Galectins and TRIMs directly interact and orchestrate autophagic response to endomembrane damage

Macroautophagy/autophagy is a homeostatic process delivering cytoplasmic targets, including damaged organelles, to lysosomes for degradation; however, it is not completely understood how compromised endomembranes are recognized by the autophagic apparatus. We have described previously that the TRIM family of proteins act as receptors for selective autophagy. In this study we uncovered the property of TRIMs to directly interact with members of the family of cytosolic lectins termed galectins. Galectins patrol the cytoplasm and recognize compromised membranes. We show that TRIM16 uses LGALS3 (galectin 3) to detect damaged lysosomes and phagosomes. TRIM16 assembles the core autophagic machinery and is found in protein complexes with MTOR and TFEB, thus regulating their activity to set in motion endomembrane quality control. The TRIM16-LGALS3 system plays a key role in autophagic homeostasis of lysosomes and in the control of Mycobacterium tuberculosis in vivo.     

Lyn Delivers Bacteria to Lysosomes for Eradication through TLR2-Initiated Autophagy Related Phagocytosis

Extracellular bacteria, such as Pseudomonas aeruginosa and Klebsiella pneumoniae, have been reported to induce autophagy; however, the role and machinery of infection-induced autophagy remain elusive. We show that the pleiotropic Src kinase Lyn mediates phagocytosis and autophagosome maturation in alveolar macrophages (AM), which facilitates eventual bacterial eradication. We report that Lyn is required for bacterial infection-induced recruitment of autophagic components to pathogen-containing phagosomes. When we blocked autophagy with 3-methyladenine (3-MA) or by depleting Lyn, we observed less phagocytosis and subsequent bacterial clearance by AM. Both morphological and biological evidence demonstrated that Lyn delivered bacteria to lysosomes through xenophagy. TLR2 initiated the phagocytic process and activated Lyn following infection. Cytoskeletal trafficking proteins, such as Rab5 and Rab7, critically facilitated early phagosome formation, autophagosome maturation, and eventual autophagy-mediated bacterial degradation. These findings reveal that Lyn, TLR2 and Rab modulate autophagy related phagocytosis and augment bactericidal activity, which may offer insight into novel therapeutic strategies to control lung infection.     

PIKfyve Inhibition Interferes with Phagosome and Endosome Maturation in Macrophages

Macrophages eliminate pathogens and cell debris through phagocytosis, a process by which particulate matter is engulfed and sequestered into a phagosome. Nascent phagosomes are innocuous organelles resembling the plasma membrane. However, through a maturation process, phagosomes are quickly remodeled by fusion with endosomes and lysosomes to form the phagolysosome. Phagolysosomes are highly acidic and degradative leading to particle decomposition. Phagosome maturation is intimately dependent on the endosomal pathway, during which diverse cargoes are sorted for recycling to the plasma membrane or for degradation in lysosomes. Not surprisingly, various regulators of the endosomal pathway are also required for phagosome maturation, including phosphatidylinositol‐3‐phosphate, an early endosomal regulator. However, phosphatidylinositol‐3‐phosphate can be modified by the lipid kinase PIKfyve into phosphatidylinositol‐3,5‐bisphosphate, which controls late endosome/lysosome functions. The role of phosphatidylinositol‐3,5‐bisphosphate in macrophages and phagosome maturation remains basically unexplored. Using Fcγ receptor‐mediated phagocytosis as a model, we describe our research showing that inhibition of PIKfyve hindered certain steps of phagosome maturation. In particular, PIKfyve antagonists delayed removal of phosphatidylinositol‐3‐phosphate and reduced acquisition of LAMP1 and cathepsin D, both common lysosomal proteins. Consistent with this, the degradative capacity of phagosomes was reduced but phagosomes appeared to still acidify. We also showed that trafficking to lysosomes and their degradative capacity was reduced by PIKfyve inhibition. Overall, we provide evidence that PIKfyve, likely through phosphatidylinositol‐3,5‐bisphosphate synthesis, plays a significant role in endolysosomal and phagosome maturation in macrophages.      

