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.     

IL-6 and IL-12 specifically regulate the expression of Rab5 and Rab7 via distinct signaling pathways

Recent studies have shown that phagosome maturation depends on the balance between pro-inflammatory and anti-inflammatory cytokines, indicating that cytokine modulates phagosome maturation. However, the mechanism of cytokine-mediated modulation of intracellular trafficking remains to be elucidated. Here, we have shown that treatment of macrophages with IL-6 specifically induce the expression of Rab5 through the activation of extracellular signal-regulated kinase, whereas IL-12 exclusively upregulate the expression of Rab7 through the activation of p38 MAPK. We have cloned the 5'-flanking regions of the rab5c or rab7 into the promoterless reporter vector. Our results have shown that cells transfected with rab5c chimera are transactivated by IL-6, and IL-12 specifically transactivates cells containing rab7 chimera. Moreover, our results also show that IL-12 induces lysosomal transport, whereas IL-6 stimulates the fusion between early compartments in macrophages and accordingly modulates Salmonella trafficking and survival in macrophages. This is the first demonstration showing that cytokine differentially regulates endocytic trafficking by controlling the expression of appropriate Rab GTPase, and provides insight into the mechanism of cytokine-mediated regulation of intracellular trafficking.     

A network of Rab GTPases controls phagosome maturation and is modulated by Salmonella enterica serovar Typhimurium

Members of the Rab guanosine triphosphatase (GTPase) family are key regulators of membrane traffic. Here we examined the association of 48 Rabs with model phagosomes containing a non-invasive mutant of Salmonella enterica serovar Typhimurium (S. Typhimurium). This mutant traffics to lysosomes and allowed us to determine which Rabs localize to a maturing phagosome. In total, 18 Rabs associated with maturing phagosomes, each with its own kinetics of association. Dominant-negative mutants of Rab23 and 35 inhibited phagosome-lysosome fusion. A large number of Rab GTPases localized to wild-type Salmonella-containing vacuoles (SCVs), which do not fuse with lysosomes. However, some Rabs (8B, 13, 23, 32, and 35) were excluded from wild-type SCVs whereas others (5A, 5B, 5C, 7A, 11A, and 11B) were enriched on this compartment. Our studies demonstrate that a complex network of Rab GTPases controls endocytic progression to lysosomes and that this is modulated by S. Typhimurium to allow its intracellular growth.     

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.     

Mycobacterial Nucleoside Diphosphate Kinase Blocks Phagosome Maturation in Murine Raw 264.7 Macrophages

Background

Microorganisms capable of surviving within macrophages are rare, but represent very successful pathogens. One of them is Mycobacterium tuberculosis (Mtb) whose resistance to early mechanisms of macrophage killing and failure of its phagosomes to fuse with lysosomes causes tuberculosis (TB) disease in humans. Thus, defining the mechanisms of phagosome maturation arrest and identifying mycobacterial factors responsible for it are key to rational design of novel drugs for the treatment of TB. Previous studies have shown that Mtb and the related vaccine strain, M. bovis bacille Calmette-Guérin (BCG), disrupt the normal function of host Rab5 and Rab7, two small GTPases that are instrumental in the control of phagosome fusion with early endosomes and late endosomes/lysosomes respectively.

Methodology/Principal Findings

Here we show that recombinant Mtb nucleoside diphosphate kinase (Ndk) exhibits GTPase activating protein (GAP) activity towards Rab5 and Rab7. Then, using a model of latex bead phagosomes, we demonstrated that Ndk inhibits phagosome maturation and fusion with lysosomes in murine RAW 264.7 macrophages. Maturation arrest of phagosomes containing Ndk-beads was associated with the inactivation of both Rab5 and Rab7 as evidenced by the lack of recruitment of their respective effectors EEA1 (early endosome antigen 1) and RILP (Rab7-interacting lysosomal protein). Consistent with these findings, macrophage infection with an Ndk knocked-down BCG strain resulted in increased fusion of its phagosome with lysosomes along with decreased survival of the mutant.

