was significantly greater than that by RO-31-8425 (P < 10^–4).

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FIG. 2. Effect of PLC mutations or PKC inhibitors on L. monocytogenes escape from the primary vacuole. L. monocytogenes was labeled with fluorescein isothiocyanate and used to infect J774 cells in the presence or absence of inhibitors. After 90 min, the cells were fixed and actin was labeled with Alexa568-phalloidin. The number of bacteria colocalizing with actin was divided by the total number of bacteria to establish the percentage of escape. (A) Cells were infected with wild-type L. monocytogenes (10403S) or the PI-PLC–, PC-PLC–, or double-mutant PI-PLC–/PC-PLC– strain. (B) Cells were infected with wild-type L. monocytogenes and left untreated (10403S, NT) or treated with the PKC inhibitor Gö 6983 or RO-31-8425. Data represent averages + standard deviations of results from four independent experiments. Asterisks indicate a P of <0.05 as determined using a two-tailed Student t test.

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TABLE 1. Bacterial strains used in this study

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, Gö 6983 (inhibiting both PKC ßI and PKC ßII) leads to greater reduction in permeabilization than RO-31-8425 (with a higher inhibitory activity toward PKC ßI than PKC ßII) (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 ß pathway is involved. Both PKC ß isoforms appear to play a role in mediating permeabilization of the phagocytic vacuole.

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FIG. 3. Effect on vacuolar permeabilization of PLC mutation or cell signaling inhibitors. Log-phase L. monocytogenes suspended in RB containing 5 mM HPTS was used to infect J774 cells at a multiplicity of infection of 25 to 30 bacteria per cell. After 10 min, the cells were washed and kept in warm RB until the end of the experiment. The number of positive cells, those displaying a change in the fluorescence absorption spectrum, was measured for a period of 30 min between 30 and 60 min postinfection. (A) Permeabilization in cells infected with wild-type L. monocytogenes strain 10403S or the PI-PLC–, PC-PLC–, or double-mutant PI-PLC–/PC-PLC– strain. (B) Permeabilization in cells infected with wild-type L. monocytogenes and left untreated (10403S, NT) or treated with the PKC inhibitor Gö 6983 or RO-31-8425. (C) Permeabilization in cells infected with wild-type L. monocytogenes and left untreated (10403S, NT) or treated with the Ca2+ signaling inhibitor thapsigargin or SK&F 96365. Data shown on each panel indicate the number of positive cells/field determined using a magnification of x100 and are representative of results from one of three different experiments. Twenty to 30 fields were counted per condition and per experiment. Each indicates the value for one field, horizontal black bars indicate the average values for the conditions, and asterisks indicate a P of <0.05 as determined using a two-tailed Student t test.

It was previously shown that inhibitors of the calcium fluxes needed for PKC ß 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 ß 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 ßI and PKC ßII 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.

 
This study was supported by NIH grant AI-45153 to H.G.

 
* Corresponding author. Mailing address: Department of Microbiology, School of Medicine, University of Pennsylvania, Philadelphia, PA 19104-6076. Phone: (215) 898-6384. Fax: (215) 898-9557. E-mail: goldfinh{at}mail.med.upenn.edu.

Editor: J. B. Bliska

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Infection and Immunity, July 2005, p. 4410-4413, Vol. 73, No. 7
0019-9567/05/$08.00+0     doi:10.1128/IAI.73.7.4410-4413.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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