Rab10 Regulates Phagosome Maturation and Its Overexpression Rescues Mycobacterium‐Containing Phagosomes Maturation

Phagosome maturation follows a defined biochemical program and, in the vast majority of cases, the microbe inside the phagosome is killed and digested. Although, an important number of pathogens, including Mycobacterium tuberculosis, which kills around two million people every year, have acquired the ability to survive, and even replicate by arresting phagosomal maturation. To identify more of the machinery involved in phagocytosis and phagosomal maturation, we investigated the function of Rab10 in engulfment and maturation of inert particles and Mycobacterium bovis bacille Calmette‐Guérin (BCG). We showed that Rab10 association with phagosomes is transient and confocal microscopy revealed detectible levels of Rab10 on phagosomal membranes at very early time‐points, occurring even before Rab5 acquisition. Rab10 recruitment had strong functional consequence, as the knockdown of endogenous Rab10 by RNA interference or overexpression of Rab10 dominant‐negative mutant delayed maturation of phagosomes of IgG‐opsonized latex beads or heat killed‐mycobacteria. These results can be explained, at least in part, by the involvement of Rab10 in recycling of some phagosomal components. More importantly, overexpression of the constitutively active mutant of Rab10 partially rescued live‐Mycobacterium‐containing phagosomes maturation. Indeed, we found that the membrane harbouring Mycobacterium acquired early endosome antigen 1 (EEA‐1), a marker excluded from phagosomes in control cells. Altogether these results indicate that Rab10, acting upstream of Rab5, plays a prominent role in phagolysosome formation and can modulate Mycobacterium‐containing phagosomes maturation.     

RNA interference in J774 macrophages reveals a role for coronin 1 in mycobacterial trafficking but not in actin-dependent processes

Macrophages are crucial for innate immunity, apoptosis, and tissue remodeling, processes that rely on the capacity of macrophages to internalize and process cargo through phagocytosis. Coronin 1, a member of the WD repeat protein family of coronins specifically expressed in leukocytes, was originally identified as a molecule that is recruited to mycobacterial phagosomes and prevents the delivery of mycobacteria to lysosomes, allowing these to survive within phagosomes. However, a role for coronin 1 in mycobacterial pathogenesis has been disputed in favor for its role in mediating phagocytosis and cell motility. In this study, a role for coronin 1 in actin-mediated cellular processes was addressed using RNA interference in the murine macrophage cell line J774. It is shown that the absence of coronin 1 in J774 macrophages expressing small interfering RNA constructs specific for coronin 1 does not affect phagocytosis, macropinocytosis, cell locomotion, or regulation of NADPH oxidase activity. However, in coronin 1-negative J774 cells, internalized mycobacteria were rapidly transferred to lysosomes and killed. Therefore, these results show that in J774 cells coronin 1 has a specific role in modulating phagosome-lysosome transport upon mycobacterial infection and that it is dispensable for most F-actin-mediated cytoskeletal rearrangements.     

Interactions of pathogenic bacteria with autophagy systems

Autophagy is a conserved cellular degradative pathway that is now established to be a vital part of the host immune response to microbial infection. Autophagy can directly eliminate intracellular pathogens by mediating their delivery to lysosomes. Canonical autophagy is characterized by the formation of a double-membrane autophagosome and the involvement of over 35 autophagy-related proteins (Atgs), including a commonly used autophagosome marker in mammalian cells, LC3. Recent studies have shown that a subset of autophagy components can lead to LC3 conjugation onto phagosomes. This process of LC3-associated phagocytosis (LAP) results in the degradation of the cargo by promoting phagosome fusion with lysosomes. Other components of the autophagy machinery also play roles in immunity that are distinct from the canonical autophagy and LAP pathways. This minireview highlights the complicated relationship between autophagy components and intracellular bacteria, including bacterial targeting mechanisms and the interaction between autophagy and effectors/toxins secreted by bacteria.     

