Rab10 Regulates Phagosome Maturation and Its Overexpression Rescues Mycobacterium-Containing

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1 0 B TRA tra Dispatch: November, 00 Journal: TRA CE: Journal Name Manuscript No. Author Received: No of pages: TS: Malaisamy Traffic 00; : John Wiley & Sons A/S doi:./j x Rab Regulates Phagosome Maturation and Its Overexpression Rescues Mycobacterium-Containing Phagosomes Maturation Carla M. P. Cardoso, Luisa Jordao and Otilia V. Vieira, Center for Neuroscience and Cell Biology, University of Coimbra, 0- Coimbra, Portugal Molecular Pathogenesis Centre, Unit of Retrovirus and Associated Infections, Faculty of Pharmacy, University of Lisbon, Av. Forças Armadas, 00-0 Lisbon, Portugal *Corresponding author: Otilia V. Vieira, vieira@cnc.cj.uc.pt Phagosome maturation follows a defined biochemical program and, in the vast majority of cases, the microbe inside the phagosome is killed and digested. Although, an important number of pathogens, including Mycobacterium tuberculosis, which kills around two million people every year, have acquired the ability to survive, and even replicate by arresting phagosomal maturation. To identify more of the machinery involved in phagocytosis and phagosomal maturation, we investigated the function of Rab in engulfment and maturation of inert particles and Mycobacterium bovis bacille Calmette-Guérin BCG. We showed that Rab association with phagosomes is transient and confocal microscopy revealed detectible levels of Rab on phagosomal membranes at very early time-points, occurring even before Rab acquisition. Rab recruitment had strong functional consequence, as the knockdown of endogenous Rab by RNA interference or overexpression of Rab dominantnegative mutant delayed maturation of phagosomes of IgG-opsonized latex beads or heat killed-mycobacteria. These results can be explained, at least in part, by the involvement of Rab in recycling of some phagosomal components. More importantly, overexpression of the constitutively active mutant of Rab partially rescued livemycobacterium-containing phagosomes maturation. Indeed, we found that the membrane harbouring Mycobacterium acquired early endosome antigen (EEA)-, a marker excluded from phagosomes in control cells. Altogether these results indicate that Rab, acting upstream of Rab, plays a prominent role in phagolysosome formation and can modulate Mycobacteriumcontaining phagosomes maturation. Key words: IgG-opsonized particles, Mycobacterium, phagosomal maturation, Rab, Rab, recycling Received April 00, revised and accepted for publication November 00 Phagocytosis allows the internalization of large particles into the cells and plays an essential role in innate immunity. After internalization, the particle surrounded by a membrane and an actin shield loses the actin and starts to interact with the components of the endocytic pathway, a process referred to as phagosome maturation. This interaction is sequential and culminates with fusion to lysosomes, which is crucial to avoid infection (for review see ( )). In the vast majority of cases, the microbe inside the phagosome is killed and digested, but a number of important pathogens, including Mycobacterium tuberculosis, which kill around two million people each year, have acquired the ability to survive, and even replicate, by subverting the host signalling machinery, thus preventing phagosome maturation (). Rab proteins (Rab GTPase family) are known to localize to specific compartments in the endocytic pathway and after recruiting their effectors have a direct role in promoting membrane transport, vesicle tethering and fusion (, ). Several reports suggest that Rab proteins are required for the interaction of phagosomes with components of the endocytic pathway (). Thus, a phagosome, normally maturing into a phagolysosome, undergoes a transition between the stages marked by early endocytic Rabs (e.g. Rab) and late endocytic GTPases (e.g. Rab) (). Indeed Rabs and are the best characterized with respect to their localization to phagosomal membrane and maturation. Rab is known to control fusion of nascent phagosomes to sorting endosomes and is required for the acquisition of Rab. Rab allows phagosomes to interact with late endosomes culminating with the formation of late phagosomes (, ). Other Rabs have been detected on phagosomal membranes, such as Rab, a GTPase involved in endocytic recycling, which appears to be involved in pseudopod extension. It is also known that inactive Rab prevents particle engulfment (). More recently, Smith et al. (), published that the expression of inactive Rabs and inhibited the fusion of phagosomes containing a Salmonella mutant with lysosomes. The function of Rab GTPases can be modulated by pathogens to allow their intracellular growth. Indeed, many studies have shown that selective inclusion or retention of Rab GTPases on vacuolar membranes could regulate the biogenesis of phagosomes inhabited by several bacteria, including Mycobacterium (). In the case of mycobacterial phagosome maturation, block occurred between the stages controlled by Rab and Rab, with no Rab acquisition by the phagosomes (). Subsequently, it has been shown that mycobacterial phagosomes retain 0

