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Role of Protein Kinase C- in the Control of Infection by Intracellular Pathogens in Macrophages¹

Anik St-Denis^*, Vassiliki Caouras, Francine Gervais and Albert Descoteaux^2,*

^* INRS-Institut Armand-Frappier, Université du Québec, Laval, Québec, Canada; and Centre for the Study of Host Resistance, McGill University, Montréal, Québec, Canada

	Abstract

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The protein kinase C (PKC) family regulates macrophage function involved in host defense against infection. In this study, we investigated the role of macrophage PKC- in the uptake and subsequent fate of Leishmania donovani promastigotes and Legionella pneumophila infections. To this end, we used clones of the murine macrophage cell line RAW 264.7 overexpressing a dominant-negative (DN) mutant of PKC-. While phagocytosis of L. donovani promastigotes was not affected by DN PKC- overexpression, their intracellular survival was enhanced by 10- to 20-fold at 48 h postinfection. Intracellular survival of a L. donovani mutant defective in lipophosphoglycan repeating units synthesis, which normally is rapidly degraded in phagolysosomes, was enhanced by 100-fold at 48 h postinfection. However, IFN--induced leishmanicidal activity was not affected by DN PKC- overexpression. Similar to macrophages from genetically resistant C57BL/6 mice, control RAW 264.7 cells were not permissive for the intracellular replication of Legionella pneumophila. In contrast, DN PKC--overexpressing RAW 264.7 clones were phenotypically similar to macrophages from genetically susceptible A/J mice, as they allowed intracellular replication of L. pneumophila. Permissiveness to L. pneumophila was not the consequence of a general defect in the microbicidal capacities because killing of a temperature-sensitive mutant of Pseudomonas aeruginosa was normal in DN PKC--overexpressing RAW 264.7 clones. Collectively, these results support a role for PKC- in the regulation of innate macrophage functions involved in the control of infection by intracellular parasites.

	Introduction

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Phagocytosis consists in the uptake and destruction of invading microorganisms, thereby representing an essential component of the host response against infections. Following their attachment to macrophage cell surface receptors, microorganisms are internalized in a vacuole, the phagosome, to be ultimately degraded in a phagolysosome. The maturation process of a phagosome into phagolysosome requires multiple fusion and fission events with the various endosomal compartments, leading to the acidification of the vacuole and the acquisition of hydrolases (1). Several signaling molecules have been implicated in the regulation of phagocytosis, including members of the protein kinase C (PKC)³ superfamily of protein serine/threonine kinases (2, 3, 4, 5). Indeed, studies with inhibitors or activators revealed that PKC activity regulates C3 receptor- and FcR-mediated phagocytosis, and, conversely, PKC is activated upon ligation of these receptors (6, 7, 8, 9, 10, 11). In particular, cross-linking of FcR causes a translocation of PKC-, -ß, -, -, and - in the membranes of monocytes (8, 11), suggesting that these isoenzymes are involved in the regulation of FcR-mediated phagocytosis. PKC may also participate in the regulation of phagosome maturation (10), as the isoenzymes and ß are associated with the phagosomal membrane (6, 12, 13). Although the precise function of these phagosome-associated PKC isoenzymes remains speculative, it has been established that PKC- phosphorylates myristoylated alanine-rich C kinase substrate (MARCKS), a membrane protein associated with actin-based motility (14) and with membrane trafficking (15). PKC-dependent phosphorylation of phagosome-associated MARCKS results in its displacement to lysosomes and may therefore participate in the movement of both phagosomes and lysosomes on microtubules (15), a process essential for their interaction.

A number of studies have implicated PKC in the control of intracellular microbial infections. In this regard, the protozoan parasite Leishmania received a great deal of attention because it impairs PKC-dependent processes in infected macrophages (16, 17, 18) by synthesizing glycolipids endowed with potent inhibitory activity toward PKC (19, 20, 21, 22, 23). Not surprisingly, inhibition or down-modulation of PKC in macrophages enhances the intracellular replication of Leishmania donovani (19, 24). The latter observations suggest a role for PKC in the control of macrophage anti-leishmanial activity and led us to propose that PKC might be considered as a host resistance determinant against Leishmania infections (19, 20). Impairment of PKC-mediated processes has also been reported in Legionella pneumophila-infected monocytes. In contrast to Leishmania, which inhibits PKC activity, L. pneumophila infection causes a down-modulation of both PKC- and PKC-ß levels in monocytes (25). Given the potential roles of PKC- and PKC-ß in the regulation of macrophage function involved in host defense (8, 26, 27, 28), it is expected that their down-modulation during Legionella infection may influence the intracellular fate of this bacterium.

