Acquisition of Hrs, an essential component of phagosomal maturation, is impaired by mycobacteria

Pathogenic mycobacteria survive within macrophages by precluding the fusion of phagosomes with late endosomes or lysosomes. Because the molecular determinants of normal phagolysosome formation are poorly understood, the sites targeted by mycobacteria remain unidentified. We found that Hrs, an adaptor molecule involved in protein sorting, associates with phagosomes prior to their fusion with late endosomes or lysosomes. Recruitment of Hrs required the interaction of its FYVE domain with phagosomal phosphatidylinositol 3-phosphate, but two other attachment sites were additionally involved. Depletion of Hrs by use of small interfering RNA impaired phagosomal maturation, preventing the acquisition of lysobisphosphatidic acid and reducing luminal acidification. As a result, the maturation of phagosomes formed in Hrs-depleted cells was arrested at an early stage, characterized by the acquisition and retention of sorting endosomal markers. This phenotype is strikingly similar to that reported to occur in phagosomes of cells infected by mycobacteria. We therefore tested whether Hrs is recruited to phagosomes containing mycobacteria. Hrs associated readily with phagosomes containing inert particles but poorly with mycobacterial phagosomes. Moreover, Hrs was found more frequently in phagosomes containing avirulent Mycobacterium smegmatis than in phagosomes with the more virulent Mycobacterium marinum. These findings suggest that the inability to recruit Hrs contributes to the arrest of phagosomal maturation induced by pathogenic mycobacteria.     

Role of COPI in phagosome maturation

Phagosomes mature by sequentially fusing with endosomes and lysosomes. Vesicle budding is presumed to occur concomitantly, mediating the retrieval of plasmalemmal components and the regulation of phagosomal size. We analyzed whether fission of vesicles from phagosomes requires COPI, a multimeric complex known to be involved in budding from the Golgi and endosomes. The role of COPI was studied using ldlF cells, that harbor a temperature-sensitive mutation in epsilon-COP, a subunit of the coatomer complex. These cells were made phagocytic toward IgG-opsonized particles by heterologous expression of human FcgammaRIIA receptors. Following incubation at the restrictive temperature, epsilon-COP was degraded in these cells and their Golgi complex dispersed. Nevertheless, phagocytosis persisted for hours in cells devoid of epsilon-COP. Retrieval of transferrin receptors from phagosomes became inefficient in the absence of epsilon-COP, while clearance of the FcgammaRIIA receptors was unaffected. This indicates that fission of vesicles from the phagosomal membrane involves at least two mechanisms, one of which requires intact COPI. Traffic of fluid-phase markers and aggregated IgG-receptor complexes along the endocytic pathway was abnormal in epsilon-COP-deficient cells. In contrast, phagosome fusion with endosomes and lysosomes was unimpaired. Moreover, the resulting phagolysosomes were highly acidic. Similar results were obtained in RAW264.7 macrophages treated with brefeldin A, which precludes COPI assembly by interfering with the activation of adenosine ribosylation factor. These data indicate that neither phagosome formation nor maturation are absolutely dependent on COPI. Our findings imply that phagosomal maturation differs from endosomal progression, which appears to be more dependent on COPI-mediated formation of carrier vesicles.     

Phagocyte meets prey: uptake, internalization, and killing of bacteria by Dictyostelium amoebae

Dictyostelium cells are professional phagocytes that avidly consume bacteria, their natural prey. Fluorescent probes have allowed us to monitor the initial steps in this process in living cells. Using probes that bind to F-actin, we have visualized the assembly and disassembly of actin filaments responsible for extending the phagocytic cup to engulf a bacterium, and, after the phagosome has sealed, the assembly of new actin filaments to propel the phagosome away from the site of uptake. Using bacteria expressing fluorescent proteins that are susceptible to proteolysis, we have monitored the loss of that fluorescent signal and the staining of the bacterial contents with neutral red, indicating permeabilization of the bacterial cell wall and acidification of the cytoplasm. We find that acidification occurs during a period of microtubule-based transport that promotes fusion of the phagosome with microtubule-associated acidic endosomes. Actin-powered phagosome internalization, transport of the phagosome along microtubules, proteolysis and acidification of bacterial contents, all typically occur within the first six or seven minutes after formation of the phagosome. Thus, tracking individual phagosomes has revealed that early steps in phagosome maturation occur much more rapidly than had been inferred from previous population studies.     

