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.     

The Protein Tyrosine Phosphatase SHP-1 Regulates Phagolysosome Biogenesis

The process of phagocytosis and phagosome maturation involves the recruitment of effector proteins that participate in phagosome formation and in the acidification and/or fusion with various endocytic vesicles. In the current study, we investigated the role of the Src homology region 2 domain-containing phosphatase 1 (SHP-1) in phagolysosome biogenesis. To this end, we used immortalized bone marrow macrophages derived from SHP-1-deficient motheaten mice and their wild-type littermates. We found that SHP-1 is recruited early and remains present on phagosomes for up to 4 h postphagocytosis. Using confocal immunofluorescence microscopy and Western blot analyses on purified phagosome extracts, we observed an impaired recruitment of lysosomal-associated membrane protein 1 in SHP-1-deficient macrophages. Moreover, Western blot analyses revealed that whereas the 51-kDa procathepsin D is recruited to phagosomes, it is not processed into the 46-kDa cathepsin D in the absence of SHP-1, suggesting a defect in acidification. Using the lysosomotropic agent LysoTracker as an indicator of phagosomal pH, we obtained evidence that in the absence of SHP-1, phagosome acidification was impaired. Taken together, these results are consistent with a role for SHP-1 in the regulation of signaling or membrane fusion events involved in phagolysosome biogenesis.     

Biogenesis of phagolysosomes: the 'kiss and run' hypothesis

Particles such as microorganisms that are taken up by the cell into phagosomes are usually ultimately degraded in phagolysosomes. However, despite its importance, phagolysosome biogenesis is poorly understood. This article presents a model for phagosome maturation into phagolysosomes that involves multiple transient fusion-fission interactions of phagosomes with endocytic organelles via a fusion-pore-like structure. This dynamic process may be modulated by the sequential appearance and disappearance of key phagosome proteins.     

Mycobacterial manipulation of the host cell

Phagosome biogenesis, the process by which macrophages neutralize ingested pathogens and initiate antigen presentation, has entered the field of cellular mycobacteriology research largely owing to the discovery 30 years ago that phagosomes harboring mycobacteria are refractory to fusion with lysosomes. In the past decade, the use of molecular genetics and biology in different model systems to study phagosome biogenesis have made significant advances in understanding subtle mechanisms by which mycobacteria inhibit the maturation of its phagosome. Thus, we are beginning to appreciate the extent to which these pathogens are able to interfere with innate immune responses and manipulate defense mechanisms to enhance their survival within the human host cell. Here, we summarize current knowledge about phagosome maturation arrest in infected macrophages and the subsequent attenuation of the macrophage-initiated adaptive anti-mycobacterial immune defenses.     

Study of Phagolysosome Biogenesis in Live Macrophages

Phagocytic cells play a major role in the innate immune system by removing and eliminating invading microorganisms in their phagosomes. Phagosome maturation is the complex and tightly regulated process during which a nascent phagosome undergoes drastic transformation through well-orchestrated interactions with various cellular organelles and compartments in the cytoplasm. This process, which is essential for the physiological function of phagocytic cells by endowing phagosomes with their lytic and bactericidal properties, culminates in fusion of phagosomes with lysosomes and biogenesis of phagolysosomes which is considered to be the last and critical stage of maturation for phagosomes. In this report, we describe a live cell imaging based method for qualitative and quantitative analysis of the dynamic process of lysosome to phagosome content delivery, which is a hallmark of phagolysosome biogenesis. This approach uses IgG-coated microbeads as a model for phagocytosis and fluorophore-conjugated dextran molecules as a luminal lysosomal cargo probe, in order to follow the dynamic delivery of lysosmal content to the phagosomes in real time in live macrophages using time-lapse imaging and confocal laser scanning microscopy. Here we describe in detail the background, the preparation steps and the step-by-step experimental setup to enable easy and precise deployment of this method in other labs. Our described method is simple, robust, and most importantly, can be easily adapted to study phagosomal interactions and maturation in different systems and under various experimental settings such as use of various phagocytic cells types, loss-of-function experiments, different probes, and phagocytic particles.     