Despite accumulating evidence that PKC regulates macrophage functions involved in host defense against infections, the involvement of individual PKC isoenzymes in phagocytosis and in the subsequent host-pathogen interaction remains to be addressed. In the present study, we have followed the fate of two intracellular pathogens, L. donovani and L. pneumophila, in RAW 264.7 macrophages overexpressing a dominant-negative (DN) mutant of PKC- (28). We obtained evidence that PKC- regulates innate macrophage functions involved in the control of infection by intracellular L. donovani and L. pneumophila.

	Materials and Methods

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Mice

Eight- to 10-wk-old mice of the C57BL/6 and A/J inbred strains were bred at the Montreal General Hospital Research Institute in a specific pathogen-free environment.

Cell lines

The murine macrophage cell line RAW 264.7 transfected with the expression vector pCIN-4 and the DN PKC--overexpressing clones A2 and C2 (29) were cultured in a 37°C incubator with 5% CO₂ in complete medium (DMEM with glutamine (Life Technologies, Mississauga, ON, Canada), containing 10% heat-inactivated FBS (HyClone, Logan, UT), 10 mM HEPES, pH 7.3, and antibiotics) supplemented with 500 µg/ml G418 (Life Technologies).

Isolation of peritoneal macrophages

Resident peritoneal macrophages were obtained by peritoneal lavages with a 10 ml volume of cold RPMI 1640 (Life Technologies)-gentamicin-10% heat-inactivated FBS. The cell suspension was centrifuged and resuspended in RPMI-10% heat-inactivated FBS. Macrophages were isolated by adherence to plastic following an incubation period of 90 min at 37°C and 5% CO₂.

Leishmania

Promastigotes of Leishmania donovani (Ethiopian strain LV9, obtained from G. Matlashewski, McGill University, Montreal, Canada) were freshly derived from amastigotes isolated from the spleen of an infected hamster and were grown in a 26°C incubator in RPMI 1640 medium supplemented with 20% heat-inactivated FCS, 100 µM adenine, 5 µM hemin, 1 µM biotin, 3 mM biopterin, 20 mM 2-[N-morpholino]ethanesulfonic acid, pH 5.5, and antibiotics (Leishmania medium). A lipophosphoglycan (LPG) repeating unit-defective mutant generated by targeted deletion of the LPG2 gene (lpg2 knockout (KO)) (30) was grown in the Leishmania medium. To generate luciferase-expressing L. donovani, both wild type (WT) and lpg2 KO promastigotes were transfected by electroporation with the luciferase expression vector pGEM72f/anealuc (kindly provided by B. Papadopoulou, Université Laval, Ste-Foy, Canada) (31). Transfectants were selected and grown in Leishmania medium in the presence of 50 µg/ml G418. In vitro amastigotes expressing luciferase were obtained by growing the WT luciferase (WT-Luc) parasites for 48 h in a 37°C incubator with 5% CO₂ in Leishmania medium.

Bacterial strain and culture conditions

A virulent strain of L. pneumophila, Philadelphia-1 strain, (serogroup 1) (32), was cultured at 37°C on buffered charcoal yeast extract agar plates for 3 days before experimentation. Bacteria were harvested by scraping the agar plate surface, were resuspended in PBS 1x, and were adjusted to 1 x 10⁸ bacteria/ml. A temperature-sensitive mutant (TSM) of Pseudomonas aeruginosa (provided by A. Morris Hooke, Miami University, Oxford, OH) was grown in trypticase soy broth at 29–30°C under agitation for 16–18 h, resuspended in PBS 1x, and adjusted to 1 x 10⁶ bacteria/ml as previously described (33).

