Interferon-gamma Listericidal Action Is Mediated by Novel Rab5a Functions at the Phagosomal Environment*

Amaya Prada-DelgadoDagger , Eugenio Carrasco-MarinDagger §, Gary M. Bokoch, and Carmen Alvarez-Dominguez||**

From the Dagger  Centro de Biología Molecular "Severo Ochoa," Universidad Autónoma de Madrid, 28049 Madrid, Spain and the  Department of Immunology-IMM14/R221, The Scripps Research Institute, La Jolla, California 92037, and the || Cell Biology Department, Washington University, St. Louis, Missouri 63110

Received for publication, February 21, 2001, and in revised form, March 21, 2001


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

Control and clearance of Listeria monocytogenes infection is an interferon-gamma -dependent process. The listericidal mechanism of action involves activation of NADPH oxidase and inducible nitric-oxide synthase to produce reactive oxygen and nitrogen intermediate radicals, respectively. Recently, we have described in a nonpathogenic model of L. monocytogenes (hemolysin negative mutant strain) that the interferon-gamma -inducible GTPase Rab5a contributed to Listeria destruction in resting macrophages. Here, we report in a pathogenic model of L. monocytogenes (hemolysin-positive strain) that Rab5a plays a central role in Listeria destruction induced by interferon-gamma and within the phagosomal environment. These findings reveal the importance of Rab5a as the responsible factor mediating the listericidal action of interferon-gamma . Active Rab5a causes remodeling of the phagosomal environment, facilitates the translocation of Rac2 to LM phagosomes, and regulates the activity of this GTPase. Rac2 activation and translocation governs the phagocyte NADPH oxidase activity and the consequent reactive oxygen intermediate production that leads to killing of the pathogen.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

Listeria monocytogenes is an intracellular facultative bacterium able to invade phagocytic cells and is responsible for severe pathologies in immunocompromised people, newborns and pregnant women (1). L. monocytogenes entry into the host cell is an active process involving several protein components. After a short phagosomal period (~30 min), L. monocytogenes escapes to the cytosol, avoids intracellular killing, and replicates (reviewed in Ref. 2). The L. monocytogenes survival mechanism involves two steps: (i) live bacteria avoid phagosome maturation by inactivation of the endosomal trafficking regulator Rab5a, which blocks the recruitment of lysosomal proteins to the phagosomes (Lamp-1 and cathepsin-D) (3) and (ii) secretion by L. monocytogenes of listeriolysin and PI-PLC lyses the phagosomal membrane, translocates L. monocytogenes to the cytoplasm, and consequently, allows for L. monocytogenes intracellular survival (4).

Control of L. monocytogenes infection and clearance is an interferon-gamma (IFN-gamma )1-dependent process. IFN-gamma priming of macrophages (MØs) recruited at the inflammatory site triggers their listericidal abilities (5). IFN-gamma signaling modulates the expression and activation of more than 200 proteins (6). However, to date, only a few of these molecules have been shown to exert a direct role in pathogen elimination (7). Among these are (i) IGTP, a GTP-binding protein relevant for Toxoplasma clearance (8) and (ii) Nramp1, a MØ-restricted lysosomal protein involved in Leishmania, Salmonella, and Mycobacterium spp. clearance (9). In addition, IFN-gamma induces the production of reactive oxygen (ROI) and nitrogen (RNI) intermediates with microbicidal activity (10). From this set of molecules, only ROI and RNI have been shown to restrict L. monocytogenes growth (10, 11), while the other two molecules (i.e. IGTP or Nramp1) play no role at all in L. monocytogenes clearance (8, 9).

Recently, we have shown that in resting MØs the inhibition of Rab5a synthesis allows for intracellular survival of a listeriolysin-defective L. monocytogenes mutant, that under normal Rab5a levels is unable to grow and fails to escape from the phagosome (12). Furthermore, we have also described that IFN-gamma signaling up-regulates Rab5a function (13). However, at this stage, no correlation between the induction of ROI and RNI by IFN-gamma and the Rab5a function has been established. Here, we show that Rab5a is a key molecule for the IFN-gamma promoted clearance of a pathogenic L. monocytogenes strain at the phagosomal stage. We show that Rab5a, in the GTP form, controls the recruitment of active Rac2 to the transformed L. monocytogenes phagolysosome and the assembly of the phagocyte NADPH oxidase with the production of toxic radicals. These Rab5a-mediated actions compromise Listeria viability within the phagolysosomes and further L. monocytogenes intracellular survival.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

Cells and Reagents-- J774 cells and proteose peptone-elicited peritoneal MØs from Balb/c mice were cultured in Dulbecco's modified Eagle's medium, 5% fetal calf serum, 2 mM L-glutamine, and 50-µg/ml gentamicin. Phosphothioate Rab5a antisense (5'-TGC GCC TCG ACT AGC CAT GT-3') and sense (5'-ACA TCG CTA GTC GAG GCG CA-3') oligonucleotides (20-mer) were from Isogen Bioscience BV (Maarseen, Holland). Mouse recombinant IFN-gamma was purchased from Roche Molecular Biochemicals; 35S translabel (10 mCi/ml) was from Amersham Pharmacia Biotech; horseradish peroxidase (HRP), superoxide dismutase, and ferricytochrome c were from Sigma; and brain heart infusion was from Difco.

Transient Overexpression of Rab5 Constructs in J774 Cells-- Rab5a:Q79L, Rab5a:S34N, and Rab5c:Q80L cDNAs were subcloned into pcDNA3 using EcoRI/BamHI sites. Cells (5 × 106) were transfected by electroporation (150 V, 800 microfarads, 129 ohms) for 24 h. Overexpression was checked on cell lysates with specific antibodies at 8 and 24 h, respectively.

