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Albert Descoteaux - One of the best experts on this subject based on the ideXlab platform.

  • The Leishmania donovani lipophosphoglycan excludes the vesicular protonATPase from phagosomes by impairing the recruitment of synaptotagmin V
    2016
    Co-Authors: Adrien F. Vinet, Mitsunori Fukuda, Salvatore J. Turco, Albert Descoteaux
    Abstract:

    We recently showed that the exocytosis regulator Synaptotagmin (Syt) V is recruited to the nascent phagosome and remains associated throughout the maturation process. In this study, we investigated the possibility that Syt V plays a role in regulating interactions between the phagosome and the endocytic organelles. Silencing of Syt V by RNA interference revealed that Syt V contributes to Phagolysosome biogenesis by regulating the acquisition of cathepsin D and the vesicular proton-ATPase. In contrast, recruitment of cathepsin B, the early endosomal marker EEA1 and the lysosomal marker LAMP1 to phagosomes was normal in the absence of Syt V. As Leishmania donovani promastigotes inhibit phagosome maturation, we investigated their potential impact on the phagosomal association of Syt V. This inhibition of Phagolysosome biogenesis is mediated by the virulence glycolipid lipophosphoglycan, a polymer of the repeating Galb1,4Mana1-PO4 units attached to the promastigote surface via an unusual glycosylphosphatidylinositol anchor. Our results showed that insertion of lipophosphoglycan into ganglioside GM1-containing microdomains excluded or caused dissociation of Syt V from phagosome membranes. As a consequence, L. donovani promatigotes established infection in a phagosome from which the vesicular proton-ATPase was excluded and which failed to acidify. Collectively, these results reveal a novel function for Syt V in Phagolysosome biogenesis and provide novel insight into the mechanism of vesicular proton-ATPase recruitment to maturing phagosomes. We also provide novel findings into the mechanism of Leishmania pathogenesis, whereby targetin

  • The protein tyrosine phosphatase SHP-1 regulates Phagolysosome biogenesis.
    Journal of Immunology, 2012
    Co-Authors: Carolina P Gómez, Marina Tiemi Shio, Pascale Duplay, Martin Olivier, Albert Descoteaux
    Abstract:

    The process of phagocytosis and phagosome maturation involves the recruitment of effector proteins that participate in phagosome formation and in the acidification and/or fusion with various endocytic vesicles. In the current study, we investigated the role of the Src homology region 2 domain-containing phosphatase 1 (SHP-1) in Phagolysosome biogenesis. To this end, we used immortalized bone marrow macrophages derived from SHP-1-deficient motheaten mice and their wild-type littermates. We found that SHP-1 is recruited early and remains present on phagosomes for up to 4 h postphagocytosis. Using confocal immunofluorescence microscopy and Western blot analyses on purified phagosome extracts, we observed an impaired recruitment of lysosomal-associated membrane protein 1 in SHP-1-deficient macrophages. Moreover, Western blot analyses revealed that whereas the 51-kDa procathepsin D is recruited to phagosomes, it is not processed into the 46-kDa cathepsin D in the absence of SHP-1, suggesting a defect in acidification. Using the lysosomotropic agent LysoTracker as an indicator of phagosomal pH, we obtained evidence that in the absence of SHP-1, phagosome acidification was impaired. Taken together, these results are consistent with a role for SHP-1 in the regulation of signaling or membrane fusion events involved in Phagolysosome biogenesis.

  • the leishmania donovani lipophosphoglycan excludes the vesicular proton atpase from phagosomes by impairing the recruitment of synaptotagmin v
    PLOS Pathogens, 2009
    Co-Authors: Adrien F. Vinet, Mitsunori Fukuda, Salvatore J. Turco, Albert Descoteaux
    Abstract:

