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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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contribution of phosphatidylserine to membrane surface charge and protein targeting during Phagosome maturation
Journal of Cell Biology, 2009Co-Authors: Tony Yeung, Gregory D. Fairn, Bryan Heit, Jean Francois Dubuisson, Basil Chiu, Robert D Inman, Andras Kapus, Michele S Swanson, Sergio GrinsteinAbstract:During phagocytosis, the phosphoinositide content of the activated membrane decreases sharply, as does the associated surface charge, which attracts polycationic proteins. The cytosolic leaflet of the plasma membrane is enriched in phosphatidylserine (PS); however, a lack of suitable probes has precluded investigation of the fate of this phospholipid during phagocytosis. We used a recently developed fluorescent biosensor to monitor the distribution and dynamics of PS during Phagosome formation and maturation. Unlike the polyphosphoinositides, PS persists on Phagosomes after sealing even when other plasmalemmal components have been depleted. High PS levels are maintained through fusion with endosomes and lysosomes and suffice to attract cationic proteins like c-Src to maturing Phagosomes. Phagocytic vacuoles containing the pathogens Legionella pneumophila and Chlamydia trachomatis, which divert maturation away from the endolysosomal pathway, are devoid of PS, have little surface charge, and fail to recruit c-Src. These findings highlight a function for PS in Phagosome maturation and microbial killing.
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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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fcγr stimulated activation of the nadph oxidase phosphoinositide binding protein p40phox regulates nadph oxidase activity after enzyme assembly on the Phagosome
Blood, 2008Co-Authors: Wei Tian, Sergio Grinstein, Xing Jun Li, Natalie D Stull, Wenyu Ming, Sarah A Bissonnette, Michael B Yaffe, Simon J Atkinson, Mary C DinauerAbstract:The phagocyte NADPH oxidase generates superoxide for microbial killing, and includes a membrane-bound flavocytochrome b558 and cytosolic p67phox, p47phox, and p40phox subunits that undergo membrane translocation upon cellular activation. The function of p40phox, which binds p67phox in resting cells, is incompletely understood. Recent studies showed that phagocytosis-induced superoxide production is stimulated by p40phox and its binding to phosphatidylinositol-3-phosphate (PI3P), a phosphoinositide enriched in membranes of internalized Phagosomes. To better define the role of p40phox in FcγR-induced oxidase activation, we used immunofluorescence and real-time imaging of FcγR-induced phagocytosis. YFP-tagged p67phox and p40phox translocated to granulocyte Phagosomes before Phagosome internalization and accumulation of a probe for PI3P. p67phox and p47phox accumulation on nascent and internalized Phagosomes did not require p40phox or PI3 kinase activity, although superoxide production before and after Phagosome sealing was decreased by mutation of the p40phox PI3P-binding domain or wortmannin. Translocation of p40phox to nascent Phagosomes required binding to p67phox but not PI3P, although the loss of PI3P binding reduced p40phox retention after Phagosome internalization. We conclude that p40phox functions primarily to regulate FcγR-induced NADPH oxidase activity rather than assembly, and stimulates superoxide production via a PI3P signal that increases after Phagosome internalization.
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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.
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.
Marcus A Horwitz - One of the best experts on this subject based on the ideXlab platform.
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The Mycobacterium bovis Bacille Calmette-Guérin Phagosome Proteome
Molecular & Cellular Proteomics, 2009Co-Authors: Deepa Jethwaney, Daniel L. Clemens, Birgit Schilling, Bradford W Gibson, Marcus A HorwitzAbstract:Mycobacterium tuberculosis and Mycobacterium bovis bacille Calmette-Guerin (BCG) alter the maturation of their Phagosomes and reside within a compartment that resists acidification and fusion with lysosomes. To define the molecular composition of this compartment, we developed a novel method for obtaining highly purified Phagosomes from BCG-infected human macrophages and analyzed the Phagosomes by Western immunoblotting and mass spectrometry-based proteomics. Our purification procedure revealed that BCG grown on artificial medium becomes less dense after growth in macrophages. By Western immunoblotting, LAMP-2, Niemann-Pick protein C1, and syntaxin 3 were readily detectable on the BCG Phagosome but at levels that were lower than on the latex bead Phagosome; flotillin-1 and the vacuolar ATPase were barely detectable on the BCG Phagosome but highly enriched on the latex bead Phagosome. Immunofluorescence studies confirmed the scarcity of flotillin on BCG Phagosomes and demonstrated an inverse correlation between bacterial metabolic activity and flotillin on M. tuberculosis Phagosomes. By mass spectrometry, 447 human host proteins were identified on BCG Phagosomes, and a partially overlapping set of 289 human proteins on latex bead Phagosomes was identified. Interestingly, the majority of the proteins identified consistently on BCG Phagosome preparations were also identified on latex bead Phagosomes, indicating a high degree of overlap in protein composition of these two compartments. It is likely that many differences in protein composition are quantitative rather than qualitative in nature. Despite the remarkable overlap in protein composition, we consistently identified a number of proteins on the BCG Phagosomes that were not identified in any of our latex bead Phagosome preparations, including proteins involved in membrane trafficking and signal transduction, such as Ras GTPase-activating-like protein IQGAP1, and proteins of unknown function, such as FAM3C. Our Phagosome purification procedure and initial proteomics analyses set the stage for a quantitative comparative analysis of mycobacterial and latex bead Phagosome proteomes.