Phagocyte meets prey: Uptake, internalization, and killing of bacteria by Dictyostelium amoebae

Dictyostelium cells are professional phagocytes that avidly consume bacteria, their natural prey. Fluorescent probes have allowed us to monitor the initial steps in this process in living cells. Using probes that bind to F-actin, we have visualized the assembly and disassembly of actin filaments responsible for extending the phagocytic cup to engulf a bacterium, and, after the phagosome has sealed, the assembly of new actin filaments to propel the phagosome away from the site of uptake. Using bacteria expressing fluorescent proteins that are susceptible to proteolysis, we have monitored the loss of that fluorescent signal and the staining of the bacterial contents with neutral red, indicating permeabilization of the bacterial cell wall and acidification of the cytoplasm. We find that acidification occurs during a period of microtubule-based transport that promotes fusion of the phagosome with microtubule-associated acidic endosomes. Actin-powered phagosome internalization, transport of the phagosome along microtubules, proteolysis and acidification of bacterial contents, all typically occur within the first six or seven minutes after formation of the phagosome. Thus, tracking individual phagosomes has revealed that early steps in phagosome maturation occur much more rapidly than had been inferred from previous population studies.     

Three sorting nexins drive the degradation of apoptotic cells in response to PtdIns(3)P signaling

Apoptotic cells are swiftly engulfed by phagocytes and degraded inside phagosomes. Phagosome maturation requires phosphatidylinositol 3-phosphate [PtdIns(3)P], yet how PtdIns(3)P triggers phagosome maturation remains largely unknown. Through a genomewide PtdIns(3)P effector screen in the nematode Caenorhabditis elegans , we identified LST-4/SNX9, SNX-1, and SNX-6, three BAR domain-containing sorting nexins, that act in two parallel pathways to drive PtdIns(3)P-mediated degradation of apoptotic cells. We found that these proteins were enriched on phagosomal surfaces through association with PtdIns(3)P and through specific protein-protein interaction, and they promoted the fusion of early endosomes and lysosomes to phagosomes, events essential for phagosome maturation. Specifically, LST-4 interacts with DYN-1 (dynamin), an essential phagosome maturation initiator, to strengthen DYN-1's association to phagosomal surfaces, and facilitates the maintenance of the RAB-7 GTPase on phagosomal surfaces. Furthermore, both LST-4 and SNX-1 promote the extension of phagosomal tubules to facilitate the docking and fusion of intracellular vesicles. Our findings identify the critical and differential functions of two groups of sorting nexins in phagosome maturation and reveal a signaling cascade initiated by phagocytic receptor CED-1, mediated by PtdIns(3)P, and executed through these sorting nexins to degrade apoptotic cells.     

Contrasting requirements for ubiquitylation during Fc receptor-mediated endocytosis and phagocytosis

Fc receptors on leukocytes mediate internalization of antibody-containing complexes. Soluble immune complexes are taken up by endocytosis, while large antibody-opsonized particles are internalized by phagocytosis. We investigated the role of ubiquitylation in internalization of the human FcgammaRIIA receptor by endocytosis and phagocytosis. A fusion of FcgammaRIIA to green fluorescent protein (GFP) was expressed in ts20 cells, which bear a temperature-sensitive mutation in the E1 ubiquitin-activating enzyme. Uptake of soluble IgG complexes mediated by FcgammaRIIA-GFP was blocked by incubation at the restrictive temperature, indicating that endocytosis requires ubiquitylation. In contrast, phagocytosis and phagosomal maturation were largely unaffected when ubiquitylation was impaired. FcgammaRIIA-GFP was ubiquitylated in response to receptor cross-linking. Elimination of the lysine residues present in the cytoplasmic domain of FcgammaRIIA impaired endocytosis, but not phagocytosis. The proteasomal inhibitor clasto-lactacystin beta-lactone strongly inhibited endocytosis, but did not affect phagocytosis. These studies demonstrate a role for ubiquitylation in the endocytosis of immune receptors, and reveal fundamental differences in the mechanisms underlying internalization of a single receptor depending on the size or multiplicity of the ligand complex.     

