A coat protein on phagosomes involved in the intracellular survival of mycobacteria.

Mycobacteria are intracellular pathogens that can survive within macrophage phagosomes, thereby evading host defense strategies by largely unknown mechanisms. We have identified a WD repeat host protein that was recruited to and actively retained on phagosomes by living, but not dead, mycobacteria. This protein, termed TACO, represents a component of the phagosome coat that is normally released prior to phagosome fusion with or maturation into lysosomes. In macrophages lacking TACO, mycobacteria were readily transported to lysosomes followed by their degradation. Expression of TACO in nonmacrophages prevented lysosomal delivery of mycobacteria and prolonged their intracellular survival. Active retention of TACO on phagosomes by living mycobacteria thus represents a mechanism preventing cargo delivery to lysosomes, allowing mycobacteria to survive within macrophages.     

Autophagy is a defense mechanism inhibiting BCG and Mycobacterium tuberculosis survival in infected macrophages

Mycobacterium tuberculosis is an intracellular pathogen persisting within phagosomes through interference with phagolysosome biogenesis. Here we show that stimulation of autophagic pathways in macrophages causes mycobacterial phagosomes to mature into phagolysosomes. Physiological induction of autophagy or its pharmacological stimulation by rapamycin resulted in mycobacterial phagosome colocalization with the autophagy effector LC3, an elongation factor in autophagosome formation. Autophagy stimulation increased phagosomal colocalization with Beclin-1, a subunit of the phosphatidylinositol 3-kinase hVPS34, necessary for autophagy and a target for mycobacterial phagosome maturation arrest. Induction of autophagy suppressed intracellular survival of mycobacteria. IFN-gamma induced autophagy in macrophages, and so did transfection with LRG-47, an effector of IFN-gamma required for antimycobacterial action. These findings demonstrate that autophagic pathways can overcome the trafficking block imposed by M. tuberculosis. Autophagy, which is a hormonally, developmentally, and, as shown here, immunologically regulated process, represents an underappreciated innate defense mechanism for control of intracellular pathogens.     

Proteomics approach to understand reduced clearance of mycobacteria and high viral titers during HIV–mycobacteria co‐infection

Environmental mycobacteria, highly prevalent in natural and artificial (including chlorinated municipal water) niches, are emerging as new threat to human health, especially to HIV‐infected population. These seemingly harmless non‐pathogenic mycobacteria, which are otherwise cleared, establish as opportunistic infections adding to HIV‐associated complications. Although immune‐evading strategies of pathogenic mycobacteria are known, the mechanisms underlying the early events by which opportunistic mycobacteria establish infection in macrophages and influencing HIV infection are unclear. Proteomics of phagosome‐enriched fractions from Mycobacterium bovis Bacillus Calmette–Guérin (BCG) mono‐infected and HIV–M. bovis BCG co‐infected THP‐1 cells by LC‐MALDI‐MS/MS revealed differential distribution of 260 proteins. Validation of the proteomics data showed that HIV co‐infection helped the survival of non‐pathogenic mycobacteria by obstructing phagosome maturation, promoting lipid biogenesis and increasing intracellular ATP equivalents. In turn, mycobacterial co‐infection up‐regulated purinergic receptors in macrophages that are known to support HIV entry, explaining increased viral titers during co‐infection. The mutualism was reconfirmed using clinically relevant opportunistic mycobacteria, Mycobacterium avium, Mycobacterium kansasii and Mycobacterium phlei that exhibited increased survival during co‐infection, together with increase in HIV titers. Additionally, the catalogued proteins in the study provide new leads that will significantly add to the understanding of the biology of opportunistic mycobacteria and HIV coalition.     