    We recently showed that the exocytosis regulator Synaptotagmin (Syt) V is recruited to the nascent phagosome and remains associated throughout the maturation process. In this study, we investigated the possibility that Syt V plays a role in regulating interactions between the phagosome and the endocytic organelles. Silencing of Syt V by RNA interference revealed that Syt V contributes to Phagolysosome biogenesis by regulating the acquisition of cathepsin D and the vesicular proton-ATPase. In contrast, recruitment of cathepsin B, the early endosomal marker EEA1 and the lysosomal marker LAMP1 to phagosomes was normal in the absence of Syt V. As Leishmania donovani promastigotes inhibit phagosome maturation, we investigated their potential impact on the phagosomal association of Syt V. This inhibition of Phagolysosome biogenesis is mediated by the virulence glycolipid lipophosphoglycan, a polymer of the repeating Galβ1,4Manα1-PO4 units attached to the promastigote surface via an unusual glycosylphosphatidylinositol anchor. Our results showed that insertion of lipophosphoglycan into ganglioside GM1-containing microdomains excluded or caused dissociation of Syt V from phagosome membranes. As a consequence, L. donovani promatigotes established infection in a phagosome from which the vesicular proton-ATPase was excluded and which failed to acidify. Collectively, these results reveal a novel function for Syt V in Phagolysosome biogenesis and provide novel insight into the mechanism of vesicular proton-ATPase recruitment to maturing phagosomes. We also provide novel findings into the mechanism of Leishmania pathogenesis, whereby targeting of Syt V is part of the strategy used by L. donovani promastigotes to prevent phagosome acidification.

  • Modulation of Phagolysosome biogenesis by the lipophosphoglycan of Leishmania.
    Clinical immunology (Orlando Fla.), 2005
    Co-Authors: Robert Lodge, Albert Descoteaux
    Abstract:

    Promastigotes of the protozoan parasite Leishmania are inoculated into the mammalian host by an infected sandfly and are phagocytosed by macrophages. There, they differentiate into amastigotes, which replicate in Phagolysosomes. A family of glycoconjugates, the phosphoglycans (PGs), plays an important role in the ability of promastigotes to survive the potentially microbicidal consequences of phagocytosis. Lipophosphoglycan (LPG), an abundant promastigote surface glycolipid, has received considerable attention over the past several years. Of interest for this review, lipophosphoglycan confers upon Leishmania donovani promastigotes the ability to inhibit Phagolysosome biogenesis. This inhibition correlates with an accumulation of periphagosomal F-actin, which may potentially form a physical barrier that prevents L. donovani promastigote-harboring phagosomes from interacting with late endosomes and lysosomes. Thus, similar to several other pathogens, Leishmania promastigotes hijack the host cell's cytoskeleton early during the infection process. Here, we review this phenomenon and discuss the potential underlying mechanisms.

  • rab5 regulates the kiss and run fusion between phagosomes and endosomes and the acquisition of phagosome leishmanicidal properties in raw 264 7 macrophages
    Journal of Cell Science, 2000
    Co-Authors: Sophie Duclos, Roberto Diez, Barbara Papadopoulou, Javier Garin, Albert Descoteaux, Harald Alfred Stenmark, Michel Desjardins
    Abstract:

    Phagolysosome biogenesis is essential for the killing and degradation of intracellular pathogens. It involves the fusion of phagosomes with various endocytic organelles, a process known to be regulated in part by Rab proteins. We generated RAW 264.7 macrophages expressing an active mutant of Rab5 (Rab5(Q79L)) to determine the role of Rab5 in phagocytosis and Phagolysosome biogenesis. Our results indicate that Rab5 stimulates phagocytosis of latex beads but not Fc or C3 receptor-mediated phagocytosis. Rab5 also acts to restrict the complete fusion of phagosomes with endosomes, a phenomenon allowing exchange of solutes from the two compartments without complete intermixing of their membrane (kiss and run). In Rab5(Q79L)-expressing macrophages, uncontrolled fusion events occurred, leading to the appearance of giant phagosomes. These phagosomes could initiate their maturation and acquire LAMP1, but failed to generate the microbicidal conditions needed to kill intracellular parasites. These results identify Rab5 as a key molecule regulating phagosome-endosome fusion and as an essential component in the innate ability of macrophages to restrict the growth of intracellular parasites. SUMMARY

Vojo Deretic - One of the best experts on this subject based on the ideXlab platform.

  • Phosphoinositides in Phagolysosome and autophagosome biogenesis.
    Biochemical Society symposium, 2007
    Co-Authors: Vojo Deretic, Sudha B Singh, Sharon Master, George B. Kyei, Alexander S Davis, John Naylor, Sergio De Haro, James S. Harris, Mónica A. Delgado, Esteban A. Roberts
    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 PtdIns3 P are critical for the default pathway of phagosomal maturation into Phagolysosomes. Mycobacteria block PtdIns3 P production and thus arrest phagosomal maturation. PtdIns3 P 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 PtdIns3 P generation dependent manner and eliminates intracellular M. tuberculosis. PtdIns3 P and PtdIns3 P -dependent processes represent an important cellular nexus where fundamental trafficking processes, disease causing host–pathogen interactions, and innate and adaptive immunity defense mechanisms meet.