Conclusion

Our findings provide evidence in support of the hypothesis that mycobacterial Ndk is a putative virulence factor that inhibits phagosome maturation and promotes survival of mycobacteria within the macrophage.     

Lysophosphatidylcholine Enhances Bactericidal Activity by Promoting Phagosome Maturation via the Activation of the NF-κB Pathway during Salmonella Infection in Mouse Macrophages

Salmonella enterica serovar Typhimurium (S. Typhimurium) is a facultative intracellular pathogen that causes salmonellosis and mortality worldwide. S. Typhimurium infects macrophages and survives within phagosomes by avoiding the phagosome-lysosome fusion system. Phagosomes sequentially acquire different Rab GTPases during maturation and eventually fuse with acidic lysosomes. Lysophosphatidylcholine (LPC) is a bioactive lipid that is associated with the generation of chemoattractants and reactive oxygen species (ROS). In our previous study, LPC controlled the intracellular growth of Mycobacterium tuberculosis by promoting phagosome maturation. In this study, to verify whether LPC enhances phagosome maturation and regulates the intracellular growth of S. Typhimurium, macrophages were infected with S. Typhimurium. LPC decreased the intracellular bacterial burden, but it did not induce cytotoxicity in S. Typhimurium-infected cells. In addition, combined administration of LPC and antibiotic significantly reduced the bacterial burden in the spleen and the liver. The ratios of the colocalization of intracellular S. Typhimurium with phagosome maturation markers, such as early endosome antigen 1 (EEA1) and lysosome-associated membrane protein 1 (LAMP-1), were significantly increased in LPC-treated cells. The expression level of cleaved cathepsin D was rapidly increased in LPC-treated cells during S. Typhimurium infection. Treatment with LPC enhanced ROS production, but it did not affect nitric oxide production in S. Typhimurium-infected cells. LPC also rapidly triggered the phosphorylation of IκBα during S. Typhimurium infection. These results suggest that LPC can improve phagosome maturation via ROS-induced activation of NF-κB pathway and thus may be developed as a therapeutic agent to control S. Typhimurium growth.     

Cutting edge: Mycobacterium tuberculosis blocks Ca2+ signaling and phagosome maturation in human macrophages via specific inhibition of sphingosine kinase

One-third of the world's population is infected with Mycobacterium tuberculosis (Mtb), and three million people die of tuberculosis each year. Following its ingestion by macrophages (MPs), Mtb inhibits the maturation of its phagosome, preventing progression to a bactericidal phagolysosome. Phagocytosis of Mtb is uncoupled from the elevation in MP cytosolic Ca(2+) that normally accompanies microbial ingestion, resulting in inhibition of phagosome-lysosome fusion and increased intracellular viability. This study demonstrates that the mechanism responsible for this failure of Ca(2+)-dependent phagosome maturation involves mycobacterial inhibition of MP sphingosine kinase. Thus, inhibition of sphingosine kinase directly contributes to survival of Mtb within human MPs and represents a novel molecular mechanism of pathogenesis.     

Rab GTPases regulating phagosome maturation are differentially recruited to mycobacterial phagosomes