Phagocytized Intracellular Microsporidian Blocks Phagosome Acidification and Phagosome-Lysosome Fusion

This study examines the relationship between phagosome acidification and phagosome-lysosome fusion events using phagocytized Glugea hertwigi spores. The incidence of lysosome fusion with Glugea spores in phagosomes of mouse peritoneal macrophages and of Tetrahymena was monitored using colloidal gold and acridine orange as labels for secondary lysosomes. Over 80% of the Glugea phagosomes remained segregated from the labeled compartments in macrophages after 60 min; this inhibition of fusion was still evident after 4 h. In Tetrahymena, Glugea spores also showed a high capacity to block fusion with secondary lysosomes (67%); however, spores coated with cationized ferritin showed an 80% fusion rate with labeled acidic compartments (i.e. lysosomes) after 60 min with both Tetrahymena and macrophages. The pH of phagosome compartments was monitored by measuring the emissions of fluorescein isothiocyanate (FITQ-labeled Glugea ingested by Tetrahymena. Tetrahymena phagosomes with FITC-Glugea did not acidify within the first hour after phagocytosis; however, phagosomes with cationized ferritin-labeled Glugea underwent acidification during this time period. This acidification took place although the capability of the host cells' lysosomes to fuse was blocked by pretreatment with poly-D-glutamic acid. The cationized ferritin bound to Glugea spores was uncoupled from the spore wall prior to fusion with colloidal gold-labeled compartments. In vitro testing showed that ferritin dissociation requires an acid pH, indicating that phagosomes acidify prior to lysosome fusion.     

Role of sulfated glycoprotein-1 (SGP-1) in the disposal of residual bodies by sertoli cells of the rat

Sulfated glycoprotein-1 (SGP-1) is a polypeptide secreted by Sertoli cells in the rat. Sequence analysis revealed a 76% sequence similarity with human prosaposin produced by various cell types. Human prosaposin is a 70 kDa protein which is cleaved in the lysosomes into four 10–15 kDa polypeptides termed saposins A, B, C, and D. The function of lysosomal saposins is to either solubilize certain membrane glycolipids or to form complexes with lysosomal enzymes and/or their glycolipid substrate to facilitate their hydrolysis. The present investigation dealt with the delivery of SGP-1 into the phagosomes of Sertoli cells; these phagosomes contain the residual bodies which detach from the late spermatids at the time of spermiation. Immunogold labeling with anti-SGP-1 antibody was found over Sertoli cell lysosomes, but was absent from phagosomes formed after phagocytosis of spermatid residual bodies in the apical Sertoli cell cytoplasm in stages VIII and early IX of the cycle of the seminiferous epithelium. The phagosomes found later in the basal Sertoli cell cytoplasm in stages IX and X of the cycle became labeled with the antibody as the components of the residual bodies rapidly underwent lysis and disappeared from the Sertoli cells. Sertoli cell lysosomes isolated by cell fractionation (estimated purity of 80%) were found to contain a 65 kDa form of SGP-1 or prosaposin, as well as the 15 kDa polypeptides or saposins. Thus, it appears that this unique lysosomal form of SGP-1 reached the Sertoli cell phagosomes and that their derived polypeptides, the saposins, must play a role in the hydrolysis of membrane glycolipids found in phagocytosed residual bodies. © 1995 Wiley-Liss, Inc.     

Changes in the composition of the lysosomes in hepatocytes cultured in vitro in modelling pathological states

The monolayer cultures of newborn rats hepatocytes were treated with substrate starvation (20 min.) anoxia accompanied with substrate starvation (1 h.), following rehabilitation (1 h.) and cooling (4 degrees C, 30 min). After the neutral red staining we have examined the alterations in lysosomes quantity and the development of phagocytosis; we also tested the hepatocytes distribution due to the amount of the second lysosomes per cell. We have shown that a short time pathological treatment caused a decrease in large lysosomes (1-3 microns) amount in significant portion of hepatocytes. There were neither increase in phagosomes amount per cell nor alterations in the quantity of cells, containing phagosomes. However a long time pathological treatment caused the increase in phagosomes and cells. We propose that initial stage of lysosomal apparatus alterations, connected with reduction of CAMP level, is accompanied by a stamping of large lysosomes.     

