Published online before print August 28, 2007
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* Division of Infectious Diseases, Department of Medicine, University of British Columbia and Vancouver Costal Health Institute, Vancouver, British Columbia, Canada; and
Dipartimento di Scienze e Tecnologie Biologiche ed Ambientali, Universita del Salento, Lecce, Italy
1Correspondence: Division of Infectious Diseases, Rm. 452D, Heather Pavilion East, 2733 Heather St., Vancouver, BC V5Z 3J5, Canada. E-mail: hmama{at}interchange.ubc.ca or deghmane{at}interchange.ubc.ca
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Key Words: lysosomes • GTPase • GTPase activating protein • latex beads • confocal microscopy
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Phagosome maturation is a series of fusion, fission, and trafficking events, which are made possible by a complex network of Rab GTPases that controls endocytic progression to lysosomes [6 , 7 ]. Specifically, Rab5 and Rab7 have been detected on early and late phagosomal membranes, respectively [8 9 10 ]. Furthermore, other emerging Rab-GTPases, such as Rab22a, have recently been shown to be up-regulated by mycobacteria infection and cause decreased Rab7 recruitment to the phagosome [11 ]. Studies focusing on molecules interacting with Rab5 using latex bead-containing phagosomes revealed the recruitment of EEA1 (early endosomal autoantigen 1) to Rab5 and the binding of its FYVE domain to phosphatidylinositol 3-phosphate (PI3P). This phospholipid is generated on phagosomal membranes by the action of another Rab5 effector, hVPS34, a type III phosphatidylinositol 3-kinase [12 ]. In contrast to the situation with latex bead-containing phagsomes, recruitment of EEA1 by Rab5 was shown to be reduced in mycobacterial phagosomes [13 ] and this implicated interference with hVPS34 on the phagosomal membrane [14 ].
Although Rab5 has been well studied and characterized, the function of Rab7 in mycobacterial phagosome is not fully understood. Rab7 has been shown to interact with RILP (Rab7-interacting lysosomal protein) in epithelial cells [15 ]. RILP possesses two distinct domains: one that binds to the GTP-bound form of Rab7 and another that recruits the dynein/dynactin complex [15 , 16 ]. By simultaneously associating with both targets, RILP promotes the interaction of vesicles bearing active Rab7 with lysosomes [15 , 16 ]. The role of RILP in phagosome biogenesis was demonstrated in epithelial cells, where infection by Salmonella typhimurium caused disruption of Rab7-RILP interaction, leading ultimately to phagosome maturation arrest [17 ]. However, there has been no investigation of this type of interaction in macrophages.
In this study, we demonstrate that the Rab7-RILP interaction is also occurring on phagosomes in the macrophage and provide evidence that live mycobacteria disrupt Rab7-RILP interaction by a mechanism dependent on mycobacterial Rab7 deactivating factor.
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-32P]-Guanosine 5'-triphosphate was purchased from Perkin Elmer (Boston, MA, USA).
Antibodies
Mouse monoclonal antibody to glutathione-S-transferase (GST), rabbit anti-GFP, and rabbit anti-Rab7 antibodies were purchased from Sigma-Aldrich. Rabbit anti-RILP antibody was described previously [15
]. Secondary antibodies were purchased from Caltag Laboratories (Burlingame, CA, USA).
Bacteria
Mycobacterium bovis BCG (Pasteur 1173P2) expressing GFP [18
] was used in this study along with M. smegmatis (mc2155) and Mtb H37Rv. BCG expressing red fluorescent protein under the control of the Hsp60 promoter (DsRed-BCG) was prepared using the plasmid construct pSMT3-Ds as described previously [19
]. pSMT3-Ds plasmid was a gift from Dr. M. Abdallah (VU Medical Centre, Amsterdam, the Netherlands). Bacteria were grown in Middlebrook 7H9 broth (Difco) supplemented with 10% (v/v) OADC (oleic acid, albumin, and dextrose solution; Difco) and 0.05% (v/v) Tween 80 (Sigma-Aldrich) at 37°C on a rotating platform (50 rpm). For macrophage infection, bacteria in mid-log phase were harvested by 5 min centrifugation in a microfuge. The culture supernatants were cleared from bacteria and debris by 30 min centrifugation and filtration through 0.22 µm pore sized filters. Killed bacteria were prepared by 2 h incubation at 37°C in the presence of 50 µg/ml gentamicin. S. enterica serovar typhimurium (SL 1344); the corresponding SifA mutant [20
] was provided by Dr. Brett Finlay (University of British Columbia, Vancouver, B.C., Canada) and grown as described previously [20
].
