[Show abstract][Hide abstract] ABSTRACT: Autophagy is a fundamental biological process of the eukaryotic cell contributing to diverse cellular and physiological functions including cell-autonomous defense against intracellular pathogens. Here, we screened the Rab family of membrane trafficking regulators for effects on autophagic elimination of Mycobacterium tuberculosis var. bovis BCG and found that Rab8b and its downstream interacting partner, innate immunity regulator TBK-1, are required for autophagic elimination of mycobacteria in macrophages. TBK-1 was necessary for autophagic maturation. TBK-1 coordinated assembly and function of the autophagic machinery and phosphorylated the autophagic adaptor p62 (sequestosome 1) on Ser-403, a residue essential for its role in autophagic clearance. A key proinflammatory cytokine, IL-1β, induced autophagy leading to autophagic killing of mycobacteria in macrophages, and this IL-1β activity was dependent on TBK-1. Thus, TBK-1 is a key regulator of immunological autophagy and is responsible for the maturation of autophagosomes into lytic bactericidal organelles.
[Show abstract][Hide abstract] ABSTRACT: mTOR (mammalian target of rapamycin) signalling and macroautophagy (henceforth autophagy) regulate numerous pathological and physiological processes, including cellular responses to altered nutrient levels. However, the mechanisms regulating mTOR and autophagy remain incompletely understood. Lysosomes are dynamic intracellular organelles intimately involved both in the activation of mTOR complex 1 (mTORC1) signalling and in degrading autophagic substrates. Here we report that lysosomal positioning coordinates anabolic and catabolic responses with changes in nutrient availability by orchestrating early plasma-membrane signalling events, mTORC1 signalling and autophagy. Activation of mTORC1 by nutrients correlates with its presence on peripheral lysosomes that are physically close to the upstream signalling modules, whereas starvation causes perinuclear clustering of lysosomes, driven by changes in intracellular pH. Lysosomal positioning regulates mTORC1 signalling, which in turn influences autophagosome formation. Lysosome positioning also influences autophagosome-lysosome fusion rates, and thus controls autophagic flux by acting at both the initiation and termination stages of the process. Our findings provide a physiological role for the dynamic state of lysosomal positioning in cells as a coordinator of mTORC1 signalling with autophagic flux.
[Show abstract][Hide abstract] ABSTRACT: IRGM, a human immunity-related GTPase, confers autophagic defence against intracellular pathogens by an unknown mechanism. Here, we report an unexpected mode of IRGM action. IRGM demonstrated differential affinity for the mitochondrial lipid cardiolipin, translocated to mitochondria, affected mitochondrial fission and induced autophagy. Mitochondrial fission was necessary for autophagic control of intracellular mycobacteria by IRGM. IRGM influenced mitochondrial membrane polarization and cell death. Overexpression of IRGMd, but not IRGMb splice isoforms, caused mitochondrial depolarization and autophagy-independent, but Bax/Bak-dependent, cell death. By acting on mitochondria, IRGM confers autophagic protection or cell death, explaining IRGM action both in defence against tuberculosis and in the damaging inflammation caused by Crohn's disease.
[Show abstract][Hide abstract] ABSTRACT: Autophagy allows cells to self-digest portions of their own cytoplasm for a multitude of physiological purposes, including innate and adaptive immunity functions. In one of its innate immunity manifestations, autophagy, is known to contribute to the killing of intracellular microbes, including Mycobacterium tuberculosis, although the molecular mechanisms have been unclear. Here, we delineated sequential steps of the autophagic pathway necessary to control intracellular M. tuberculosis and found that in addition to autophagy initiation and maturation, an accessory autophagy-targeting molecule p62 (A170 or SQSTM1) was required for mycobactericidal activity. The p62 adaptor protein delivered specific ribosomal and bulk ubiquitinated cytosolic proteins to autolysosomes where they were proteolytically converted into products capable of killing M. tuberculosis. Thus, p62 brings cytosolic proteins to autolysosomes where they are processed from innocuous precursors into neo-antimicrobial peptides, explaining in part the unique bactericidal properties of autophagic organelles.