2 0 AQ Cardoso et al. several Rab effectors, but prevent phosphatidylinositol -phosphate (PIP) formation and, consequently, the recruitment of PI()P-binding proteins to phagosome maturation, thus contributing to the arrest of phagosomal maturation (,, ). More recently, Rab and Rab were identified as contributors to the arrest of mycobacterial phagosomes by promoting phagosomal fusion with early endocytic organelles and thus playing a role in the maintenance of M. tuberculosis-phagosome in its immature early endosomal-like stage (). There are over 0 Rabs identified in mammalian cells and more than 0 on phagosomal membranes (,, ) but only a few of them, as mentioned above, have been studied in terms of phagocytosis and phagolysosome biogenesis. Proteomic studies have detected Rab on model latex beads phagosomes (, ). Furthermore, the recent, two studies have provided evidence that interfering with Rab function leads to defects in endocytosis/recycling in polarized cells (, 0). This Rab may also play a role in exocytic trafficking in polarized MDCK cells (). Rab has a single isoform, is conserved throughout metazoan evolution and is ubiquitously expressed in mouse and human tissues and belongs to a family of Rab proteins that includes Rab and Rab (, ). Because it is unclear if and how most Rabs function during phagosome maturation, we decided to elucidate the role of Rab on phagocytosis and phagosomal maturation. Here, we examined the requirement of Rab on the engulfment of IgG-opsonized latex beads and Mycobacterium and then on the interaction of the two different phagosomes with components of the endocytic pathway. The former particle, a phagocytosis model system, is internalized via the Fc-receptors and undergoes a canonical maturation process. The latter, a gram-positive acid fast bacteria, is an example of an organism that can inhibit normal phagosomal maturation in order to survive within an infected cell (). We demonstrate that Rab is not required for internalization of phagocytic particles but plays a prominent role in phagosome maturation being acquired upstream of Rab. Thus, this work defines Rab as a new component of the phagosomal maturation machinery. Our results also suggest that overexpression of the activated Rab changes the properties of the Mycobacterium-containing phagosomes. Results Rab associates transiently with phagosomes and A cells stably expressing the FcγRIIA are a suitable model to study Fc-mediated phagocytosis Professional phagocytes can present a challenge to research. Though macrophages, monocytes and predifferentiated neutrophile cell lines exist and are useful models, they are usually refractory to DNA transfection. In addition, it is difficult to study the signalling consequences of a single phagocytic receptor because professional phagocytes express an array of receptors, some of which recognize the same ligand (, ). By contrast, non-professional phagocyte cell lines such as CHO and COS cells are easier to maintain in culture, amenable to genetic manipulation and are suitable to study phagosome maturation (). However, the information on Chinese hamster and African Green monkey genomes is still very limited. Therefore, we decided to generate an engineered human phagocyte cell line to study the involvement of Rab on Fc-mediated phagocytosis and on phagosomal maturation. We decided to use the human A cell line (American Type Culture Collection (ATCC) CRL-, established from an epidermoid carcinoma) and stably expressed the FcγRIIA, an abundant and widely expressed phagocytic receptor, using a retroviral system. We chose opsonized inert particles (latex beads and sheep-red blood cells) as our model to generate phagosomes. As shown in Figure A,B and as previously published for some non-human cell lines (), the expression of Fc receptors on the surface of non-phagocytic cells confers upon them the ability to internalize IgG-coated particles. We then tracked the association of green fluorescent protein GFP-Rab with the model phagosomes in A cells by doing pulse-chase experiments (Figure A C). In parallel, we transfected RAW cells (Figure D F), professional phagocytes, with GFP-Rab and monitored Rab association with phagosomes. The dynamics of Rab in phagosomes of both cell lines was analysed by fluorescence confocal microscopy. In both professional and engineered phagocytes, Rab associated transiently with phagosomes (Figure C,F). The maximal association occurred after the pulse, of min for RAW and min for A cells (=0 min chase), and Rab remained associated with latex-beads phagosomes for a few minutes, in RAW cells (Figures C,F and ). We counted a positive association of Rab with the model phagosome as a distinct ring around the particles (enlargements in Figure A,D). At each time-point investigated, we determined Rab association for at least 0 internalized particles, ensuring we only counted cells where expression of Rab was relatively low. Additionally, to ensure that we counted only internalized beads, we immunostained for human IgG on ice before chase in order to exclude the phagosomes that were formed after the pulse. Rab has been previously identified on purified latex beads phagosomes () and on phagosomes of a Salmonella mutant (). The results we obtained correlated well with previous data describing the association of this Rab with phagosomes, both in professional and non-professional phagocytes. We then decided to follow maturation of phagosomes formed in our engineered phagocyte model and address the functional relevance of Rab acquisition by the phagosome. 0 AQ Traffic 00; : 0 0

3 0 Both the inactive Rab expression and the absence of Rab delay phagolysosome formation The normal nucleotide cycle of Rabs can often be inhibited by the expression of activated or inactive mutant variants. In general, the constitutively active Rab mutants cannot be turned OFF because of their inability to hydrolyse GTP, while expression of dominant-negative mutants sequesters nucleotide exchange factors thus inactivating endogenous Rabs (). To explore the possible role of Rab in phagocytosis and/or phagosomal maturation, we introduced RabTN (inactive form) and RabQL (activated form) () by transfecting them into cells. Expression of the GFP tagged inactive RabTN, activated RabQL and wild-type Rab caused no obvious effect on the internalization of opsonized IgG-particles both in professional and non-professional phagocytes (results not shown). We then analysed the trafficking of phagosomes. Again phagosomal maturation and the functional role of Rab were studied in both professional and engineered phagocytes. The effect of Rab mutants on phagosomal maturation was assessed by measuring the Rab is a Component of the Phagosomal Machinery Figure : Rab associates transiently with phagosomes. A-FcγRIIA and RAW cells were transfected with GFP-Rab and allowed to ingest IgGopsonized latex beads for and min, respectively. A), Distribution of GFP-Rab in A cells. D), Distribution of GFP-Rab in RAW cells; B and E) are the corresponding differential interference contrast (DIC) images. Arrows indicate co-localization between latex vacuoles and Rab. The insets are enlargements of the areas outlined with the squares. Scale bars are μm. Acquisition of Rab by phagosomes in A (C) and RAW. (F) cells. IgG-opsonized beads were added to the cells and allowed to phagocytose for and min, respectively in (C) and (F), (chase time = 0 min), at which time-point external beads were stained using antibodies and excluded from the calculations. The phagosomes were allowed to mature for the times indicated in the graphs abscissa. Data shows the percentages of Rab-positive phagosomes and are means ± SD of three independent experiments (0 phagosomes for each condition). Samples were analysed by fluorescence confocal microscopy. acquisition of the early endosomal marker early endosome antigen (EEA)-, and the late endosomal protein Lamp-. At each time, cells were fixed and processed for immunofluorescence microscopy. For EEA- assessment, A and RAW cells were challenged with IgG-opsonized particles for and min, respectively, and then chased for and min (Figure ). In A cells, at zero min chase there was a decrease in EEA- acquisition (Figure C) in cells expressing RabTN (. ±.%) compared with the control cells (. ±.0%, cells not expressing the inactive Rab, Figure C, black columns). Conversely, the activated RabQL (Figure B) and the wild-type Rab (Figure A) did not affect the acquisition of this early phagosomal marker. Though, the acquisition of EEA- was not attenuated in RAW cells expressing the inactive Rab (Figure E). To examine whether the effect of the inactive Rab was specific for fusion with early endosomes, we next tested Rab effects on phagosome-late endosome/lysosome fusion (Figure ). A cells were challenged with 0 Traffic 00; : 0 0

4 Cardoso et al. 0 Figure : Inactive RabTN attenuates EEA- acquisition in engineered phagocytes. Cells were transfected with wild-type (A and D), activated (B) and inactive Rab (C and E). The transfected A (A-C) and RAW cells (D and E) with the indicated Rab variants were pulsed with IgG-opsonized latex beads for or min, respectively. The external beads were stained and the cells chased for the times indicated in the graphs. Then the cells were fixed, stained for EEA- and examined by confocal microscopy. Black columns represent control cells and empty columns, the cells expressing Rab variants. Results are means (± SD) of three to four independent experiments.*, p < 0.0 comparing differences between non-transfected and transfected cells. IgG-opsonized particles for min and RAW cells for min and then chased for the times indicated on the graphs axis. Acquisition of Lamp-, both in A and RAW cells, was delayed in cells expressing inactive Rab (Figure C F,H). For example, at min chase time, only. ±.% of the phagosomes were positive for Lamp- in contrast with. ±.% in control A cells. At h chase, this difference was attenuated. Similar values were obtained in RAW cells, where at min chase. ±.% of the phagosomes were positive for Lamp- versus. ± % in control cells. In the same way as for EEA- acquisition, the expression of wild-type and activated Rab did not affect Lamp- acquisition (Figure A,B,G). Though Rab is required, both in RAW and A cells for Lamp- acquisition, this requirement was not observed for EEA- in RAW cells (Figure ). RAW are professional phagocytes and phagosomal maturation is faster than in A cells. Furthermore, EEA- association with phagosomal membranes is transient. Thus, taking into account these two parameters (kinetics and association time) it is more difficult to assess effects on EEA- acquisition in RAW cells than in A cells. This can explain the discrepancy observed on the effect of the inactive Rab overexpression on EEA- acquisition. The effect of inactive Rab was a strong indication that Rab function is required for phagosome maturation. To fully demonstrate this, we used RNA interference (RNAi) to deplete the endogenous protein in A cells (Figure ). Retrovirus-mediated expression of short hairpin RNAs followed by elimination of non-transduced cells by antibiotic selection reduced Rab messenger RNA mrna levels by.0 ±.0% [mean ± standard error of the mean standard deviations (SD), n = ]. We could not measure Rab protein levels directly because we did not have a suitable antibody. Yet, expression of transfected GFP-Rab was almost completely suppressed in Rab knockdown cells (Rab KD cells) (Figure A C), suggesting that 0 AQ Traffic 00; : 0 0