Antibodies and Proteins-- The following antibodies were used. Mouse monoclonal anti-Rab5a (4F11) was described in Ref. 3. Polyclonal rabbit anti-Rab5c was a gift from M. Zerial (EMBL, Heidelberg, Germany). Rabbit anti-Rab7 was a generous gift from A. Wandinger-Ness (University of New Mexico, Alburquerque, NM). Rabbit anti-Rac2 was developed in rabbits (R786/9) (14), and rabbit anti-cathepsin-D (3) was a gift from P. D. Stahl (Washington University. St. Louis, MO). Rabbit anti-Limp-II was a gift from I. V. Sandoval (Centro de Biología Molecular "Severo Ochoa," Madrid, Spain). Rat anti-mouse Lamp-1 monoclonal antibody (1G11) was a gift from D. G. Russell, (Washington University, St. Louis, MO). Biotinylated rat anti-mouse TfR (CD71) was purchased from Caltag, and secondary peroxidase-conjugated antibodies (goat anti-mouse, anti-rabbit, or anti-rat) were from Amersham Pharmacia Biotech. Streptoavidin-peroxidase-conjugated antibody was purchased from Roche Molecular Biochemicals.

GST-PBD, the p21-activated kinase-derived binding domain for activated Rac2 proteins, was expressed in E. coli BL-21 strain. Recombinant proteins were induced with 5 mM isopropyl-beta -D-thiogalactoside for 3 h at 37 °C and purified with glutathione-Sepharose according to the manufacturer's instructions (CLONTECH, Palo Alto, CA).

Antisense and IFN-gamma Treatment of Cells-- Introduction of antisense and sense phosphothioate oligonucleotides onto J774 cells or peritoneal MØs (5 × 106/ml) was performed as described (12) with a Baxter BTX-603 electroporator and the following settings: 220 V, 800 microfarads, 75 ohms. Cells were set onto culture plates for 6 h at 37 °C and washed and incubated (+) or not (-) with 100 units/ml of IFN-gamma for 16 h.

Bacterial Infection and Intracellular Assays-- D. A. Portnoy (University of California, CA) kindly provided the pathogenic L. monocytogenes strain (10403S). L. monocytogenes infection was performed according to standard protocols (3) at a 10:1 bacteria/cell ratio. After 15 min of uptake, cells were incubated for 45 min in medium containing 5 µg/ml gentamicin to kill extracellular bacteria. This time period was considered 0 h. Infected cells were then incubated at 37 °C in complete medium containing 5 µg/ml gentamicin for 16 h in the presence or absence of 100 units/ml IFN-gamma , washed, lysed, and plated onto brain heart infusion agar plates (37 °C, 36 h). The number of live bacteria was estimated by counting CFU. To estimate the percentage of growth, the ratio of CFU recovered at 16 h divided by the CFU recovered at 0 h was expressed as the replication index.

Isolation of Phagosomes-- Phagosomes were purified from 30-min (Western experiments) or 1-h (viability experiments) infection protocols. L. monocytogenes-infected cells were pretreated or not with IFN-gamma (16 h). For phagosome-lysosome fusion assays, cells were offered HRP (100 µg/ml, 5 min, washed and chased for 2 h). Cells were infected with L. monocytogenes for 30 min. Postnuclear supernatants from these cells were applied to a 8.8-20-40% discontinuous sucrose gradient and L. monocytogenes phagosomes recovered from the 20-40% interface and lower 20% sucrose fraction as previously reported (13). Phagosomes were solubilized with PBS-0.05% Triton X-100 and plated onto brain heart infusion-agar plates to count CFU or added to 4× Laemmli sample buffer for Western blots. For phagosome-lysosome fusion assays, HRP reaction was evaluated with o-dianisidine as a substrate. HRP was measured in postnuclear supernatants and considered the total HRP. Results were expressed as the percentage of total absorbance values of HRP activity per mg of protein.

Western Blot Assays-- 30 µg of phagosomal proteins per lane were loaded for Western blots. Phagosomes were normalized using anti-Rab5c marker as a standard, since this marker does not vary upon IFN-gamma or antisense treatment. Normalization was also performed after analysis with a rabbit anti-Listeria protein antiserum, to check the same level of Listeria proteins onto phagosomes. Antibody dilutions were as follows: mouse anti-Rab5a and rabbit anti-Rab5c, anti-Rac2, and anti-Listeria antibodies (1:1000); rabbit anti-cathepsin-D and anti-Limp-II and rat anti-mouse Lamp-1 and anti-mouse TfR (1:500); and rabbit anti-Rab7 (1:300).

Immunoprecipitation-- Cells (5 × 106/ml) were labeled with 50 µCi of [35S]Met/Cys promix for 2 h. Immunoprecipitations were performed as described (13). Beads were washed three times with radioimmune precipitation buffer (1% PBS, 0.1% Triton X-100, 0.5% SDS), radioimmune precipitation buffer plus 500 mM NaCl, and PBS. Elution was performed with 1× Laemmli sample buffer for 1 h at room temperature.

Membrane and Cytosolic Distribution of Rab5a-- J774 cells treated with antisense/sense oligonucleotides, incubated with (+) or without (-) IFN-gamma for 16 h and labeled with 50 µCi of [35S]Met/Cys promix as above, were homogenized in HBE buffer (250 mM sucrose, 0.5 mM EGTA, 20 mM Hepes-KOH, pH 7.2) with a ball bearing homogenizer. Homogenates were spun down at 2000 × g to remove nuclei and large organelles, and supernatants were centrifuged at 100,000 × g for 60 min to obtain total membranes (M) and cytosols (C) (supernatants) (see Fig. 2). Pellets were resuspended in HBE buffer, and supernatants were precipitated with 10% trichloroacetic acid and resuspended in the same volume as pellets. All samples were quickly frozen in liquid nitrogen, and immunoprecipitations were performed as above.