Infections

For infections with Leishmania, 2.5 x 10⁵ adherent macrophages were incubated with 2.5 x 10⁶ parasites for 1 h. Uningested Leishmania were then removed by four washes with warm medium without serum. The absence of nonphagocytized parasites was ensured by microscopic visualization. Macrophages were then incubated in complete medium in the presence of 200 µg/ml G418, and infection levels were determined after 1, 24, and 48 h postinfection by measuring luciferase activity in cell extracts. When indicated, N^G-monomethyl-L-arginine monoacetate (L-NMMA; Alexis, San Diego, CA) was used at 500 µM, and superoxide dismutase (Sigma, St. Louis, MO) was used at 100 U/ml. For infections with bacteria, 5 x 10⁵ macrophages grown for 3 days in the absence of antibiotics were plated in 48-well tissue culture plates. Cells were allowed to adhere for 90 min at 37°C and 5% CO₂. Nonadherent cells were removed by washing with warm RPMI 1640 without antibiotics. Adherent macrophages were then infected with 5 x 10⁷ L. pneumophila (bacteria:macrophage ratio of 100:1) or 5 x 10⁵ TSM P. aeruginosa (bacteria:cell ratio of 1:1) suspended in RPMI 1640 supplemented with 10% FCS (without antibiotics). After infection, the plates were washed twice with warm RPMI 1640 without antibiotics to remove nonphagocytosed bacteria. Then, 500 µl of medium without antibiotics was added to each well, and plates were incubated for 72 h (L. pneumophila) or 90 min (TSM P. aeruginosa). The number of intracellular viable bacteria (following phagocytosis or bactericidal period) was determined by lysing infected macrophages with a sterile solution of 0.01% (w/v) BSA in distilled water. Serial dilutions of the cells lysates were diluted and plated on buffered charcoal yeast extract agar (L. pneumophila) or TSA agar (TSM P. aeruginosa). CFU were determined after 3 days of incubation at 37°C for L. pneumophila or 16–18 h at room temperature for TSM P. aeruginosa.

Measurement of luciferase activity

Luciferase activity was measured in Leishmania or Leishmania-infected cells extracts using the Promega luciferase assay system as recommended by the manufacturer (Promega, Madison, WI). Briefly, cells were lysed in 100 µl of 1x cell culture lysis reagent. Then, 20 µl of cells extracts were mixed with 100 µl luciferase assay reagent at room temperature, and light emission was quantified in a luminometer (Berthold, Nashua, NH).

Assay for superoxide anion generation

Superoxide was measured by the reduction of cytochrome c as previously described (29). Briefly, adherent macrophages were incubated in 160 µM cytochrome c (Sigma) in HBSS (without phenol red) in the presence or absence of superoxide dismutase (300 U/ml). Cells were then incubated with either PMA (10 and 100 ng/ml) or Leishmania promastigotes for 90 min at 37°C. Controls were incubated in the absence of stimuli. OD was determined spectrophotometrically at 550 nm, and the concentration of superoxide anion was determined using the equation: O₂^- nmol/ml = [A₅₅₀ without superoxide dismutase x 158.73] - [A₅₅₀ with superoxide dismutase x 158.73] (29). Each reaction was performed in triplicate.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Increased intracellular survival of L. donovani in macrophages overexpressing a DN mutant of PKC-

Previous studies showed that down-modulation or inhibition of PKC activity in macrophages enhances the intracellular survival of L. donovani (19, 24, 34), thereby raising the possibility that PKC regulates the ability of macrophages to control infection by this parasite. To determine whether the PKC- isoenzyme plays a role in this process, we first compared the survival of L. donovani promastigotes in normal RAW 264.7 cells and in two clones of RAW 264.7 cells overexpressing a DN mutant of PKC- (clones A2 and C2; Ref. 28). To facilitate quantification of infection levels, we have generated WT L. donovani promastigotes expressing the luciferase gene (designated WT-Luc). Using this approach, as little as 100 parasites can be detected, and luciferase activity measured in extracts prepared from 10² to 10⁸ parasites is within a linear range (not shown). After 1 h infection, the initial uptake of L. donovani promastigotes was similar in the three cell lines (normal RAW 264.7 cells, clone A2, and clone C2) (Fig. 1A). By 48 h postinfection, 1% of the ingested parasites survived in normal RAW 264.7 cells, whereas 9% survived in clone A2 and 27% survived in clone C2 (Fig. 1A). These results indicate that while DN PKC- overexpression had no effect on the phagocytosis of promastigotes, it significantly enhanced their intramacrophage survival. The increased survival of L. donovani correlates with DN PKC- expression levels (28). Similar results were obtained with two other DN PKC--overexpressing clones (clones B1 and D1; Ref. 28) (data not shown). In vitro amastigotes also displayed an increased survival in clones A2 and C2 with respect to normal RAW 264.7 cells (not shown).