5 Rab is a Component of the Phagosomal Machinery Color 0 Figure : Inactive RabTN attenuates phagolysosome formation. Cells were transfected with wild-type (A and G), activated (B) or inactive Rab (C F and H). The transfected A (A F) and RAW cells (G and H) with the indicated Rab variants were pulsed with IgG-opsonized latex beads for and min, respectively. After the indicated chase time the cells were fixed, stained for Lamp- and examined by scanning confocal fluorescence microscopy. Black columns represent control cells and empty columns, the cells expressing Rab variants. Results are means (± SD) of three to four independent experiments. Note that the 0% was set as the value for the latest chase time-point in the Lamp- acquisition experiments. h **, p < 0.0 and ***, p < 0.00 comparing differences between non-transfected and transfected cells. D), shows Lamp- staining at min chase. E), shows cells expressing the inactive GFP-Rab. F), shows the respective DIC image. *, indicates a RabTN transfected cell. Solid and open arrows point to Lamp--positive or -negative phagosomes, respectively. Scale bars is μm. the endogenous Rab had largely been removed. As shown in Figure H K,L (empty columns), the percentage of positive Lamp- phagosomes in Rab-depleted cells was lowered when compared with that of control cells (Figure D G and black columns in Figure L). At min chase, in cells infected with control empty virus, 0 Traffic 00; : 0 0

6 Cardoso et al. Color Figure : Legend on next page. 0 0 Traffic 00; : 0 0

7 Rab is a Component of the Phagosomal Machinery 0 AQ the acquisition of Lamp- was. ±.% compared with. ±.0% in Rab KD cells. The effect of Rab- depletion on the association of Lamp- with engulfed particles was also verified by immunoblotting of purified phagosomes obtained from A cells (Figure M O). Phagosomes isolated from Rab KD cells exhibited less Lamp- on their membranes. The density of the Lamp- band dropped to. and.% in phagosomes from Rab depleted cells, respectively, after a and min chase period when compared with those from control cells (considered 0%). However, at h chase a negligible difference was observed between control and KD cells, suggesting once more that in absence of Rab phagosomal maturation is delayed and not blocked. Taken together, the results presented in Figures showed that phagosomes in engineered phagocytes proceed to mature in a manner that is indistinguishable from that of professional phagocytes. More importantly, we demonstrated that depletion of functional Rab, either through RNA depletion or by overexpression of inactive Rab, delayed phagosome maturation. Rab is acquired before Rab Because Rab plays a prominent role in phagolysosome formation and, in A cells, affects the phagosomal recruitment of the Rab-effector, EEA-, we then asked whether this small G protein acts upstream or in parallel with Rab. To answer this question, we co-transfected RAW cells with Rabs and, challenged these cells with opsonized particles and subjected them to live-confocal microscopy. Live confocal microscopy applied to RAW cells expressing both GFP-Rab and mrfp-rab, a GTPase known to enhance early endosomes fusion (), revealed that Rab was recruited earlier than Rab during phagosome formation (Figure ). Soon after addition of opsonized particles, pseudopods labeled with GFP-Rab extended from the macrophage surface and made contact with the IgG-opsonized particles (Figure ). This is best illustrated in enlargement of the inset zero min, green colour of Figure. A few minutes after recording, Rab was no longer associated with the phagosomal membrane (Figure and enlargement of the inset min). In contrast with what was observed for Rab, mrfp-rab on IgGopsonized particles phagosomes became visible min post-recording and remained associated with the phagosomal membranes for - min (Figure, enlargement of insets, red colour). Of note, Rab and Rab also showed some degree of co-localization (Figure and enlargements of the insets 0. and min). Some co-localization was also observed between Rab and the Rab-effector, EEA- on phagosomal membranes (results not shown). To better define the stage of the phagocytic sequence of Rab acquisition, we examined whether Rab was colocalizing with actin. As can be observed in Figure A F pseudopods and nascent phagosomes decorated with actin (rhodamine-phalloidin staining) also had GFP-Rab, confirming that this small G protein is localized to the phagocytic cup. Because the effect of the inactive Rab on acquisition of Rab-effector, EEA-, can be because of inhibition of the recruitment and/or activation of Rab we decided to monitor the phagosomal association of Rab and two Rab- GEFs, Rabex- and RIN-, with phagosomal membranes in the presence and absence of Rab (Figure H). The overexpression of both wild-type and activated Rab did not seem to affect Rab recruitment but the expression of inactive Rab decreased Rab acquisition (Figure G). However, this effect cannot be attributed to the absence of Rabex- or RIN- since at 0 min chase the phagosomal association of Rabex- and RIN- was not significantly reduced in cells depleted of Rab. Thus, Rab does not seem to be involved in the recruitment of these two Rab-GTP exchange factor (GEFs), although it affected Rab acquisition to the phagosomal membranes. Figure : Depletion of Rab by RNAi delays phagolysosome formation. Effects of Rab depletion by RNAi on phagosomal maturation. Knockdown cells were generated by retrovirus-mediated RNAi and A cells transduced with empty retrovirus were used as control. Rab knockdown (Rab KD) cells suppress expression of GFP-Rab (A C). Control and Rab KD cells were transfected with GFP-Rab using Lipofectamine 000. Equivalent amounts of cell lysate were separated by SDS PAGE and subjected to immunobloting with antibodies to GFP and Lamp- (loading control). The table below shows the downregulation efficiency in terms of mrna, assessed by RT-PCR, and in terms of protein quantified by western blot, using Quantity One Software (BioRad). (B, C) fluorescence images showing the expression of GFP-Rab in control and Rab KD cells, respectively. Green represents GFP-Rab and blue, nuclei visualized with DAPI. Rab knockdown affects Lamp- acquisition (D-O). Lamp- acquisition was assayed in control and Rab KD cells by immunofluorescence, as described in the legend of Figure A typical experiment is illustrated in panels D K. D G represent control cells and H-K, Rab KD cells. (E, I) Show the distribution of Lamp-. (G, K) Show the particles internalized after the pulse, stained with secondary antibodies conjugated with Cy (green colour). Panels D and H show the respective merge images. Panels F and J show the corresponding DIC images. Solid and open arrows point to Lamp--positive and -negative phagosomes after a chase of min, respectively (E and I). Scale bars are μm. (L) Quantification of the effect of Rab depletion on Lamp- acquisition. Black columns represent control and empty columns, Rab KD cells. The presence of Lamp- on phagosomes (L) was assessed after a min pulse of phagocytosis followed by or 0 min chase. Data are means ± SD of three separate experiments (00 cells were counted in each). *, p < 0.0 comparing differences between control and Rab-depleted cells. The effect of Rab depletion on Lamp- acquisition was also assessed by western blot of isolated phagosomes from cells exposed to opsonized particles for min and then chased for, and 0 min (M O), as indicated. μg of protein were loaded in each lane. 0 AQ Traffic 00; : 0 0