Assays for Rac2 Activation in J774 Cells-- These assays were performed as previously reported for Rac2 (15). In brief, cells (2 × 107 cells/assay) were treated with Rab5a antisense/sense oligonucleotides for 6 h and incubated with 100 units/ml IFN-gamma 16 h. Accordingly, similar assays were performed in transient overexpressed cells with Rab5a:Q79L, Rab5a:S34N, Rab5c:Q80L, or pcDNA3 cDNAs. L. monocytogenes phagosomes or whole cells were then washed in HBSS-g and lysed in a 2× lysis buffer (50 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 5 mM EDTA, 200 mM NaCl, 2% Nonidet P-40, 10% glycerol, 2 mM phenylmethylsulfonyl fluoride, 2 µg/ml leupeptin, 2 µg/ml aprotinin, and 2 mM sodium orthovanadate). Lysates placed on ice were clarified by 20,000 × g centrifugation at 4 °C, and 8 µg of GST-PBD was added. Binding buffer (25 mM Tris-HCl, pH 7.5, 1 mM dithiothreitol, 30 mM MgCl2, 40 mM NaCl, 0.5% Nonidet P-40), and glutathione-Sepharose were added for 90 min at 4 °C with shaking (a total of 300 µl). Washings were performed with binding buffer. The bead pellets were resuspended in 1× Laemmli sample buffer. Proteins were separated by 15% SDS-PAGE, transferred to membranes, and blotted for the appropriate GTPase using antibody R786/9 (Rac2). ECL detected immunoblots. Total putative activated protein from each sample was obtained by treatment with 100 µM GTPgamma S for 15 min at 30 °C before GST-PBD incubation.

O&cjs1138;2 Production Assay-- This assay was performed essentially as previously described (16). In brief, proteose peptone-elicited peritoneal MØs (or J774 cells) treated with Rab5a antisense/sense oligonucleotides were plated onto 96-well plates at 3 × 105 cells/well in Dulbecco's modified Eagle's medium without phenol red plus 50 µg/ml gentamicin. A solution of 80 µM ferricytochrome c in HBSS-g containing the corresponding stimulus was added (10 or 100 units/ml IFN-gamma ). Cells were incubated for 60 min at 37 °C. The change in absorbance (A550 nm) obtained in well samples was subtracted from those wells incubated with the same stimulus in the presence of superoxide dismutase (10 µg/ml). Results were expressed as nmol of O&cjs1138;2 produced/106 cells in quadruplicate wells. Results were representative of at least seven independent experiments.

    RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

L. monocytogenes is a facultative intracellular parasite able to infect and replicate inside nonactivated MØs. Once in the phagosome, the bacterium secretes listeriolysin and PI-PLC to lyse the phagosome and escape to the cytoplasm, where it replicates (4). The activation of MØs by IFN-gamma provokes an increase in the phagocytic rate, partially prevents the escape of L. monocytogenes to the cytosol (17), and triggers the production of toxic radicals (i.e. ROI and RNI), which lead to the elimination of the microorganism (10).

Analysis of phagosomes from infected MØs pulsed with IFN-gamma has established that this lymphokine promotes phagosome maturation (18, 19). These observations show that phagosome maturation is an active process that involves the interaction of phagosomes with several organelles (reviewed in Ref. 20).

At the molecular level, IFN-gamma signaling up-regulates the levels of endosomal/lysosomal proteins, such as cathepsin-D (21) and Rab5a (13), and down-regulates endosomal markers, such as the mannose and the transferrin receptors, TfR (22, 23), while other Rabs remain unaffected (i.e. Rab5b, Rab5c, Rab7, or Rab11) (13). However, until now, the link between the IFN-gamma -induced microbicidal effects and the up-regulation of Rab5a has remained elusive.

IFN-gamma Reduces Intraphagosomal Listeria Viability and Promotes Phagosome Maturation-- Since IFN-gamma triggers the listericidal activity of MØs, we first investigated its action in infected MØs at the phagosomal stage. As shown in Fig. 1, among the typical endosomal markers analyzed in L. monocytogenes isolated phagosomes, Rab5a was clearly increased after IFN-gamma treatment (+), whereas Rab5c or Rab7 was unmodified and TfR was down-regulated. Similar data have been reported in studies of whole cell extracts (13, 23). Isolated phagosomes from MØs not treated with IFN-gamma (-) lacked lysosomal markers such as cathepsin-D, Lamp-1 and Limp-II. In contrast, all of these markers were increased in phagosomes from MØs previously pulsed with IFN-gamma (+). These results suggested that IFN-gamma up-regulated the interactions of phagosomes with late endosomes and lysosomes rather than with early endosomes. IFN-gamma promotion of phagosome maturation was accompanied by a significant reduction of the pathogen viability inside the phagosomes (1-h phagosomes in Fig. 1, bar graphs). These results argue that a more rapid entry of this lysosomal environment into the phagosomal space contributes to the killing of the pathogen.


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Fig. 1.   Effects of IFN-gamma on L. monocytogenes survival and phagosomal composition. J774 cells were incubated (+) or not (-) with 100 units/ml IFN-gamma for 16 h before infection with L. monocytogenes (1 h). Phagosomes were isolated, and viable bacteria (CFU) were determined after plating onto brain heart infusion-agar plates. Results shown are the mean ± S.D. of triplicate cultures (A, graphic bar). Lanes corresponded to Western blots of solubilized isolated phagosomes (30-min infection) and incubated with the following antibodies: monoclonal anti-Rab5a antibody; rabbit anti-Rab5c, anti-Rab7, anti-Limp-II, and anti-cathepsin-D (cat-D); and rat anti-mouse TfR and anti-mouse Lamp-1.

