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.     

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.     

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.     

Mycobacterium tuberculosis phagosome

The arrest of Mycobacterium tuberculosis phagosome maturation in infected macrophages is a phenomenon of dual significance both for the pathogenesis of tuberculosis and as a model system to study interference of microbes with membrane trafficking and organelle biogenesis in host cells. Among other factors, compartment-specialized regulators of vesicular trafficking and other parts of membrane fusion machinery are likely to play a role in these processes. Here we summarize the emerging view of mycobacterial phagosome maturation arrest in the context of the dynamic processes of intracellular membrane trafficking.     

Autophagy in immune defense against Mycobacterium tuberculosis

Autophagy is a newly recognized innate and adaptive immunity defense against intracellular pathogens, in keeping with its role as a cytoplasmic maintenance pathway. Induction of autophagy by physiological, pharmacological or immunological means can eliminate intracellular Mycobacterium tuberculosis, providing one of the first examples of the immunological role of autophagy. Under normal circumstances, M. Tuberculosis survives in macrophages by inhibiting phagolysosome biogenesis. Induction of autophagy overcomes the mycobacterial phagosome maturation block, and delivers the tubercle bacilli to degradative compartments where they are eliminated.     

Autophagy in Immune Defense Against Mycobacterium tuberculosis

Autophagy is a newly recognized innate and adaptive immunity defense against intracellular pathogens, in keeping with its role as a cytoplasmic maintenance pathway. Induction of autophagy by physiological, pharmacological or immunological means can eliminate intracellular Mycobacterium tuberculosis, providing one of the first examples of the immunological role of autophagy. Under normal circumstances, M. tuberculosis survives in macrophages by inhibiting phagolysosome biogenesis. Induction of autophagy overcomes the mycobacterial phagosome maturation block, and delivers the tubercle bacilli to degradative, compartments, where they are eliminated.     

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.     

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.     

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.     

LRRK2 is a negative regulator of Mycobacterium tuberculosis phagosome maturation in macrophages

Mutations in the leucine‐rich repeat kinase 2 (LRRK2) are associated with Parkinson's disease, chronic inflammation and mycobacterial infections. Although there is evidence supporting the idea that LRRK2 has an immune function, the cellular function of this kinase is still largely unknown. By using genetic, pharmacological and proteomics approaches, we show that LRRK2 kinase activity negatively regulates phagosome maturation via the recruitment of the Class III phosphatidylinositol‐3 kinase complex and Rubicon to the phagosome in macrophages. Moreover, inhibition of LRRK2 kinase activity in mouse and human macrophages enhanced Mycobacterium tuberculosis phagosome maturation and mycobacterial control independently of autophagy. In vivo, LRRK2 deficiency in mice resulted in a significant decrease in M. tuberculosis burdens early during the infection. Collectively, our findings provide a molecular mechanism explaining genetic evidence linking LRRK2 to mycobacterial diseases and establish an LRRK2‐dependent cellular pathway that controls M. tuberculosis replication by regulating phagosome maturation.     

ESX-1-mediated translocation to the cytosol controls virulence of mycobacteria

Mycobacterium species, including Mycobacterium tuberculosis and Mycobacterium leprae, are among the most potent human bacterial pathogens. The discovery of cytosolic mycobacteria challenged the paradigm that these pathogens exclusively localize within the phagosome of host cells. As yet the biological relevance of mycobacterial translocation to the cytosol remained unclear. In this current study we used electron microscopy techniques to establish a clear link between translocation and mycobacterial virulence. Pathogenic, patient-derived mycobacteria species were found to translocate to the cytosol, while non-pathogenic species did not. We were further able to link cytosolic translocation with pathogenicity by introducing the ESX-1 (type VII) secretion system into the non-virulent, exclusively phagolysosomal Mycobacterium bovis BCG. Furthermore, we show that translocation is dependent on the C-terminus of the early-secreted antigen ESAT-6. The C-terminal truncation of ESAT-6 was shown to result in attenuation in mice, again linking translocation to virulence. Together, these data demonstrate the molecular mechanism facilitating translocation of mycobacteria. The ability to translocate from the phagolysosome to the cytosol is with this study proven to be biologically significant as it determines mycobacterial virulence.     