Phosphatidylinositol 3-kinases in carcinoembryonic antigen-related cellular adhesion molecule-mediated internalization of Neisseria gonorrhoeae

Neisseria gonorrhoeae can be internalized by mammalian cells through interactions between bacterial opacity-associated (Opa) adhesins and members of the human carcinoembryonic antigen-related cellular adhesion molecule (CEACAM) family. We examined the role of phosphatidylinositol 3-kinases (PI3Ks) in gonococcal invasion of epithelial cell lines expressing either CEACAM1 or CEACAM3. CEACAM3-mediated internalization, but not that mediated by CEACAM1, was accompanied by localized and transient accumulation of the class I PI3K product phosphatidylinositol 3,4,5-trisphosphate at sites of bacterial engulfment. Inhibition of phosphatidylinositol 3-kinases reduced CEACAM3-mediated uptake but, paradoxically, led to an increase in intracellular survival of bacteria internalized via either CEACAM1 or CEACAM3, suggesting additional roles for PI3K products. Consistent with this finding, the class III PI3K product phosphatidylinositol 3-phosphate accumulated and persisted in the membrane of gonococcal phagosomes after internalization. Inhibition of PI3K blocked phagosomal acquisition of the late endosomal marker lysosome-associated membrane protein 2 and reduced phagosomal acidification. Inhibiting phagosomal acidification with concanamycin A also increased survival of intracellular gonococci. These results suggest two modes of action of phosphatidylinositol 3-kinases during internalization of gonococci: synthesis of phosphatidylinositol 3,4,5-trisphosphate is important for CEACAM3-mediated uptake, while phosphatidylinositol 3-phosphate is needed for phagosomal maturation and acidification, which are required for optimal bacterial killing.     

Phagosomes fuse with late endosomes and/or lysosomes by extension of membrane protrusions along microtubules: role of Rab7 and RILP

Nascent phagosomes must undergo a series of fusion and fission reactions to acquire the microbicidal properties required for the innate immune response. Here we demonstrate that this maturation process involves the GTPase Rab7. Rab7 recruitment to phagosomes was found to precede and to be essential for their fusion with late endosomes and/or lysosomes. Active Rab7 on the phagosomal membrane associates with the effector protein RILP (Rab7-interacting lysosomal protein), which in turn bridges phagosomes with dynein-dynactin, a microtubule-associated motor complex. The motors not only displace phagosomes in the centripetal direction but, strikingly, promote the extension of phagosomal tubules toward late endocytic compartments. Fusion of tubules with these organelles was documented by fluorescence and electron microscopy. Tubule extension and fusion with late endosomes and/or lysosomes were prevented by expression of a truncated form of RILP lacking the dynein-dynactin-recruiting domain. We conclude that full maturation of phagosomes requires the retrograde emission of tubular extensions, which are generated by activation of Rab7, recruitment of RILP, and consequent association of phagosomes with microtubule-associated motors.     

Membrane trafficking along the phagocytic pathway

Phagosome maturation involves extensive remodelling of the phagosomal membrane as a result of intracellular transport events. Newly formed phagosomes exchange membrane-associated and soluble proteins with early endosomes by fusion. Budding of vesicles from the phagosome and fusion with Golgi-derived vesicles may also contribute to the remodelling of the phagosomal compartment. As a consequence of changes in membrane composition, phagosomes acquire the ability to fuse with late endocytic compartments. In vitro reconstitution and other studies suggest that the trafficking events underlying phagosome maturation require several GTP-binding proteins, including Rab5 and Galphas', NSF-SNAP-SNARE complexes and coatomers.     

Mycobacteria and the intraphagosomal environment: take it with a pinch of salt(s)!