基于自噬溶酶体形成机制调控缺血性脑卒中后神经元自噬流

脑卒中是由脑血管阻塞或出血引发的急性脑血管病，约84%的临床脑卒中患者由脑缺血引起。研究表明，自噬广泛参与并显著影响脑卒中病理生理进程。自噬是一个将陈旧蛋白质、损伤细胞器及多余胞质组分等呈递给溶酶体进行降解的代谢过程，其包括自噬的激活、自噬体的形成和成熟、自噬体与溶酶体融合、自噬产物在自噬溶酶体内消化和降解等过程。自噬流通常被定义为自噬/溶酶体信号机制。最近发现，自噬流障碍是导致缺血性脑卒中后神经元损伤的重要原因，而在自噬过程中任一步骤发生障碍均可导致自噬流损伤。本文重点对自噬体-溶酶体融合的机制，以及该机制在缺血性脑卒中后发生障碍的致病机理进行详细阐述，以期基于自噬体-溶酶体融合机制对神经元自噬流进行调节，进而诱导缺血性脑卒中后的神经保护。本文可为脑卒中病理机制研究指明方向，为脑卒中治疗探寻新的线索。     

Activation of the macroautophagy pathway by Yersinia enterocolitica promotes intracellular multiplication and egress of yersiniae from epithelial cells

The virulence strategy of pathogenic Yersinia spp. involves cell‐invasive as well as phagocytosis‐preventing tactics to enable efficient colonisation of the host organism. Enteropathogenic yersiniae display an invasive phenotype in early infection stages, which facilitates penetration of the intestinal mucosa. Here we show that invasion of epithelial cells by Yersinia enterocolitica is followed by intracellular survival and multiplication of a subset of ingested bacteria. The replicating bacteria were enclosed in vacuoles with autophagy‐related characteristics, showing phagophore formation, xenophagy, and recruitment of cytoplasmic autophagosomes to the bacteria‐containing compartments. The subsequent fusion of these vacuoles with lysosomes and concomitant vesicle acidification were actively blocked by Yersinia. This resulted in increased intracellular proliferation and detectable egress of yersiniae from infected cells. Notably, deficiency of the core autophagy machinery component FIP200 impaired the development of autophagic features at Yersinia‐containing vacuoles as well as intracellular replication and release of bacteria to the extracellular environment. These results suggest that Y. enterocolitica may take advantage of the macroautophagy pathway in epithelial cells to create an autophagosomal niche that supports intracellular bacterial survival, replication, and, eventually, spread of the bacteria from infected cells.     

Ca2+ and synaptotagmin VII-dependent delivery of lysosomal membrane to nascent phagosomes

Synaptotagmin (Syt) VII is a ubiquitously expressed member of the Syt family of Ca2+ sensors. It is present on lysosomes in several cell types, where it regulates Ca2+-dependent exocytosis. Because [Ca2+]i and exocytosis have been associated with phagocytosis, we investigated the phagocytic ability of macrophages from Syt VII-/- mice. Syt VII-/- macrophages phagocytose normally at low particle/cell ratios but show a progressive inhibition in particle uptake under high load conditions. Complementation with Syt VII rescues this phenotype, but only when functional Ca2+-binding sites are retained. Reinforcing a role for Syt VII in Ca2+-dependent phagocytosis, particle uptake in Syt VII-/- macrophages is significantly less dependent on [Ca2+]i. Syt VII is concentrated on peripheral domains of lysosomal compartments, from where it is recruited to nascent phagosomes. Syt VII recruitment is rapidly followed by the delivery of Lamp1 to phagosomes, a process that is inhibited in Syt VII-/- macrophages. Thus, Syt VII regulates the Ca2+-dependent mobilization of lysosomes as a supplemental source of membrane during phagocytosis.     