The Leishmania donovani Lipophosphoglycan Excludes the Vesicular Proton-ATPase from Phagosomes by Impairing the Recruitment of Synaptotagmin V

We recently showed that the exocytosis regulator Synaptotagmin (Syt) V is recruited to the nascent phagosome and remains associated throughout the maturation process. In this study, we investigated the possibility that Syt V plays a role in regulating interactions between the phagosome and the endocytic organelles. Silencing of Syt V by RNA interference revealed that Syt V contributes to phagolysosome biogenesis by regulating the acquisition of cathepsin D and the vesicular proton-ATPase. In contrast, recruitment of cathepsin B, the early endosomal marker EEA1 and the lysosomal marker LAMP1 to phagosomes was normal in the absence of Syt V. As Leishmania donovani promastigotes inhibit phagosome maturation, we investigated their potential impact on the phagosomal association of Syt V. This inhibition of phagolysosome biogenesis is mediated by the virulence glycolipid lipophosphoglycan, a polymer of the repeating Galβ1,4Manα1-PO₄ units attached to the promastigote surface via an unusual glycosylphosphatidylinositol anchor. Our results showed that insertion of lipophosphoglycan into ganglioside GM1-containing microdomains excluded or caused dissociation of Syt V from phagosome membranes. As a consequence, L. donovani promatigotes established infection in a phagosome from which the vesicular proton-ATPase was excluded and which failed to acidify. Collectively, these results reveal a novel function for Syt V in phagolysosome biogenesis and provide novel insight into the mechanism of vesicular proton-ATPase recruitment to maturing phagosomes. We also provide novel findings into the mechanism of Leishmania pathogenesis, whereby targeting of Syt V is part of the strategy used by L. donovani promastigotes to prevent phagosome acidification.     

Cellubrevin alterations and Mycobacterium tuberculosis phagosome maturation arrest

The intracellular trafficking processes controlling phagosomal maturation remain to be fully delineated. Mycobacterium tuberculosis var. bovis BCG, an organism that causes phagosomal maturation arrest, has emerged as a tool for dissection of critical phagosome biogenesis events. In this work, we report that cellubrevin, a v-SNARE functioning in endosomal recycling and implicated in endosomal interactions with post-Golgi compartments, plays a role in phagosomal maturation and that it is altered on mycobacterial phagosomes. Both mycobacterial phagosomes, which undergo maturation arrest, and model phagosomes containing latex beads, which follow the normal pathway of maturation into phagolysosomes, acquired cellubrevin. However, the mycobacterial and model phagosomes differed, as a discrete proteolytic degradation of this SNARE was detected on mycobacterial phagosomes. The observed cellubrevin alteration on mycobacterial phagosomes was not a passive event secondary to a maturation arrest at another checkpoint of the phagosome maturation pathway, since pharmacological inhibitors of phagosomal/endosomal pathways blocking phagosomal maturation did not cause cellubrevin degradation on model phagosomes. Cellubrevin status on phagosomes had consequences on phagosomal membrane and lumenal content trafficking, involving plasma membrane marker recycling and delivery of lysosomal enzymes. These results suggest that cellubrevin plays a role in phagosomal maturation and that it is a target for modification by mycobacteria or by infection-induced processes in the host cell.     

The phagosome proteome: insight into phagosome functions

Phagosomes are key organelles for the innate ability of macrophages to participate in tissue remodeling, clear apoptotic cells, and restrict the spread of intracellular pathogens. To understand the functions of phagosomes, we initiated the systematic identification of their proteins. Using a proteomic approach, we identified >140 proteins associated with latex bead-containing phagosomes. Among these were hydrolases, proton pump ATPase subunits, and proteins of the fusion machinery, validating our approach. A series of unexpected proteins not previously described along the endocytic/phagocytic pathways were also identified, including the apoptotic proteins galectin3, Alix, and TRAIL, the anti-apoptotic protein 14-3-3, the lipid raft-enriched flotillin-1, the anti-microbial molecule lactadherin, and the small GTPase rab14. In addition, 24 spots from which the peptide masses could not be matched to entries in any database potentially represent new phagosomal proteins. The elaboration of a two-dimensional gel database of >160 identified spots allowed us to analyze how phagosome composition is modulated during phagolysosome biogenesis. Remarkably, during this process, hydrolases are not delivered in bulk to phagosomes, but are instead acquired sequentially. The systematic characterization of phagosome proteins provided new insights into phagosome functions and the protein or groups of proteins involved in and regulating these functions.     