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deviant expression of rab5 on Phagosomes containing the intracellular pathogens mycobacterium tuberculosis and legionella pneumophila is associated with altered phagosomal fate
Infection and Immunity, 2000Co-Authors: Daniel L. Clemens, Marcus A HorwitzAbstract:The intracellular human pathogens Legionella pneumophila and Mycobacterium tuberculosis reside in altered Phagosomes that do not fuse with lysosomes and are only mildly acidified. The L. pneumophila Phagosome exists completely outside the endolysosomal pathway, and the M. tuberculosis Phagosome displays a maturational arrest at an early endosomal stage along this pathway. Rab5 plays a critical role in regulating membrane trafficking involving endosomes and Phagosomes. To determine whether an alteration in the function or delivery of Rab5 could play a role in the aberrant development of L. pneumophila and M. tuberculosis Phagosomes, we have examined the distribution of the small GTPase, Rab5c, in infected HeLa cells overexpressing Rab5c. Both pathogens formed Phagosomes in HeLa cells with molecular characteristics similar to their Phagosomes in human macrophages and multiplied in these host cells. Phagosomes containing virulent wild-type L. pneumophila never acquired immunogold staining for Rab5c, whereas Phagosomes containing an avirulent mutant L. pneumophila (which ultimately fused with lysosomes) transiently acquired staining for Rab5c after phagocytosis. In contrast, M. tuberculosis Phagosomes exhibited abundant staining for Rab5c throughout its life cycle. To verify that the overexpressed, recombinant Rab5c observed on the bacterial Phagosomes was biologically active, we examined the Phagosomes in HeLa cells expressing Rab5c Q79L, a fusion-promoting mutant. Such HeLa cells formed giant vacuoles, and after incubation with various particles, the giant vacuoles acquired large numbers of latex beads, M. tuberculosis, and avirulent L. pneumophila but not wild-type L. pneumophila, which consistently remained in tight Phagosomes that did not fuse with the giant vacuoles. These results indicate that whereas Rab5 is absent from wild-type L. pneumophila Phagosomes, functional Rab5 persists on M. tuberculosis Phagosomes. The absence of Rab5 on the L. pneumophila Phagosome may underlie its lack of interaction with endocytic compartments. The persistence of functional Rab5 on the M. tuberculosis Phagosomes may enable the Phagosome to retard its own maturation at an early endosomal stage.
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the mycobacterium tuberculosis Phagosome interacts with early endosomes and is accessible to exogenously administered transferrin
Journal of Experimental Medicine, 1996Co-Authors: Daniel L. Clemens, Marcus A HorwitzAbstract:Previous studies have demonstrated that the Mycobacterium tuberculosis Phagosome in human monocyte-derived macrophages acquires markers of early and late endosomes, but direct evidence of interaction of the M. tuberculosis Phagosome with the endosomal compartment has been lacking. Using the cryosection immunogold technique, we have found that the M. tuberculosis Phagosome acquires exogenously added transferrin in a time-dependent fashion. Near-maximal acquisition of transferrin occurs within 15 min, kinetics of acquisition consistent with interaction of the M. tuberculosis Phagosome with early endosomes. Transferrin is chased out of the M. tuberculosis Phagosome by incubation of the infected macrophages in culture medium lacking human transferrin. Phagosomes containing latex beads or heat-killed M. tuberculosis, on the other hand, do not acquire staining for transferrin. These and other findings demonstrate that M. tuberculosis arrests the maturation of its Phagosome at a stage at which the Phagosome interacts with early and late endosomes, but not with lysosomes. The transferrin endocytic pathway potentially provides a novel route for targeting antimicrobials to the M. tuberculosis Phagosome.
