To see the other types of publications on this topic, follow the link: Phagosomal acidification.
Journal articles on the topic 'Phagosomal acidification'
Create a spot-on reference in APA, MLA, Chicago, Harvard, and other styles
Consult the top 50 journal articles for your research on the topic 'Phagosomal acidification.'
Next to every source in the list of references, there is an 'Add to bibliography' button. Press on it, and we will generate automatically the bibliographic reference to the chosen work in the citation style you need: APA, MLA, Harvard, Chicago, Vancouver, etc.
You can also download the full text of the academic publication as pdf and read online its abstract whenever available in the metadata.
Browse journal articles on a wide variety of disciplines and organise your bibliography correctly.
1
Steinberg, B. E., K. K. Huynh, and S. Grinstein. "Phagosomal acidification: measurement, manipulation and functional consequences." Biochemical Society Transactions 35, no. 5 (October 25, 2007): 1083–87. http://dx.doi.org/10.1042/bst0351083.
Full textAbstract:
Phagocytosis holds a central position in the development of a successful innate immune response and in the initiation of the corresponding adaptive response. The destruction of invading pathogens and the presentation of their antigens to lymphoid cells require acidification of the phagosomal lumen. The present review discusses the mechanism of phagosome acidification, with particular reference to the two components of the protonmotive force: the chemical (pH) gradient and the electrical potential across the phagosomal membrane. A method for the in situ measurement of the electrical potential across the phagosomal membrane is described. In addition, we discuss the finding that acidification is not only a consequence, but also a critical determinant of phagosome maturation. Luminal acidification appears to function as a timing device controlling the transition between early and late phagosomes.
2
Clemens, Daniel L., Bai-Yu Lee, and Marcus A. Horwitz. "Francisella tularensis Phagosomal Escape Does Not Require Acidification of the Phagosome." Infection and Immunity 77, no. 5 (February 23, 2009): 1757–73. http://dx.doi.org/10.1128/iai.01485-08.
Full textAbstract:
ABSTRACT Following uptake, Francisella tularensis enters a phagosome that acquires limited amounts of lysosome-associated membrane glycoproteins and does not acquire cathepsin D or markers of secondary lysosomes. With additional time after uptake, F. tularensis disrupts its phagosomal membrane and escapes into the cytoplasm. To assess the role of phagosome acidification in phagosome escape, we followed acidification using the vital stain LysoTracker red and acquisition of the proton vacuolar ATPase (vATPase) using immunofluorescence within the first 3 h after uptake of live or killed F. tularensis subsp. holarctica live vaccine strain (LVS) by human macrophages. Whereas 90% of the phagosomes containing killed LVS stained intensely for the vATPase and were acidified, only 20 to 30% of phagosomes containing live LVS stained intensely for the vATPase and were acidified. To determine whether transient acidification might be required for phagosome escape, we assessed the impact on phagosome permeabilization of the proton pump inhibitor bafilomycin A. Using electron microscopy and an adenylate cyclase reporter system, we found that bafilomycin A did not prevent phagosomal permeabilization by F. tularensis LVS or virulent type A strains (F. tularensis subsp. tularensis strain Schu S4 and a recent clinical isolate) or by “F. tularensis subsp. novicida,” indicating that F. tularensis disrupts its phagosomal membrane by a mechanism that does not require acidification.
3
Tranchemontagne, Zachary R., Ryan B. Camire, Vanessa J. O'Donnell, Jessfor Baugh, and Kristin M. Burkholder. "Staphylococcus aureus Strain USA300 Perturbs Acquisition of Lysosomal Enzymes and Requires Phagosomal Acidification for Survival inside Macrophages." Infection and Immunity 84, no. 1 (October 26, 2015): 241–53. http://dx.doi.org/10.1128/iai.00704-15.
Full textAbstract:
Methicillin-resistantStaphylococcus aureus(MRSA) causes invasive, drug-resistant skin and soft tissue infections. Reports thatS. aureusbacteria survive inside macrophages suggest that the intramacrophage environment may be a niche for persistent infection; however, mechanisms by which the bacteria might evade macrophage phagosomal defenses are unclear. We examined the fate of theS. aureus-containing phagosome in THP-1 macrophages by evaluating bacterial intracellular survival and phagosomal acidification and maturation and by testing the impact of phagosomal conditions on bacterial viability. Multiple strains ofS. aureussurvived inside macrophages, and in studies using the MRSA USA300 clone, the USA300-containing phagosome acidified rapidly and acquired the late endosome and lysosome protein LAMP1. However, fewer phagosomes containing live USA300 bacteria than those containing dead bacteria associated with the lysosomal hydrolases cathepsin D and β-glucuronidase. Inhibiting lysosomal hydrolase activity had no impact on intracellular survival of USA300 or otherS. aureusstrains, suggesting thatS. aureusperturbs acquisition of lysosomal enzymes. We examined the impact of acidification onS. aureusintramacrophage viability and found that inhibitors of phagosomal acidification significantly impaired USA300 intracellular survival. Inhibition of macrophage phagosomal acidification resulted in a 30-fold reduction in USA300 expression of the staphylococcal virulence regulatoragrbut had little effect on expression ofsarA,saeR, orsigB. Bacterial exposure to acidic pHin vitroincreasedagrexpression. Together, these results suggest thatS. aureussurvives inside macrophages by perturbing normal phagolysosome formation and that USA300 may sense phagosomal conditions and upregulate expression of a key virulence regulator that enables its intracellular survival.