Mycobacterium tuberculosis (M. tb) is an intracellular pathogen that can replicate within infected macrophages. The ability of M. tb to arrest phagosome maturation is believed to facilitate its intracellular multiplication. Rab GTPases regulate membrane trafficking, but details of how Rab GTPases regulate phagosome maturation and how M. tb modulates their localization during inhibiting phagolysosome biogenesis remain elusive. We compared the localization of 42 distinct Rab GTPases to phagosomes containing either Staphylococcus aureus or M. tb. The phagosomes containing S. aureus were associated with 22 Rab GTPases, but only 5 of these showed similar localization kinetics as the phagosomes containing M. tb. The Rab GTPases responsible for phagosome maturation, phagosomal acidification and recruitment of cathepsin D were examined in macrophages expressing the dominant-negative form of each Rab GTPase. LysoTracker staining and immunofluorescence microscopy revealed that Rab7, Rab20 and Rab39 regulated phagosomal acidification and Rab7, Rab20, Rab22b, Rab32, Rab34, Rab38 and Rab43 controlled the recruitment of cathepsin D to the phagosome. These results suggest that phagosome maturation is achieved by a series of interactions between Rab GTPases and phagosomes and that differential recruitment of these Rab GTPases, except for Rab22b and Rab43, to M. tb-containing phagosomes is involved in arresting phagosome maturation and inhibiting phagolysosome biogenesis.     

Leishmania promastigotes require lipophosphoglycan to actively modulate the fusion properties of phagosomes at an early step of phagocytosis

The lipophosphoglycan (LPG) of Leishmania promastigotes plays key roles in parasite survival in both insect and mammalian hosts. Evidence suggests that LPG decreases phagosome fusion properties at the onset of infection in macrophages. The mechanisms of action of this molecule are, however, poorly understood. In the present study, we used a panoply of Leishmania mutants displaying modified LPG structures to determine more precisely how LPG modulates phagosome-endosome fusion. Using an in vivo fusion assay measuring, at the electron microscope, the transfer of solute materials from endosomes to phagosomes, we provided further evidence that the repeating Gal(beta1,4)Man(alpha1-PO4) units of LPG are responsible for the alteration in phagosome fusion. The inhibitory effect of LPG on phagosome fusion was shown to be more potent towards late endocytic organelles and lysosomes than early endosomes, explaining how Leishmania promastigotes can avoid degradation in hydrolase-enriched compartments. The involvement of other repeating unit-containing molecules, including the secreted acid phosphatase, in the inhibition process was ruled out, as an LPG-defective mutant (Ipg1-) which secretes repeating unit-containing glycoconjugates was present in highly fusogenic phagosomes. In L. major, oligosaccharide side-chains of LPG did not contribute to the inhibition process, as Spock, an L. major mutant lacking LPG side-chains, blocked fusion to the same extent as wild-type parasites. Finally, dead parasites internalized from the culture medium were not as efficient as live parasites in altering phagosome-endosome fusion, despite the presence of LPG. However, the killing of parasites with vital dyes after their sequestration in phagosomes had no effect on the fusion properties of this organelle. Collectively, these results suggest that living promastigotes displaying full-length cell surface LPG can actively influence macrophages at an early stage of phagocytosis to generate phagosomes with poor fusogenic properties.     

Transport of phagosomal components to an endosomal compartment.

The participation of phagosomes in interorganellar protein and membrane exchange is important to the maturation of phagosomes into phagolysosomes. To investigate this process, we have developed an assay to measure protein transport from phagosomes to other vesicle populations. J774-E clone macrophages phagocytosed 125I-anti-dinitrophenol IgG-coated Staphylococcus aureus for 3 min followed by chase for intervals of 0-30 min. Following cell fractionation, the intracellular distribution of radioiodinated protein was assayed. We observed a time-dependent increase radioiodinated protein in a non-phagosome vesicle fraction which displayed endosome characteristics. Concomitantly, radioiodinated protein within phagosomes decreased over the chase period. As assessed via Percoll density gradient fractionation, the phagocytosed radioiodinated protein migrated to both heavy (lysosome density) and light (endosome density) vesicle populations. Characterization of the fusogenic properties of the transport vesicles demonstrated that they are capable of in vitro fusion with early endosomes. Furthermore, this fusion event shares many of the biochemical requirements identified for phagosome-endosome and endosome-endosome fusion. Morphological analysis of phagosome maturation provides additional evidence for phagosome to endosome transport. These results suggest phagocytosed material is transferred from phagosomes to endosomes and then recycled out of the cell.     