The Francisella tularensis pathogenicity island protein IglC and its regulator MglA are essential for modulating phagosome biogenesis and subsequent bacterial escape into the cytoplasm

The Francisella tularensis subsp. novicida-containing phagosome (FCP) matures into a late endosome-like stage that acquires the late endosomal marker LAMP-2 but does not fuse to lysosomes, for the first few hours after bacterial entry. This modulation in phagosome biogenesis is followed by disruption of the phagosome and bacterial escape into the cytoplasm where they replicate. Here we examined the role of the Francisella pathogenicity island (FPI) protein IglC and its regulator MglA in the intracellular fate of F. tularensis subsp. novicida within human macrophages. We show that F. tularensis mglA and iglC mutant strains are defective for survival and replication within U937 macrophages and human monocyte-derived macrophages (hMDMs). The defect in intracellular replication of both mutants is associated with a defect in disruption of the phagosome and failure to escape into the cytoplasm. Approximately, 80-90% of the mglA and iglC mutants containing phagosomes acquire the late endosomal/lysosomal marker LAMP-2 similar to the wild-type (WT) strain. Phagosomes harbouring the mglA or iglC mutants acquire the lysosomal enzyme Cathepsin D, which is excluded from the phagosomes harbouring the WT strain. In hMDMs in which the lysosomes are preloaded with BSA-gold or Texas Red Ovalbumin, phagosomes harbouring the mglA or the iglC mutants acquire both lysosomal tracers. We conclude that the FPI protein IglC and its regulator MglA are essential for modulating phagosome biogenesis and subsequent bacterial escape into the cytoplasm. Therefore, acquisition of the FPI, within which iglC is contained, is essential for the pathogenic evolution of F. tularensis to evade lysosomal fusion within human macrophages and cause tularemia. This is the first example of specific virulence factors of F. tularensis that are essential for evasion of fusion of the FCP to lysosomes.     

Higher order Rab programming in phagolysosome biogenesis

Phagosomes offer kinetically and morphologically tractable organelles to dissect the control of phagolysosome biogenesis by Rab GTPases. Model phagosomes harboring latex beads undergo a coordinated Rab5-Rab7 exchange, which is akin to the process of endosomal Rab conversion, the control mechanisms of which are unknown. In the process of blocking phagosomal maturation, the intracellular pathogen Mycobacterium tuberculosis prevents Rab7 acquisition, thus, providing a naturally occurring tool to study Rab conversion. We show that M. tuberculosis inhibition of Rab7 acquisition and arrest of phagosomal maturation depends on Rab22a. Four-dimensional microscopy revealed that phagosomes harboring live mycobacteria recruited and retained increasing amounts of Rab22a. Rab22a knockdown in macrophages via siRNA enhanced the maturation of phagosomes with live mycobacteria. Conversely, overexpression of the GTP-locked mutant Rab22aQ64L prevented maturation of phagosomes containing heat-killed mycobacteria, which normally progress into phagolysosomes. Moreover, Rab22a knockdown led to Rab7 acquisition by phagosomes harboring live mycobacteria. Our findings show that Rab22a defines the critical checkpoint for Rab7 conversion on phagosomes, allowing or disallowing organellar transition into a late endosomal compartment. M. tuberculosis parasitizes this process by actively recruiting and maintaining Rab22a on its phagosome, thus, preventing Rab7 acquisition and blocking phagolysosomal biogenesis.     

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.     

A tale of two lipids: Mycobacterium tuberculosis phagosome maturation arrest

Mycobacterium tuberculosis persistence in human populations relies on its ability to inhibit phagosomal maturation. M. tuberculosis resides in a pathogen-friendly phagosome escaping lysosomal bactericidal mechanisms and efficient antigen presentation in the host phagocytic cell. M. tuberculosis phagosome maturation arrest includes the action of mycobacterial lipid products, which mimic mammalian phosphatidylinositols, targeting host cell membrane trafficking processes. These products interfere with membrane trafficking and organelle biogenesis processes initiated by Ca(2+) fluxes, and ending with host cell Rab GTP-binding proteins and their effectors. The block includes phosphatidylinositol 3-kinase and membrane tethering molecules that prepare phagosomes for fusion with other organelles. Understanding these processes could provide new targets for pharmacological intervention in tuberculosis.     