View larger version (12K): [in this window] [in a new window]   FIGURE 1. Effect of DN PKC- overexpression on intracellular survival of L. donovani. Adherent RAW 264.7 macrophages (vector alone, ; clone A2, ; and clone C2, ) were infected with either WT-Luc promastigotes (A) or lpg2 KO-Luc mutants (B) for 1 h. Uningested parasites were removed by four washes and infected macrophages were further incubated for 1, 24, and 48 h. Infection levels were determined by measuring luciferase activity in Leishmania-infected cells extracts as described in Materials and Methods. The data shown is representative of three independent experiments, and each infection was performed in triplicate. The error bars indicate the SDs.

Production of NO and superoxide anion by DN PKC--overexpressing macrophages

To determine whether the increased survival of L. donovani in DN PKC--overexpressing clones A2 and C2 was the consequence of an impaired production of NO, we measured nitrite levels in the supernatants of WT-Luc-infected RAW 264.7 cells and clones A2 and C2. Consistent with previous observations (37), L. donovani promastigotes (WT-Luc) failed to induce NO production in all cell lines (data not shown). In addition, the inducible NO synthase (iNOS) inhibitor L-NMMA (500 µM) had no effect on the survival of L. donovani in both control RAW 264.7 cells and clone C2 (Fig. 2A). We also measured the release of superoxide anion in control and in DN PKC--overexpressing RAW 264.7 cells. As shown in Table I, both cell lines released equivalent amounts of superoxide anion in response to 10 ng/ml PMA. At 100 ng/ml PMA, there was a 30% reduction in the amount of superoxide anion produced by clone C2 with respect to control RAW 264.7 cells. Phagocytosis of L. donovani promastigotes triggered the release of low levels of superoxide anion in both control RAW 264.7 cells and clone C2 (Table I). The impact of superoxide production on L. donovani survival was minimal, as by 48 h postinfection, the presence of superoxide dismutase (100 U/ml) increased parasite survival by 3.5-fold in control RAW 264.7 and by 1.5-fold in clone C2 (Fig. 2B). Collectively, these results suggest that the increased survival of L. donovani promastigotes in DN PKC--overexpressing macrophages cannot be attributed to an impaired production of NO or superoxide anion.

View larger version (13K): [in this window] [in a new window]   FIGURE 2. Effect of L-NMMA and superoxide dismutase on intracellular survival of L. donovani. Adherent RAW 264.7 macrophages (vector alone, squares; clone C2, circles) were infected with WT-Luc promastigotes for 1 h, in the absence (open symbols) or the presence (closed symbols) of either 500 µM L-NMMA (A) or 100U/ml superoxide dismutase (B). Uningested parasites were removed by four washes and infected macrophages were further incubated for 1, 24, and 48 h in the presence of either 500 µM L-NMMA (A) or 100 U/ml superoxide dismutase (B). Infection levels were determined by measuring luciferase activity in Leishmania-infected cells extracts as described in Materials and Methods. The data shown is representative of two independent experiments, and each infection was performed in triplicate. The error bars indicate the SDs.

View this table: [in this window] [in a new window]   Table I. Effect of DN PKC- overexpression on superoxide production in RAW 264.7 macrophages1

IFN--primed DN PKC--overexpressing macrophages are leishmanicidal

Pretreatment of macrophages with IFN- induces potent leishmanicidal activity (38). We thus investigated whether DN PKC- overexpression would affect the ability of IFN--primed cells to kill L. donovani promastigotes. Normal RAW 264.7 cells and clones A2 and C2 were preincubated with 100 U/ml IFN- for 24 h before infection with WT-Luc promastigotes. As shown in Fig. 3, the initial uptake of L. donovani was similar in the three IFN--activated cell lines, and by 48 h postinfection most parasites were killed. In contrast to unprimed macrophages, infection of IFN--pretreated RAW 264.7 and clones A2 and C2 induced the production of high levels of NO (data not shown), the main mediator of the macrophage anti-leishmanial arsenal (39, 40). These data clearly indicate that PKC- does not participate in IFN--induced leishmanicidal activity.