IFN-gamma Promotion of Phagosomal Maturation and Lysosomal Protein Transport Is Regulated by Rab5a-- Our earlier data showed that within the phagosome, Listeria avoided phagosome maturation by blocking the Rab5a activity (3, 12). Since IFN-gamma signaling increases the synthesis and enhances the activity of this small GTPase (13), we next studied whether promotion of phagosome maturation by IFN-gamma was mediated by Rab5a and designed the following experimental strategy. Rab5a synthesis was first blocked using phosphothioate antisense oligonucleotides targeted to the Rab5a translation initiation codon, and then an IFN-gamma pulse was given (12).

Analysis of phagosomes from MØs treated with Rab5a antisense oligonucleotide (A) showed that this treatment effectively blocked Rab5a expression on phagosomes (A- versus S- cells in Fig. 2A). Moreover, Rab5a antisense treatment also blocked Rab5a expression even in phagosomes of MØs pulsed with IFN-gamma (+) (compare A+ with S+ cells and S- cells with S+ cells, respectively; Fig. 2A). The inhibition was thus effective at the level of protein synthesis; as shown in Fig. 2B, this reduction mainly affected membrane-bound Rab5a.


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Fig. 2.   Effect of Rab5a antisense/sense and IFN-gamma treatment onto L. monocytogenes intracellular survival, phagosomal viability, and composition. A, J774 were treated with Rab5a antisense oligonucleotide (A) or with Rab5a sense oligonucleotide (S) for 6 h. The cells were pulsed (+) or not (-) with 100 units/ml IFN-gamma for 16 h and then infected with L. monocytogenes for 30 min. Finally, phagosomes (Phg) were isolated and solubilized, and material was assayed for the presence of different proteins: Rab5a, Rab5c, Rab7, TfR, Lamp-1, Limp-II, and cathepsin-D. B, J774 cells treated as above were metabolically labeled for 2 h. After that, cytosol (C) and membrane extracts (M) were obtained, and Rab5a was immunoprecipitated (IP) to test the distribution of newly synthesized Rab5a in both fractions. C, L. monocytogenes viability was analyzed on J774 cells treated as before. Results shown are the mean of triplicates ± S.D. and expressed as the replication index after 16 h of infection for total viability of L. monocytogenes (white bars) or as CFU in 1-h obtained phagosomes (filled bars).

Parallel studies of Rab5c and Rab7 levels showed that their expression in phagosomes was not affected by the Rab5a antisense treatment, either alone or in combination with a pulse of IFN-gamma (Fig. 2A). On the other hand, the increase in the levels of Lamp-1, Limp-II, and cathepsin-D in phagosomes induced by IFN-gamma in control cells (S+), were almost completely abolished in cells treated with Rab5a antisense oligonucleotides (A+). Experiments performed with lysosensor green as a pH indicator showed that IFN-gamma treatment decreased MØ vesicle pH, but independently of Rab5a antisense or sense treatment (data not shown).

These observations highlighted two important conclusions: (i) Rab5a acquires a novel function upon IFN-gamma stimulation that affects the transport of lysosomal proteins to phagosomes, and (ii) the normal L. monocytogenes strategy of blocking transport of newly synthesized lysosomal proteins to phagosomes (3) can be overcome by the increase in Rab5a function promoted by IFN-gamma .

IFN-gamma induction of Rab5a synthesis has been shown to promote the binding of the GTP form of Rab5a to membranes (13). This argues that the Rab5a form that controls the transport of lysosomal proteins to the phagosomes should be the GTP form. Our data are also in agreement with a recent report showing a role for rabenosyn-5, a Rab5-GTP-interacting effector protein, in the transport of cathepsin-D from the Golgi complex to lysosomes (24).

Rab5a Mediates the IFN-gamma -induced Listericidal Abilities at the Phagosomal Stage-- When intracellular growth of L. monocytogenes was studied, there was an inverse correlation between viability, expressed as the replication index, and Rab5a levels. The highest replication index corresponded to Rab5a antisense-treated cells (A-) (Fig. 2C) that showed no detectable Rab5a levels (Fig. 2B, Rab5a-IP lanes). A lower replication index was found in Rab5a antisense-treated cells also pulsed with IFN-gamma (A+) relative to the controls (S-); the replication index values were 22.5 in A- cells, 9 in A+ cells, and 6.2 in S- cells (Fig. 2C, white bars). Interestingly, these Rab5a antisense-treated cells pulsed with IFN-gamma (A+) expressed detectable Rab5a protein levels (Fig. 2B, Rab5a-IP lanes). The Rab5a protein expressed in these A+ cells efficiently bound to intracellular membranes, with no detectable pool in the cytosolic fraction (see M/C distributions on IP-Rab5a lanes; Fig. 2B). Rab5a antisense treatment blocks the listericidal effect of IFN-gamma as shown by the replication index values of 9 in A+ cells compared with a replication index value of 0.18 in S+ cells (Fig. 2C, white bars). This Rab5a antisense treatment also prevented the expression and induction of Rab5a observed in control cells (Fig. 2B, Rab5a-IP lanes). These results cannot be explained by different L. monocytogenes ingestion rates on Rab5a antisense- or Rab5a sense-treated cells (12). Similarly, intraphagosomal L. monocytogenes viability, expressed as CFU from isolated phagosomes after 1 h of infection, inversely correlated with Rab5a expression on L. monocytogenes phagosomes. The highest viability corresponded to A- phagosomes with the lowest Rab5a levels. The lowest viability corresponded to S+ phagosomes with the highest Rab5a levels, as shown by the viability values ranging from 29 × 104 CFU in A- cells to 16 × 104 CFU in A+ cells, 9 × 104 CFU in S- cells, and 2.7 × 104 CFU in S+ cells (Fig. 2C, filled bars).