4
Chong, Audrey, Tara D. Wehrly, Vinod Nair, Elizabeth R. Fischer, Jeffrey R. Barker, Karl E. Klose, and Jean Celli. "The Early Phagosomal Stage of Francisella tularensis Determines Optimal Phagosomal Escape and Francisella Pathogenicity Island Protein Expression." Infection and Immunity 76, no. 12 (October 13, 2008): 5488–99. http://dx.doi.org/10.1128/iai.00682-08.
Full textAbstract:
ABSTRACT Francisella tularensis is an intracellular pathogen that can survive and replicate within macrophages. Following phagocytosis and transient interactions with the endocytic pathway, F. tularensis rapidly escapes from its original phagosome into the macrophage cytoplasm, where it eventually replicates. To examine the importance of the nascent phagosome for the Francisella intracellular cycle, we have characterized early trafficking events of the F. tularensis subsp. tularensis strain Schu S4 in a murine bone marrow-derived macrophage model. Here we show that early phagosomes containing Schu S4 transiently interact with early and late endosomes and become acidified before the onset of phagosomal disruption. Inhibition of endosomal acidification with the vacuolar ATPase inhibitor bafilomycin A1 or concanamycin A prior to infection significantly delayed but did not block phagosomal escape and cytosolic replication, indicating that maturation of the early Francisella-containing phagosome (FCP) is important for optimal phagosomal escape and subsequent intracellular growth. Further, Francisella pathogenicity island (FPI) protein expression was induced during early intracellular trafficking events. Although inhibition of endosomal acidification mimicked the early phagosomal escape defects caused by mutation of the FPI-encoded IglCD proteins, it did not inhibit the intracellular induction of FPI proteins, demonstrating that this response is independent of phagosomal pH. Altogether, these results demonstrate that early phagosomal maturation is required for optimal phagosomal escape and that the early FCP provides cues other than intravacuolar pH that determine intracellular induction of FPI proteins.
5
Levin, Roni, Gerald R. V. Hammond, Tamas Balla, Pietro De Camilli, Gregory D. Fairn, and Sergio Grinstein. "Multiphasic dynamics of phosphatidylinositol 4-phosphate during phagocytosis." Molecular Biology of the Cell 28, no. 1 (January 2017): 128–40. http://dx.doi.org/10.1091/mbc.e16-06-0451.
Full textAbstract:
We analyzed the distribution, fate, and functional role of phosphatidylinositol 4-phosphate (PtdIns4P) during phagosome formation and maturation. To this end, we used genetically encoded probes consisting of the PtdIns4P-binding domain of the bacterial effector SidM. PtdIns4P was found to undergo complex, multiphasic changes during phagocytosis. The phosphoinositide, which is present in the plasmalemma before engagement of the target particle, is transiently enriched in the phagosomal cup. Soon after the phagosome seals, PtdIns4P levels drop precipitously due to the hydrolytic activity of Sac2 and phospholipase C, becoming undetectable for ∼10 min. PtdIns4P disappearance coincides with the emergence of phagosomal PtdIns3P. Conversely, the disappearance of PtdIns3P that signals the transition from early to late phagosomes is accompanied by resurgence of PtdIns4P, which is associated with the recruitment of phosphatidylinositol 4-kinase 2A. The reacquisition of PtdIns4P can be prevented by silencing expression of the kinase and can be counteracted by recruitment of a 4-phosphatase with a heterodimerization system. Using these approaches, we found that the secondary accumulation of PtdIns4P is required for proper phagosomal acidification. Defective acidification may be caused by impaired recruitment of Rab7 effectors, including RILP, which were shown earlier to displace phagosomes toward perinuclear lysosomes. Our results show multimodal dynamics of PtdIns4P during phagocytosis and suggest that the phosphoinositide plays important roles during the maturation of the phagosome.
6
Steele-Mortimer, Olivia, Maryse St-Louis, Martin Olivier, and B. Brett Finlay. "Vacuole Acidification Is Not Required for Survival ofSalmonella enterica Serovar Typhimurium within Cultured Macrophages and Epithelial Cells." Infection and Immunity 68, no. 9 (September 1, 2000): 5401–4. http://dx.doi.org/10.1128/iai.68.9.5401-5404.2000.