Ancient protozoan phagocytes and modern professional phagocytes of metazoans, such as macrophages, employ evolutionarily conserved mechanisms to kill microbes. These mechanisms rely on microbial ingestion, followed by maturation of the phagocytic vacuole, or so-called phagosome. Phagosome maturation includes a series of fusion and fission events with the host cell endosomes and lysosomes, leading to a rapid increase of the degradative properties of the vacuole and to the destruction of the ingested microbe within a very hostile intracellular compartment, the phagolysosome. Historically, the mechanisms and weapons used by phagocytes to kill microbes have been separated into different classes. Phagosomal acidification, together with the production of reactive oxygen and nitrogen species, the selective manipulation of various ions in the phagosomal lumen, and finally the engagement of a battery of acidic hydrolases, are well-recognized players in this process. However, it is relatively recently that interconnections among these mechanisms have become apparent. In this review, we will focus on some emerging concepts about these interconnected aspects of the warfare at the host-pathogen interface, using mostly Mycobacterium tuberculosis as an example of intracellular pathogen. In particular, recent discoveries on the role of phagosomal ions and other chemicals in the control of pathogens, as well as mechanisms evolved by intracellular pathogens to circumvent or even exploit the weapons of the host cell will be discussed.     

Expression of the protein kinase C substrate pleckstrin in macrophages: association with phagosomal membranes.

Despite evidence suggesting that protein kinase C (PKC) isoforms are important in phagocytosis by Fcgamma receptors, the mechanisms by which the substrates of these kinases act are largely unknown. We have investigated the role of one PKC substrate, pleckstrin, in cells of the monocyte/macrophage lineage. Pleckstrin expression in mouse macrophages was induced severalfold in response to bacterial LPS and IFN-gamma. In unstimulated cells, the protein was largely confined to the cytosol. Upon ingestion of IgG-opsonized zymosan particles (OPZ), however, pleckstrin accumulated on the phagosomal membrane. This association was transient, being maximal after 15 min and declining thereafter. Similar kinetics of association was also seen for both filamentous actin and the delta isoform of PKC. Ingestion of OPZ was found to induce phosphorylation of pleckstrin. To examine whether phosphorylation was required for phagosomal association, pleckstrin was expressed in CHO-IIA cells that stably express the FcgammaRIIA receptor and are competent for phagocytosis of OPZ. In these cells, both wild-type pleckstrin and mutants in which the phosphoacceptor sites had been mutated to either alanine (nonphosphorylatable) or glutamine (pseudophosphorylated) were found to accumulate on OPZ phagosomes. Thus, association of pleckstrin with phagosomes is independent of its phosphorylation. Our findings suggest that pleckstrin may serve as an intracellular adaptor/targeting protein in response to particulate stimuli. By targeting interacting ligands to the phagosomal compartment, pleckstrin may serve to regulate phagocytosis and/or early steps during maturation of the phagosome.     

Phagocytosis and phagosome maturation are regulated by calcium in J774 macrophages interacting with unopsonized prey

Phagocytosis by neutrophils, macrophages, and other professional phagocytes requires rapid remodeling of actin. Early phagosomes are surrounded by a rim of F-actin that is disassembled during phagosomoal maturation. Breakdown of periphagosomal F-actin and phagolysosome fusion are calcium dependent processes in neutrophils interacting with serum-opsonized prey, but appears to be calcium independent in macrophages interacting with serum- or IgG-opsonized prey. In the present study, we found that calcium was necessary for phagocytosis, breakdown of periphagosomal F-actin, and phagosomal maturation in J774 macrophages interacting with unopsonized prey. We also observed that lipophosphoglycan (LPG) from Leishmania donovani promastigotes required calcium to exert its inhibitory effect on macrophage phagocytosis and periphagosomal F-actin breakdown. We conclude that calcium is essential for phagocytosis, depolymerization of periphagosomal F-actin, and phagosomal maturation in J774 macrophages interacting with unopsonized prey, as well as for proper functioning of LPG.     