8 Color 0 Cardoso et al. The effects of RabTN on EEA- and Rab acquisition suggest that Rab functions upstream of Rab, though it is not known the role of Rab on Rab acquisition but it is possible that the former supplies signals for acquisition of the latter. Overexpression of activated Rab changes the properties of Mycobacterium containing phagosome Some pathogens regulate host trafficking pathways by the selective inclusion or retention of Rab GTPases on membranes of the vacuoles that they occupy in host cells during infection. Furthermore, Rab is acquired very early and the phenotype of phagosomes in Rab-depleted cells bears some resemblance to that of mycobacterial phagosomes. Both are deficient in Lamp proteins and appearing to be affected at an early stage of maturation. We therefore tested whether Rab is recruited to phagosomes containing intracellular surviving mycobacteria. As shown in Figure A,C Rab associated poorly with mycobacterial phagosomes. Indeed, Rab was found more frequently in phagosomes containing Mycobacterium smegmatis, a species completely killed within macrophages by h, (results not shown) or with phagosomes containing inert particles (Figure ) than in phagosomes with Mycobacterium bovis bacille Calmette Guérin (BCG). This raises the possibility that impairment of Rab recruitment is part of the mechanism used by pathogenic mycobacteria to prevent phagolysosome formation. Interestingly, we observed Figure : Distribution of Rab and Rab during phagocytosis. Recruitment of Rab and Rab to phagosomes. RAW cells were co-transfected with GFP-Rab and mrfp-rab challenged with opsonized sheep-red blood cells (RCBs) and imaged by live confocal microscopy. The time-lapse live imaging shows that recruitment of Rab occurs before Rab. First panel is the DIC image corresponding to the fluorescence image at time = 0 min. The indicated times refer to the time elapsed after the onset of recording and not to the time after the addition of the RBCs. The insets show separately the fluorescence of GFP-Rab and mrfp-rab of the areas boxed by the dotted line. Scale bars are μm. The images are representative of 0 cells from five independent experiments. that activated Rab had greater association with live Mycobacterium-containing phagosomes (Figure A, light grey columns and B). Therefore, we decided to address whether the overexpression of wild-type and the Rab mutants could change the fate of the Mycobacteriumcontaining phagosome. As can be observed in the graphs D and E of the Figure, expression of activated Rab partially overcame phagosome maturation block imposed by live mycobacteria. Indeed, we found that the membrane harbouring live Mycobacterium acquired PI()P, as judged by the association of the -FYVE construct that binds specifically to this inositide, and EEA-, markers excluded from the phagosomes in control cells (cells not transfected, black columns) (Figure D E). However, no effect of the activated Rab overexpression on Lamp- acquisition was observed within the period investigated (Figure G). Concerning Rab-acquisition by the live BCG phagosomes, the overexpression of the dominant-negative Rab did not seem to have major effects on the kinetics of association nor on the percentage of Rab-positive phagosomes but overexpression of activated Rab increased the percentage of Rab-positive phagososmes (Figure F). Thus, Rab overexpression changed the properties of the live Mycobaterium-containing phagosomes, though it was not sufficient for fusion with lysosomes. All the experiments to assess the involvement of Rab in maturation described above were mainly performed 0 AQ Traffic 00; : 0 0

9 Color 0 with IgG-opsonized particles that are engulfed via the Fc-receptors. Mycobacterium recognition by phagocytes is likely to involve more than one phagocytic receptor. Thus, we decided to assess whether Rab had any functional relevance in maturation of phagosomes containing heat-killed Mycobacterium that, like IgG-opsonizedred Rab is a Component of the Phagosomal Machinery Figure : Rab is recruited earlier than Rab to phagosomal membranes. RAW (A C) and A (D F) cells expressing GFP-Rab (A, D) were allowed to initiate phagocytosis of RBCs (A) or latex beads (B), followed by fixation and staining for F-actin with rhodamine-phalloidin (B and E); C and F are the corresponding DIC images. Arrows point to phagocytic cups/phagosomes positive for actin and Rab. Scale bars are μm. Quantification of the effect of RabTN on Rab acquisition by phagosomes (G). Acells were co-transfected with the indicated Rab constructs and Rab for h. Quantification of the effect of Rab-depletion on RIN- and Rabex- acquisition byphagosomes (H). Control cells, Black columns. Rab KD cells, empty columns. In G and H the cells were challenged with IgG-opsonized particles for min. Then the cells were fixed and examined by confocal microscopy. Data are means ± SD of three separate experiments (00 cells were counted in each); *, p < 0.0 comparing differences between cells expressing wild-type and Rab variants. blood cells (RBC) or latex beads, follows the canonical maturation process, meaning that the heat-killed Mycobacterium phagosome fuses with lysosomes. As shown in Figure H, overexpression of the dominant negative Rab mutant also delayed fusion of heatkilled Mycobacterium phagosomes with late endocytic 0 Traffic 00; : 0 0

10 Cardoso et al. Color 0 Figure : Rab overexpression modulates Mycobacterium-containing phagosomes maturation. Acquisition of Rab, EEA-, PI()P, Rab and Lamp- by BCG-containing phagosomes. RAW macrophages (control) or RAW macrophages transfected with wild-type mrfp-rab, mrfp-rabql or mrfp-rabtn were infected with live (A G) or fed with heat killed BCG (H) for min. The recruitment of wild-type Rab (empty columns) and activated Rab (light grey columns) by phagosomes was assessed (A). In macrophages infected with live BCG we observed less recruitment of Rab to phagosomal membranes. A representative picture of the recruitment of activated Rab is shown (B) and the arrow points to a positive phagosome. A representative picture of absence of recruitment of wild-type Rab is shown (C) and the arrow points to a negative phagosome. In red, mrfp-rabql (B) or mrfp-wildtype Rab (C) and in green, live BCG. Scale bars are μm. Insets show a magnification of the boxed areas showing a Rab-positive or negative phagosomes. Quantification of the acquisition of EEA- (D), PI()P (E), Rab (F) and Lamp- (G) by phagosomes containing live BCG. Both control and transfected macrophages were followed and compared. In E and F, RAW macrophages were co-transfected with a construct consisting of two tandem PI()P-binding FYVE domains of EEA- fused with GFP (FYVE-GFP) or with Rab-GFP and the indicated Rab constructs. A delay in Lamp- acquisition by phagosomes containing heat killed BCG was observed in macrophages transfected with mrfp-rabtn (H). Black columns represent control; light grey columns, RabQL; empty columns, wild-type Rab and dark grey, RabTN. Data are means ± SD of three separate experiments (00 cells were counted in each); *, p < 0.0; **, p < 0.0. Figure continued on next Page compartments. Taken together our results suggest that Rab is a central and general regulator of the transition of the nascent phagosome to early phagosome, affecting the transition of this organelle into a degradative compartment. Rab affects recycling from phagosomal membranes We next examined by which mechanism(s) Rab contributes to the phagosomal maturation delay. Because maturing phagosomes undergo both fusion with other organelles and concomitant fission and because Rab 0 Traffic 00; : 0 0