Mycobacterium requires an all-around closely apposing phagosome membrane to maintain the maturation block and this apposition is re-established when it rescues itself from phagolysosomes

Pathogenic mycobacteria survive in macrophages of the host organism by residing in phagosomes which they prevent from undergoing maturation and fusion with lysosomes. Several molecular mechanisms have been associated with the phagosome maturation block. Here we show for Mycobacterium avium in mouse bone marrow-derived macrophages that the maturation block required an all-around close apposition between the mycobacterial surface and the phagosome membrane. When small (0.1 μm) latex beads were covalently attached to the mycobacterial surface to act as a spacer that interfered with a close apposition, phagosomes rapidly acquired lysosomal characteristics as indicators for maturation and fusion with lysosomes. As a result, several mycobacteria were delivered into single phagolysosomes. Detailed electron-microscope observations of phagosome morphology over a 7-day post-infection period showed a linear correlation between bead attachment and phagosome–lysosome fusion. After about 3 days post infection, conditions inside phagolysosomes caused a gradual release of beads. This allowed mycobacteria to re-establish a close apposition with the surrounding membrane and sequester themselves into individual, non-maturing phagosomes which had lost lysosomal characteristics. By rescuing themselves from phagolysosomes, mycobacteria remained fully viable and able to multiply at the normal rate. In order to unify the present observations and previously reported mechanisms for the maturation block, we discuss evidence that they may act synergistically to interfere with 'Phagosome Membrane Economics' by causing relative changes in incoming and outgoing endocytic membrane fluxes.     

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.     

Effect of Nramp1 on bacterial replication and on maturation of Mycobacterium avium-containing phagosomes in bone marrow-derived mouse macrophages

Pathogenic mycobacteria prevent maturation of the phagosomes in which they reside inside macrophages and this is thought to be a major strategy allowing them to survive and multiply within macrophages. The molecular basis for this inhibition is only now beginning to emerge with the molecular characterization of the phagosome membrane enclosing these pathogens. We have used here several electron microscopy approaches in combination with counts of bacterial viability to analyse how expression of Nramp1 at the phagosomal membrane may influence survival of Mycobacterium avium and affect its ability to modulate the fusogenic properties of the phagosome in which it resides. The experiments were carried out in bone marrow-derived macrophages from wild-type 129sv (Nramp1(G169)) mice and from isogenic 129sv carrying a null mutation at Nramp1 (Nramp(1-/-)) following infection with a virulent strain of M. avium. We show here that Nramp1 expression has a bacteriostatic effect and that abrogation of Nramp1 restores the bacteria's capacity to replicate within macrophages. The combined analyses of the acquisition of endocytic contents markers delivered to early endosomes and/or lysosomes either prior to or after phagocytic uptake showed that in Nramp1-positive macrophages, M. avium was unable to prevent phagosome maturation and fusion with lysosomes but that in Nramp1-negative macrophages this capacity was restored. Several hypotheses are proposed to explain how Nramp1 could affect survival of M. avium. We also propose how the present observations could relate to the model according to which mycobacteria can prevent phagosome maturation by establishing a tight interaction with constituents of the phagosome membrane. Furthermore, these results show the importance of the choice of macrophages used as a model to study intracellular survival strategies of pathogens.     

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.     

Delay of phagosome maturation by a mycobacterial lipid is reversed by nitric oxide

Mycobacterium tuberculosis is a facultative intracellular pathogen that inhibits phagosome maturation in macrophages thereby securing survival and growth. Mycobacteria reside in an early endocytic compartment of near-neutral pH where they upregulate production of complex glycolipids such as trehalose dimycolate. Here, we report that trehalose dimycolate coated onto beads increased the bead retention in early phagosomes, i.e. at a similar stage as viable mycobacteria. Thus, a single mycobacterial lipid sufficed to divert phagosome maturation and likely contributes to mycobacterial survival in macrophages. Previous studies showed that activated macrophages promote maturation of mycobacterial phagosomes and eliminate mycobacteria through bactericidal effectors including nitric oxide generated by inducible nitric-oxide synthase. We show that deceleration of bead phagosome maturation by trehalose dimycolate was abolished in immune-activated wild type, but not in activated nitric-oxide synthase-deficient macrophages, nor when hydroxyl groups of trehalose dimycolate were chemically modified by reactive nitrogen intermediates. Thus, specific host defence effectors of activated macrophages directly target a specific virulence function of mycobacteria.     

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.     