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characterization of the mycobacterium tuberculosis Phagosome and evidence that phagosomal maturation is inhibited
Journal of Experimental Medicine, 1995Co-Authors: Daniel L. Clemens, Marcus A HorwitzAbstract:We have used the cryosection immunogold technique to study the composition of the Mycobacterium tuberculosis Phagosome. We have used quantitative immunogold staining to determine the distribution of several known markers of the endosomal-lysosomal pathway in human monocytes after ingestion of either M. tuberculosis, Legionella pneumophila, or polystyrene beads. Compared with the other phagocytic particles studied, the M. tuberculosis Phagosome exhibits delayed clearance of major histocompatibility complex (MHC) class I molecules, relatively intense staining for MHC class II molecules and the endosomal marker transferrin receptor, and relatively weak staining for the lysosomal membrane glycoproteins, CD63, LAMP-1, and LAMP-2 and the lysosomal acid protease, cathepsin D. In contrast to M. tuberculosis, the L. pneumophila Phagosome rapidly clears MHC class I molecules and excludes all endosomal-lysosomal markers studied. In contrast to both live M. tuberculosis and L. pneumophila Phagosomes, Phagosomes containing either polystyrene beads or heat-killed M. tuberculosis stain intensely for lysosomal membrane glycoproteins and cathepsin D. These findings suggest that (a) M. tuberculosis retards the maturation of its Phagosome along the endosomal-lysosomal pathway and resides in a compartment with endosomal, as opposed to lysosomal, characteristics; and (b) the intraphagosomal pathway, i.e., the pathway followed by several intracellular parasites that inhibit Phagosome-lysosome fusion, is heterogeneous.
Joel A. Swanson - One of the best experts on this subject based on the ideXlab platform.
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The uniformity of Phagosome maturation in macrophages.
Journal of Cell Biology, 2004Co-Authors: Rebecca M. Henry, Adam D. Hoppe, Nikhil Joshi, Joel A. SwansonAbstract:Many studies of endocytosis and phagocytosis presume that organelles containing a single kind of internalized particle exhibit invariant patterns of protein and phospholipid association as they mature inside cells. To test this presumption, fluorescent protein chimeras were expressed in RAW 264.7 macrophages, and time-lapse ratiometric fluorescence microscopy was used to measure the maturation dynamics of individual Phagosomes containing IgG-opsonized erythrocytes. Quantitative analysis revealed consistent patterns of association for YFP chimeras of β-actin, Rab5a, Rab7, and LAMP-1, and no association of YFP chimeras marking endoplasmic reticulum or Golgi. YFP-2xFYVE, recognizing phosphatidylinositol 3-phosphate (PI(3)P), showed two patterns of Phagosome labeling. Some Phagosomes increased labeling quickly after Phagosome closure and then lost the label within 20 min, whereas others labeled more slowly and retained the label for several hours. The two patterns of PI(3)P on otherwise identical Phagosomes indicated that organelle maturation does not necessarily follow a single path and that some features of Phagosome maturation are integrated over the entire organelle.
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rapid and complete fusion of macrophage lysosomes with Phagosomes containing salmonella typhimurium
Infection and Immunity, 1996Co-Authors: Y K Oh, Celia Alpuchearanda, E P Berthiaume, T Jinks, Samuel I Miller, Joel A. SwansonAbstract:The virulence of Salmonella typhimurium for mice results, in part, from its ability to survive after phagocytosis by macrophages. Although it is generally agreed that intracellular bacteria persist in membrane-bound Phagosomes, there remains some question as to whether these Phagosomes fuse with macrophage lysosomes. This report describes the maturation of Phagosomes containing S. typhimurium inside mouse bone marrow-derived macrophages. Macrophages were infected briefly and incubated for various intervals; then they were examined by fluorescence microscopy for colocalization of bacteria with lysosomal markers. These markers included LAMP-1, cathepsin L, and fluorescent proteins or dextrans preloaded into lysosomes by endocytosis. By all measures, Phagosomes containing S. typhimurium merged completely with the lysosomal compartment within 20 min of phagocytosis. The rate of Phagosome-lysosome fusion was similar to the rate for phagocytosed latex beads. Phagolysosomes remained accessible to fluid-phase probes and contained lysosomal markers for many hours. Moreover, a large percentage of the wild-type bacteria that were viable 20 min after infection survived longer incubations inside macrophages, indicating that the survivors were not a minor subpopulation that avoided Phagosome-lysosome fusion. Therefore, we conclude that S. typhimurium survives within the lysosomal compartments of macrophages.