View larger version (17K): [in this window] [in a new window]   FIGURE 3. Effect of IFN--primed DN PKC- overexpression on leishmanicidal activity. Adherent RAW 264.7 cells (vector alone, ; clone A2, ; and clone C2, ) were preincubated with 100 U/ml IFN- for 24 h before infection with WT-Luc promastigotes for 1 h. Uningested parasites were removed by four washes. Infected macrophages were further incubated for 1, 24, and 48 h. Infection levels were determined by measuring luciferase activity in Leishmania-infected cells extracts as described in Materials and Methods. Similar results were obtained in three independent experiments, and each infection was performed in triplicate. The error bars indicate the SDs.

Overexpression of DN PKC- renders macrophages permissive for the replication of L. pneumophila

To determine whether overexpression of DN PKC- would affect the fate of another intracellular pathogen, we infected the normal RAW 264.7 cells and the DN PKC--overexpressing clones A2 and C2 with Legionella pneumophila. As controls, we used peritoneal macrophages from the genetically resistant C57BL/6 mice and from the genetically susceptible A/J mice (32). No differences were observed between the normal RAW 264.7 cells and the DN PKC--overexpressing clones A2 and C2 for the initial uptake of L. pneumophila (Fig. 4). Thus, as was the case with L. donovani, phagocytosis of L. pneumophila was not affected by DN PKC- overexpression. Similar to peritoneal macrophages from the genetically resistant C57BL/6 mice, control RAW 264.7 cells, which are derived from the genetically resistant BALB/c mice, were not permissive for the intracellular replication of L. pneumophila (Fig. 4). In contrast, over 72 h, intracellular Legionella increased 35-fold in the DN PKC--overexpressing clone A2 and 145-fold in the clone C2, while macrophages from the genetically susceptible A/J mice allowed a 310-fold increase in L. pneumophila replication (Fig. 3). To determine whether a reduced production of NO accounted for the inability of clones A2 and C2 to control Legionella replication, we measured nitrite levels in the supernatants of macrophages infected for 3 days. No significant differences were observed among the various Legionella-infected macrophages (14.1 ± 3.1 µM for control RAW 264.7; 12.4 ± 2.4 µM for clone A2; and 12.6 ± 3.5 µM for clone C2). Collectively, these data suggest a role for PKC- in the control of intramacrophage replication of L. pneumophila through a NO-independent mechanism.

View larger version (12K): [in this window] [in a new window]   FIGURE 4. Effect of DN PKC- overexpression on the phagocytosis and replication of L. pneumophila. Adherents peritoneal macrophages from A/J and C57BL/6 mice and RAW 264.7 macrophages (vector alone, clone A2, clone C2) were infected with L. pneumophila for 90 min, as described in Materials and Methods. Uptake of L. pneumophila was determined after the 90 min phagocytosis period (). Intracellular replication was determined 3 days postinfection (). Similar results were obtained in three separate experiments, which were done in triplicate. The error bars indicate the SD.

Effect of DN PKC- on macrophage bactericidal activity

To determine whether DN PKC- impaired the ability of macrophages to kill ingested microbes, we measured the bactericidal activity of DN PKC--overexpressing clones A2 and C2 using a TSM of P. aeruginosa (33). This mutant does not grow at 37°C, thus allowing one to measure only the killing ability of the assayed cells. As shown in Fig. 5, clones A2 and C2 displayed normal phagocytic and bactericidal functions toward TSM P. aeruginosa, indicating that overexpression of DN PKC- did not alter the anti-Pseudomonas capacities of RAW 264.7 cells.