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.     

Modulation of Rab5 and Rab7 recruitment to phagosomes by phosphatidylinositol 3-kinase

Phagosomal biogenesis is central for microbial killing and antigen presentation by leukocytes. However, the molecular mechanisms governing phagosome maturation are poorly understood. We analyzed the role and site of action of phosphatidylinositol 3-kinases (PI3K) and of Rab GTPases in maturation using both professional and engineered phagocytes. Rab5, which is recruited rapidly and transiently to the phagosome, was found to be essential for the recruitment of Rab7 and for progression to phagolysosomes. Similarly, functional PI3K is required for successful maturation. Remarkably, inhibition of PI3K did not preclude Rab5 recruitment to phagosomes but instead enhanced and prolonged it. Moreover, in the presence of PI3K inhibitors Rab5 was found to be active, as deduced from measurements of early endosome antigen 1 binding and by photobleaching recovery determinations. Though their ability to fuse with late endosomes and lysosomes was virtually eliminated by wortmannin, phagosomes nevertheless recruited a sizable amount of Rab7. Moreover, Rab7 recruited to phagosomes in the presence of PI3K antagonists retained the ability to bind its effector, Rab7-interacting lysosomal protein, suggesting that it is functionally active. These findings imply that (i) dissociation of Rab5 from phagosomes requires products of PI3K, (ii) PI3K-dependent effectors of Rab5 are not essential for the recruitment of Rab7 by phagosomes, and (iii) recruitment and activation of Rab7 are insufficient to induce fusion of phagosomes with late endosomes and lysosomes. Accordingly, transfection of constitutively active Rab7 did not bypass the block of phagolysosome formation exerted by wortmannin. We propose that Rab5 activates both PI3K-dependent and PI3K-independent effectors that act in parallel to promote phagosome maturation.     

Alterations in recruitment and activation of Rab proteins during mycobacterial infection

At the phagosome level, Mycobacterium spp. alters activation and recruitment of several "Ras gene from rat brain" proteins, commonly known as Rab. Mycobacterial phagosomes have a greater and sustained expression of Rab5, Rab11, Rab14 and Rab22a, and lowered or no expression of Rab7, Rab9 and Rab6. This correlates with increased fusion of the phagosomes with early and recycling endosomes acquiring some features of early phagosomes, allowing the bacteria to gain access to nutrients and preventing the activation of anti-mycobacterial mechanisms. The expression of constitutively active mutants of Rab from the early stage endosomes prevents the maturation of phagosomes containing latex beads or heat-inactivated mycobacteria. Silencing of these mutants by interference RNA or dominant negative forms induces the maturation of mycobacterial phagosomes. The mechanisms have not been established by which mycobacteria alter the expression of these GTPases and thereby shift the phagolysosomal maturation. The problem can be explained by alterations in the recruitment of proteins that interact with Rab, such as phosphoinositide 3-kinases and early endosomal antigen 1. Identifying the mechanisms used by Mycobacterium spp. to disrupt the cycle of Rab activation will be essential to understand the pathophysiology of mycobacterial infections and usefully to potential drug targets.     

Mycobacterium tuberculosis phagosomes exhibit altered calmodulin-dependent signal transduction: contribution to inhibition of phagosome-lysosome fusion and intracellular survival in human macrophages