  • Higher order Rab programming in Phagolysosome biogenesis.
    The Journal of cell biology, 2006
    Co-Authors: Esteban A. Roberts, Jennifer Chua, George B. Kyei, Vojo Deretic
    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.

  • autophagy is a defense mechanism inhibiting bcg and mycobacterium tuberculosis survival in infected macrophages
    Cell, 2004
    Co-Authors: Sudha B Singh, Maximiliano G Gutierrez, Sharon Master, Maria I. Colombo, Gregory A Taylor, Vojo Deretic
    Abstract:

    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.

  • Mycobacterium tuberculosis Reprograms Waves of Phosphatidylinositol 3-Phosphate on Phagosomal Organelles
    Journal of Biological Chemistry, 2004
    Co-Authors: Jennifer Chua, Vojo Deretic
    Abstract:

    Abstract The potent human pathogen Mycobacterium tuberculosis persists in macrophages within a specialized, immature phagosome by interfering with the pathway of Phagolysosome biogenesis. The molecular mechanisms underlying this process remain to be fully elucidated. Here, using four-dimensional microscopy, we detected on model phagosomes, which normally mature into Phagolysosomes, the existence of cyclical waves of phosphatidylinositol 3-phosphate (PI3P), a membrane trafficking regulatory lipid essential for phagosomal acquisition of lysosomal characteristics. We show that mycobacteria interfere with the dynamics of PI3P on phagosomal organelles by altering the timing and characteristics of the PI3P waves on phagosomes. The default program of cyclical PI3P waves on model phagosomes is composed of an initial stage (phase I), represented by a strong PI3P burst occurring only upon the completion of phagosome formation, and a subsequent stage (phase II) of recurring PI3P waves on maturing phagosomes with the average periodicity of 20 min. Mycobacteria alter this program in two ways: (i) by inducing, in a cholesterol-dependent fashion, a neophase I* of premature PI3P production, coinciding with the process of mycobacterial entry into the macrophage, and (ii) by inhibiting the calmodulin-dependent phase II responsible for the acquisition of lysosomal characteristics. We conclude that the default pathway of phagosomal maturation into the Phagolysosome includes temporally organized cyclical waves of PI3P on phagosomal membranes and that this process is targeted for reprogramming by mycobacteria as they prevent Phagolysosome formation.

Lutz Thilo - One of the best experts on this subject based on the ideXlab platform.

  • 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
    Cellular Microbiology, 2006
    Co-Authors: Chantal De Chastellier, Lutz Thilo
    Abstract:

    Summary 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-β-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.

  • 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.
    Cellular microbiology, 2006
    Co-Authors: Chantal De Chastellier, Lutz Thilo
    Abstract:

    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.

Carolina P Gómez - One of the best experts on this subject based on the ideXlab platform.

  • The protein tyrosine phosphatase SHP-1 regulates Phagolysosome biogenesis.
    Journal of Immunology, 2012
    Co-Authors: Carolina P Gómez, Marina Tiemi Shio, Pascale Duplay, Martin Olivier, Albert Descoteaux
    Abstract:

    The process of phagocytosis and phagosome maturation involves the recruitment of effector proteins that participate in phagosome formation and in the acidification and/or fusion with various endocytic vesicles. In the current study, we investigated the role of the Src homology region 2 domain-containing phosphatase 1 (SHP-1) in Phagolysosome biogenesis. To this end, we used immortalized bone marrow macrophages derived from SHP-1-deficient motheaten mice and their wild-type littermates. We found that SHP-1 is recruited early and remains present on phagosomes for up to 4 h postphagocytosis. Using confocal immunofluorescence microscopy and Western blot analyses on purified phagosome extracts, we observed an impaired recruitment of lysosomal-associated membrane protein 1 in SHP-1-deficient macrophages. Moreover, Western blot analyses revealed that whereas the 51-kDa procathepsin D is recruited to phagosomes, it is not processed into the 46-kDa cathepsin D in the absence of SHP-1, suggesting a defect in acidification. Using the lysosomotropic agent LysoTracker as an indicator of phagosomal pH, we obtained evidence that in the absence of SHP-1, phagosome acidification was impaired. Taken together, these results are consistent with a role for SHP-1 in the regulation of signaling or membrane fusion events involved in Phagolysosome biogenesis.

David E Heinrichs - One of the best experts on this subject based on the ideXlab platform.