On the killing of mycobacteria by macrophages

Both pathogenic and non-pathogenic mycobacteria are internalized into macrophage phagosomes. Whereas the non-pathogenic types are invariably killed by all macrophages, the pathogens generally survive and grow. Here, we addressed the survival, production of nitrogen intermediates (RNI) and intracellular trafficking of the non-pathogenic Mycobacterium smegmatis , the pathogen-like, BCG and the pathogenic M. bovis in different mouse, human and bovine macrophages. The bacteriocidal effects of RNI were restricted for all bacterial species to the early stages of infection. EM analysis showed clearly that all the mycobacteria remained within phagosomes even at late times of infection. The fraction of BCG and M. bovis found in mature phagolysosomes rarely exceeded 10% of total, irrespective of whether bacteria were growing, latent or being killed, with little correlation between the extent of phagosome maturation and the degree of killing. Theoretical modelling of our data identified two different potential sets of explanations that are consistent with our results. The model we favour is one in which a small but significant fraction of BCG is killed in an early phagosome, then maturation of a small fraction of phagosomes with both live and killed bacteria, followed by extremely rapid killing and digestion of the bacteria in phago-lysosomes.     

Label-free proteomics and systems biology analysis of mycobacterial phagosomes in dendritic cells and macrophages

Proteomics has been applied to study intracellular bacteria and phagocytic vacuoles in different host cell lines, especially macrophages (Mφs). For mycobacterial phagosomes, few studies have identified over several hundred proteins for systems assessment of the phagosome maturation and antigen presentation pathways. More importantly, there has been a scarcity in publication on proteomic characterization of mycobacterial phagosomes in dendritic cells (DCs). In this work, we report a global proteomic analysis of Mφ and DC phagosomes infected with a virulent, an attenuated, and a vaccine strain of mycobacteria. We used label-free quantitative proteomics and bioinformatics tools to decipher the regulation of phagosome maturation and antigen presentation pathways in Mφs and DCs. We found that the phagosomal antigen presentation pathways are repressed more in DCs than in Mφs. The results suggest that virulent mycobacteria might co-opt the host immune system to stimulate granuloma formation for persistence while minimizing the antimicrobial immune response to enhance mycobacterial survival. The studies on phagosomal proteomes have also shown promise in discovering new antigen presentation mechanisms that a professional antigen presentation cell might use to overcome the mycobacterial blockade of conventional antigen presentation pathways.     

The sodium proton exchanger NHE9 regulates phagosome maturation and bactericidal activity in macrophages

Acidification of phagosomes is essential for the bactericidal activity of macrophages. Targeting machinery that regulates pH within the phagosomes is a prominent strategy employed by various pathogens that have emerged as major threats to public health. Nascent phagosomes acquire the machinery for pH regulation through a graded maturation process involving fusion with endolysosomes. Meticulous coordination between proton pumping and leakage mechanisms is crucial for maintaining optimal pH within the phagosome. However, relative to mechanisms involved in acidifying the phagosome lumen, little is known about proton leakage pathways in this organelle. Sodium proton transporter NHE9 is a known proton leakage pathway located on the endosomes. As phagosomes acquire proteins through fusions with endosomes during maturation, NHE9 seemed a promising candidate for regulating proton fluxes on the phagosome. Here, using genetic and biophysical approaches, we show NHE9 is an important proton leakage pathway associated with the maturing phagosome. NHE9 is highly expressed in immune cells, specifically macrophages; however, NHE9 expression is strongly downregulated upon bacterial infection. We show that compensatory ectopic NHE9 expression hinders the directed motion of phagosomes along microtubules and promotes early detachment from the microtubule tracks. As a result, these phagosomes have shorter run lengths and are not successful in reaching the lysosome. In accordance with this observation, we demonstrate that NHE9 expression levels negatively correlate with bacterial survival. Together, our findings show that NHE9 regulates lumenal pH to affect phagosome maturation, and consequently, microbicidal activity in macrophages.     

