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Sergio Grinstein - One of the best experts on this subject based on the ideXlab platform.
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Autophagy proteins are not universally required for phagosome maturation
Autophagy, 2016Co-Authors: Marija Cemma, Sergio Grinstein, John Hunter BrumellAbstract:ABSTRACTPhagocytosis plays a central role in immunity and tissue homeostasis. After internalization of cargo into single-membrane Phagosomes, these compartments undergo a maturation sequences that terminates in lysosome fusion and cargo degradation. Components of the autophagy pathway have recently been linked to phagosome maturation in a process called LC3-associated phagocytosis (LAP). In this process, autophagy machinery is thought to conjugate LC3 directly onto the phagosomal membrane to promote lysosome fusion. However, a recent study has suggested that ATG proteins may in fact impair phagosome maturation to promote antigen presentation. Here, we examined the impact of ATG proteins on phagosome maturation in murine cells using FCGR2A/FcγR-dependent phagocytosis as a model. We show that phagosome maturation is not affected in Atg5-deficient mouse embryonic fibroblasts, or in Atg5- or Atg7-deficient bone marrow-derived macrophages using standard assays of phagosome maturation. We propose that ATG prote...
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recruitment of ocrl and inpp5b to Phagosomes by rab5 and appl1 depletes phosphoinositides and attenuates akt signaling
Molecular Biology of the Cell, 2012Co-Authors: Michal Bohdanowicz, Daniel M Balkin, Pietro De Camilli, Sergio GrinsteinAbstract:Sealing of Phagosomes is accompanied by the disappearance of phosphatidylinositol (4,5)-bisphosphate (PtdIns(4,5)P2) from their cytoplasmic leaflet. Elimination of PtdIns(4,5)P2, which is required for actin remodeling during phagosome formation, has been attributed to hydrolysis by phospholipase C and phosphorylation by phosphatidylinositol 3-kinase. We found that two inositol 5-phosphatases, OCRL and Inpp5B, become associated with nascent Phagosomes. Both phosphatases, which are Rab5 effectors, associate with the adaptor protein APPL1, which is recruited to the Phagosomes by active Rab5. Knockdown of APPL1 or inhibition of Rab5 impairs association of OCRL and Inpp5B with Phagosomes and prolongs the presence of PtdIns(4,5)P2 and actin on their membranes. Even though APPL1 can serve as an anchor for Akt, its depletion accentuated the activation of the kinase, likely by increasing the amount of PtdIns(4,5)P2 available to generate phosphatidylinositol (3,4,5)-trisphosphate. Thus, inositol 5-phosphatases are important contributors to the phosphoinositide remodeling and signaling that are pivotal for phagocytosis.
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Membrane Trafficking during Phagosome Formation and Maturation
Phagocyte-Pathogen Interactions, 2009Co-Authors: Gregory D. Fairn, Elena Gershenzon, Sergio GrinsteinAbstract:This chapter addresses the vesicular trafficking events involved in the formation and maturation of the phagosome. The reported increase in surface area during phagocytosis is most readily explained by delivery of an internal pool of membranes to the plasmalemma. Recent studies demonstrated that specific and tertiary granules contain VAMP1 and VAMP2, while azurophilic granules contain VAMP1 and VAMP7, with STX4 and SNAP23 involved in the exocytosis of these distinct populations of granules. Lysobisphosphatidic acid (LBPA) is found in late Phagosomes, where it is likely to complex ALIX and direct membrane fission and the intermediate stages of maturation. Phagosome formation and maturation are impressive microbicidal tools. For this reason pathogens have developed a remarkable variety of strategies to subvert this process. Some of the most virulent and persistent bacterial pathogens such as Mycobacterium and Leishmania in fact take advantage of the phagocytic machinery to gain access into the host cell interior, where they are able to circumvent the sophisticated killing mechanism of the maturing phagosome. At present the knowledge of phagosome maturation is rudimentary and largely extrapolated from that garnered for the endocytic pathway. Some extrapolation is probably warranted, but many unique features of Phagosomes will only be revealed by direct studies of particle, preferably microbial, ingestion.