Flotillin-1-enriched lipid raft domains accumulate on maturing phagosomes

Flotillin-1 was recently shown to be enriched on detergent-resistant domains of the plasma membrane called lipid rafts. These rafts, enriched in sphingolipids and cholesterol, sequester certain proteins while excluding others. Lipid rafts have been implicated in numerous cellular processes including signal transduction, membrane trafficking, and molecular sorting. In this study, we demonstrate both morphologically and biochemically that lipid rafts are present on phagosomes. These structures are enriched in flotillin-1 and devoid of the main phagosomes membrane protein lysosomal-associated membrane protein (LAMP1). The flotillin-1 present on phagosomes does not originate from the plasma membrane during phagocytosis but accumulates gradually on maturing phagosomes. Treatment with bafilomycin A1, a compound that inhibits the proton pump ATPase and prevents the fusion of phagosomes with late endocytic organelles, prevents the acquisition of flotillin-1 by phagosomes, indicating that this protein might be recruited on phagosomes from endosomal organelles. A proteomic characterization of the lipid rafts of phagosomes indicates that actin, the alpha- and beta-subunits of heterotrimeric G proteins, as well as subunits of the proton pump V-ATPase are among the constituents of these domains. Remarkably, the intracellular parasite Leishmania donovani can actively inhibit the acquisition of flotillin-1-enriched lipid rafts by phagosomes and the maturation of these organelles. These results indicate that specialized functions required for phagolysosome biogenesis may occur at focal points on the phagosome membrane, and therefore represent a potential target of intracellular pathogens.     

Induction of p38 mitogen-activated protein kinase reduces early endosome autoantigen 1 (EEA1) recruitment to phagosomal membranes

Mycobacterium tuberculosis survives in the infected host by parasitizing macrophages in which the bacillus resides in a specialized phagosome sequestered from the phagolysosomal degradative pathway. Here we report a role of the stress-induced p38 mitogen-activated protein kinase (p38 MAPK) in the component of M. tuberculosis phagosome maturation arrest that has been linked previously to the reduced recruitment of the endosomal and phagosomal membrane-tethering molecule called early endosome autoantigen 1 (EEA1; Fratti, R. A., Backer, J. M., Gruenberg, J., Corvera, S., and Deretic, V. (2001) J. Cell Biol. 154, 631-644). A pharmacological inhibition of M. tuberculosis var. bovis Bacillus Calmette-Guérin-induced p38 MAPK activity caused a marked increase in EEA1 colocalization with mycobacterial phagosomes. Consistent with the increase in EEA1 association and its role in phagosomal maturation, the pharmacological block of p38 activity caused phagosomal acidification and enrichment of the late endocytic markers lysobisphosphatidic acid and CD63 (lysosomal integral membrane protein 1) on mycobacterial phagosomes. A negative regulatory role of p38 MAPK activation in phagosome maturation was further demonstrated by converse experiments with latex bead phagosomes. Artificial activation of p38 MAPK caused a decrease in EEA1 colocalization with model latex bead phagosomes, which normally acquire EEA1 and subsequently mature into the phagolysosome. These findings show that p38 MAPK activity contributes to the arrest of M. tuberculosis phagosome maturation and demonstrate a negative regulatory role of p38 in phagolysosome biogenesis.     