Decoupling Internalization,Acidification and Phagosomal-Endosomal/lysosomal Fusion during Phagocytosis of InlA Coated Beads in Epithelial Cells

Background

Phagocytosis has been extensively examined in ‘professional’ phagocytic cells using pH sensitive dyes. However, in many of the previous studies, a separation between the end of internalization, beginning of acidification and completion of phagosomal-endosomal/lysosomal fusion was not clearly established. In addition, very little work has been done to systematically examine phagosomal maturation in ‘non-professional’ phagocytic cells. Therefore, in this study, we developed a simple method to measure and decouple particle internalization, phagosomal acidification and phagosomal-endosomal/lysosomal fusion in Madin-Darby Canine Kidney (MDCK) and Caco-2 epithelial cells.

Methodology/Principal Findings

Our method was developed using a pathogen mimetic system consisting of polystyrene beads coated with Internalin A (InlA), a membrane surface protein from Listeria monocytogenes known to trigger receptor-mediated phagocytosis. We were able to independently measure the rates of internalization, phagosomal acidification and phagosomal-endosomal/lysosomal fusion in epithelial cells by combining the InlA-coated beads (InlA-beads) with antibody quenching, a pH sensitive dye and an endosomal/lysosomal dye. By performing these independent measurements under identical experimental conditions, we were able to decouple the three processes and establish time scales for each. In a separate set of experiments, we exploited the phagosomal acidification process to demonstrate an additional, real-time method for tracking bead binding, internalization and phagosomal acidification.

Conclusions/Significance

Using this method, we found that the time scales for internalization, phagosomal acidification and phagosomal-endosomal/lysosomal fusion ranged from 23–32 min, 3–4 min and 74–120 min, respectively, for MDCK and Caco-2 epithelial cells. Both the static and real-time methods developed here are expected to be readily and broadly applicable, as they simply require fluorophore conjugation to a particle of interest, such as a pathogen or mimetic, in combination with common cell labeling dyes. As such, these methods hold promise for future measurements of receptor-mediated internalization in other cell systems, e.g. pathogen-host systems.     

LAMP proteins are required for fusion of lysosomes with phagosomes

Lysosome-associated membrane proteins 1 and 2 (LAMP-1 and LAMP-2) are delivered to phagosomes during the maturation process. We used cells from LAMP-deficient mice to analyze the role of these proteins in phagosome maturation. Macrophages from LAMP-1- or LAMP-2-deficient mice displayed normal fusion of lysosomes with phagosomes. Because ablation of both the lamp-1 and lamp-2 genes yields an embryonic-lethal phenotype, we were unable to study macrophages from double knockouts. Instead, we reconstituted phagocytosis in murine embryonic fibroblasts (MEFs) by transfection of FcgammaIIA receptors. Phagosomes formed by FcgammaIIA-transfected MEFs obtained from LAMP-1- or LAMP-2- deficient mice acquired lysosomal markers. Remarkably, although FcgammaIIA-transfected MEFs from double-deficient mice ingested particles normally, phagosomal maturation was arrested. LAMP-1 and LAMP-2 double-deficient phagosomes acquired Rab5 and accumulated phosphatidylinositol 3-phosphate, but failed to recruit Rab7 and did not fuse with lysosomes. We attribute the deficiency to impaired organellar motility along microtubules. Time-lapse cinematography revealed that late endosomes/lysosomes as well as phagosomes lacking LAMP-1 and LAMP-2 had reduced ability to move toward the microtubule-organizing center, likely precluding their interaction with each other.     

Ezrin promotes actin assembly at the phagosome membrane and regulates phago-lysosomal fusion