Endocytic SNAREs are involved in optimal Coxiella burnetii vacuole development

Coxiella burnetii is a Gram‐negative intracellular bacterium. As previously described, both the endocytic and the autophagic pathways contribute to the maturation of Coxiella replicative vacuoles (CRVs). The large CRVs share the properties of both phagolysosomal and autophagolysosomal compartments. Vamp3, Vamp7 and Vamp8 are v‐SNAREs involved in the endocytic pathway which participate mainly in the fusion between endosomes and lysosomes. In the present study we observed that Vamp7 interacts with C. burnetii at different infection times (1 h–48 h p.i.). We have determined that a truncated mutant of Vamp7 (Vamp7 NT) and a siRNA against this SNARE protein affects the optimal development of CRVs, suggesting that Vamp7 mediates fusion events that are required for the biogenesis of CRVs. Indeed, we have observed that overexpression of Vamp7 NT inhibited the heterotypic fusion with lysosomes and the homotypic fusion between individual Coxiella phagosomes and CRVs. Moreover, we have detected in the vacuole membrane, at different infection times, the Vamp7 partners (Vti1a and Vti1b). Interestingly, treatment with chloramphenicol reduced the colocalization between C. burnetii and Vamp7, Vti1a or Vti1b, indicating that the recruitment of these SNAREs proteins is a bacteria‐driven process that favours the CRV biogenesis, likely by facilitating the interaction with the endolysosomal compartment.     

Beclin1-binding UVRAG targets the class C Vps complex to coordinate autophagosome maturation and endocytic trafficking

Autophagic and endocytic pathways are tightly regulated membrane rearrangement processes that are crucial for homeostasis, development and disease. Autophagic cargo is delivered from autophagosomes to lysosomes for degradation through a complex process that topologically resembles endosomal maturation. Here, we report that a Beclin1-binding autophagic tumour suppressor, UVRAG, interacts with the class C Vps complex, a key component of the endosomal fusion machinery. This interaction stimulates Rab7 GTPase activity and autophagosome fusion with late endosomes/lysosomes, thereby enhancing delivery and degradation of autophagic cargo. Furthermore, the UVRAG-class-C-Vps complex accelerates endosome-endosome fusion, resulting in rapid degradation of endocytic cargo. Remarkably, autophagosome/endosome maturation mediated by the UVRAG-class-C-Vps complex is genetically separable from UVRAG-Beclin1-mediated autophagosome formation. This result indicates that UVRAG functions as a multivalent trafficking effector that regulates not only two important steps of autophagy - autophagosome formation and maturation - but also endosomal fusion, which concomitantly promotes transport of autophagic and endocytic cargo to the degradative compartments.     

Drosophila Rab2 controls endosome-lysosome fusion and LAMP delivery to late endosomes

Rab2 is a conserved Rab GTPase with a well-established role in secretory pathway function and phagocytosis. Here we demonstrate that Drosophila Rab2 is recruited to late endosomal membranes, where it controls the fusion of LAMP-containing biosynthetic carriers and lysosomes to late endosomes. In contrast, the lysosomal GTPase Gie/Arl8 is only required for late endosome-lysosome fusion, but not for the delivery of LAMP to the endocytic pathway. We also find that Rab2 is required for the fusion of autophagosomes to the endolysosomal pathway, but not for the biogenesis of lysosome-related organelles. Surprisingly, Rab2 does not rely on HOPS-mediated vesicular fusion for recruitment to late endosomal membranes. Our work suggests that Drosophila Rab2 is a central regulator of the endolysosomal and macroautophagic/autophagic pathways by controlling the major heterotypic fusion processes at the late endosome.     