  • staphylococcus aureus uses the graxrs regulatory system to sense and adapt to the acidified Phagolysosome in macrophages
    Mbio, 2018
    Co-Authors: Ronald S Flannagan, Robert C Kuiack, Martin J Mcgavin, David E Heinrichs
    Abstract:

    ABSTRACT Macrophages are critical to innate immunity due to their ability to phagocytose bacteria. The macrophage Phagolysosome is a highly acidic organelle with potent antimicrobial properties, yet remarkably, ingested Staphylococcus aureus replicates within this niche. Herein we demonstrate that S. aureus requires the GraXRS regulatory system for growth within this niche, while the SaeRS and AgrAC two-component regulatory systems and the α-phenol soluble modulins are dispensable. Importantly, we find that it is exposure to acidic pH that is required for optimal growth of S. aureus inside fully acidified macrophage Phagolysosomes. Exposure of S. aureus to acidic pH evokes GraS signaling, which in turn elicits an adaptive response that endows the bacteria with increased resistance to antimicrobial effectors, such as antimicrobial peptides, encountered inside macrophage Phagolysosomes. Notably, pH-dependent induction of antimicrobial peptide resistance in S. aureus requires the GraS sensor kinase. GraS and MprF, a member of the GraS regulon, play an important role for bacterial survival in the acute stages of systemic infection, where in murine models of infection, S. aureus resides within liver-resident Kupffer cells. We conclude that GraXRS represents a vital regulatory system that functions to allow S. aureus to evade killing, prior to commencement of replication, within host antibacterial immune cells. IMPORTANCE S. aureus can infect any site of the body, including the microbicidal Phagolysosome of the macrophage. The ability of S. aureus to infect diverse niches necessitates that the bacteria be highly adaptable. Here we show that S. aureus responds to Phagolysosome acidification to evoke changes in gene expression that enable the bacteria to resist phagolysosomal killing and to promote replication. Toxin production is dispensable for this response; however, the bacteria require the sensor kinase GraS, which transduces signals in response to acidic pH. GraS is necessary for phagolysosomal replication and survival of S. aureus in the acute stage of systemic infection. Disruption of this S. aureus adaptation would render S. aureus susceptible to phagocyte restriction.

  • Staphylococcus aureus Uses the GraXRS Regulatory System To Sense and Adapt to the Acidified Phagolysosome in Macrophages
    American Society for Microbiology, 2018
    Co-Authors: Ronald S Flannagan, Robert C Kuiack, Martin J Mcgavin, David E Heinrichs
    Abstract:

    Macrophages are critical to innate immunity due to their ability to phagocytose bacteria. The macrophage Phagolysosome is a highly acidic organelle with potent antimicrobial properties, yet remarkably, ingested Staphylococcus aureus replicates within this niche. Herein we demonstrate that S. aureus requires the GraXRS regulatory system for growth within this niche, while the SaeRS and AgrAC two-component regulatory systems and the α-phenol soluble modulins are dispensable. Importantly, we find that it is exposure to acidic pH that is required for optimal growth of S. aureus inside fully acidified macrophage Phagolysosomes. Exposure of S. aureus to acidic pH evokes GraS signaling, which in turn elicits an adaptive response that endows the bacteria with increased resistance to antimicrobial effectors, such as antimicrobial peptides, encountered inside macrophage Phagolysosomes. Notably, pH-dependent induction of antimicrobial peptide resistance in S. aureus requires the GraS sensor kinase. GraS and MprF, a member of the GraS regulon, play an important role for bacterial survival in the acute stages of systemic infection, where in murine models of infection, S. aureus resides within liver-resident Kupffer cells. We conclude that GraXRS represents a vital regulatory system that functions to allow S. aureus to evade killing, prior to commencement of replication, within host antibacterial immune cells.S. aureus can infect any site of the body, including the microbicidal Phagolysosome of the macrophage. The ability of S. aureus to infect diverse niches necessitates that the bacteria be highly adaptable. Here we show that S. aureus responds to Phagolysosome acidification to evoke changes in gene expression that enable the bacteria to resist phagolysosomal killing and to promote replication. Toxin production is dispensable for this response; however, the bacteria require the sensor kinase GraS, which transduces signals in response to acidic pH. GraS is necessary for phagolysosomal replication and survival of S. aureus in the acute stage of systemic infection. Disruption of this S. aureus adaptation would render S. aureus susceptible to phagocyte restriction