Rab7 and RILP vector constructs
Plasmid vector expressing RILP-GST, Rab7wt-GST, Rab7Q67L-GST (constitutively active), or Rab7T22N-GST (dominant negative) were described previously [15
, 21
]. Plasmids were transformed into Escherichia coli strain BL21 and grown to an OD600 of 0.6 at 37°C, and expression was induced with 0.2 mM IPTG at 22°C overnight. After centrifugation, bacteria were resuspended in PBS containing 1 mM DTT, 0.1 mM PMSF, 1 mg/ml lysozyme for 30 min and lysed by sonication. Bacterial lysates were clarified by high-speed centrifugation, then purified on glutathione-agarose resin (Sigma-Aldrich). Fusion proteins were eluted by 10 mM reduced glutathione in 50 mM Tris-HCl, pH 9.5. Rab7wt-GFP, Rab7Q67L-GFP, and Rab7T22N-GFP plasmids were generated as described previously [9
, 15
]. RILP-DsRed plasmid was provided by Dr. Brett Finlay. These plasmids were prepared using endotoxin-free plasmid maxiprep kit (Sigma-Aldrich).
Cell culture, transfection, and infection
J774A.1 and RAW 264.7 (American Type Culture Collection, Manassas, VA, USA) were maintained in 10 cm diameter culture dishes (Corning Inc., Corning, NY, USA) at a density of 
105 per cm2 in DMEM containing 5% FCS and 1% each of L-glutamine, HEPES, and nonessential amino acids (100x solution, StemCell). Bone marrow-derived macrophages from C57BL/6 mice were generated as described [22
]. In brief, bone marrow cells were differentiated with 50 ng/ml of M-CSF for 3 days in complete DMEM culture medium.
Prior to transfection, RAW cells were washed extensively and harvested by scraping. Approximately 3.5 x 106 cells were seeded per 6 cm diameter culture dish and allowed to grow overnight. Cells were then transfected with the GFP and/or DsRed constructs described above using the Lipofectamine 2000 reagent (Invitrogen) as recommended by the manufacturer. Twenty-four h post-transfection, cells were washed and infected with mycobacteria at multiplicity of infection (MOI) 20:1. Infection was completed with 2 h of pulse, followed by a 4 h chase period.
Intracellular Rab7 staining
The immunofluorescence staining was performed as described previously [23
, 24
]. Briefly, infected cells were fixed in 2.5% paraformaldehyde in PBS, permeabilized with 0.2% saponin, and blocked with 1% normal goat serum in PBS. Rabbit anti-Rab7 was used at 10 µg/ml in 0.2% saponin, and 1% normal goat serum and Texas Red-conjugated goat anti-rabbit IgG was used at a dilution of 1:1000.
Fluorescence microscopy
Transfected or immunostained cells adherent to coverslips were mounted on microscope slides in FluorSaveTM (Calbiochem-Novabiochem, La Jolla, CA, USA) to minimize photobleaching. Slides were then examined by digital confocal microscopy using an Axioplan II epifluorescence microscope (Carl Zeiss Inc., Thornwood, NY, USA) equipped with 63x/1.4 Plan-Apochromat objective (Carl Zeiss Inc.). Images were recorded using a CCD digital camera (Retiga EX, QImaging, Burnaby, BC, Canada) coupled to the Northern Eclipse software (Empix Imaging, Inc., Mississauga, ON, Canada).
Coating of latex microspheres with RILP
Latex beads were coated with RILP protein as described earlier [25
]. In brief, 108 beads were washed twice with 25 mM MES buffer (pH 5.8) and resuspended in 500 µl of the same buffer containing 250 µg/ml of either GST-RILP or GST. After overnight incubation at room temperature on a shaker, latex beads were washed three times with PBS and resuspended in 1 ml of PBS, 1% BSA, 0.1% NaN3. Protein coating was routinely verified prior to each experiment by immunostaining with anti-GST antibodies and FACS analysis.