Phagosome maturation is defined as the process by which phagosomes fuse sequentially with endosomes and lysosomes to acquire an acidic pH and hydrolases that degrade ingested particles. While the essential role of actin cytoskeleton remodeling during particle internalization is well established, its role during the later stages of phagosome maturation remains largely unknown. We have previously shown that purified mature phagosomes assemble F-actin at their membrane, and that the ezrin-radixin-moesin (ERM) proteins ezrin and moesin participate in this process. Moreover, we provided evidence that actin assembly on purified phagosomes stimulates their fusion with late endocytic compartments in vitro. In this study, we further investigated the role of ezrin in phagosome maturation. We engineered a structurally open form of ezrin and demonstrated that ezrin binds directly to the actin assembly promoting factor N-WASP (Neural Wiskott-Aldrich Syndrome Protein) by its FERM domain. Using a cell-free system, we found that ezrin stimulates F-actin assembly on purified phagosomes by recruiting the N-WASP-Arp2/3 machinery. Accordingly, we showed that the down-regulation of ezrin activity in macrophages by a dominant-negative approach caused reduced F-actin accumulation on maturing phagosomes. Furthermore, using fluorescence and electron microscopy, we found that ezrin is required for the efficient fusion between phagosomes and lysosomes. Live-cell imaging analysis supported the notion that ezrin is necessary for the fusogenic process itself, promoting the transfer of the lysosome content into the phagosomal lumen.     

Fusion of newly formed phagosomes with endosomes in intact cells and in a cell-free system

Phagosomes are membrane-bound vesicles, formed by the receptor-mediated internalization of particulate ligands, which exchange soluble and membrane proteins with other endocytic compartments as a part of their maturation process. This exchange of material is undoubtedly mediated by fusion of phagosomes with other membrane-bound compartments of the endocytic pathway. By using a particulate probe (fixed Staphylococcus aureus coated with mouse anti-dinitrophenol monoclonal antibody) localized in phagosomes and a soluble probe (dinitrophenol-derivitized beta-glucuronidase) internalized by receptor-mediated endocytosis, we have studied phagosome-endosome and phagosome-lysosome fusion in intact cells and in a cell-free system. Vesicle fusion was assessed by measuring beta-glucuronidase activity associated with S. aureus particles after lysis of the membranes. In intact macrophages, newly formed phagosomes fused with early endosomes and with lysosomes. Fusion with lysosomes was observed to commence after a short lag period of about 5 min. In broken-cell preparations, phagosomes were able to fuse with early endosomes. It was not possible to reconstitute phagosome-lysosome fusion in vitro. In vitro phagosome-endosome fusion required energy and cytosolic- and membrane-associated proteins. A nonhydrolyzable analog of GTP stimulated fusion at low cytosol concentrations and inhibited fusion at high cytosol concentrations. These observations indicate that the mechanisms mediating phagosome-endosome fusion are similar to those described for endosome-endosome fusion. Our results suggest that exchange of material with endosomes is an important step in the process of phagosome maturation.     

Mycobacterium's arrest of phagosome maturation in macrophages requires Rab5 activity and accessibility to iron

Many mycobacteria are intramacrophage pathogens that reside within nonacidified phagosomes that fuse with early endosomes but do not mature to phagolysosomes. The mechanism by which mycobacteria block this maturation process remains elusive. To gain insight into whether fusion with early endosomes is required for mycobacteria-mediated inhibition of phagosome maturation, we investigated how perturbing the GTPase cycles of Rab5 and Rab7, GTPases that regulate early and late endosome fusion, respectively, would affect phagosome maturation. Retroviral transduction of the constitutively activated forms of both GTPases into primary murine macrophages had no effect on Mycobacterium avium retention in an early endosomal compartment. Interestingly, expression of dominant negative Rab5, Rab5(S34N), but not dominant negative Rab7, resulted in a significant increase in colocalization of M. avium with markers of late endosomes/lysosomes and increased mycobacterial killing. This colocalization was specific to mycobacteria since Rab5(S34N) expressing cells showed diminished trafficking of endocytic tracers to lysosomes. We further demonstrated that maturation of M. avium phagosomes was halted in Rab5(S34N) expressing macrophages supplemented with exogenous iron. These findings suggest that fusion with early endosomes is required for mycobacterial retention in early phagosomal compartments and that an inadequate supply of iron is one factor in mycobacteria's inability to prevent the normal maturation process in Rab5(S34N)-expressing macrophages.     