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salmonella typhimurium activates virulence gene transcription within acidified macrophage Phagosomes
Proceedings of the National Academy of Sciences of the United States of America, 1992Co-Authors: C Alpuche M Aranda, Joel A. Swanson, Wendy P Loomis, Samuel I MillerAbstract:Abstract Survival of Salmonella typhimurium within macrophage Phagosomes requires the coordinate expression of bacterial gene products. This report examines the contribution of phagosomal pH as a signal for expression of genes positively regulated by the S. typhimurium virulence regulators PhoP and PhoQ. Several hours after bacterial phagocytosis by murine bone marrow-derived macrophages, PhoP-activated gene transcription increased 50- to 77-fold. In contrast, no difference in PhoP-activated gene expression was observed after infection of cultured epithelial cells, suggesting that the membrane sensor PhoQ recognized signals unique to macrophage Phagosomes. The increase in PhoP-regulated gene expression was abolished when macrophage culture medium contained NH4Cl or chloroquine, weak bases that raise the pH of acidic compartments. Measurements of pH documented that S. typhimurium delayed and attenuated acidification of its intracellular compartment. Phagosomes containing S. typhimurium required 4-5 hr to reach pH < 5.0. In contrast, within 1 hr vacuoles containing heat-killed bacteria were measured at pH < 4.5. The eventual acidification of Phagosomes to pH < 5.0 correlated with the period of maximal PhoP-dependent gene expression. These observations implicate Phagosome acidification as an intracellular inducer of PhoP-regulated gene expression and suggest that Salmonella survival is dependent on its ability to attenuate Phagosome acidification.
Philip D. Stahl - One of the best experts on this subject based on the ideXlab platform.
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modulation of rab5 and rab7 recruitment to Phagosomes by phosphatidylinositol 3 kinase
Molecular and Cellular Biology, 2003Co-Authors: Otilia V Vieira, Letizia Lanzetti, Alan D Schreiber, Philip D. Stahl, Jean Gruenberg, William S. Trimble, Rene E. Harrison, Cecilia Bucci, Sergio GrinsteinAbstract:Phagosomal biogenesis is central for microbial killing and antigen presentation by leukocytes. However, the molecular mechanisms governing Phagosome maturation are poorly understood. We analyzed the role and site of action of phosphatidylinositol 3-kinases (PI3K) and of Rab GTPases in maturation using both professional and engineered phagocytes. Rab5, which is recruited rapidly and transiently to the Phagosome, was found to be essential for the recruitment of Rab7 and for progression to phagolysosomes. Similarly, functional PI3K is required for successful maturation. Remarkably, inhibition of PI3K did not preclude Rab5 recruitment to Phagosomes but instead enhanced and prolonged it. Moreover, in the presence of PI3K inhibitors Rab5 was found to be active, as deduced from measurements of early endosome antigen 1 binding and by photobleaching recovery determinations. Though their ability to fuse with late endosomes and lysosomes was virtually eliminated by wortmannin, Phagosomes nevertheless recruited a sizable amount of Rab7. Moreover, Rab7 recruited to Phagosomes in the presence of PI3K antagonists retained the ability to bind its effector, Rab7-interacting lysosomal protein, suggesting that it is functionally active. These findings imply that (i) dissociation of Rab5 from Phagosomes requires products of PI3K, (ii) PI3K-dependent effectors of Rab5 are not essential for the recruitment of Rab7 by Phagosomes, and (iii) recruitment and activation of Rab7 are insufficient to induce fusion of Phagosomes with late endosomes and lysosomes. Accordingly, transfection of constitutively active Rab7 did not bypass the block of phagolysosome formation exerted by wortmannin. We propose that Rab5 activates both PI3K-dependent and PI3K-independent effectors that act in parallel to promote Phagosome maturation.