Dissection of Rab7 localization on Mycobacterium tuberculosis phagosome

The late endosomal marker Rab7 has been long believed to be absent from the phagosome containing Mycobacterium tuberculosis (M.tb) in macrophage, but the detail kinetics remains elusive. Here, we found that Rab7 is transiently recruited to and subsequently released from M.tb phagosomes. For further understanding of the effect of Rab7 dissociation from the phagosome, we examined the localization of lysosomal markers on the phagosome in the macrophage expressing a dominant-negative Rab7. The localization of lysosomal associated membrane protein-2 (LAMP-2) on the phagosome was Rab7-independent, while that of cathepsin D was Rab7-dependent. These results agree with the localization of each lysosomal marker on M.tb phagosome at 6 h postinfection-i.e., LAMP-2, but not cathepsin D localized on the majority of M.tb phagosomes. These results suggest that the dissociation of Rab7 from M.tb phagosome is the important process in inhibition of phagolysosome biogenesis.     

Role of phosphatidylinositol 3-kinase and Rab5 effectors in phagosomal biogenesis and mycobacterial phagosome maturation arrest

Phagosomal biogenesis is a fundamental biological process of particular significance for the function of phagocytic and antigen-presenting cells. The precise mechanisms governing maturation of phagosomes into phagolysosomes are not completely understood. Here, we applied the property of pathogenic mycobacteria to cause phagosome maturation arrest in infected macrophages as a tool to dissect critical steps in phagosomal biogenesis. We report the requirement for 3-phosphoinositides and acquisition of Rab5 effector early endosome autoantigen (EEA1) as essential molecular events necessary for phagosomal maturation. Unlike the model phagosomes containing latex beads, which transiently recruited EEA1, mycobacterial phagosomes excluded this regulator of vesicular trafficking that controls membrane tethering and fusion processes within the endosomal pathway and is recruited to endosomal membranes via binding to phosphatidylinositol 3-phosphate (PtdIns[3]P). Inhibitors of phosphatidylinositol 3'(OH)-kinase (PI-3K) activity diminished EEA1 recruitment to newly formed latex bead phagosomes and blocked phagosomal acquisition of late endocytic properties, indicating that generation of PtdIns(3)P plays a role in phagosomal maturation. Microinjection into macrophages of antibodies against EEA1 and the PI-3K hVPS34 reduced acquisition of late endocytic markers by latex bead phagosomes, demonstrating an essential role of these Rab5 effectors in phagosomal biogenesis. The mechanism of EEA1 exclusion from mycobacterial phagosomes was investigated using mycobacterial products. Coating of latex beads with the major mycobacterial cell envelope glycosylated phosphatidylinositol lipoarabinomannan isolated from the virulent Mycobacterium tuberculosis H37Rv, inhibited recruitment of EEA1 to latex bead phagosomes, and diminished their maturation. These findings define the generation of phosphatidylinositol 3-phosphate and EEA1 recruitment as: (a) important regulatory events in phagosomal maturation and (b) critical molecular targets affected by M. tuberculosis. This study also identifies mycobacterial phosphoinositides as products with specialized toxic properties, interfering with discrete trafficking stages in phagosomal maturation.     

Early trafficking and intracellular replication of Legionella longbeachaea within an ER-derived late endosome-like phagosome

Legionella pneumophila is the predominant cause of Legionnaires' disease in the USA and Europe in contrast to Legionella longbeachaea, which is the leading cause of the disease in Western Australia. The ability of L. pneumophila to replicate intracellularly is triggered at the post-exponential phase along with expression of other virulence traits, such as motility. We show that while motility of L. longbeachaea is triggered upon growth transition into post-exponential phase, its ability to proliferate intracellularly is totally independent of the bacterial growth phase. Within macrophages, L. pneumophila replicates in a phagosome that excludes early and late endocytic markers and is surrounded by the rough endoplasmic reticulum (RER). In contrast, the L. longbeachaea phagosome colocalizes with the early endosomal marker early endosomal antigen 1 (EEA1) and the late endosomal markers lysosomal associated membrane glycoprotein 2 (LAMP-2) and mannose 6-phosphate receptor (M6PR), and is surrounded by the RER. The L. longbeachaea phagosome does not colocalize with the vacuolar ATPase (vATPase) proton pump, and the lysosomal luminal protease Cathepsin D, or the lysosomal tracer Texas red Ovalbumin (TROV). Intracellular proliferation of L. longbeachaea occurs in LAMP-2-positive phagosomes that are remodelled by the RER. Despite their distinct trafficking, both L. longbeachaea and L. pneumophila can replicate in communal phagosomes whose biogenesis is predominantly modulated by L. longbeachaea into LAMP-2-positive phagosomes. In addition, the L. pneumophila dotA mutant is rescued for intracellular replication if it co-inhabits the phagosome with L. longbeachaea. During late stages of infection, L. longbeachaea escape into the cytoplasm, prior to lysis of the macrophage, similar to L. pneumophila. We conclude that the L. longbeachaea phagosome matures to a non-acidified late endosome-like stage that is remodelled by the RER, indicating an idiosyncratic trafficking of L. longbeachaea compared with other intracellular pathogens, and a divergence in its intracellular lifestyle from L. pneumophila. In addition, re-routing biogenesis of the L. pneumophila phagosome into a late endosome controlled by L. longbeachaea has no effect on intracellular replication.     