We also estimated lysosomal protein synthesis after antisense or sense treatment in the presence or absence of IFN-gamma . As shown in Fig. 3, synthesis of Lamp-1 or Limp-II was not affected by any of the treatments. IFN-gamma increased the synthesis of cathepsin-D, as previously reported (21), independently of Rab5a antisense or sense treatments. These results confirmed that Rab5a regulated the transport of lysosomal proteins to phagosomes induced by IFN-gamma but not the synthesis of lysosomal proteins.


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Fig. 3.   Rab5a antisense/sense treatment did not affect lysosomal protein synthesis. J774 cells were treated with Rab5a antisense oligonucleotide (A) or with Rab5a sense oligonucleotide (S) for 6 h. Cells were then pulsed (+) or not (-) with 100 units/ml IFN-gamma for 16 h. Cells were metabolically labeled for 2 h, and then cell lysates were immunoprecipitated with 1G11 (anti-Lamp-1), rabbit anti-Limp-II, or rabbit anti-cathepsin-D antibody.

Taken together, these results show that IFN-gamma -promoted phagosomal maturation depends on Rab5a that more importantly mediated the listericidal effect of IFN-gamma on phagosomes, leading to a significant decrease in L. monocytogenes viability.

Rab5a Regulates the IFN-gamma -promoted Interactions of L. monocytogenes Phagosomes with Lysosomes-- Until now, transport from late endosomes/lysosomes to phagosomes has been suggested to be Rab5a-mediated in resting phagosomes (25). However, no report has implicated Rab5a in these transport events from activated MØs; nor has the effect of IFN-gamma in stimulating this process been shown. To analyze the role of Rab5a and IFN-gamma in this event, we studied the transfer of HRP from preloaded early endosomes (100 µg/ml, HRP uptake for 5 min) or lysosomes (100 µg/ml, HRP uptake for 5 min and 2 h chase) into L. monocytogenes phagosomes. L. monocytogenes phagosomes were isolated from cells treated with antisense/sense oligonucleotides and pulsed with IFN-gamma as above. The results shown in Fig. 4 indicated that the Rab5a antisense treatment significantly blocked the transfer of HRP from early endosomes to phagosomes. However, under these conditions, IFN-gamma treatment did not promote the fusion between early endosomes and L. monocytogenes phagosomes (Fig. 4, white bars). In contrast, IFN-gamma clearly promoted the transfer of lysosomal HRP to phagosomes as shown by the increase in HRP values that ranged from 1.8 in S- cells to 4.9 in S+ cells. The most striking finding was that Rab5a antisense treatment strongly blocked the ability of IFN-gamma to enhance the transfer of lysosomal HRP to L. monocytogenes phagosomes, as shown by the decrease in HRP values from 4.9 in S+ cells to 2.5 in A+ cells (Fig. 4, filled bars).


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Fig. 4.   Rab5a regulates the IFN-gamma promoted interactions of L. monocytogenes phagosomes with lysosomes. For phagosome-endosome fusion experiments (P-E fusion), cells were treated with Rab5a antisense/sense oligonucleotides and IFN-gamma as above and then offered HRP (100 µg/ml, 5 min) and infected with L. monocytogenes for 15 min. For phagosome-lysosome fusion experiments (P-L fusion), cells treated as above were offered HRP (100 µg/ml, 5 min, washed and chased for 2 h). Cells were infected with L. monocytogenes for 15 min, and phagosomes were isolated, solubilized, and HRP reaction-evaluated with o-dianisidine as a substrate. Results are expressed as the ratio of absorbance value per mg of protein. Results are the mean ± S.D. of triplicates.

In summary, the experiments shown in Figs. 2-4 indicated that IFN-gamma promoted the delivery of lysosomal proteins to L. monocytogenes phagosomes via a process that was clearly dependent on Rab5a. These results link Rab5a action to the clearance of L. monocytogenes by phagocytes. Moreover, the simplest model to explain the IFN-gamma effect on L. monocytogenes phagosome maturation is that it promotes the Rab5a-mediated fusion of phagosomes with late endosomes/lysosomes. These data were in accordance with those reported with the yeast homologue, Ypt51p, or the allelic form, Vps21p, on late transport events (26-28) as well as in fusion events of latex bead phagosomes with lysosomes from resting MØs (25).