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Cholesterol accumulation by macrophages impairs phagosome maturation.
Journal of Biological Chemistry, 2008Co-Authors: Kassidy K. Huynh, Elena Gershenzon, Sergio GrinsteinAbstract:Abstract Macrophages are key to the pathogenesis of atherosclerosis. They take up and store excessive amounts of cholesterol associated with modified low density lipoprotein, eventually becoming foam cells that display altered immune responsiveness. We studied the effects of cholesterol accumulation on phagosome formation and maturation, using lipid transport antagonists and cholesterol transport-deficient mutants. In macrophages treated with U18666A, a transport antagonist that prevents cholesterol exit from late endosomes/lysosomes, the early stages of maturation proceeded normally; Phagosomes acquired Rab5, phosphatidylinositol 3-phosphate, and EEA1 and merged with LAMP-containing vesicles. However, fusion with lysosomes was impaired. Rab7, which is required for phagolysosome formation, was acquired by Phagosomes but remained inactive. Maturation was also studied in fibroblasts from Niemann-Pick type C individuals that have defective cholesterol transport. Transfection of FcγIIA receptors was used to confer phagocytic capability to these fibroblasts. Niemann-Pick type C Phagosomes failed to fuse with lysosomes, whereas wild type fibroblasts formed normal phagolysosomes. These findings indicate that cholesterol accumulation can have a detrimental effect on phagosome maturation by impairing the activation of Rab7, sequestering it and its effectors in cholesterol-enriched multilamellar compartments.
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LAMP proteins are required for fusion of lysosomes with Phagosomes
EMBO Journal, 2007Co-Authors: Kassidy K. Huynh, Eeva Liisa Eskelinen, Cameron C. Scott, Anatoly Malevanets, Paul Saftig, Sergio GrinsteinAbstract:Lysosome-associated membrane proteins 1 and 2 (LAMP-1 and LAMP-2) are delivered to Phagosomes during the maturation process. We used cells from LAMP-deficient mice to analyze the role of these proteins in phagosome maturation. Macrophages from LAMP-1- or LAMP-2-deficient mice displayed normal fusion of lysosomes with Phagosomes. Because ablation of both the lamp-1 and lamp-2 genes yields an embryonic-lethal phenotype, we were unable to study macrophages from double knockouts. Instead, we reconstituted phagocytosis in murine embryonic fibroblasts (MEFs) by transfection of FcgammaIIA receptors. Phagosomes formed by FcgammaIIA-transfected MEFs obtained from LAMP-1- or LAMP-2- deficient mice acquired lysosomal markers. Remarkably, although FcgammaIIA-transfected MEFs from double-deficient mice ingested particles normally, phagosomal maturation was arrested. LAMP-1 and LAMP-2 double-deficient Phagosomes acquired Rab5 and accumulated phosphatidylinositol 3-phosphate, but failed to recruit Rab7 and did not fuse with lysosomes. We attribute the deficiency to impaired organellar motility along microtubules. Time-lapse cinematography revealed that late endosomes/lysosomes as well as Phagosomes lacking LAMP-1 and LAMP-2 had reduced ability to move toward the microtubule-organizing center, likely precluding their interaction with each other.
Gareth Griffiths - One of the best experts on this subject based on the ideXlab platform.