Phosphorylation of mycobacterial PcaA inhibits mycolic acid cyclopropanation: consequences for intracellular survival and for phagosome maturation block

Pathogenic mycobacteria survive within macrophages by residing in phagosomes, which they prevent from maturing and fusing with lysosomes. Although several bacterial components were seen to modulate phagosome processing, the molecular regulatory mechanisms taking part in this process remain elusive. We investigated whether the phagosome maturation block (PMB) could be modulated by signaling through Ser/Thr phosphorylation. Here, we demonstrated that mycolic acid cyclopropane synthase PcaA, but not MmaA2, was phosphorylated by mycobacterial Ser/Thr kinases at Thr-168 and Thr-183 both in vitro and in mycobacteria. Phosphorylation of PcaA was associated with a significant decrease in the methyltransferase activity, in agreement with the strategic structural localization of these two phosphoacceptors. Using a BCG ΔpcaA mutant, we showed that PcaA was required for intracellular survival and prevention of phagosome maturation in human monocyte-derived macrophages. The physiological relevance of PcaA phosphorylation was further assessed by generating PcaA phosphoablative (T168A/T183A) or phosphomimetic (T168D/T183D) mutants. In contrast to the wild-type and phosphoablative pcaA alleles, introduction of the phosphomimetic pcaA allele in the ΔpcaA mutant failed to restore the parental mycolic acid profile and cording morphotype. Importantly, the PcaA phosphomimetic strain, as the ΔpcaA mutant, exhibited reduced survival in human macrophages and was unable to prevent phagosome maturation. Our results add new insight into the importance of mycolic acid cyclopropane rings in the PMB and provide the first evidence of a Ser/Thr kinase-dependent mechanism for modulating mycolic acid composition and PMB.     

The human macrophage sodium channel NaV1.5 regulates mycobacteria processing through organelle polarization and localized calcium oscillations

Phagocytosis and intracellular processing of mycobacteria by macrophages are complex cellular processes that require spatial and temporal coordination of particle uptake, organelle movement, activation of signaling pathways, and channel-mediated ionic flux. Recent work demonstrated that human macrophage NaV1.5, an intracellular voltage-gated sodium channel expressed on late endosomes, enhances endosomal acidification and phagocytosis. Here, using bacillus Camille-Guerin (BCG) as a model of mycobacterial infection, we examined how this channel regulates phagocytosis and phagosome maturation in human macrophages. Knockdown of NaV1.5 reduced high capacity uptake of labeled BCG. BCG-containing, NaV1.5-expressing cells demonstrated localization of NaV1.5 and Rab-7 positive endosomes and mitochondria to periphagosome regions that was not observed in NaV1.5-deficient cells. Knockdown of the channel reduced the initial calcium response following bacterial challenge and prevented the generation of prolonged and localized calcium oscillations during phagosome maturation. Inhibition of the mitochondrial Na(+) /Ca(2+) exchanger also prevented prolonged calcium oscillations during phagosome maturation. These results suggest that NaV1.5 and mitochondrial-dependent calcium signaling regulate mycobacteria phagocytosis and phagosome maturation in human macrophages through spatial-temporal coordination of calcium signaling within a unique subcellular region.     

Effective generation of reactive oxygen species in the mycobacterial phagosome requires K+ efflux from the bacterium

Efficient killing of mycobacteria by host macrophages depends on a number of mechanisms including production of reactive oxygen species (ROS) by the phagosomal NADPH oxidase, NOX2. Survival of pathogenic mycobacteria in the phagosome relies on the ability to control maturation of the phagosome such that it is biologically and chemically altered in comparison to phagosomes containing non‐pathogenic bacteria. In this study we show that the action of NOX2 to produce ROS in the mycobacterial phagosome is paradoxically dependent on a bacterial potassium transporter. We show that a Mycobacterium bovis BCG mutant (BCGΔkef), deficient in a Kef‐type K+ transporter, exhibits an increased intracellular survival phenotype in resting and activated macrophages, yet retains the ability to inhibit phagosome acidification, and does not show increased resistance to acidic conditions or ROS. Addition of a ROS scavenger replicates this phenotype in macrophages infected with wild‐type BCG, and the production of ROS by macrophages infected with BCGΔkef is substantially decreased compared with those infected with wild‐type BCG. Our results suggest that increased intracellular survival of BCGΔkef is mediated by inducing a decreased macrophage oxidative burst, and are consistent with Kef acting to alter the ionic contents of the phagosome and promoting NOX2 production of ROS.     