Pull-down assays
Double transfected (GFP-Rab7/DsRed-RILP) and infected macrophages were lysed in extraction buffer (25 mM Tris-HCl, pH, 7.5; 1 mM EDTA; 1 mM EGTA; 100 mM NaCl; 1% Triton X-100; 0.5% NP-40; 0.2 mM PMSF, and protease inhibitor cocktail) for 20 min at 4°C and debris were removed by high-speed centrifugation. Soluble proteins were mixed with rabbit anti-GFP (1:100) and incubated for 3 h at 4°C. Protein A-agarose beads were added to the mixtures and the samples were incubated an additional 30 min at 4°C. Agarose beads were washed extensively and protein complexes were solubilized in 2 x Laemmeli buffer, then subjected to SDS-PAGE and Western blotting with rabbit anti-RILP to detect associated RILP to Rab7.
GAP (GTPase-activating protein) activity assay
Rab7-GST was resolved by 12% SDS-PAGE and transferred to nitrocellulose membrane. Thereafter, membranes were incubated in renaturation buffer (50 mM HEPES pH 7.2, 5 mM MgAc, 100 mM KAc, 3 mM DTT, 10 mg/ml BSA, 0.1% Triton X-100, 0.3% Tween-20) overnight at 4°C with gentle agitation. Membrane strips were then loaded with [-32P]GTP by incubation with 10 µCi of [-32P]GTP in 100 µl of reaction buffer (50 mM HEPES pH 7.4, 50 mM NaCl, 0.1 mM DTT, 5 mM EDTA and 1 mg/ml BSA) for 10 min at 37°C. Unbound radioactivity was washed extensively with cold reaction buffer and membrane strips were incubated in the presence of bacterial culture supernatants and protease inhibitor cocktails for 4 h at room temperature. After three washes with cold buffer, membrane-associated radioactivity was determined by autoradiography.
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Figure 1. Rab7-RILP interaction in macrophages. (A) RAW cells were cotransfected with RILP-DsRed and Rab7-GFP or Rab7T22N-GFP as described in Materials and Methods, then exposed to 4 µm latex beads for 1 h at 37°C. Partially attached, noningested beads were removed by a 5 min treatment with trypsin-EDTA and extensive washing with HBSS; cells were replenished with culture medium and cultured at 37°C. At 4 h post-phagocytosis, cells were fixed and analyzed by digital confocal microscopy. The yellow signal corresponds to colocalization of Rab7 (green fluorescence) with RILP-DsRed (red fluorescence). (B) Quantification of the confocal data shown in panel A.
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Figure 2. Live BCG disrupts phagosomal recruitment of RILP. (A) Panels a and c: Adherent RAW cells to coverslips were transfected with RILP-DsRed, then infected with either live or gentamicin-killed GFP-BCG as described in Materials and Methods. Cells were then washed extensively, fixed, and examined by confocal microscopy. Panels b and d: RAW cells were infected with live or killed GFP-BCG then fixed/permeabilized and stained for intracellular Rab7 as described in Materials and Methods. Samples were then examined by digital confocal microscopy. The yellow signal in panels a, b, and d corresponds to colocalization of RILP (red fluorescence) with BCG (green fluorescence) and Rab7 (red fluorescence) with BCG (green fluorescence), respectively. (B) Quantification of the confocal data shown in panel A. (C) RILP-transfected RAW cells were loaded with CB-DXT (0.5 mg/ml) for 2 h, washed and chased in culture medium for an additional 2 h. The DXT-loaded cells were then infected with either live or gentamicin-killed GFP-BCG as described in Materials and Methods. Cells were then washed extensively, fixed, and examined by confocal microscopy. The white signal in the top panel (see also the magnification insert a) corresponds to colocalization of RILP (red fluorescence) with BCG (green fluorescence) and dextran (blue fluorescence). (D) Quantification of the confocal data shown in panel C. (E) RAW cells were transfected with Rab7-GFP and RILP-DsRed as indicated, then infected with live BCG, killed BCG, S. typhimurium (wild-type), or S. typhimurium ( SifA). At 4 h post-phagocytosis, cells were lysed and immunoprecipitation with anti-GFP was performed as described in Material and Methods. Membranes were then probed with anti-RILP antibodies (upper panel), striped and reprobed with anti-Rab7 antibodies (lower panel).