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transient assembly of f actin by Phagosomes delays phagosome fusion with lysosomes in cargo overloaded macrophages
Journal of Cell Science, 2009Co-Authors: David Liebl, Gareth GriffithsAbstract:Dynamic remodelling of the cortical actin cytoskeleton is required for phagocytic uptake of pathogens and other particles by macrophages. Actin can also be nucleated de novo on membranes of nascent Phagosomes, a process that can stimulate or inhibit phagosome fusion with lysosomes. Recently, Phagosomes were shown to polymerize actin in transient pulses, called actin `flashing9, whose function remains unexplained. Here, we investigated phagosomal actin dynamics in live macrophages expressing actin tagged with green fluorescent protein (GFP). We show that only immature Phagosomes can transiently induce assembly of actin coat, which forms a barrier preventing phagosome-lysosome docking and fusion. The capacity of Phagosomes to assemble actin is enhanced in cells exposed to increased phagocytic load, which also exhibit a delay in phagosome maturation. Parallel analysis indicated that polymerization of actin on macropinosomes also induces compression and propulsion. We show that dynamic interactions between membrane elastic tension and compression forces of polymerizing actin can also lead to macropinosome constriction and scission – a process that is obstructed on rigid Phagosomes. We hypothesize that the rate of individual phagosome maturation, as well as the biogenesis and remodelling of macropinosomes, can be regulated by the extent and manner of actin assembly on their membrane.
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Lipids regulate P2X7-receptor-dependent actin assembly by Phagosomes via ADP translocation and ATP synthesis in the phagosome lumen.
Journal of Cell Science, 2009Co-Authors: Mark P Kuehnel, Elsa Anes, Vladimir Rybin, Paras Anand, Gareth GriffithsAbstract:Latex bead Phagosomes isolated from J774 macrophages polymerize actin. We show here that five lipids – phosphatidylinositol-4-phosphate, phosphatidylinositol-(4,5)-bisphosphate, sphingosine-1-phosphate (S1P), ceramide-1-phosphate and phosphatidic acid – stimulate both actin assembly and transport of ADP across the phagosomal membrane into the lumen. Once there, this ADP is converted to ATP by adenylate kinase activity. High luminal ATP concentrations correlated well with phagosome actin assembly under different conditions. The ATP-binding P2X7 receptor (P2X7R) was detected in Phagosomes. Although S1P stimulated actin assembly by Phagosomes from P2X7R-containing bone marrow macrophages, S1P-stimulated actin assembly was inhibited in Phagosomes from cells lacking P2X7R. We propose that luminal ATP accumulates in response to selected lipids and activates the P2X7R that signals across the phagosomal membrane to trigger actin assembly on the cytoplasmic membrane surface. In the accompanying paper by Kuehnel et al. (doi:10.1242/jcs.034207), more evidence is provided in support of this model from the analysis of actin assembly at the plasma membrane of intact macrophages.
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Light and electron microscopy of Phagosomes
EMC 2008 14th European Microscopy Congress 1–5 September 2008 Aachen Germany, 2008Co-Authors: Gareth Griffiths, David Liebl, Christopher K. E. Bleck, Andreas Merz, Paul Walther, Jacques Dubochet, Benoit ZuberAbstract:Phagocytosis is the process whereby cells such as macrophages take up particles larger than 0.3 uM. The ingested particle can range from inert material such as carbon or asbestos to live pathogens such as Mycobacterium tuberculosis. After engulfment by the plasma membrane of the phagocytic cell the engulfed particle becomes surrounded by a membrane in a specialized intracellular compartment - the phagosome. This compartment fuses sequentially with early endosomes, late endosomes and lysosomes, a process referred to as phagosome maturation, in which the phagosome acquires acid hydrolases and a low pH that together facilitate pathogen clearance. Actin assembly by Phagosomes plays an important role in phagosome maturation. Once internalized into the phagocytic cell M.tuberculosis has evolved the ability to block actin assembly and maturation of phagosome, that facilitates the ability of this pathogen to survive and grow within macrophages. This presentation will focus on two aspects of phagosome biology, in order to emphasize the importance of light microscopy and EM in analyzing the cell biology of Phagosomes. In the first part the use of live cell video microscopy will be described in order to reveal striking dynamic process of transient assembly of actin by latex bead Phagosomes in GFP-actin expressing mouse macrophages. The second part will address the use of different EM sectioning approaches to visualize different mycobacteria in macrophage Phagosomes and will conclude that the best sectioning method to monitor the native structure of free mycobacteria and mycobacteria within phaagosomes is the Cryo EM of Vitrified Sections (CEMOVIS).