Selected lipids activate phagosome actin assembly and maturation resulting in killing of pathogenic mycobacteria

Pathogenic mycobacteria such as Mycobacterium tuberculosis and Mycobacterium avium facilitate disease by surviving intracellularly within a potentially hostile environment: the macrophage phagosome. They inhibit phagosome maturation processes, including fusion with lysosomes, acidification and, as shown here, membrane actin assembly. An in vitro assay developed for latex bead phagosomes (LBPs) provided insights into membrane signalling events that regulate phagosome actin assembly, a process linked to membrane fusion. Different lipids were found to stimulate or inhibit actin assembly by LBPs and mycobacterial phagosomes in vitro. In addition, selected lipids activated actin assembly and phagosome maturation in infected macrophages, resulting in a significant killing of M. tuberculosis and M. avium. In contrast, the polyunsaturated sigma-3 lipids behaved differently and stimulated pathogen growth. Thus, lipids can be involved in both stimulatory and inhibitory signalling networks in the phagosomal membrane.     

Mycobacterial mutants with defective control of phagosomal acidification

The pathogenesis of mycobacterial infection is associated with an ability to interfere with maturation of the phagosomal compartment after ingestion by macrophages. Identification of the mycobacterial components that contribute to this phenomenon will allow rational design of novel approaches to the treatment and prevention of tuberculosis. Microarray-based screening of a transposon library was used to identify mutations that influence the fate of Mycobacterium bovis bacille Calmette-Guérin (BCG) following uptake by macrophages. A screen based on bacterial survival during a 3-d infection highlighted genes previously implicated in growth of Mycobacterium tuberculosis in macrophages and in mice, together with a number of other virulence genes including a locus encoding virulence-associated membrane proteins and a series of transporter molecules. A second screen based on separation of acidified and non-acidified phagosomes by flow cytometry identified genes involved in mycobacterial control of early acidification. This included the KefB potassium/proton antiport. Mutants unable to control early acidification were significantly attenuated for growth during 6-d infections of macrophages. Early acidification of the phagosome is associated with reduced survival of BCG in macrophages. A strong correlation exists between genes required for intracellular survival of BCG and those required for growth of M. tuberculosis in mice. In contrast, very little correlation exists between genes required for intracellular survival of BCG and those that are up-regulated during intracellular adaptation of M. tuberculosis. This study has identified targets for interventions to promote immune clearance of tuberculosis infection. The screening technologies demonstrated in this study will be useful to the study of pathogenesis in many other intracellular microorganisms.     

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.     

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.     

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.     

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.     

Mobility of late endosomal and lysosomal markers on phagosomes analyzed by fluorescence recovery after photobleaching

During phagosome maturation, the late endosomal marker Rab7 and the lysosomal marker LAMP1 localize to the phagosomes. We investigated the mobility of Rab7 and LAMP1 on the phagosomes in macrophages by fluorescence recovery after photobleaching (FRAP) analysis. Rab7 was mobile between the phagosomal membrane and the cytosol in macrophages that ingested latex beads during phagosome maturation. The addition of interferon-γ (IFN-γ) restricted this mobility, suggesting that Rab7 is forced to bind to the phagosomal membrane by IFN-γ-mediated activation. Immobilization of LAMP1 on the phagosomes was observed irrespective of IFN-γ-activation. We further examined the mobility of Rab7 on the phagosomes containing Mycobacterium bovis BCG by FRAP analysis. The rate of fluorescence recovery for Rab7 on mycobacterial phagosomes was lower than that on the phagosomes containing latex beads, suggesting that mycobacteria impaired the mobility of Rab7 and arrested phagosome maturation.     

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.