[Show abstract][Hide abstract] ABSTRACT: The majority of studies on autophagy, a cytoplasmic homeostasis pathway of broad biological and medical significance, have been hitherto focused on the phosphatidylinositol 3-kinases as the regulators of autophagy. Here, we addressed the reverse process driven by phosphoinositide phosphatases and uncovered a key negative regulatory role in autophagy of a phosphatidylinositol 3-phosphate (PI3P) phosphatase Jumpy (MTMR14). Jumpy associated with autophagic isolation membranes and early autophagosomes, defined by the key factor Atg16 necessary for proper localization and development of autophagic organelles. Jumpy orchestrated orderly succession of Atg factors by controlling recruitment to autophagic membranes of the sole mammalian Atg factor that interacts with PI3P, WIPI-1 (Atg18), and by affecting the distribution of Atg9 and LC3, the two Atg factors controlling organization and growth of autophagic membranes. A catalytically inactive Jumpy mutant, R336Q, found in congenital disease centronuclear myopathy, lost the ability to negatively regulate autophagy. This work reports for the first time that initiation of autophagy is controlled not only by the forward reaction of generating PI3P through a lipid kinase but that its levels are controlled by a specific PI3P phosphatase, which when defective can lead to human disease.
The EMBO Journal 08/2009; 28(15):2244-58. · 9.82 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Autophagy is a cytoplasmic degradative pathway that can participate in biosynthetic processes, as in the yeast Cvt pathway, but is more commonly known for its functions in removing damaged or surplus organelles and macromolecular complexes. Here, we find that autophagy intersects with human immunodeficiency virus (HIV) biogenesis, mirroring the above dichotomy. Early, nondegradative stages of autophagy promoted HIV yields. HIV Gag-derived proteins colocalized and interacted with the autophagy factor LC3, and autophagy promoted productive Gag processing. Nevertheless, when autophagy progressed through maturation stages, HIV was degraded. This, however, does not occur, as the HIV protein Nef acts as an antiautophagic maturation factor through interactions with the autophagy regulatory factor Beclin 1, thus protecting HIV from degradation. The dual interaction of HIV with the autophagy pathway enhances viral yields by using the early stages while inhibiting the late stages of autophagy. The role of Nef in the latter process enhances yields of infectious HIV and may be of significance for progression to clinical AIDS.
The Journal of Cell Biology 08/2009; 186(2):255-68. · 10.82 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Autophagy is a major intracellular pathway for the lysosomal degradation of long-lived cytoplasmic macromolecules and damaged or surplus organelles. More recently, autophagy has also been linked with innate and adaptive immune responses against intracellular pathogens, including Mycobacterium tuberculosis, which can survive within macrophages by blocking fusion of the phagosome with lysosomes. Induction of autophagy by the Th1 cytokine IFN-gamma enables infected macrophages to overcome this phagosome maturation block and inhibit the intracellular survival of mycobacteria. Conversely, the Th2 cytokines IL-4 and IL-13 inhibit autophagy in murine and human macrophages. We discuss how differential modulation of autophagy by Th1 and Th2 cytokines may represent an important feature of the host response to mycobacteria.
Veterinary Immunology and Immunopathology 11/2008; 128(1-3):37-43. · 1.88 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: Autophagy is a cellular homeostasis pathway used to sustain cellular anabolic needs during times of nutrient or energy deprivation. Autophagosomes sequester cytoplasmic constituents, including macromolecules such as long-lived proteins. Upon fusion of autophagosomes with lysosomes, the engulfed cargo is degraded. The proteolysis of longlived proteins by macroautophagy is a standard, specific measure of autophagic degradation and represents an end-point assay for the pathway. The assay is based on a pulse-chase approach, whereby cellular proteins are radiolabeled by an isotopically marked amino acid, the short-lived, rapidly turned over, proteins are allowed to be degraded during a long chase period, and then the remaining, stable radiolabeled proteins are subjected to autophagic degradation. The classical application of this method has been in hepatocytes, but the recent growth of interest in autophagy has necessitated adaptation of this method in nonliver cells. Here we describe a protocol to quantify autophagic degradation of longlived proteins in macrophages. This chapter details the method of analyzing autophagic proteolysis in RAW264.7 mouse macrophages.