11 Rab is a Component of the Phagosomal Machinery Color 0 Figure : Continued from previous page was found to be involved in recycling, we tested whether it also contributes to the recycling of phagosomal components back to the plasma membrane. In this context the effect of Rab depletion on the detachment of a plasma membrane marker GFP-GL-GPI (glycosyl phosphatidyl inositol-anchored green fluorescent protein) and on transferrin receptor recycling on phagosomes was assessed. The results of these studies are shown in Figure. The plasma membrane protein, GFP-GL-GPI that demarcated the forming and early phagosomes (results not shown) was eliminated with time from maturing phagosomes both in control (Figure A, black columns) and Rab-depleted (Figure A, empty columns) cells. However, the percentage of GFP-GL-GPI-positive phagosomes was higher in Rab-depleted than in control cells. This effect was more pronounced at min chase where in control cells only. ±.% of the phagosomes were positive for GFP-GL-GPI versus. ±.0% for Rab KD cells. Similar results were obtained in cells expressing the inactive Rab mutant (results not shown). Rab depletion showed some tendency to slow transferrin receptor recycling from phagosomal membranes (Figure B) but it was less pronounced than in GFP-GL-GPI recycling (Figure A). Jointly, these observations indicate that the loss of components of the phagosomal membranes is affected in the absence of Rab, which can explain, at least in part, the delay in phagolysosome formation. Discussion Intracellular bacterial pathogens have evolved highly specialized mechanisms to enter and survive within their eukaryotic hosts resulting in devastating diseases such as tuberculosis, food poisoning, etc. In order to do this, bacterial pathogens need to avoid host cell degradation and obtain nutrients and biosynthetic precursors, as well as evade detection by the host immune system. To create an intracellular niche that is favorable for replication, some intracellular pathogens inhibit the maturation of the phagosome or exit the endocytic pathway by modifying the identity of their phagosome through the exploitation of the host cell trafficking pathways. If we take into account that in eukaryotic cells, organelle identity is determined, in part, by the composition of active Rab GTPases on their membranes, the retention or exclusion of Rab proteins from the phagosomal membranes can explain, at least in part, their escape from the degradative endosomal or lysosomal 0 Traffic 00; : 0 0

12 0 Cardoso et al. pathways. Indeed, evidence indicates that a number of intracellular bacterial pathogens, including M.tuberculosis, Salmonella enterica serovar Typhimurium, Legionella pneumophila and Chlamydia pneumoniae exploit Rab GTPases to regulate the biogenesis of the phagosomes and to ensure their intracellular survival (,,, ). In this context, the aim of this study was to better understand the requirement of Rab for phagosomal maturation in professional and engineered phagocytes with two different types of phagocytic particles. We focused on the small G protein, Rab for two reasons: i) it has been identified in phagosomes by proteomic and other approaches (,, ); ii) and it has been implicated in membrane trafficking ( ). Our results indicate that Rab is required for phagosome maturation, both in professional and non-professional phagocytes. More Figure : Rab is involved in recycling. Control and Rab- depleted A cells stably expressing the FcγRIIA were plated on glass coverslips and then challenged with opsonized IgG-particles. Effect of Rab-depletion on the disappearance of GFP-GL-GPI from the phagosomes (A). The cells were transfected with GFP-GL-GP h before the phagocytosis assays. Effect of Rab-depletion on transferrin receptor recycling from the phagosomes (B). The experiment was performed as described in the Material and Methods section. Control cells, black columns, Rab- depleted cells, empty columns. The cells were exposed to latex beads for min and then chased for the times indicated on the graphs.then the cells were fixed and examined by confocal microscopy. Data are means ± SD of three separate experiments (00 cells were counted in each); ***, p < 0.00 comparing differences between control and Rab-depleted A cells. importantly, the overexpression of its activated mutant changes, at least in part, the properties of Mycobacterium containing phagosome. The functional involvement of Rab on phagocytosis and phagosomal maturation was tested by overexpression of Rab mutants and by RNAi. In cells expressing the inactive RabTN or in Rab KD cells phagolysosome formation was delayed, indicating a role of Rab on phagolysosome biogenesis. In earlier proteomic studies Rab was detected along with Rab in purified latex beads phagosomal preparations (). Here, confocal live microscopy showed that Rab is acquired even before Rab, suggesting that the kinetic aspects of bulk biochemical studies () and video microscopy cannot be directly compared. However, it is difficult to determine where exactly Rab may act. We observed that in 0 Traffic 00; : 0 0