Mycobacterium tuberculosis successfully parasitizes macrophages by disrupting the maturation of its phagosome, creating an intracellular compartment with endosomal rather than lysosomal characteristics. We have recently demonstrated that live M. tuberculosis infect human macrophages in the absence of an increase in cytosolic Ca(2+) ([Ca(2+)](c)), which correlates with inhibition of phagosome-lysosome fusion and intracellular viability. In contrast, killed M. tuberculosis induces an elevation in [Ca(2+)](c) that is coupled to phagosome-lysosome fusion. We tested the hypothesis that defective activation of the Ca(2+)-dependent effector proteins calmodulin (CaM) and CaM-dependent protein kinase II (CaMKII) contributes to the intracellular pathogenesis of tuberculosis. Phagosomes containing live M. tuberculosis exhibited decreased levels of CaM and the activated form of CaMKII compared with phagosomes encompassing killed tubercle bacilli. Furthermore, ionophore-induced elevations in [Ca(2+)](c) resulted in recruitment of CaM and activation of CaMKII on phagosomes containing live M. tuberculosis. Specific inhibitors of CaM or CaMKII blocked Ca(2+) ionophore-induced phagosomal maturation and enhanced the bacilli's intracellular viability. These results demonstrate a novel role for CaM and CaMKII in the regulation of phagosome-lysosome fusion and suggest that defective activation of these Ca(2+)-activated signaling components contributes to the successful parasitism of human macrophages by M. tuberculosis.     

Cholesterol accumulation by macrophages impairs phagosome maturation

Macrophages are key to the pathogenesis of atherosclerosis. They take up and store excessive amounts of cholesterol associated with modified low density lipoprotein, eventually becoming foam cells that display altered immune responsiveness. We studied the effects of cholesterol accumulation on phagosome formation and maturation, using lipid transport antagonists and cholesterol transport-deficient mutants. In macrophages treated with U18666A, a transport antagonist that prevents cholesterol exit from late endosomes/lysosomes, the early stages of maturation proceeded normally; phagosomes acquired Rab5, phosphatidylinositol 3-phosphate, and EEA1 and merged with LAMP-containing vesicles. However, fusion with lysosomes was impaired. Rab7, which is required for phagolysosome formation, was acquired by phagosomes but remained inactive. Maturation was also studied in fibroblasts from Niemann-Pick type C individuals that have defective cholesterol transport. Transfection of FcgammaIIA receptors was used to confer phagocytic capability to these fibroblasts. Niemann-Pick type C phagosomes failed to fuse with lysosomes, whereas wild type fibroblasts formed normal phagolysosomes. These findings indicate that cholesterol accumulation can have a detrimental effect on phagosome maturation by impairing the activation of Rab7, sequestering it and its effectors in cholesterol-enriched multilamellar compartments.     

The fungal pathogen Cryptococcus neoformans manipulates macrophage phagosome maturation

Phagocytosis by cells of the innate immune system, such as macrophages, and the subsequent successful maturation of the phagosome, is key for the clearance of pathogens. The fungal pathogen Cryptococcus neoformans is known to overcome killing by host phagocytes and both replicate within these cells and also escape via a non‐lytic process termed vomocytosis. Here we demonstrate that, during intracellular growth, cryptococci modify phagolysosome maturation. Live cryptococci, but not heat‐killed pathogens or inert targets, induce the premature removal of the early phagosome markers Rab5 and Rab11. In addition, significant acidification of the phagosome, calcium flux and protease activity is hindered, thus rendering the phagosome permissive for cryptococcal proliferation. Interestingly, several attenuated cryptococcal mutants retain this ability to subvert phagosomal maturation, suggesting that hitherto unidentified pathogen mechanisms regulate this process.     

Drosophila Rab14 mediates phagocytosis in the immune response to Staphylococcus aureus

Drosophila haemocytes are essential for the animal to resist Staphylococcus aureus infections. Phagocytosis is a central component of the haemocyte‐mediated immune response. It involves regulated interaction between the phagocytic and the endocytic compartments. RabGTPases are pivotal for the membrane trafficking and fusion events, and thus are often targets of intracellular pathogens that subvert phagocytosis. An in vivo screen identified Rab2 and Rab14 as candidates for proteins regulating phagosome maturation. Since Rab14 is often targeted by intracellular pathogens, an understanding of its function during phagocytosis and the overall immune response can give insight into the pathogenesis of intracellular microbes. We generated a Drosophila Rab14 mutant and characterized the resulting immune defects in animals and specifically in haemocytes in response to an S. aureus infection. Haemocyte based immunofluorescence studies indicate that Rab14 is recruited to the phagosome and like Rab7, a well‐characterized regulator of the phagocytic pathway, is essential for progression of phagosome maturation. Rab14 mutant haemocytes show impaired recruitment of Rab7 and of a lysosomal marker onto S. aureus phagosomes. The defect in phagocytosis is associated with higher bacterial load and increased susceptibility to S. aureus in the animal.     