View larger version (15K): [in this window] [in a new window]   FIGURE 5. Effect of DN PKC- overexpression on the killing of a TSM P. aeruginosa. Adherents peritoneal macrophages from A/J and C57BL/6 mice and RAW 264.7 macrophages (vector alone, clone A2, clone C2) were infected with P. aeruginosa for 30 min, as described in Materials and Methods. The extent of P. aeruginosa uptake was determined following 30 min phagocytosis period (). Bactericidal activity was determined 90 min postphagocytosis (). Similar results were obtained in three separate experiments, which were done in triplicate. The error bars indicate the SD.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Leishmania and Legionella are two intracellular pathogens that target host macrophage PKC, with the consequence that PKC-mediated events are impaired during infection (16, 17, 19, 20, 24, 25, 43). In addition, inhibition or down-modulation of PKC in macrophages significantly enhances survival and replication of the intracellular parasite L. donovani (19, 24). Collectively, these observations led us to propose that PKC might be considered as a host resistance determinant in the context of Leishmania infection (19, 20). However, the identity and mechanism of action of individual PKC isoenzymes in this process remain to be established. A role for PKC- in the control of Leishmania infection is suggested by the finding that intracellular survival of L. donovani promastigotes was increased by 10- to 20-fold in clones of DN PKC--overexpressing RAW 264.7 macrophages when compared with control RAW 264.7 cells. Previous studies based on pharmacological inhibitors suggested a requirement for PKC in the induction of macrophage functions by IFN- (44, 45, 46). The observation that DN PKC--overexpressing macrophages can be fully activated by IFN- for leishmanicidal activity indicates that PKC- does not participate in this pathway. In addition, while PKC- regulates LPS-induced NO production (26, 28), it is not required for Leishmania-induced NO secretion by IFN--primed macrophages. It has been previously demonstrated that high NO levels produced by IFN--primed macrophages in response to Leishmania are responsible for parasite killing (39, 47). It is thus likely that this pathway, which remains intact in DN PKC--overexpressing RAW 264.7 macrophages, is responsible for the efficient killing of Leishmania promastigotes by IFN--primed normal and DN PKC--overexpressing RAW 264.7 macrophages. These observations, coupled with the finding that PKC- is required for selective LPS-induced responses (26, 28), raise the possibility that PKC- might be involved in the regulation of innate macrophage functions.

Peritoneal macrophages from most mouse strains and the RAW 264.7 macrophage line (derived from BALB/c) are not permissive for the intracellular replication of L. pneumophila, whereas macrophages from A/J mice allow over an 100-fold replication of the bacteria within three days (48, 49). This natural resistance of inbred mouse strains to Legionella infection is controlled by Lgn1, a single dominant gene that controls the absence or presence of intracellular replication inside host macrophages (32, 50). Interestingly, our DN PKC--overexpressing RAW 264.7 clones are phenotypically similar to macrophages from the genetically susceptible A/J mice, as they allow intracellular replication of Legionella. This striking observation clearly supports a role for PKC- in the control of intramacrophage Legionella replication, and raises the possibility that both PKC- and the protein encoded by Lgn1 act within the same pathway. Evidence that the effect of DN PKC- overexpression on Legionella replication is not a generalized defect in the microbicidal capacities was provided by the fact that phagocytosis and killing of a TSM of Pseudomonas aeruginosa were normal in DN PKC--overexpressing RAW 264.7 clones. In addition, the negligible induction of NO production by Legionella, as well as the previously reported lack of effect of an inhibitor of NO synthase on Legionella survival in RAW 264.7 cells (48), indicate that the replication of L. pneumophila in DN PKC--overexpressing RAW 264.7 macrophages is not related to a defect in NO production.