13 0 coverslip-grown A cells GFP-tagged wild-type Rab localizes mostly at the Golgi and partially with early endosomal compartments (supporting information Figure S). The constitutively active mutant (RabQL) localized mainly in the early endosomal vesicles as shown by pronounced co-localization with Rab (Figure S), while the inactive mutant of Rab (RabTN) localized to the Golgi and distributed in the cytosol. These results are consistent with previous studies of Rab localization, which showed that GFP-Rab is distributed between the Golgi and endosomes in polarized intestinal cells in Caenorhabditis elegans (). Rab proteins usually localize to the sites at which they regulate membrane trafficking events. Based on the general principle that Rab proteins interact with their effector proteins only after GDP-to-GTP exchange, it is likely that the GTP-bound Rab mutants accumulate on their target membrane domains in association with their effectors, whereas the inactive GDP-bound state indicate where membrane recruitment takes place (). Thus, in A cells it is likely that Rab is involved in transport of carrier membranes between these two compartments, Golgi and endosomes. How exactly Rab acts, remains to be answered. It is thought that Rab acts in a similar recycling pathway as Raba because both Rabs share a common GTPase activating protein (GAP), As0 () and can interact with Myosin V (), a motor protein implicated in membrane recycling. However, the exact role(s) of Rab is/are not well established. Recent work from three groups has placed Rab within the endocytic/recycling system in C. elegans () intestinal cells, MDCK cells and in human gastric parietal cells (0, ). Another study suggests that Rab controls exocytic transport in polarized MDCK cells, but the authors do not rule out that it may also be needed, directly or indirectly, for endocytic trafficking (). Indeed, this phenotype was obtained only by expression of the activated Rab, which is likely to sequester and thus inactivate a number of effector proteins, whereas the corresponding inactive mutant or Rab-depletion by RNAi had subtle or no effects (). Furthermore, Secp, the yeast homologue of Rab, is involved in the exocytic pathway (). However, it is possible that the multiple functions of Secp in yeast are carried out by separate Rabs in higher organisms, so that Rab, Rab and possibly Rab could represent split versions of Secp. Many parallels are likely to exist between endocytosis and phagocytosis. Based on our own data Rab seems to promote phagosome maturation by mediating recycling from phagosomal components, which is necessary for phagolysosome biogenesis. In fact, Rab and Rab, known to be involved in the recycling pathway, are present on phagosomal membranes, suggesting that recycling processes might also be important for phagolysosome biogenesis (, ). Alternatively, Rab may affect interactions between phagosomes and early endosomal organelles because of its involvement in the biosynthetic pathway. In its absence, Golgi to endosome transport Rab is a Component of the Phagosomal Machinery might be affected, disrupting the supply of components to the endocytic machinery, indirectly affecting maturation. Several lines of evidence indicate that the mycobacterial phagosome maintains an early endosomal niche in infected macrophages. First, the phagosome associates with early endosomal Rabs or transferrin receptor but not with late endosomal GTPase, Rab, or late endosomal tetraspanin, CD, (, ). Second, mycobacterial phagosomes are known to have diminished generation of PIP, leading to exclusion of PIP-binding effectors, such as Hrs and EEA-, thus preventing membrane tethering necessary for onward progression to late endosomes (, ). Third, it has been well established that mycobacterial phagosomes do not acquire the vacuolar proton pump necessary for acidification of phagosomes (). Hitherto, the studies of early endocytic Rabs in bacterial pathogenesis have been restricted to only a few Rabs, mainly Rab (, ). Furthermore, recent work has identified more two host GTPases, Rab and Raba, involved in the blockade of normal phagolysosome biogenesis during mycobacterial infection (, ). Here, we identified Rab, a protein that plays an essential role in early phagosome-to-phagolysosome transition, as part of the host machinery that is also targeted by Mycobacterium. Indeed, we found that the recruitment of Rab is considerably lower in the case of live BCG than in that of opsonized latex particles or heat-killed BCG. These findings suggest that the inability to recruit Rab may contribute to the arrest of phagosomal maturation induced by pathogenic mycobacteria. More importantly, this arrest can be partially rescued by overexpressing the activated Rab. Thus, here we identified Rab as a potential attractive target that should be taken into account for the development of therapeutics that can restore normal phagosome maturation. With over 0 Rabs identified in the human genome, much remains to be learned regarding the role of these regulatory GTPases in general intracellular trafficking, and in phagosomal and endosomal organelles, as well as in microbial pathogenesis. Our data indicate that the process of phagosome maturation is far more complex than a single Rab to Rab transition. Based on the inhibitory effect of the inactive Rab on EEA- phagosomal acquisition we suggest that Rab is a central regulator of the transition of a nascent phagosome to an early phagosome, affecting the transition of this organelle into a degradative compartment. Furthermore, we were able to monitor the trafficking of a pathogenic bacterium and characterize the divergence of its vacuolar compartment from the normal degradative pathway. Future studies are required to define the molecular mechanisms by which Rab controls phagosome maturation and how intracellular pathogens, like Mycobacterium, modulate their function. Understanding the precise function of Rab and how it is connected with other parts of the transport machinery, such as cargo adaptors, molecular 0 Traffic 00; : 0 0

14 0 Cardoso et al. motors, vesicle fusion proteins and other Rab GTPases, will require the identification of all Rab-effector proteins. Materials and Methods Antibodies Monoclonal were anti-human (HB), anti-mouse (ABL-) Lamp- (Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA) and anti-mouse (M) Flag (Sigma-Aldrich). Polyclonal were goat anti-eea- (Santa Cruz Biotechnology) and goat anti-gfp (D. Drechsel, MPI-CBG). Secondary antibodies were from Molecular Probes or from Jackson Immunoresearch. Rhodamin-phalloidin was from Molecular Probes and DAPI from Fluka. Plasmids and generation of A cells stably expressing the FcγRIIA GFP-GL-GPI, Canine pgfp-rab-wt (wild-type), pgfp-rabtn (inactive) and pgfp-rabql (active) plasmid constructs, were kindly provided by Kai Simons (Max-Planck Institute for Molecular Cell Biology and Genetics, Dresden, Germany) and were described elsewhere (). Tagged- Rab and -FYVE-GFP were kindly provided by Marino Zerial (Max-Planck Institute for Molecular Cell Biology and Genetics). Flag-mRIN- and FlagmRabex- plasmid constructs were kindly provided by Dr Sayaka Yoshiki (Kyoto University, Japan). Human FcγRIIA tagged with myc was subcloned into the retroviral vector pbabe-puro. In order to generate A cells stably expressing FcγRIIA, retrovirus production, cells infection and selection were done as described before (). Cells and transfection Mouse macrophages RAW. and A cells (ATCC) were grown in Dulbecco s Modified Eagle Medium (DMEM) high glucose, supplemented with % fetal calf serum (FCS), mm L-glutamine, 0 U/mL of penicillin, and 0 μg/ml of streptomycin (Life Technologies). The cells were transiently transfected with Lipofectamine 000 (Invitrogen), according to the manufacturer s recommendations. Phagocytosis and bacterial infection Fresh sheep-red blood cells (RBC) were opsonized with rabbit anti-sheep RBC antibody (:). Latex beads were opsonized with mg of human IgG/mL. Opsonization was for either h at room temperature or overnight at C. The onset of phagocytosis was synchronized by allowing the particles to bind to cells on ice for min, and ingestion was then initiated by incubation at C. Excess particles were washed away with phosphate-buffered saline (PBS) and, when indicated, the cells were incubated in culture medium at C for the specified additional chase period. To identify adherent particles that were not internalized, the cells were incubated at C with Cy Cy/Cy-labeled donkey anti-rabbit IgG (:00) or Cy/Cy/Cy-labeled donkey anti-human IgG (:00) for min. For live cell imaging, RAW macrophages grown on circular coverslips were transferred to a live cell imaging chamber and bathed in Hepesbuffered RPMI (Sigma). In these experiments phagocytosis was not synchronized. M. bovis BCG harbouring a pmn plasmid (a kind gift from Michael Niederweis, Department of Microbiology, University of Alabama at Birmingham, Birmingham) was grown until exponential phase on Middlebrook s H broth medium (Difco) supplemented with % Middlebrook OADC (Oleic acid, albumin, dextrose, catalase) (Difco) (v/v) and 0.0% Tween 0 (v/v) at C/% CO. Media were supplemented with μg/ml hygromycin (Roche) for selection of recombinant mycobacteria. Bacteria were prepared for macrophage infection as described previously (). A bacterial suspension with an optical density OD 00 nm of 0. was added to RAW macrophages, centrifuged at 0 g for minat C and incubated at Cina%CO atmosphere for min. Extracellular mycobacteria were removed as described previously () and the protocol described for latex beads was followed. Immunofluorescence and confocal microscopy Cells were grown on -well plates and fixed with Methanol or % PFA for min, followed by quenching of the aldehyde groups with glycine or ammonium chloride and permeabilization with Triton-X0. Cells were then incubated with the primary antibodies (EEA- or Lamp-) for h at room temperature, washed and finally incubated with the secondary antibodies conjugated with a fluorophore for another h. The antibody dilutions used were EEA- (:0), Lamp- (:0) and Flag (:0). Flag antibody was incubated for h at room temperature, washed and finally incubated with the secondary antibody anti-mouse conjugated with a Cy-fluorophore for another h. DAPI at a final concentration of nm was incubated for 0 min at room temperature. Live cell imaging was performed in CO - independent medium at C. Both live and fixed samples were analysed by using the lymphocyte separation medium LSM 0 META point-scan confocal laser microscope (Zeiss) with a oil immersion objective. Transferrin uptake and recycling from phagosomal membranes Control and Rab-depleted A cells stably expressing the FcγRIIA were plated on glass coverslips, washed with cell culture medium, and incubated for h at C in serum-free DMEM. Then 0.0 μg/ml rhodaminconjugated transferrin (Molecular Probes) and opsonized latex-beads were added to the cells followed by incubation at C for min. After this incubation time (pulse) the cells were extensively washed and the extracellular beads stained with secondary antibodies. Finally the cells were chased for various time intervals, fixed with % PFA and imaged. RNAi The Rab target sequence has been described previously (). Retrovirusmediated RNAi and quantitative reverse transcription polymerase chain reaction RT-PCR were done as before (). Phagosomes isolation and western blot Phagosomes were isolated by the method of Desjardins et al. () from A cells grown on cm diameter petri dishes to 0 0% confluence. The protein concentration of the phagosomal preparation was determined with the bicinchoninic acid assay (Pierce), using albumin as the standard. Isolated phagosomes or whole-cell extracts were solubilized in Laemmli s sample buffer, resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (GE), and transferred onto polyvinylidene difluoride membranes. The membranes were blocked for min with % milk in PBS and 0.% Tween 0. Antibodies to GFP were used at :000 dilution, and Lamp- antibody was used at a :00. Immunoreactive bands were visualized by ECF reagent (GE healthcare). Statistical analysis Results were expressed as the means ± (SD). Statistical significance was assessed by the Student t-test (two-tailed). A p value of <0.0 was considered to be statistically significant. Acknowledgments This work was supported by the Research grant PTDC/SAU- MII//00 from the Foundation for Science and Technology of the Portuguese Ministry of Science and Higher Education (FCT). CMPC and LJ are holders of post-doctoral fellowships from the FCT, ref.: SFRH/BPD//00 and ref.: SFRH/BPD//00, respectively. We 0 AQ Traffic 00; : 0 0