Dectin-1 Activation Controls Maturation of β-1,3-Glucan-containing Phagosomes

Elimination of fungal pathogens by phagocytes requires phagosome maturation, a process that involves the recruitment and fusion of intracellular proteins. The role of Dectin-1, a β-1,3-glucan receptor, critical for fungal recognition and triggering of Th17 responses, to phagosomal maturation has not been defined. We show that GFP-Dectin-1 translocates to the fungal phagosome, but its signal decays after 2 h. Inhibition of acidification results in retention of GFP-Dectin-1 to phagosome membranes highlighting the requirement for an acidic pH. Following β-1,3-glucan recognition, GFP-Dectin-1 undergoes tyrosine phosphorylation by Src kinases with subsequent Syk activation. Our results demonstrate that Syk is activated independently of intraphagosomal pH. Inhibition of Src or Syk results in prolonged retention of GFP-Dectin-1 to the phagosome signifying a link between Syk and intraphagosomal pH. β-1,3-glucan phagosomes expressing a signaling incompetent Dectin-1 failed to mature as demonstrated by prolonged Dectin-1 retention, presence of Rab5B, failure to acquire LAMP-1 and inability to acidify. Phagosomes containing Candida albicans also require Dectin-1-dependent Syk activation for phagosomal maturation. Taken together, these results support a model where Dectin-1 not only controls internalization of β-1,3-glucan containing cargo and triggers proinflammatory cytokines, but also acts as a master regulator for subsequent phagolysosomal maturation through Syk activation.     

Live Salmonella modulate expression of Rab proteins to persist in a specialized compartment and escape transport to lysosomes

We investigated the intracellular route of Salmonella in macrophages to determine a plausible mechanism for their survival in phagocytes. Western blot analysis of isolated phagosomes using specific antibodies revealed that by 5 min after internalization dead Salmonella-containing phagosomes acquire transferrin receptors (a marker for early endosomes), whereas by 30 min the dead bacteria are found in vesicles carrying the late endosomal markers cation-dependent mannose 6-phosphate receptors, Rab7 and Rab9. In contrast, live Salmonella-containing phagosomes (LSP) retain a significant amount of Rab5 and transferrin receptor until 30 min, selectively deplete Rab7 and Rab9, and never acquire mannose 6-phosphate receptors even 90 min after internalization. Retention of Rab5 and Rab18 and selective depletion of Rab7 and Rab9 presumably enable the LSP to avoid transport to lysosomes through late endosomes. The presence of immature cathepsin D (48 kDa) and selective depletion of the vacuolar ATPase in LSP presumably contributes to the less acidic pH of LSP. In contrast, proteolytically processed cathepsin D (M(r) 17,000) was detected by 30 min on the dead Salmonella-containing phagosomes. Morphological analysis also revealed that after uptake by macrophages, the dead Salmonella are transported to lysosomes, whereas the live bacteria persist in compartments that avoid fusion with lysosomes, indicating that live Salmonella bypass the normal endocytic route targeted to lysosomes and mature in a specialized compartment.     

Rab14 is critical for maintenance of Mycobacterium tuberculosis phagosome maturation arrest

Mycobacterium tuberculosis arrests phagosomal maturation in infected macrophage, and, apart from health significance, provides a superb model system to dissect the phagolysosomal biogenesis pathway. Here, we demonstrate a critical role for the small GTPase Rab14 in maintaining mycobacterial phagosome maturation block. Four-dimensional microscopy showed that phagosomes containing live mycobacteria accumulated Rab14 following phagocytosis. The recruitment of Rab14 had strong functional consequence, as a knockdown of endogenous Rab14 by siRNA or overexpression of Rab14 dominant-negative mutants (Rab14S25N and Rab14N125I) released the maturation block and allowed phagosomes harboring live mycobacteria to progress into phagolysosomes. Conversely, overexpression of the wild-type Rab14 and the constitutively active mutant Rab14Q70L prevented phagosomes with dead mycobacteria from undergoing default maturation into phagolysosomal organelles. Mechanistic studies demonstrated a role for Rab14 in stimulating organellar fusion between phagosomes and early endosomes but not with late endosomes. Rab14 enables mycobacterial phagosomes to maintain early endosomal characteristics and avoid late endosomal/lysosomal degradative components.