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Isolation of Latex Bead-and Mycobacteria-Containing Phagosomes
Cell Biology, 2006Co-Authors: Mark Kühnel, Elsa Anes, Gareth GriffithsAbstract:Publisher Summary This chapter describes how bead- and mycobacteria-containing Phagosomes are isolated for in vitro research work. This chapter also summarizes recent approaches developed for the more difficult problem of isolating Phagosomes containing live and killed mycobacteria. Phagocytosis is a crucial mechanism by which specialized cells, especially macrophages, neutophils, and dendritic cells, remove ingested debris, dead cells, and pathogens from the body. An enormous advantage of the use of latex beads is the ease by which they can be conjugated to selected proteins, such as IgG, or complement. Latex beads, either naked or coated with substances of interest, can be used for the generation of Phagosomes. Latex bead Phagosomes (LBP) have also been used extensively as control Phagosomes for analyses of pathogen-enclosing Phagosomes; the latter usually modify the intracellular behavior of the phagosome in a manner that facilitates the survival and growth of the pathogens.
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On phagosome individuality and membrane signalling networks
Trends in Cell Biology, 2004Co-Authors: Gareth GriffithsAbstract:Abstract Cells such as macrophages take up pathogens into specialized membrane organelles (Phagosomes) that fuse with other organelles, including lysosomes, in a process termed maturation. The fully matured phagolysosome is a low-pH, hydrolase-rich killing device that some pathogens can bypass. One might expect that Phagosomes containing a given type of particle that entered cells simultaneously via the same receptor would behave the same, at least in a single cell. Surprisingly, however, recent data show that Phagosomes formed via the same receptors can find themselves in different chemical states even within the same macrophage. Here, I argue that each phagosome is an individual entity whose behaviour depends on a finite number of stable equilibrium states in its membrane signalling networks.
James A. Cardelli - One of the best experts on this subject based on the ideXlab platform.
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p110-related PI 3-kinases regulate phagosome-phagosome fusion and phagosomal pH through a PKB/Akt dependent pathway in Dictyostelium.
Journal of cell science, 2020Co-Authors: Adam C. Rupper, Juan M. Rodriguez-paris, B D Grove, James A. CardelliAbstract:The Dictyostelium p110-related PI 3-kinases, PIK1 and PIK2, regulate the endosomal pathway and the actin cytoskeleton, but do not significantly regulate internalization of particles in D. discoideum. Bacteria internalized into delta ddpik1/ddpik2 cells or cells treated with PI 3-kinase inhibitors remained intact as single particles in Phagosomes with closely associated membranes after 2 hours of internalization, while in control cells, bacteria appeared degraded in multi-particle spacious Phagosomes. Addition of LY294002 to control cells, after 60 minutes of chase, blocked formation of spacious Phagosomes, suggesting PI 3-kinases acted late to regulate spacious phagosome formation. Phagosomes purified from control and drug treated cells contained equivalent levels of lysosomal proteins, including the proton pump complex, and were acidic, but in drug treated cells and delta ddpik1/ddpik2 cells phagosomal pH was significantly more acidic during maturation than the pH of control Phagosomes. Inhibition of phagosomal maturation by LY294002 was overcome by increasing phagosomal pH with NH(4)Cl, suggesting that an increase in pH might trigger homotypic phagosome fusion. A pkbA null cell line (PKB/Akt) reproduced the phenotype described for cells treated with PI 3-kinase inhibitors and delta ddpik1/ddpik2 cells. We propose that PI 3-kinases, through a PKB/Akt dependent pathway, directly regulate homotypic fusion of single particle containing Phagosomes to form multi-particle, spacious Phagosomes, possibly through the regulation of phagosomal pH.