Rab5a Acts Upstream of Rac2 and ROI Production in the IFN-gamma Signaling Pathway-- The results presented above clearly indicate that Rab5a regulated the IFN-gamma -induced listericidal abilities of MØs at the phagosomal stage. To date, the listericidal mechanisms induced by different signals including IFN-gamma rely on the production of ROI and RNI toxic molecules (5, 11, 29, 30). Production of ROI radicals requires translocation of Rac2 to the membranes to assemble an active phagocyte NADPH oxidase complex (31), a process triggered by IFN-gamma . Only active Rac2 (GTP form) is known to be required to activate the phagocyte NADPH oxidase to produce ROI (32). We next analyzed whether Rab5a was involved in the activation of Rac2 and production of ROI radicals regulated by IFN-gamma . First, we observed that IFN-gamma promoted the translocation of Rac2 to the phagosomes, and this was almost completely abolished by treatment of MØs with Rab5a antisense (Fig. 5A). These results strongly suggested that in the pathway of IFN-gamma signaling, Rab5a action acted upstream of rac2. For ROI production, the NADPH oxidase enzyme needs to bind and activate onto the phagosomal membranes (31). NADPH oxidase activation correlates with the recruitment of the active form of Rac2 to the membranes (32). Recently, a protocol has been described to quantify activated Rac2 in whole cells using the binding domain of the p21-activated kinase 1 that exclusively binds Rac2-GTP (Fig. 5B, GST-PBD lanes) (15). Using this protocol, we observed that the amount of activated Rac2 in Rab5a antisense-treated cells was significantly lower than in sense-treated cells, both in the absence and presence of IFN-gamma (Fig. 5B, GST-PBD-IP lanes), while the total Rac2 levels remained constant (Fig. 5B, +GTPgamma s lanes and Rac2 lanes). The same results were observed when the study was repeated in isolated L. monocytogenes phagosomes after Rab5a antisense/sense and IFN-gamma treatment (data not shown). This observation suggests that measurement of Rac2 activation on whole cell extracts was a valid indicator of the translocation of GTP-active Rac2 to the phagosomes. More interestingly, these findings indicate that Rab5a mediates the IFN-gamma -induced Rac2 activation and translocation to the phagosomes. With respect to this, it is not inconceivable that Rab5a-Rac2 may act in conjunction. In fact, Rab5a and the Rac family have been reported to act together in coordinating the process of (re)assembly of stress fibers and focal adhesions (33) as well as in coordinating EGF receptor signaling and trafficking (34).


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Fig. 5.   Rab5a acts upstream of Rac2 and ROI production in the IFN-gamma signaling pathway. A, J774 cells were treated with Rab5a antisense/sense and IFN-gamma as above. Isolated phagosomes were solubilized, and Western blots were developed with rabbit R786/9 anti-Rac2 antibody. B, J774 cells treated as above and assayed for Rac2 activation (GST-PBD lanes) (lanes labeled as -GTPgamma S). Controls (lanes labeled as +GTPgamma S) corresponded to total Rac2 protein able to be activated. Whole cell Rac2 levels are shown in lanes labeled as rac2. C, proteose peptone-elicited peritoneal MØs (or J774 cells) were treated with Rab5a antisense/sense oligonucleotides as above and assayed for O&cjs1138;2 production as under "Experimental Procedures." Stimuli were 10 or 100 units/ml of IFN-gamma /well for 60 min at 37 °C. Absorbance (A550) of each value was subtracted from superoxide dismutase values, and results are expressed as nmol of O&cjs1138;2/106 cells in quadruplicate wells. Results corresponded to a representative experiment out of seven.

Activation of the phagocyte NADPH oxidase leads to a functional enzyme able to produce ROI toxic molecules (31). Next, we studied the role of Rab5a on the IFN-gamma -induced production of ROI radicals by a functional phagocyte NADPH oxidase. For this purpose, we used the same Rab5a antisense/sense strategy in the presence and absence of an IFN-gamma pulse as above. For ROI production, we used peritoneally elicited MØs due to their higher ROI production levels. Nonetheless, experiments performed in the J774 MØ cell line showed similar results (an average of five different assays were performed (data not shown). As shown in Fig. 5C, which shows one representative experiment (out of seven), production of ROI correlated perfectly with Rac2 activation and was inhibited by Rab5a antisense treatment. A 1.4-fold inhibition was observed in A- cells compared with S- cells. As expected, IFN-gamma treatment induced ROI production in control cells, but interestingly, this ROI induction was 1.6-fold inhibited by the Rab5a antisense treatment. The effect of Rab5a antisense treatment on ROI production correlated well with the effect shown on Rac2 activation (Fig. 5B) and translocation to the phagosomes (Fig. 5A). The data show that Rab5a regulates the IFN-gamma -mediated Rac2 activation, both by enhancing its translocation from the cytosol to the phagosomes and by locking Rac2 in its active GTP conformation.

To address whether Rab5a synthesis alone was sufficient to trigger the effects observed on phagosome-lysosome fusion (Fig. 4) and Rac2-GTP recruitment (Fig. 5), we transiently overexpressed both the active and inactive forms of Rab5a into J774 cells; we also included the active form of another Rab5 isoform expressed onto L. monocytogenes phagosomes, Rab5c, as a control (5a:Q79L, 5a:S34N, and 5c:Q80L, respectively).

Overexpression protocols after 24 h of transfection gave 5-7-fold increased levels above controls (cells transfected with vector alone) (Fig. 6A). Phagosomes (after 30 min of infection) from these cells showed that transfer of HRP from lysosomes was particularly enhanced in Rab5a:Q79L-transfected cells (Fig. 6B). These results were in accordance with those previously reported for Rab5a:WT-transfected cells (13). Overexpression with Rab5a-inactive form (5a:S34N) inhibited the phagosome-lysosome fusion enhancement, even below control levels. However, overexpression with the active form of Rab5c, Q80L, showed a very low levels of phagosome-lysosome fusion, similar to control cells and to results reported previously (13). These data argue that Rab5a synthesis and its activation in the GTP form were sufficient to promote phagosome-lysosome fusion.


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Fig. 6.   Rab5a-GTP form promoted the interactions of L. monocytogenes phagosomes with lysosomes and the recruitment of active Rac2-GTP. J774 cells were transiently transfected as described under "Experimental Procedures" with Rab5a:Q79L, Rab5a:S34N, and Rab5c:Q80L subcloned into pcDNA3 vector or with vector alone for 24 h. A, cells were metabolically labeled for 2 h, and then lysates were immunoprecipitated with 4F11 (anti-Rab5a) or rabbit anti-Rab5c antibody, respectively. Controls corresponded to Rab5a levels. Rab5c levels were similar to Rab5a levels in control cells (data not shown). B, phagosome-lysosome fusion was performed after offering HRP to the cells (100 µg/ml, 5 min, washed and chased for 2 h). Cells were infected with L. monocytogenes for 30 min, and phagosomes were isolated, solubilized, and HRP reaction-evaluated with o-dianisidine as a substrate. HRP was also analyzed in postnuclear supernatants to give total values. Results are expressed as the percentage of total absorbance values per mg of protein. Results are the mean ± S.D. of triplicates. C, phagosomes from transfected cells as in A were solubilized and assayed for Rac2 activation and translocation (GST-PBD/IP lanes). After immunoprecipitation, Westerns blots were developed with rabbit R786/9 anti-Rac2 antibody (whole cell extracts from these cells showed similar results; data not shown).