The Phosphoinositide‐Gated Lysosomal Ca2+ Channel,TRPML1, Is Required for Phagosome Maturation

Macrophages internalize and sequester pathogens into a phagosome. Phagosomes then sequentially fuse with endosomes and lysosomes, converting into degradative phagolysosomes. Phagosome maturation is a complex process that requires regulators of the endosomal pathway including the phosphoinositide lipids. Phosphatidylinositol‐3‐phosphate and phosphatidylinositol‐3,5‐bisphosphate (PtdIns(3,5)P₂), which respectively control early endosomes and late endolysosomes, are both required for phagosome maturation. Inhibition of PIKfyve, which synthesizes PtdIns(3,5)P₂, blocked phagosome–lysosome fusion and abated the degradative capacity of phagosomes. However, it is not known how PIKfyve and PtdIns(3,5)P₂ participate in phagosome maturation. TRPML1 is a PtdIns(3,5)P₂‐gated lysosomal Ca²⁺ channel. Because Ca²⁺ triggers membrane fusion, we postulated that TRPML1 helps mediate phagosome–lysosome fusion. Using Fcγ receptor‐mediated phagocytosis as a model, we describe our research showing that silencing of TRPML1 hindered phagosome acquisition of lysosomal markers and reduced the bactericidal properties of phagosomes. Specifically, phagosomes isolated from TRPML1‐silenced cells were decorated with lysosomes that docked but did not fuse. We could rescue phagosome maturation in TRPML1‐silenced and PIKfyve‐inhibited cells by forcible Ca²⁺ release with ionomycin. We also provide evidence that cytosolic Ca²⁺ concentration increases upon phagocytosis in a manner dependent on TRPML1 and PIKfyve. Overall, we propose a model where PIKfyve and PtdIns(3,5)P₂ activate TRPML1 to induce phagosome–lysosome fusion.      

Involvement of heterotrimeric G proteins in phagocytosis and recycling from the phagosomal compartment.

Phagocytosis is a receptor-mediated process by which specialized cell types engulf large extracellular particles. Phagosome maturation involves a series of intracellular membrane fusion and budding events resulting in the delivery of particles to compartments enriched in lysosomal hydrolases where they are digested. Substantial amounts of plasma membrane and many phagosomal proteins, such as receptors, rapidly recycle to the plasma membrane following phagosome formation. Despite the importance of this recycling pathway in phagosome maturation and in the retrieval of immunogenic peptides from phagosomes, the molecular machinery involved is largely unknown. To assess the participation of GTPases in phagocytosis and recycling from phagosomes we used aluminum fluoride (AIF(-)(4)), which activates the GDP-bound form of stimulatory and inhibitory trimeric G proteins. AlF(-)(4) inhibited both the uptake to and the recycling from the phagosomal compartment. Cholera toxin, which activates Galphas, and pertussis toxin, which uncouples Gi and Go from receptors, were effective inhibitors of phagocytosis. However, both toxins stimulated recycling from phagosomes. These results suggest that more than one GTP-binding protein participates either directly or indirectly not only in phagocytosis, but also in maturation and recycling from phagosomes, and thereby assign a role for heterotrimeric G proteins in controlling traffic through the phagocytic pathway.     

Virulence‐associated protein A from Rhodococcus equi is an intercompartmental pH‐neutralising virulence factor