15 AQ 0 thank Profs Kai Simons, Marino Zerial and Sayaka Yoshiki for plasmids. We are grateful to Matadouro da Beira Serra for providing the sheep blood for the experiments. We are very grateful to Dr Marta Miaczynska, International Institute of Molecular and Cell Biology, Warsaw, Poland, and Dr Elsa Anes, Faculty of Pharmacy, University of Lisbon, Portugal, for critical reading of the manuscript. Supporting Information Additional Supporting Information may be found in the online version of this article: Figure S: Rab localizes primarily to the Golgi and partially in early endosomes. GFP-Rab was introduced into A cells grown on coverslips by transient transfection and analysed by immunofluorescence confocal microscopy. Weakly expressing cells were chosen for imaging to avoid mislocalization because of high levels of expression. (A G) After methanol or PFA fixation and Triton-X0 permeabilization, cells were stained as described in Material and Methods section. Wild-type mrfp- Rab and GFP-FAPP-PH (A), wild-type GFP-Rab and mrfp-rab (C), GFPRabQL and mrfprab (D), mrfp-rabtn and GFP- FAPP-PH (E), or GPF-GL-GPI and mrfp-rabwt (G) were co-expressed transiently in A cells. (B) Wild-type Rab and endogenous EEA- or (F) wild-type Rab and Lamp-. Scale bars are μm. Please note: Wiley-Blackwell are not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article. References. Aderem A, Underhill DM. Mechanisms of phagocytosis in macrophages. Annu Rev Immunol ;:.. Greenberg S, Grinstein S. Phagocytosis and innate immunity. Curr Opin Immunol 00;:.. Vieira OV, Botelho RJ, Grinstein S. Phagosome maturation: aging gracefully. Biochem J 00;: 0.. Swanson JA. Shaping cups into phagosomes and macropinosomes. Nat Rev Mol Cell Biol 00;:.. Via LE, Deretic D, Ulmer RJ, Hibler NS, Huber LA, Deretic V. Arrest of mycobacterial phagosome maturation is caused by a block in vesicle fusion between stages controlled by rab and rab. J Biol Chem ;:.. Zerial M, McBride H. Rab proteins as membrane organizers. Nat Rev Mol Cell Biol 00;:.. Grosshans BL, Ortiz D, Novick P. Rabs and their effectors: achieving specificity in membrane traffic. Proc Natl Acad Sci U S A 00;:.. Desjardins M, Huber LA, Parton RG, Griffiths G. Biogenesis of phagolysosomes proceeds through a sequential series of interactions with the endocytic apparatus. J Cell Biol ;:.. Vieira OV, Bucci C, Harrison RE, Trimble WS, Lanzetti L, Gruenberg J, Schreiber AD, Stahl PD, Grinstein S. Modulation of Rab and Rab recruitment to phagosomes by phosphatidylinositol -kinase. Mol Cell Biol 00;:.. Meresse S, Steele-Mortimer O, Finlay BB, Gorvel JP. The rab GTPase controls the maturation of Salmonella typhimurium-containing vacuoles in HeLa cells. Embo J ;:.. Cox D, Lee DJ, Dale BM, Calafat J, Greenberg S. A Rab-containing rapidly recycling compartment in macrophages that promotes phagocytosis. Proc Natl Acad Sci U S A 000;:0.. Smith AC, Heo WD, Braun V, Jiang X, Macrae C, Casanova JE, Scidmore MA, Grinstein S, Meyer T, Brumell JH. A network of Rab GTPases controls phagosome maturation and is modulated by Salmonella enterica serovar Typhimurium. J Cell Biol 00;:. Rab is a Component of the Phagosomal Machinery. Kyei GB, Vergne I, Chua J, Roberts E, Harris J, Junutula JR, Deretic V. Rab is critical for maintenance of Mycobacterium tuberculosis phagosome maturation arrest. Embo J 00;:.. Fratti RA, Backer JM, Gruenberg J, Corvera S, Deretic V. Role of phosphatidylinositol -kinase and Rab effectors in phagosomal biogenesis and mycobacterial phagosome maturation arrest. J Cell Biol 00;:.. Vieira OV, Harrison RE, Scott CC, Stenmark H, Alexander D, Liu J, Gruenberg J, Schreiber AD, Grinstein S. Acquisition of Hrs, an essential component of phagosomal maturation, is impaired by mycobacteria. Mol Cell Biol 00;: 0.. Stuart LM, Boulais J, Charriere GM, Hennessy EJ, Brunet S, Jutras I, Goyette G, Rondeau C, Letarte S, Huang H, Ye P, Morales F, Kocks C, Bader JS, Desjardins M, et al. A systems biology analysis of the Drosophila phagosome. Nature 00;:.. Rogers LD, Foster LJ. The dynamic phagosomal proteome and the contribution of the endoplasmic reticulum. Proc Natl Acad Sci U S A 00;:0.. Garin J, Diez R, Kieffer S, Dermine JF, Duclos S, Gagnon E, Sadoul R, Rondeau C, Desjardins M. The phagosome proteome: insight into phagosome functions. J Cell Biol 00;: 0.. Chen CC, Schweinsberg PJ, Vashist S, Mareiniss DP, Lambie EJ, Grant BD. RAB- is required for endocytic recycling in the Caenorhabditis elegans intestine. Mol Biol Cell 00;:. 0. Babbey CM, Ahktar N, Wang E, Chen CC, Grant BD, Dunn KW. Rab regulates membrane transport through early endosomes of polarized Madin-Darby canine kidney cells. Mol Biol Cell 00;:.. Schuck S, Gerl MJ, Ang A, Manninen A, Keller P, Mellman I, Simons K. Rab is involved in basolateral transport in polarized Madin-Darby canine kidney cells. Traffic 00;: 0.. Pereira-Leal JB, Seabra MC. Evolution of the Rab family of small GTP-binding proteins. J Mol Biol 00;: 0.. Gurkan C, Lapp H, Alory C, Su AI, Hogenesch JB, Balch WE. Largescale profiling of Rab GTPase trafficking networks: the membrome. Mol Biol Cell 00;:.. Jordao L, Bleck CK, Mayorga L, Griffiths G, Anes E. On the killing of mycobacteria by macrophages. Cell Microbiol 00;:.. Mukhopadhyay S, Gordon S. The role of scavenger receptors in pathogen recognition and innate immunity. Immunobiology 00;0:.. Underhill DM, Gantner B. Integration of Toll-like receptor and phagocytic signaling for tailored immunity. Microbes Infect 00;:.. Downey GP, Botelho RJ, Butler JR, Moltyaner Y, Chien P, Schreiber AD, Grinstein S. Phagosomal maturation, acidification, and inhibition of bacterial growth in nonphagocytic cells transfected with FcgammaRIIA receptors. J Biol Chem ;:.. Indik ZK, Park JG, Hunter S, Mantaring M, Schreiber AD. Molecular dissection of Fc gamma receptor-mediated phagocytosis. Immunol Lett ;:.. Stenmark H, Parton RG, Steele-Mortimer O, Lutcke A, Gruenberg J, Zerial M. Inhibition of rab GTPase activity stimulates membrane fusion in endocytosis. Embo J ;:.. Cortes C, Rzomp KA, Tvinnereim A, Scidmore MA, Wizel B. Chlamydia pneumoniae inclusion membrane protein Cpn0 interacts with multiple Rab GTPases. Infect Immun 00;:.. Derre I, Isberg RR. Legionella pneumophila replication vacuole formation involves rapid recruitment of proteins of the early secretory system. Infect Immun 00;:.. Junutula JR, De Maziere AM, Peden AA, Ervin KE, Advani RJ, van Dijk SM, Klumperman J, Scheller RH. Rab is involved in membrane trafficking between the Golgi complex and endosomes. Mol Biol Cell 00;:.. Sano H, Eguez L, Teruel MN, Fukuda M, Chuang TD, Chavez JA, Lienhard GE, McGraw TE. Rab, a target of the AS0 Rab GAP, is required for insulin-stimulated translocation of GLUT to the adipocyte plasma membrane. Cell Metab 00;:.. Roland JT, Lapierre LA, Goldenring JR. Alternative splicing in class V myosins determines association with Rab. J Biol Chem 00;:.. Lapierre LA, Avant KM, Caldwell CM, Ham AJ, Hill S, Williams JA, Smolka AJ, Goldenring JR. Characterization of immunoisolated 0 Traffic 00; : 0 0