We also checked whether Rab5a synthesis was able to control the observed rac2-GTP recruitment to phagosomes (Fig. 5B). To do this, we isolated phagosomes from cells overexpressed with Rab5a or Rab5c cDNAs, as in Fig. 6A, and analyzed the induction of Rac2 activation and translocation to the phagosomes, using the same protocol as used in Fig. 5B. As shown in Fig. 6C, Rac2 recruitment was promoted in Rab5a:Q79L-overexpressed cells and was diminished below control levels in Rab5a:S34N-overexpressing cells. It should be noted that cells overexpressing the active Rab5c:Q80L form showed levels similar to the controls. These results clearly indicate that Rab5a-GTP, exclusively, controls the recruitment of Rac2-GTP and that Rab5a action is upstream of Rac2.

It is also interesting that another cytokine, the granulocyte colony-stimulating factor, could control the growth of the pathogen Brucella abortus also by regulating the interactions with the endosomal compartment (35). This interaction may transfer bacteria from a relative nonhostile environment to one that contains reducing agents, acid hydrolases, and oxygen radicals (35). It can be speculated that these ROI, elements of the respiratory burst, also present in the endocytic compartments, can then reach both the Listeria- and Brucella-containing phagosomes under each cytokine situation and compromise the bacterial growth inside the cells.

In summary, our results are the first to implicate a small GTPase, Rab5a, in pathogen clearance by phagocytes and to show that this function is induced by IFN-gamma action. The novelty of this Rab5a-IFN-gamma -mediated function resides in the regulation by this GTPase of two sequential processes in the phagosomes. First, Rab5a-GTP promotes phagosomal maturation by regulating the transport of lysosomal proteins to the phagosomes. Second, it regulates Rac2 activation and the assembly of the phagocyte NADPH oxidase to produce toxic free radicals. The combined effects of these Rab5a actions are a more effective destruction of pathogens. Finally, these Rab5a novel functions acquired by the IFN-gamma treatment acted together with another IFN-gamma -mediated feature on L. monocytogenes phagosome (i.e. the blockage of the action of the two membrane lytic L. monocytogenes proteins, listeriolysin and PI-PLC).

    ACKNOWLEDGEMENTS

We are indebted to P. D. Stahl, A. Wandinger-Ness, and M. Zerial for generous gifts of reagents. We especially acknowledge the critical review and suggestions of G. Griffiths, J. P. Gorvel, and G. Li and the encouragement, research facilities, financial support and critical reading of the manuscript by I. V. Sandoval.

    FOOTNOTES

* This was supported in part by Spanish DGCICYT Grant PB94-0035, INCO-DEV program of the European Union Grant ICA4-CT-10001, and National Institutes of Health Grant GM44428.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Supported by a contract from the MEC-Universidad Autónoma de Madrid program to reincorporate doctors.

** Supported by a contract from the MEC-Consejo Superior de Investigaciones Científicas program to reincorporate doctors. To whom correspondence should be addressed: Centro de Biología Molecular "Severo Ochoa," Universidad Autónoma de Madrid, Cantoblanco, 28049 Madrid, Spain. Tel.: 34-91-3978455; Fax: 34-91-3974799; E-mail: calvarez@cbm.uam.es.

Published, JBC Papers in Press, March 21, 2001, DOI 10.1074/jbc.M101639200

    ABBREVIATIONS

The abbreviations used are: IFN-gamma , interferon-gamma ; CFU, colony-forming units; GST, glutathione S-transferase; HBE, homogenization buffer; HBSS-g, Hank's balanced salt solution containing 10 mM glucose; RNI, reactive nitrogen intermediates; ROI, reactive oxygen intermediates; MØ, macrophage; HRP, horseradish peroxidase; PBD, p21-activated kinase-derived binding domain; GTPgamma S, guanosine 5'-3-O-(thio) triphosphate or guanosine 5'-O-(3-thiotriphosphate); PI-PLC, phosphatidyl inositol phospholipase C.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