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The regulation of phagosome maturation in Dictyostelium
Journal of Muscle Research and Cell Motility, 2002Co-Authors: Damian Duhon, James A. CardelliAbstract:Macropinocytosis (fluid uptake) and phagocytosis (particle uptake) are processes that result in the formation of intracellular membrane enclosed vacuoles termed macropinosomes and Phagosomes, respectively. Macropinosomes and Phagosomes are modified by fission and fusion reactions with the endo-lysosomal pathway that eventually transform these vacuoles into a lysosomal environment. Many human bacterial pathogens, including species of Mycobacteria, Legionella, and Chlamydia, are thought to survive by disrupting the normal membrane trafficking events that usually result in the formation of phago-lysosomes and death of the microorganism. In addition, a number of important pathogens facilitate homotypic phagosome fusion in order to generate an intracellular environment conducive for survival. A greater understanding of the regulation of phagosomal maturation and fusion will be critical in designing new therapies to treat infections caused by intracellular pathogens. The genetically tractable phagocyte, D. discoideum, has proven extremely useful in dissecting the signaling pathways regulating macropinocytosis, phagocytosis, phagosomal maturation and phagosome–phagosome fusion. A body of knowledge has accumulated and demonstrates important roles for Rab GTPases, the cytoskeleton, phosphoinositide metabolism and pH regulation in regulating phagosome maturation. This review will summarize the current state of knowledge.
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p110-related PI 3-kinases regulate phagosome-phagosome fusion and phagosomal pH through a PKB/Akt dependent pathway in Dictyostelium
Journal of Cell Science, 2001Co-Authors: Adam C. Rupper, Juan M. Rodriguez-paris, Bryon Grove, James A. CardelliAbstract:The Dictyostelium p110-related PI 3-kinases, PIK1 and PIK2, regulate the endosomal pathway and the actin cytoskeleton, but do not significantly regulate internalization of particles in D. discoideum. Bacteria internalized into (Δ)ddpik1/ddpik2 cells or cells treated with PI 3-kinase inhibitors remained intact as single particles in Phagosomes with closely associated membranes after 2 hours of internalization, while in control cells, bacteria appeared degraded in multi-particle spacious Phagosomes. Addition of LY294002 to control cells, after 60 minutes of chase, blocked formation of spacious Phagosomes, suggesting PI 3-kinases acted late to regulate spacious phagosome formation. Phagosomes purified from control and drug treated cells contained equivalent levels of lysosomal proteins, including the proton pump complex, and were acidic, but in drug treated cells and (Δ)ddpik1/ddpik2 cells phagosomal pH was significantly more acidic during maturation than the pH of control Phagosomes. Inhibition of phagosomal maturation by LY294002 was overcome by increasing phagosomal pH with NH(4)Cl, suggesting that an increase in pH might trigger homotypic phagosome fusion. A pkbA null cell line (PKB/Akt) reproduced the phenotype described for cells treated with PI 3-kinase inhibitors and (Δ)ddpik1/ddpik2 cells. We propose that PI 3-kinases, through a PKB/Akt dependent pathway, directly regulate homotypic fusion of single particle containing Phagosomes to form multi-particle, spacious Phagosomes, possibly through the regulation of phagosomal pH.
David J Kusner - One of the best experts on this subject based on the ideXlab platform.
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sphingosine kinase 1 sk1 is recruited to nascent Phagosomes in human macrophages inhibition of sk1 translocation by mycobacterium tuberculosis
Journal of Immunology, 2005Co-Authors: Christopher Thompson, Shankar S Iyer, Natalie A Melrose, Rebecca L Vanoosten, Korey R Johnson, Stuart M Pitson, Lina M Obeid, David J KusnerAbstract:Mycobacterium tuberculosis ( M.tb ) is a leading cause of global infectious mortality. The pathogenesis of tuberculosis involves inhibition of phagosome maturation, leading to survival of M.tb within human macrophages. A key determinant is M.tb -induced inhibition of macrophage sphingosine kinase (SK) activity, which normally induces Ca 2+ signaling and phagosome maturation. Our objective was to determine the spatial localization of SK during phagocytosis and its inhibition by M.tb . Stimulation of SK activity by killed M.tb , live Staphylococcus aureus , or latex beads was associated with translocation of cytosolic SK1 to the phagosome membrane. In contrast, SK1 did not associate with Phagosomes containing live M.tb . To characterize the mechanism of phagosomal translocation, live cell confocal microscopy was used to compare the localization of wild-type SK1, catalytically inactive SK1 G82D , and a phosphorylation-defective mutant that does not undergo plasma membrane translocation (SK1 S225A ). The magnitude and kinetics of translocation of SK1 G82D and SK1 S225A to latex bead Phagosomes were indistinguishable from those of wild-type SK1, indicating that novel determinants regulate the association of SK1 with nascent Phagosomes. These data are consistent with a model in which M.tb inhibits both the activation and phagosomal translocation of SK1 to block the localized Ca 2+ transients required for phagosome maturation.