The following mechanisms might be considered to explain the increased survival of both the LPG repeating unit-defective mutant and the WT L. donovani in DN PKC--overexpressing RAW 264.7 macrophages. First, overexpression of DN PKC- impaired the production of NO and superoxide anion, two potent leishmanicidal molecules (51). However, the presence of superoxide dismutase throughout the infection process only had a minor effect on the Leishmania survival in both the control RAW 264.7 cells and clone C2, and promastigotes induced low levels of superoxide in both control and DN PKC--overexpressing RAW 264.7 macrophages. Similarly, inhibition of the iNOS throughout the infection had no effect on the survival of Leishmania in control RAW 264.7 cells and clone C2, and promastigotes failed to induce significant levels of NO. These data argue that increased survival of the parasite in the DN PKC--overexpressing macrophages cannot be attributed to a defective production of these two microbicidal molecules. Second, the antimicrobial mechanism independent of superoxide and NO, which was observed in macrophages from mice doubly deficient in both phagocyte oxidase and iNOS (52), may require PKC-. However, analysis of L. donovani infection in these mice indicated that this phagocyte oxidase- and iNOS-independent microbicidal mechanism does not contribute to the control of L. donovani infection (51). Third, phagosome maturation is impaired in DN PKC--overexpressing macrophages. Indeed, based on the observation that PKC- is associated with the phagosomal membrane, Allen and Aderem proposed that PKC- plays a role in phagosome maturation (12). A similar role has been recently proposed for PKC-ß, which is localized to the membrane of Mycobacterium bovis-containing phagosomes (13). While no direct evidence exists to support a role for neither PKC- nor PKC-ß in phagosome maturation, the presence of PKC substrates on the phagosomal membrane raises the possibility that PKC may regulate their activity. One such substrate is MARCKS, a membrane protein associated with actin-based motility and with membrane trafficking. The observation that PKC-dependent phosphorylation of phagosome-associated MARCKS results in its displacement to lysosomes suggests a mechanism by which PKC- participates in the movement of both phagosomes and endosomes on microtubules (15), a process essential for their interaction. This hypothesis is supported by the observation that acquisition of the lysosomal marker Lamp-1 by LPG-defective mutant-containing phagosomes is impaired in DN PKC--overexpressing clones (A. Breton and A. Descoteaux, unpublished observation). We have recently reported that WT L. donovani promastigotes efficiently inhibit the maturation process of the phagosome in which they are present after phagocytosis (53). This inhibition may provide an environment propitious to the differentiation from promastigote to amastigote, thereby representing a strategy to establish infection. Indeed, L. donovani mutants defective in the cell-surface expression of LPG repeating units, which are rapidly destroyed by the macrophages (Refs. 19 and 36 and this study), are unable to inhibit the maturation process of the phagosome in which they were ingested (53). The inability of these mutants to inhibit phagosomal maturation may contribute to their rapid destruction after phagocytosis (53).

It is notewhorthy that both L. donovani promastigotes and L. pneumophila have in common the ability to inhibit the maturation process of the phagosome in which they reside (53, 54). This inhibition process, which takes place within minutes after phagocytosis, requires the modification of the phagosomal membrane by pathogen-derived molecules (53, 55). One may envision that the extent and efficiency by which this modification takes place may be crucial for the subsequent survival of the pathogen. In contrast, an efficient phagosomal maturation process may provide an advantage to the host. It will thus be of interest to determine whether the increased survival of L. donovani promastigotes and permissiveness to L. pneumophila replication observed in DN PKC--overexpressing RAW 264.7 macrophages is related to an altered phagosomal maturation process.

	Acknowledgments

 
We thank G. Matlashewski for providing amastigotes of the LV9 strain of L. donovani, B. Papadopoulou for providing the pGEM72f/anealuc plasmid, and A. Morris Hooke for the TSM of P. aeruginosa. We also thank P. Duplay and F. Chano for critical comments on this manuscript.

	Footnotes

 
¹ This work was supported partly by the Medical Research Council of Canada (Grant MT-12933 to A.D.) and by the Fonds pour la Formation de Chercheurs et l’Aide à la Recherche of Québec (to A.D.). A.D. is a Medical Research Council Scholar.

² Address correspondence and reprint requests to Dr. Albert Descoteaux, Institut Armand-Frappier, Université du Québec, 531 des Prairies, Laval, Québec, Canada H7V 1B7. E-mail address:

³ Abbreviations used in this paper: PKC, protein kinase C; LPG, lipophosphoglycan; iNOS, inducible NO synthase; MARCKS, myristoylated alanine-rich C kinase substrate; DN, dominant-negative; KO, knockout; WT, wild type; Luc, luciferase; TSM, temperature-sensitive mutant; L-NMMA, N^G-monomethyl-L-arginine monoacetate.

Received for publication May 26, 1999. Accepted for publication August 25, 1999.

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