1. Lorber, B. (1997) Clin. Infect. Dis. 24, 1-11[Medline] [Order article via Infotrieve]
2. Portnoy, D. A. (1992) Curr. Opin. Immunol. 4, 20-24[CrossRef][Medline] [Order article via Infotrieve]
3. Alvarez-Dominguez, C., Roberts, R., and Stahl, P. D. (1997) J. Cell Sci. 110, 731-743[Abstract/Free Full Text]
4. Portnoy, D. A., Chakraborty, T., Goebel, W., and Cossart, P. (1992) Infect. Immun. 60, 1263-1267[Medline] [Order article via Infotrieve]
5. Unanue, E. R. (1997) Immunol. Rev. 158, 11-25[Medline] [Order article via Infotrieve]
6. Der, S. D., Zhou, A., Williams, BRG, and Silverman, R. H. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 15623-15628[Abstract/Free Full Text]
7. Bach, E., Auguet, M., and Schreiber, R. D. (1997) Annu. Rev. Immunol. 15, 563-591[CrossRef][Medline] [Order article via Infotrieve]
8. Taylor, G. A., Collazo, C. M., Yap, G., Nguyen, K., Gregorio, T. A., Taylor, L. S., Eagleson, B., Secrest, L., Southeon, E. A., Reid, S. N., Tessarrollo, L., Bray, M., McVicor, D. N., Komshlies, K. L., Young, H. A., Biron, C. A., Sher, A., and Vande Wonde, G. F. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 751-755[Abstract/Free Full Text]
9. Searle, S., Bright, N. A., Roach, T. I. A., Atkinson, P. G. P., Barton, C. H., Meloen, R. H., and Blackwell, J. M. (1998) J. Cell Sci. 111, 2855-2866[Abstract/Free Full Text]
10. Shiloh, M. U., MacMicking, J. D., Nicholson, S., Brause, J. E., Potter, S., Marino, M., Fang, F., Dinauer, M., and Nathan, C. (1999) Immunity 10, 29-38[Medline] [Order article via Infotrieve]
11. Alvarez-Dominguez, C., Carrasco-Marin, E., Paz-Miguel, J. E., Lopez-Mato, P., and Leyva-Cobian, F. (2000) Immunology 100, 83-89[CrossRef]
12. Alvarez-Dominguez, C., and Stahl, P. D. (1999) J. Biol. Chem. 274, 11459-11462[Abstract/Free Full Text]
13. Alvarez-Dominguez, C., and Stahl, P. D. (1998) J. Biol. Chem. 273, 33901-33904[Abstract/Free Full Text]
14. Quinn, M. T., Evans, T., Loetterle, L. R., Jesiatis, A. J., and Bokoch, G. M. (1993) J. Biol. Chem. 268, 20983-20987[Abstract/Free Full Text]
15. Bernard, V., Bohl, B. P., and Bokoch, G. M. (1999) J. Biol. Chem. 274, 13198-13204[Abstract/Free Full Text]
16. Leyva-Cobian, F., and Carrasco-Marin, E. (1994) Immunol. Lett. 43, 29-37[Medline] [Order article via Infotrieve]
17. Portnoy, D. A., Schreiber, R. D., Connelly, P., and Tilney, L. G. (1989) J. Exp. Med. 170, 2141-2146[Abstract]
18. Schaible, U. E., Sturgill-Koszycki, S., Schlesinger, P. H., and Russell, D. G. (1998) J. Immunol. 160, 1290-1296[Abstract/Free Full Text]
19. Via, L. E., Fratti, R. A., McFalone, M., Pagan-Ramos, E., Deretic, D., and Deretic, V. (1998) J. Cell Sci. 111, 897-905[Abstract/Free Full Text]
20. Meresse, S., Steele-Mortimer, O., Moreno, E., Desjardins, M., Finlay, B., and Gorvel, J. P. (1999) Nat. Cell Biol. 1, E183-E188[CrossRef][Medline] [Order article via Infotrieve]
21. Rossman, M. D., Maida, B. T., and Douglas, S. D. (1990) Cell. Immunol. 126, 268-277[Medline] [Order article via Infotrieve]
22. Montaner, L. J., da Silva, R. P., Sun, J., Sutlerwala, S., Holliushead, M., Vaux, D., and Gordon, S. (1999) J. Immunol. 162, 4606-4613[Abstract/Free Full Text]
23. Byrd, T. F., and Horwitz, M. A. (1993) J. Clin. Invest. 91, 969-976[Medline] [Order article via Infotrieve]
24. Jahraus, A., Tjelle, T. E., Berg, T., Habermann, A., Storrie, B., Ullrich, O., and Griffiths, G. (1998) J. Biol. Chem. 273, 30379-30390[Abstract/Free Full Text]
25. Nielsen, E., Christoforidis, S., Uttenweiler-Joseph, S., Miaczynska, M., Dewitte, F., Wilson, M., Hoflack, B., and Zerial, Z. (2000) J. Cell Biol. 151, 601-612[Abstract/Free Full Text]
26. Singer-Kruger, B., Stenmark, H., Dusterhoft, A., Philippsen, P., Yoo, J. S., Gallwitz, D., and Zerial, M. (1994) J. Cell Biol. 125, 282-298
27. Horazdovsky, B. F., Busch, G. R., and Emr, S. D. (1994) EMBO J. 13, 1297-1309[Abstract]
28. Gerrard, S. R., Bryant, N. J., and Stevens, T. H. (2000) Mol. Biol. Cell 11, 613-626[Abstract/Free Full Text]
29. Bortolussi, R., Vanderbroucke-Grauls, C. M. J. E, Asbeck, B. S., and Verhoef, J. (1987) Infect. Immun. 55, 3197-3203[Medline] [Order article via Infotrieve]
30. Higginbotham, J. N., Lin, T. L., and Pruett, S. B. (1992) Clin. Exp. Immunol. 88, 492-498[Medline] [Order article via Infotrieve]
31. Bastian, N. R., and Hibbs, J. B. (1994) Curr. Opin. Immunol. 6, 131-139[CrossRef][Medline] [Order article via Infotrieve]
32. Bokoch, G. M. (1995) Trends Cell Biol. 5, 109-113[CrossRef]
33. Imamura, H., Takaishi, K., Nakano, K., Kodama, A., Oishi, H., Shiozaki, H., Monden, M., Sasaki, T., and Takai, Y. (1998) Mol. Biol. Cell 9, 2561-2575[Abstract/Free Full Text]
34. Lanzetti, L., Rybin, V., Malabarba, M. G., Christoforidis, S., Scita, G., Zerial, M., and Di Fiore, P. P. (2000) Nature 408, 374-377[CrossRef][Medline] [Order article via Infotrieve]
35. Pizarro-Cerda, J., Desjardins, M., Moreno, E., Akira, S., and Gorvel, J. P. (1999) J. Immunol. 162, 3519-3526[Abstract/Free Full Text]


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