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To seek additional evidence to support disruption of Rab7-RILP interaction in BCG-infected macrophages, RAW cells were simultaneously transfected with Rab7-GFP and RILP-DsRed prior to infection with mycobacteria. Cell lysates were then prepared and subjected to Rab7 pull-down with anti-GFP antibodies and Western blotting with anti-RILP. A comparison was made to infection with S. typhimurium, which was reported to secrete an effector, SifA (salmonella induced filaments), responsible for uncoupling Rab7 from RILP in epithelial cells [17 ]. The results obtained (Fig. 2E) showed significant pull-down of Rab7 associated RILP in control uninfected cells and in cells infected with killed BCG or SifA defective Salmonella. In contrast, there was no detectable Rab7-RILP association in cells infected with live BCG and wild-type S. typhimurium. These finding were consistent with the immunofluorescence data (Fig. 2A) and demonstrated that live BCG disrupts the Rab7-RILP interaction in macrophages.
Live BCG inactivates Rab7
The failure of RILP recruitment by phagosomes containing live mycobacteria suggested that Rab7 on phagosomes is likely inactive (i.e., in the GDP-locked form). To address this hypothesis, we took advantage of the findings that RILP interacts only with active Rab7 (GTP-bound form) (Fig. 1)
[15
, 26
] and developed a novel Rab7 activation assay based on Rab7 pull-down with RILP-coated latex beads. To validate this assay, lysate were prepared from RAW cells transfected with GFP-tagged constructs of either wild-type Rab7, Rab7Q67L (constitutively active) or Rab7T22N (constitutively inactive) and incubated with RILP-coated latex beads overnight at 4°C. Beads were then extensively washed and subjected to Western blotting analysis with anti-GFP antibodies. Results in Fig. 3A
showed that only the wild-type and constitutively active mutant interacted with RILP-beads, whereas the inactive Rab7T22N did not. The specificity of Rab7-RILP association was demonstrated by the absence of Rab7 pull-down with the control GST-coated beads.
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Figure 3. Live BCG inactivates Rab7. (A) Raw cells were transfected with wild-type Rab7-GFP (WT), Rab7Q67L-GFP (constitutively active), or Rab7T22N (dominant negative). Cells were lysed 24 h post-transfection and incubated with GST or GST-RILP-coated latex beads. Attached material to the beads was then subjected to SDS-PAGE and Western blotting with anti-GFP antibodies, as described in Material and Methods. Membranes were then stripped and reprobed with anti-RILP antibodies. Lower panel shows 10% of whole-cell lysate (WCL) from each sample used in the assays to show that similar levels of Rab7 constructs were subjected to pull-down with anti-GFP antibodies in each treatment sample. (B) J774 and mouse bone marrow macrophages (BMMØ) were infected with M. smegmatis, gentamicin-killed BCG, or live BCG at MOI 20:1. At 4 h after phagocytosis, cells were lysed and soluble proteins were mixed with RILP-beads overnight at 4°C to pull-down endogenous active Rab7. The beads were then washed and attached material was subjected to Western analysis with anti-Rab7 antibodies (top panel). The membrane was then stripped and reprobed with anti-GST to control for the amounts of RILP-beads used in each treatment sample (middle panel). In the bottom panel, 10% of WCL used in pull-down assay were submitted to Western analysis with anti-Rab7 antibodies to ensure that equal amounts of Rab7 were subjected to pull-down with RILP-beads in each treatment sample. (C) J774 cells were infected with M. smegmatis, gentamicin-killed BCG, or live BCG at MOI 20:1. At 4 h post-phagocytosis, cells were washed extensively with HBSS, detached with cold PBS plus 5 mM EDTA, and fixed with 2% paraformaldehyde. Samples were then subjected to FACS analysis along with the control uninfected cells to determine autofluorescence. Results are expressed as histograms of fluorescence intensity. The percentages of phagocytosis were determined with the WinMDI 2.8 software.
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Live BCG produces a Rab7-inactivating factor
The observation that RILP recruitment to phagosomes in macrophages is dependent on Rab7 activation (switch of GDP form into GTP form) [26
] (Fig. 1)
along with the failure of RILP to interact with Rab7 from cells infected with live, but not killed, BCG suggested that metabolically active bacteria secrete an inhibitor of Rab7 activation. To examine this possibility, J774 cell lysates (source of endogenous Rab7) were incubated with culture filtrate proteins (CFP) of exponentially growing mycobacteria in the presence of protease inhibitors, then assayed with RILP-beads. The results obtained (Fig. 4A
) showed that BCG CFP completely inhibited the binding of Rab7 to immobilized RILP. In contrast, M. smegmatis CFP and the control culture medium alone had no effect on Rab7-RILP interaction. These results suggested that mycobacterial CFP might contain a GAP-like activity that catalyzes the conversion of Rab7-GTP into an inactive GDP form. To examine this hypothesis, Rab7 inactivation was further examined with a GAP activity assay using []32P-GTP. Recombinant Rab7-GST was resolved by SDS-PAGE, transferred to nitrocellulose membranes, and renatured. Membrane strips were then loaded with []32P-GTP and incubated with bacterial CFPs as described in Materials and Methods. Consistent with the RILP pull-down data, the results shown in Fig. 4B
and 4C
indicated that CFP from BCG and Mtb, but not M. smegmatis, contain a factor that catalyzes 75% hydrolysis of the -phosphate from Rab7-GTP on the membrane. Taken together, these results demonstrated that the block of Rab7-RILP interaction in infected macrophages is dependent at least in part on a GAP-like activity.