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mycobacterium tuberculosis Phagosomes exhibit altered calmodulin dependent signal transduction contribution to inhibition of phagosome lysosome fusion and intracellular survival in human macrophages
Journal of Immunology, 2001Co-Authors: Zulfiqar A Malik, Shankar S Iyer, David J KusnerAbstract:Mycobacterium tuberculosis successfully parasitizes macrophages by disrupting the maturation of its phagosome, creating an intracellular compartment with endosomal rather than lysosomal characteristics. We have recently demonstrated that live M. tuberculosis infect human macrophages in the absence of an increase in cytosolic Ca 2+ ([Ca 2+ ] c ), which correlates with inhibition of phagosome-lysosome fusion and intracellular viability. In contrast, killed M. tuberculosis induces an elevation in [Ca 2+ ] c that is coupled to phagosome-lysosome fusion. We tested the hypothesis that defective activation of the Ca 2+ -dependent effector proteins calmodulin (CaM) and CaM-dependent protein kinase II (CaMKII) contributes to the intracellular pathogenesis of tuberculosis. Phagosomes containing live M. tuberculosis exhibited decreased levels of CaM and the activated form of CaMKII compared with Phagosomes encompassing killed tubercle bacilli. Furthermore, ionophore-induced elevations in [Ca 2+ ] c resulted in recruitment of CaM and activation of CaMKII on Phagosomes containing live M. tuberculosis . Specific inhibitors of CaM or CaMKII blocked Ca 2+ ionophore-induced phagosomal maturation and enhanced the bacilli’s intracellular viability. These results demonstrate a novel role for CaM and CaMKII in the regulation of phagosome-lysosome fusion and suggest that defective activation of these Ca 2+ -activated signaling components contributes to the successful parasitism of human macrophages by M. tuberculosis .
Vojo Deretic - One of the best experts on this subject based on the ideXlab platform.
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In vitro phagosome-endosome fusion.
Methods of Molecular Biology, 2008Co-Authors: Isabelle Vergne, Vojo DereticAbstract:Phagolysosome biogenesis plays a pivotal role in elimination of foreign particles and pathogens by leukocytes. This process is achieved by multiple cycles of membrane fusion between the phagosome and the endosomal system. In vitro reconstitution of phagosome fusion with endosomes is instrumental in studying this intricate process. Such an assay is also invaluable for the identification of effectors produced by intracellular pathogens that may hamper the pathway. In this chapter we describe a highly sensitive and relatively rapid method to measure fusion between Phagosomes and early, as well as late, endosomal compartments. The assay is based on the formation of a stable biotin-streptavidin complex upon fusion between biotinylated-peroxidase loaded endosomes and magnetic streptavidin conjugated bead-containing Phagosomes. Fusion is quantified using a fluorescence-based detection method that measures the peroxidase activity associated with the beads.