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Sequential maturation of Phagosomes provides unique targets for pathogens
Advances in Cellular and Molecular Biology of Membranes and Organelles, 1999Co-Authors: Carmen Alvarez-dominguez, Philip D. Stahl, Luis S. MayorgaAbstract:Publisher Summary This chapter provides new insights on Phagosome maturation. The hypothesis, suggested by the work of several groups, is that Phagosome maturation is a highly ordered, sequential process. Specifically, the interaction of Phagosomes with early endocytic compartments sets into motion a process whose completion is required for interaction of Phagosomes with later compartments (late endosomes and lysosomes). The sequential nature of Phagosome maturation and the requirement for obligatory intermediates might well create special opportunities for intracellular pathogens. Indeed, in the immediate future, Phagosome maturation might be defined in terms of the mechanisms utilized by specific pathogens to create biological niches for intracellular survival. Interference might be achieved by elaborating lipids or factors that modulate the function of key regulatory proteins in the host. Just as virus assembly was used to dissect the secretory pathway in mammalian cells, intracellular pathogens will guide the way to identify the steps required for Phagosome maturation.
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phagocytosed live listeria monocytogenes influences rab5 regulated in vitro Phagosome endosome fusion
Journal of Biological Chemistry, 1996Co-Authors: Carmen Alvarezdominguez, Alejandro M Barbieri, Walter Beron, Angela Wandingerness, Philip D. StahlAbstract:Abstract Survival or destruction of a pathogen following phagocytosis depends, in part, on fusion events between the Phagosome and the endosomal or lysosomal compartments. Here we use an in vitro assay to show that Phagosome-endosome fusion is regulated by the small GTPase rab5 and that fusion events are influenced by an internalized live organism, Listeria monocytogenes (LM). We compare the in vitro fusion of Phagosomes containing heat-killed organisms (dead LM) with that of Phagosomes containing a live nonhemolytic mutant (live LMhly−). Unlike the wild-type organism, LMhly− remains trapped inside the Phagosome. Phagosome-endosome fusion was reconstituted using biotinylated organisms and endosomes containing horseradish peroxidase conjugated with avidin. With both live LMhly− and dead LM preparations, in vitro Phagosome-endosome fusion was time-, temperature-, and cytosol-dependent. Live LMhly− Phagosomes exhibited a faster rate of fusion. Fusion in both preparations was regulated by rab5 and possibly by other GTPases. Anti-rab5 antibodies and immunodepletion of cytosolic rab5 inhibited fusion. Addition of glutatione S-transferase-rab5 in the GTP form stimulated Phagosome-endosome fusion, whereas addition of a dominant negative mutant of rab5 blocked fusion. Purified live LMhly− phagosomal membranes were enriched in rab5 as revealed by Western blotting, compared with dead LM Phagosomes. Fusion of endosomes with dead LM containing Phagosomes required ATP and was inhibited by ATP depletion and by N-ethylmaleimide (NEM) and anti-NEM-sensitive factor (NSF) antibodies. Unexpectedly, Phagosome-endosome fusion with live LMhly−-containing Phagosomes was not inhibited by ATP depletion nor by NEM or anti-NSF antibodies. Western blot analysis revealed that live LMhly−-containing Phagosomes were enriched for membrane-bound NSF, while dead LM containing Phagosomes contained low or undetectable quantities. Washing live LMhly−-containing Phagosomes with 0.5 M KCl removed NSF associated with the membranes and rendered them NEM, ATP, anti-NSF antibody sensitive for fusion. We conclude that rab5 regulates Phagosome-endosome fusion and that live microorganisms can up-regulate this process by recruiting rab5 to the membrane.
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alterations in the protein composition of maturing Phagosomes
Journal of Clinical Investigation, 1992Co-Authors: A Pitt, Luis S. Mayorga, Philip D. Stahl, Alan L SchwartzAbstract:We investigated the protein composition of J774-E clone macrophage Phagosomes isolated at different stages of phagolysosome biogenesis. Phagosomes formed by internalizing antibody-coated Staphylococcus aureus for 3 min followed by chase for 0, 4, 9, or 15 min were isolated by density gradient centrifugation. Enrichment and purity of the Phagosome preparations were quantitated by radiolabeled ligand recovery, enzyme markers, and electron microscopy. One-dimensional SDS-PAGE analyses of the isolated Phagosomes revealed virtually identical protein compositions. However, Western blot analyses with antibodies directed against selected proteins of known itineraries along the endocytic pathway demonstrated distinct differences in Phagosome protein compositions. Accumulating within the maturing Phagosome were the 31-kD subunit of the vacuolar proton pump, cathepsin D,beta-glucuronidase, the cation dependent mannose 6-phosphate receptor, and LAMP-1. Decreasing within the maturing Phagosome were the FcII receptor, the mannose receptor, and alpha-adaptin. These results indicate that although the macrophage Phagosome's total protein composition changes little during phagolysosome formation, the maturing Phagosome both receives and eliminates, possibly by protein recycling, specific membrane and sequestered proteins.