16 Cardoso et al. 0 human gastric parietal cells tubulovesicles: identification of regulators of apical recycling. Am J Physiol Gastrointest Liver Physiol 00;:G G.. Salminen A, Novick PJ. A ras-like protein is required for a post-golgi event in yeast secretion. Cell ;:.. Damiani MT, Pavarotti M, Leiva N, Lindsay AJ, McCaffrey MW, Colombo MI. Rab coupling protein associates with phagosomes and regulates recycling from the phagosomal compartment. Traffic 00;:.. Sonnichsen B, De Renzis S, Nielsen E, Rietdorf J, Zerial M. Distinct membrane domains on endosomes in the recycling pathway visualized by multicolor imaging of Rab, Rab, and Rab. J Cell Biol 000;:0.. Sturgill-Koszycki S, Schlesinger PH, Chakraborty P, Haddix PL, Collins HL, Fok AK, Allen RD, Gluck SL, Heuser J, Russell DG. Lack of acidification in Mycobacterium phagosomes produced by exclusion of the vesicular proton-atpase. Science ;:.. Roberts EA, Chua J, Kyei GB, Deretic V. Higher order Rab programming in phagolysosome biogenesis. J Cell Biol 00;:.. Schuck S, Manninen A, Honsho M, Fullekrug J, Simons K. Generation of single and double knockdowns in polarized epithelial cells by retrovirus-mediated RNA interference. Proc Natl Acad Sci U S A 00;:.. Desjardins M, Celis JE, van Meer G, Dieplinger H, Jahraus A, Griffiths G, Huber LA. Molecular characterization of phagosomes. J Biol Chem ;: Traffic 00; : 0 0

17 QUERIES TO BE ANSWERED BY AUTHOR IMPORTANT NOTE: Please mark your corrections and answers to these queries directly onto the proof at the relevant place. DO NOT mark your corrections on this query sheet. Queries from the Copyeditor: AQ. AQ. AQ. AQ. AQ. AQ. AQ. AQ. Please confirm if this abbreviation needs to be spelt out. If yes, please provide the expansion. Please confirm if this abbreviation needs to be spelt out. If yes, please provide the expansion. Please confirm if this abbreviation needs to be spelt out. If yes, please provide the expansion. Please confirm if this abbreviation needs to be spelt out. If yes, please provide the expansion. Please confirm if this abbreviation needs to be spelt out. If yes, please provide the expansion. Please confirm if this abbreviation needs to be spelt out. If yes, please provide the expansion. Please confirm if this abbreviation needs to be spelt out. If yes, please provide the expansion. As per journal style, Supporting information data will only be published online, so we have deleted the supplementary data provided and retained the Supporting Information figure caption. Please check and confirm.

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