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mechanism of inducible nitric oxide synthase exclusion from mycobacterial Phagosomes
PLOS Pathogens, 2007Co-Authors: Alexander S Davis, Jennifer Chua, Isabelle Vergne, Sharon Master, George B Kyei, Vojo DereticAbstract:Mycobacterium tuberculosis is sensitive to nitric oxide generated by inducible nitric oxide synthase (iNOS). Consequently, to ensure its survival in macrophages, M. tuberculosis inhibits iNOS recruitment to its phagosome by an unknown mechanism. Here we report the mechanism underlying this process, whereby mycobacteria affect the scaffolding protein EBP50, which normally binds to iNOS and links it to the actin cytoskeleton. Phagosomes harboring live mycobacteria showed reduced capacity to retain EBP50, consistent with lower iNOS recruitment. EBP50 was found on purified Phagosomes, and its expression increased upon macrophage activation, paralleling expression changes seen with iNOS. Overexpression of EBP50 increased while EBP50 knockdown decreased iNOS recruitment to Phagosomes. Knockdown of EBP50 enhanced mycobacterial survival in activated macrophages. We tested another actin organizer, coronin-1, implicated in mycobacterium-macrophage interaction for contribution to iNOS exclusion. A knockdown of coronin-1 resulted in increased iNOS recruitment to model latex bead Phagosomes but did not increase iNOS recruitment to Phagosomes with live mycobacteria and did not affect mycobacterial survival. Our findings are consistent with a model for the block in iNOS association with mycobacterial Phagosomes as a mechanism dependent primarly on reduced EBP50 recruitment.
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autophagy is a defense mechanism inhibiting bcg and mycobacterium tuberculosis survival in infected macrophages
Cell, 2004Co-Authors: Gregory A. Taylor, Maximiliano G Gutierrez, Sharon Master, Sudha B Singh, Maria I Colombo, Vojo DereticAbstract:Abstract 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-γ induced autophagy in macrophages, and so did transfection with LRG-47, an effector of IFN-γ 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.
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Cellubrevin Alterations and Mycobacterium tuberculosis Phagosome Maturation Arrest
Journal of Biological Chemistry, 2002Co-Authors: Rutilio A. Fratti, Jennifer Chua, Vojo DereticAbstract:Abstract 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.
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Effects of cytokines on mycobacterial phagosome maturation
Journal of Cell Science, 1998Co-Authors: Rutilio A. Fratti, M. Mcfalone, Eileen Pagán-ramos, Dusanka Deretic, Vojo DereticAbstract:One of the major mechanisms permitting intracellular pathogens to parasitize macrophages is their ability to alter maturation of the phagosome or affect its physical integrity. These processes are opposed by the host innate and adaptive immune defenses, and in many instances mononuclear phagocytes can be stimulated with appropriate cytokines to restrict the growth of the microorganisms within the phagosomal compartment. Very little is known about the effects that cytokines have on phagosome maturation. Here we have used green fluorescent protein (GFP)-labeled mycobacteria and a fixable acidotropic probe, LysoTracker Red DND-99, to monitor maturation of the mycobacterial phagosome. The macrophage compartments that stained with the LysoTracker probe were examined first. This dye was found to colocalize preferentially with the late endosomal and lysosomal markers rab7 and Lamp1, and with a fluid phase marker chased into the late endosomal compartments. In contrast, LysoTracker showed only a minor overlap with the early endosomal marker rab5. Pathogenic mycobacteria are believed to reside in nonacidified vacuoles sequestered away from late endosomal compartments as a part of their intracellular survival strategy. We examined the status of mycobacterial Phagosomes in macrophages from IL-10 knockout mice, in quiescent cells, and in mononuclear phagocytes stimulated with the macrophage-activating cytokine IFN-(gamma). When macrophages were derived from the bone marrow of transgenic IL-10 mice lacking this major deactivating cytokine, colocalization of GFP-fluorescing mycobacteria with the LysoTracker staining appeared enhanced, suggestive of increased acidification of the mycobacterial phagosome relative to macrophages from normal mice. When bone marrow-derived macrophages from normal mice or a J774 murine macrophage cell line were stimulated with IFN-(gamma) and LPS, this resulted in increased colocalization of mycobacteria and LysoTracker, but no statistically significant enhancement was observed in IL-10 transgenic animals. These studies are consistent with the interpretation that proinflammatory and anti-inflammatory cytokines affect maturation of mycobacterial Phagosomes. Although multiple mechanisms are likely to be at work, we propose the existence of a direct link between cytokine effects on the host cell and phagosome maturation in the macrophage.