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Figure 4. BCG secretes a Rab7-inactivating factor. (A) Equal amounts of proteins from a pooled source of J774 cell lysates were incubated for 1 h at room temperature with either BCG, or M. smegmatis culture filtrate proteins (CFP), or culture media (7H9-Tween) alone or left untreated (control). The mixtures were then incubated with GST or GST-RILP-coated latex beads overnight at 4°C. The beads were washed and attached material was subjected to Western analysis with anti-Rab7 antibodies (upper panel). Membranes were then stripped and reprobed with anti-GST to control for the amounts of coated beads used in each treatment sample (lower panel). (B) Rab7-GST was resolved by SDS-PAGE and transferred to nitrocellulose membrane. Rab7 protein bands were then renatured and loaded with 10 µCi of [-32P]-GTP at 37°C. Membranes were extensively washed and either left untreated (control) or incubated with mycobacterial CFP in the presence of protease inhibitors at room temperature for 4 h. Membranes were washed dried and exposed to X-ray film. After film development, membranes were probed with anti-Rab7 antibodies to ensure equal loading of Rab7 protein (lower panel). (C) Band intensities were quantified by densitometry using the ImageJ software (http://rsb.info.nih.gov/ij/) and shown as percent of bound GTP relative to the control untreated Rab7wt strips.
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Figure 5. Rab7-inactivating factor is diffusible. (A) RAW cells were transfected with RILP-DsRed and exposed to live or killed BCG (MOI 20:1) mixed with 4 µm latex beads, particle to cell ratio: 2:1. Noningested bacteria and beads were removed by extensive washing with HBSS; cells were replenished with culture medium and cultured at 37°C. At 4 h post-phagocytosis, cells were fixed and analyzed by digital confocal microscopy. The yellow signal in the top panel corresponds to colocalization of BCG (green fluorescence) with RILP-DsRed (red fluorescence). The red signal (RILP-DsRed) is seen on bead-containing phagosomes of cells infected with killed but not live BCG. (B) Quantification of the confocal data shown in panel A.
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One of the well-characterized downstream effector of Rab7 is the RILP molecule [15 , 16 ]. Accumulated data using epithelial cell line models showed that RILP interacts with activated Rab7 in order to bridge phagosomes with dynein-dynactin, a microtubule-associated motor complex [33 ]. The motors displace phagosomes in the centripetal direction as well as promote the extension of phagosomal tubules toward late endocytic compartments such as lysosomes [33 ].
The results presented in this study demonstrated that the Rab7-RILP interaction is also occurring on latex phagosomes in the macrophage when Rab7 is bound to GTP. However, this interaction is disrupted in macrophages infected with live M. bovis BCG or wild-type Salmonella enterica typhimurium. These data recapitulated previous observations of RILP exclusion from Salmonella phagosomes in infected epithelial cells [17 ]. The same study showed normal acquisition of active Rab7 and explained RILP exclusion by direct interaction between Salmonella virulence factor SifA [17 ] and Rab7, resulting in competitive displacement of RILP. Our observations that BCG phagosomes recruit Rab7 but fail to recruit the marker RILP along with absence of fusion with DXT-loaded lysosomes are consistent with the findings of Clemens et al. [34 ], who reported that in Rab7 transfected HeLa cells, phagosomes containing live Mtb acquired little or no LAMP-1, a strong lysosomal marker [35 , 36 ], despite their tendency to carry more Rab7 than phagosomes of heat-killed Mtb. Our data also show that beyond the BCG phagosome, the RILP molecule colocalizes quite uniformly with lysosomes identified with CB-DXT, in agreement with earlier studies showing abundance of RILP on lysosomes in HeLa cells [15 ].