Stage-specific pathways of Leishmania infantum chagasi entry and phagosome maturation in macrophages

The life stages of Leishmania spp. include the infectious promastigote and the replicative intracellular amastigote. Each stage is phagocytosed by macrophages during the parasite life cycle. We previously showed that caveolae, a subset of cholesterol-rich membrane lipid rafts, facilitate uptake and intracellular survival of virulent promastigotes by macrophages, at least in part, by delaying parasitophorous vacuole (PV)-lysosome fusion. We hypothesized that amastigotes and promastigotes would differ in their route of macrophage entry and mechanism of PV maturation. Indeed, transient disruption of macrophage lipid rafts decreased the entry of promastigotes, but not amastigotes, into macrophages (P<0.001). Promastigote-containing PVs were positive for caveolin-1, and co-localized transiently with EEA-1 and Rab5 at 5 minutes. Amastigote-generated PVs lacked caveolin-1 but retained Rab5 and EEA-1 for at least 30 minutes or 2 hours, respectively. Coinciding with their conversion into amastigotes, the number of promastigote PVs positive for LAMP-1 increased from 20% at 1 hour, to 46% by 24 hours, (P<0.001, Chi square). In contrast, more than 80% of amastigote-initiated PVs were LAMP-1+ at both 1 and 24 hours. Furthermore, lipid raft disruption increased LAMP-1 recruitment to promastigote, but not to amastigote-containing compartments. Overall, our data showed that promastigotes enter macrophages through cholesterol-rich domains like caveolae to delay fusion with lysosomes. In contrast, amastigotes enter through a non-caveolae pathway, and their PVs rapidly fuse with late endosomes but prolong their association with early endosome markers. These results suggest a model in which promastigotes and amastigotes use different mechanisms to enter macrophages, modulate the kinetics of phagosome maturation, and facilitate their intracellular survival.     

Inhibition of Rab5a exchange activity is a key step for Listeria monocytogenes survival

Listeria monocytogenes (LM) modifies the phagocytic compartment by targeting Rab5a function through an unknown mechanism. Inhibition of Rab5a exchange by LM can be considered the main virulence mechanism as it favours viability of the parasite within the phagosome as well as the exclusion of putative listericidal lysosomal proteases such as cathepsin-D. The significance of this survival mechanism is evidenced by the overexpression of Rab5a mutants in CHO cells that promoted GDP exchange on Rab5a and eliminated pathogenic LM. The following mutants showed listericidal effects: Rab5a:Q79L, a constitutively active mutant with accelerated GDP exchange and Rab5a GEF, Vps9, which overactivates the endogenous protein. Clearance of LM from these phagosomes was controlled by the hydrolytic action of cathepsin-D as suggested by the lysosomal protease inhibitor chloroquine, or the cathepsin-D inhibitor, pepstatin A, which caused a reversion of listericidal activity. Moreover, the effects of LM on Rab5a phagocytic function mimics those reported for the GDP locked dominant negative Rab5a mutant, S34N. Transfection of these mutants into CHO cells increased pathogen survival as they showed higher numbers of viable bacteria, complete inhibition of GDP exchange on Rab5a and impairment of the listericidal action probably exerted by cathepsin-D. We cotransfected functional Rab5a GEF into this dominant negative mutant and restored normal LM intraphagosomal viability, Rab5a exchange and listericidal action of cathepsin-D.