Mycobacterium tuberculosis reprograms waves of phosphatidylinositol 3-phosphate on phagosomal organelles

The potent human pathogen Mycobacterium tuberculosis persists in macrophages within a specialized, immature phagosome by interfering with the pathway of phagolysosome biogenesis. The molecular mechanisms underlying this process remain to be fully elucidated. Here, using four-dimensional microscopy, we detected on model phagosomes, which normally mature into phagolysosomes, the existence of cyclical waves of phosphatidylinositol 3-phosphate (PI3P), a membrane trafficking regulatory lipid essential for phagosomal acquisition of lysosomal characteristics. We show that mycobacteria interfere with the dynamics of PI3P on phagosomal organelles by altering the timing and characteristics of the PI3P waves on phagosomes. The default program of cyclical PI3P waves on model phagosomes is composed of an initial stage (phase I), represented by a strong PI3P burst occurring only upon the completion of phagosome formation, and a subsequent stage (phase II) of recurring PI3P waves on maturing phagosomes with the average periodicity of 20 min. Mycobacteria alter this program in two ways: (i) by inducing, in a cholesterol-dependent fashion, a neophase I* of premature PI3P production, coinciding with the process of mycobacterial entry into the macrophage, and (ii) by inhibiting the calmodulin-dependent phase II responsible for the acquisition of lysosomal characteristics. We conclude that the default pathway of phagosomal maturation into the phagolysosome includes temporally organized cyclical waves of PI3P on phagosomal membranes and that this process is targeted for reprogramming by mycobacteria as they prevent phagolysosome formation.     

Mycobacterium tuberculosis inhibition of phagolysosome biogenesis and autophagy as a host defence mechanism

A marquee feature of the powerful human pathogen Mycobacterium tuberculosis is its macrophage parasitism. The intracellular survival of this microorganism rests upon its ability to arrest phagolysosome biogenesis, avoid direct cidal mechanisms in macrophages, and block efficient antigen processing and presentation. Mycobacteria prevent Rab conversion on their phagosomes and elaborate glycolipid and protein trafficking toxins that interfere with Rab effectors and regulation of specific organellar biogenesis in mammalian cells. One of the major Rab effectors affected in this process is the type III phosphatidylinositol 3-kinase hVPS34 and its enzymatic product phosphatidylinositol 3-phosphate (PI3P), a regulatory lipid earmarking organellar membranes for specific trafficking events. PI3P is also critical for the process of autophagy, recently recognized as an effector of innate and adaptive immunity. Induction of autophagy by physiological, pharmacological or immunological signals, including the major antituberculosis Th1 cytokine IFN-gamma and its downstream effector p47 GTPase LRG-47, can overcome mycobacterial phagosome maturation block and inhibit intracellular M. tuberculosis survival. This review summarizes the findings centred around the PI3P-nexus where the mycobacterial phagosome maturation block and execution stages of autophagy intersect.     

Proteomic analysis of phagocytosis in the enteric protozoan parasite Entamoeba histolytica

Proteomic analysis of phagosomes isolated from Entamoeba histolytica by liquid chromatography and mass spectrometry identified 85 proteins involved in surface recognition, actin cytoskeleton rearrangement, vesicular trafficking, and degradation. Phagosome localization of representative proteins was verified by immunofluorescence assay. This study should provide a basis for molecular identification and characterization of phagosome biogenesis.