The results reported in this study suggested that Rab7 is likely converted into Rab7-GDP but remains attached to the membrane of phagosomes containing live BCG, indicating that cytosolic Rab-GDIs [37 , 38 ] is unable to completely deplete Rab7-GDP from the phagosome. These findings are consistent with the observation of significant colocalization of Rab7-T22N (GDP locked form) with latex bead phagosomes (Fig. 1) . Furthermore, the ability of Rab-GDI to extract Rab7-GDP from phagosomes has been shown to be regulated by other chaperone factors such as Hsp90 [39 , 40 ]. Therefore, in our model the possibility of BCG interference with Rab-GDI regulators is an additional mechanism that might contribute to the retention of Rab7-GDP on the phagosome membrane.
The findings presented in this study differ markedly, but do not entirely contradict, those of Via et al. [41 ], who reported that Rab7 level is significantly reduced on BCG phagosomes isolated form J774 macrophages. We believe that Rab7-GDP (low-affinity binding GTPase) dissociated easily from the phagosomal membrane during cell homogenization and phagosome purification by Via et al. Indeed, these authors showed high concentrations of Rab7 (presumably GTP locked form and thus high-affinity binding GTPase) on latex bead phagosomes. Consistent with this hypothesis, a previous study from our group reported a reduced Rab7 staining on phagosomes containing latex beads mechanically isolated from cells that were exposed to mycobacterial lysates [28 ].
The earlier observation that mycobacteria arrested phagosome maturation despite the presence of constitutively active mutant Rab7Q67L [34 ] suggested that Rab7 GTPase is not the only key regulator of phagosome maturation. In fact, Rab5 recruitment has been shown to play a prominent role in phagosome maturation [13 , 42 ], as well as VPS34, which is a common effector of both Rab5 and Rab7 [43 ]. In this context, the finding that anti-Rab7 activity is trafficking beyond the mycobacterial phagosome suggested the possibility of extended activity toward Rab5 and other GTPases within the host cell. Nevertheless, this should not decrease the significance of the Rab7-RILP interaction in macrophages, which is clearly demonstrated here as an important mechanisms regulating phagosome biogenesis targeted by pathogenic mycobacteria to block fusion with lysosomes.
The finding that live, but not killed, BCG reproduces the effect of Salmonella suggested the possibility of an active inhibitory mechanism mediated by expression of a SifA-like protein by Mycobacterium. However, our search in BCG and Mtb genome databases did not reveal any predicted SifA ortholog. This was not entirely unexpected as, in contrast to Salmonella [44
], Mycobacterium does not induce filament formation within the host cell. Meanwhile, our Rab7 activation assay based on affinity binding of Rab7-GTP to RILP-beads clearly demonstrated the predominance of inactive Rab7 (GDP-bound form) in macrophage infected with live but not killed BCG. This inactive form of Rab7 cannot associate with and recruit RILP intracellularly [15
]. Such findings suggested that live mycobacteria express a GAP-like activity inhibiting the function of Rab7 that has been recruited to the phagosome. Indeed, analysis of BCG CFP revealed a secreted activity that catalyzes the hydrolysis of the -phosphate from GTP, leading to the switch of GTP into GDP on the Rab7 molecule. The likelihood of this mechanism within the host cell is supported by previous findings that several pathogenic bacteria secrete proteins that act as either GAP or GEF, and eventually facilitate their pathogenesis [45
]. For instance, secreted ExoS cytotoxin by Pseudomonas aeruginosa disrupts the actin cytoskeleton by acting as GAP for Rho-GTPases [46
]. Similarly, Yersinia pseudotuberculosis secretes a cytotoxic factor, YopE, which depolymerizes the actin stress fiber through its GAP activity for Rho-GTPases [47
]. A recent study identified a nucleoside diphosphate kinase (Ndk) in mycobacteria that acts as a GAP for Rho-GTPases [48
].
In summary, this study provides evidence that mycobacteria actively disrupt Rab7-RILP association as a part of multiple mechanisms to prevent phagosome maturation to fusion with lysosomes. The results suggest that mycobacteria express within the macrophage a diffusible GAP-like protein for Rab7 GTPase, and experiments are in progress to examine whether mycobacterial Ndk or another specific GAP is deactivating Rab7.
Received May 6, 2007; revised July 22, 2007; accepted August 7, 2007.
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