Professional phagocytic cells such as macrophages are a central part of innate immune defence. They ingest microorganisms into membrane‐bound compartments (phagosomes), which acidify and eventually fuse with lysosomes, exposing their contents to a microbicidal environment. Gram‐positive Rhodococcus equi can cause pneumonia in young foals and in immunocompromised humans. The possession of a virulence plasmid allows them to subvert host defence mechanisms and to multiply in macrophages. Here, we show that the plasmid‐encoded and secreted virulence‐associated protein A (VapA) participates in exclusion of the proton‐pumping vacuolar‐ATPase complex from phagosomes and causes membrane permeabilisation, thus contributing to a pH‐neutral phagosome lumen. Using fluorescence and electron microscopy, we show that VapA is also transferred from phagosomes to lysosomes where it permeabilises the limiting membranes for small ions such as protons. This permeabilisation process is different from that of known membrane pore formers as revealed by experiments with artificial lipid bilayers. We demonstrate that, at 24 hr of infection, virulent R. equi is contained in a vacuole, which is enriched in lysosome material, yet possesses a pH of 7.2 whereas phagosomes containing a vapA deletion mutant have a pH of 5.8 and those with virulence plasmid‐less sister strains have a pH of 5.2. Experimentally neutralising the macrophage endocytic system allows avirulent R. equi to multiply. This observation is mirrored in the fact that virulent and avirulent R. equi multiply well in extracts of purified lysosomes at pH 7.2 but not at pH 5.1. Together these data indicate that the major function of VapA is to generate a pH‐neutral and hence growth‐promoting intracellular niche. VapA represents a new type of Gram‐positive virulence factor by trafficking from one subcellular compartment to another, affecting membrane permeability, excluding proton‐pumping ATPase, and consequently disarming host defences.     

Mitophagy in cells with mtDNA mutations

Despite the emergence of autophagy as a key process for mitochondrial quality control, the existence and persistence of pathogenic mtDNA mutations in human disease suggests that the degradation of dysfunctional mitochondria does not occur widely in vivo. During macroautophagy, a double-membraned cup-shaped structure engulfs cytosolic content. This autophagic vesicle then fuses with lysosomes, allowing hydrolytic enzymes to degrade the contents. Mitochondrial autophagy, or mitophagy, is thought to degrade damaged or nonfunctioning mitochondria specifically. The Parkinson disease-related proteins PINK1 (a mitochondrially localized kinase) and PARK2 (PARKIN, a cytosolically-localized E3 ubiquitin ligase) are essential for targeting mitochondria for mitophagy. Upon chemical uncoupling of the mitochondrial transmembrane potential (Δψ_m), PINK1 located in the mitochondrial outer membrane recruits PARK2 from the cytosol to the mitochondria, followed by delivery of the organelle to the autophagic machinery for degradation.     

Cholesterol depletion in Mycobacterium avium-infected macrophages overcomes the block in phagosome maturation and leads to the reversible sequestration of viable mycobacteria in phagolysosome-derived autophagic vacuoles

Phagocytic entry of mycobacteria into macrophages requires the presence of cholesterol in the plasma membrane. This suggests that pathogenic mycobacteria may require cholesterol for their subsequent intra-cellular survival in non-maturing phagosomes. Here we report on the effect of cholesterol depletion on pre-existing phagosomes in mouse bone marrow-derived macrophages infected with Mycobacterium avium. Cholesterol depletion with methyl-beta-cyclodextrin resulted in a loosening of the close apposition between the phagosome membrane and the mycobacterial surface, followed by fusion with lysosomes. The resulting phagolysosomes then autonomously executed autophagy, which did not involve the endoplasmic reticulum. After 5 h of depletion, intact mycobacteria had accumulated in large auto-phagolysosomes. Autophagy was specific for phagolysosomes that contained mycobacteria, as it did not involve latex bead-containing phagosomes in infected cells. Upon replenishment of cholesterol, mycobacteria became increasingly aligned to the lysosomal membrane, from where they were individually sequestered in phagosomes with an all-around closely apposed phagosome membrane and which no longer fused with lysosomes. These observations indicate that, cholesterol depletion (i) resulted in phagosome maturation and fusion with lysosomes and (ii) caused mycobacterium-containing phagolysosomes to autonomously undergo autophagy. Furthermore, (iii) mycobacteria were not killed in auto-phagolysosomes, and (iv) cholesterol replenishment enabled mycobacterium to rescue itself from autophagic phagolysosomes to again reside individually in phagosomes which no longer fused with lysosomes.