Modulation of cellular phosphatidylinositol 3-phosphate levels in primary macrophages affects heat-killed but not viable Mycobacterium avium's transport through the phagosome maturation process

Most disease causing mycobacteria are intramacrophage pathogens which replicate within nonacidified phagosomes that can interact with the early endosomal network but fail to mature to a phagolysosome. The mycobacterial phagosome retain some proteins required for fusion with endocytic vesicles including Rab5 but lack others such as early endosomal autoantigen 1 (EEA1). As the membrane lipid phosphatidylinositol 3-phosphate (PtdIns-3-P) is required for EEA1 membrane association and phagosome maturation, it may be a potential target of pathogenic mycobacteria. To test this hypothesis, macrophage cellular levels of PtdIns-3-P were altered by retroviral introduction of the type III Phosphoinositide 3-Kinase (VPS34) and the PtdIns-3-P phosphatase myotubularin 1 (MTM1). By utilizing the PtdIns-3-P-specific probes FYVE and PX coupled to EGFP (EGFP-2-FYVE and EGFP-PX, respectively), the expression of PtdIns-3-P on the mycobacterial phagosome was addressed. All phagosomes containing viable Mycobacterium avium stained positive for EGFP-2-FYVE and EGFP-PX despite obvious differences in PtdIns-3-P concentrations in cells expressing MTM1 or VPS34. Altering PtdIns-3-P cellular concentrations did not affect trafficking of live bacilli. However, a significant increase in the transport of killed bacilli to a late endosomal/lysosomal compartment was observed in VPS34-compared to MTM1-transduced macrophages. Therefore, although overexpression of PdtIns-3-P in macrophages can facilitate phagosome maturation, its effect on phagosomes containing viable M. avium was negligible.     

Cytokine effects on maturation of the phagosomes containing Mycobacteria avium subspecies paratuberculosis in J774 cells

Mycobacterium avium subspecies paratuberculosis (M. a. ptb) is an intracellular pathogen of macrophages. Intracellular survival of several species of pathogenic mycobacteria is dependent on inhibition of maturation of the phagosomes containing these pathogens into functional phagolysosomes. In activated macrophages, however, this capacity is reduced, leading to increased bacterial killing. It is the hypothesis of this study that there is increased acidification and maturation of the phagosome containing M. a. ptb in interferon gamma and lipopolysaccharide (IFN-gamma/LPS) activated macrophages. In activated macrophages colocalization of M. a. ptb with either a marker of acidic compartments (Lysotracker Red) or compartments containing a late phagosome maturation marker lysosome-associated membrane protein-1 (Lamp-1) were evaluated by laser confocal microscopy. Intracellular survival of M. a. ptb in activated macrophages was evaluated directly using differential fluorescent live/dead staining. The results of this study demonstrated increased colocalization of both Lysotracker Red and Lamp-1 with FITC labeled M. a. ptb, which correlated with decreased survival of M. a. ptb within activated macrophages.     

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.     

The facultative intracellular pathogen Candida glabrata subverts macrophage cytokine production and phagolysosome maturation

Although Candida glabrata is an important human pathogenic yeast, its pathogenicity mechanisms are largely unknown. Immune evasion strategies seem to play key roles during infection, since very little inflammation is observed in mouse models. Furthermore, C. glabrata multiplies intracellularly after engulfment by macrophages. In this study, we sought to identify the strategies that enable C. glabrata to survive phagosome biogenesis and antimicrobial activities within human monocyte-derived macrophages. We show that, despite significant intracellular proliferation, macrophage damage or apoptosis was not apparent, and production of reactive oxygen species was inhibited. Additionally, with the exception of GM-CSF, levels of pro- and anti-inflammatory cytokines were only marginally increased. We demonstrate that adhesion to and internalization by macrophages occur within minutes, and recruitment of endosomal early endosomal Ag 1 and lysosomal-associated membrane protein 1 indicates phagosome maturation. However, phagosomes containing viable C. glabrata, but not heat-killed yeasts, failed to recruit cathepsin D and were only weakly acidified. This inhibition of acidification did not require fungal viability, but it had a heat-sensitive surface attribute. Therefore, C. glabrata modifies the phagosome into a nonacidified environment and multiplies until the host cells finally lyse and release the fungi. Our results suggest persistence of C. glabrata within macrophages as a possible immune evasion strategy.     

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.