[Show abstract][Hide abstract] ABSTRACT: Tuberculosis is currently the most devastating human bacterial disease, causing millions of deaths annually and infecting an overwhelming percentage of the global population. Its success as a scourge lies in the ability of Mycobacterium tuberculosis to prevent normal phagolysosome biogenesis, essential to the destruction of invading microorganisms, inside macrophages. Recent work has identified host GTPases involved in the block of normal phagolysosome biogenesis during mycobacterial infection and has provided a set of methods, in particular efficient macrophage transfection, which will prove essential in examining the role of host effectors in this process.
[Show abstract][Hide abstract] ABSTRACT: Autophagy is a recently recognized immune effector mechanism against intracellular pathogens. The role of autophagy in innate immunity has been well established, but the extent of its regulation by the adaptive immune response is less well understood. The T helper 1 (Th1) cell cytokine IFN-gamma induces autophagy in macrophages to eliminate Mycobacterium tuberculosis. Here, we report that Th2 cytokines affect autophagy in macrophages and their ability to control intracellular M. tuberculosis. IL-4 and IL-13 abrogated autophagy and autophagy-mediated killing of intracellular mycobacteria in murine and human macrophages. Inhibition of starvation-induced autophagy by IL-4 and IL-13 was dependent on Akt signaling, whereas the inhibition of IFN-gamma-induced autophagy was Akt independent and signal transducer and activator of transcription 6 (STAT6) dependent. These findings establish a mechanism through which Th1-Th2 polarization differentially affects the immune control of intracellular pathogens.
[Show abstract][Hide abstract] ABSTRACT: Interconversions of phosphoinositides play a pivotal role during phagocytosis and at the subsequent stages of phagosomal maturation into the phagolysosome. Several model systems have been used to study the role of phosphoinositides in phagosomal membrane remodelling. These include phagosomes formed by inanimate objects such as latex beads, or pathogenic bacteria, e.g. Mycobacterium tuberculosis. The latter category provides naturally occurring tools to dissect membrane trafficking processes governing phagolysosome biogenesis. M. tuberculosis persists in infected macrophages by blocking Rab conversion and affecting Rab effectors. One of the major Rab effectors involved in this process is the type III phosphatidylinositol 3-kinase hVPS34. The lipid kinase hVPS34 and its enzymatic product PtdIns3P are critical for the default pathway of phagosomal maturation into phagolysosomes. Mycobacteria block PtdIns3P production and thus arrest phagosomal maturation. PtdIns3P is also critical for the process of autophagy, recently recognized as an effector of innate immunity defenses. Induction of autophagy by pharmacological, physiological, or immunological means, overcomes mycobacterial phagosome maturation block in a PtdIns3P generation dependent manner and eliminates intracellular M. tuberculosis. PtdIns3P and PtdIns3P-dependent processes represent an important cellular nexus where fundamental trafficking processes, disease causing host-pathogen interactions, and innate and adaptive immunity defense mechanisms meet.
Biochemical Society Symposium 02/2007; · 2.74 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
The EMBO Journal 12/2006; 25(22):5250-9. · 9.82 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
The Journal of Cell Biology 10/2006; 174(7):923-9. · 10.82 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: Mycobacterium tuberculosis is an intracellular pathogen of mononuclear phagocytes and is highly adapted to the human host. This bacterium enters macrophages by the phagocytic process using a defined subset of receptors and subsequently multiplies within a unique phagosomal compartment. M. tuberculosis has developed mul-tiple strategies to circumvent the normal fate of ingested pathogens both during and following phagocytosis. Evidence is emerging that surface mannosylation of M. tuberculosis bacilli is an important host adaptive mechanism for directing the phagocytic and post-phagocytic processes, especially within the unique microenvi-ronment of lung alveoli. Key biochemical pathways and mycobacterial determinants in the development and maintenance of the mycobacterial phagosome are beginning to be identified. In addition, recently it has been shown that the induction of autophagy in the macrophage plays an important role in the innate immune response to M. tuberculosis. Major developments in these areas are the focus of this chapter.
[Show abstract][Hide abstract] ABSTRACT: 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.