Full textAbstract:
ABSTRACT Phagosome acidification is an important component of the microbicidal response by infected eukaryotic cells. Thus, intracellular pathogens that reside within phagosomes must either block phagosome acidification or be able to survive at low pH. In this work, we studied the effect of phagosomal acidification on the survival of intracellular Salmonella enterica serovar Typhimurium in different cell types. Bafilomycin A1, a specific inhibitor of the vacuolar proton-ATPases, was used to block acidification of salmonella-containing vacuoles. We found that in several epithelial cell lines, treatment with bafilomycin A1 had no effect on intracellular survival or replication. Furthermore, although acidification was essential for Salmonella intracellular survival in J774 cultured macrophages, as reported previously (13), it is not essential in other macrophage cell lines. These data suggest that vacuolar acidification may play a role in intracellular survival of salmonellae only under certain conditions and in specific cell types.
7
Riazanski, Vladimir, Aida G. Gabdoulkhakova, Lin S. Boynton, Raphael R. Eguchi, Ludmila V. Deriy, D. Kyle Hogarth, Nadège Loaëc, et al. "TRPC6 channel translocation into phagosomal membrane augments phagosomal function." Proceedings of the National Academy of Sciences 112, no. 47 (November 10, 2015): E6486—E6495. http://dx.doi.org/10.1073/pnas.1518966112.
Full textAbstract:
Defects in the innate immune system in the lung with attendant bacterial infections contribute to lung tissue damage, respiratory insufficiency, and ultimately death in the pathogenesis of cystic fibrosis (CF). Professional phagocytes, including alveolar macrophages (AMs), have specialized pathways that ensure efficient killing of pathogens in phagosomes. Phagosomal acidification facilitates the optimal functioning of degradative enzymes, ultimately contributing to bacterial killing. Generation of low organellar pH is primarily driven by the V-ATPases, proton pumps that use cytoplasmic ATP to load H+ into the organelle. Critical to phagosomal acidification are various channels derived from the plasma membrane, including the anion channel cystic fibrosis transmembrane conductance regulator, which shunt the transmembrane potential generated by movement of protons. Here we show that the transient receptor potential canonical-6 (TRPC6) calcium-permeable channel in the AM also functions to shunt the transmembrane potential generated by proton pumping and is capable of restoring microbicidal function to compromised AMs in CF and enhancement of function in non-CF cells. TRPC6 channel activity is enhanced via translocation to the cell surface (and then ultimately to the phagosome during phagocytosis) in response to G-protein signaling activated by the small molecule (R)-roscovitine and its derivatives. These data show that enhancing vesicular insertion of the TRPC6 channel to the plasma membrane may represent a general mechanism for restoring phagosome activity in conditions, where it is lost or impaired.
8
Hackam, David J., Ori D. Rotstein, Wei-jian Zhang, Samantha Gruenheid, Philippe Gros, and Sergio Grinstein. "Host Resistance to Intracellular Infection: Mutation of Natural Resistance-associated Macrophage Protein 1 (Nramp1) Impairs Phagosomal Acidification." Journal of Experimental Medicine 188, no. 2 (July 20, 1998): 351–64. http://dx.doi.org/10.1084/jem.188.2.351.
Full textAbstract:
The mechanisms underlying the survival of intracellular parasites such as mycobacteria in host macrophages remain poorly understood. In mice, mutations at the Nramp1 gene (for natural resistance-associated macrophage protein), cause susceptibility to mycobacterial infections. Nramp1 encodes an integral membrane protein that is recruited to the phagosome membrane in infected macrophages. In this study, we used microfluorescence ratio imaging of macrophages from wild-type and Nramp1 mutant mice to analyze the effect of loss of Nramp1 function on the properties of phagosomes containing inert particles or live mycobacteria. The pH of phagosomes containing live Mycobacterium bovis was significantly more acidic in Nramp1- expressing macrophages than in mutant cells (pH 5.5 ± 0.06 versus pH 6.6 ± 0.05, respectively; P <0.005). The enhanced acidification could not be accounted for by differences in proton consumption during dismutation of superoxide, phagosomal buffering power, counterion conductance, or in the rate of proton “leak”, as these were found to be comparable in wild-type and Nramp1-deficient macrophages. Rather, after ingestion of live mycobacteria, Nramp1-expressing cells exhibited increased concanamycin-sensitive H+ pumping across the phagosomal membrane. This was associated with an enhanced ability of phagosomes to fuse with vacuolar-type ATPase–containing late endosomes and/or lysosomes. This effect was restricted to live M. bovis and was not seen in phagosomes containing dead M. bovis or latex beads. These data support the notion that Nramp1 affects intracellular mycobacterial replication by modulating phagosomal pH, suggesting that Nramp1 plays a central role in this process.
9
Hackam, David J., Ori D. Rotstein, Wei-Jian Zhang, Nicolas Demaurex, Michael Woodside, Olivia Tsai, and Sergio Grinstein. "Regulation of Phagosomal Acidification." Journal of Biological Chemistry 272, no. 47 (November 21, 1997): 29810–20. http://dx.doi.org/10.1074/jbc.272.47.29810.
Full text
10
Mangahas, Paolo M., Xiaomeng Yu, Kenneth G. Miller, and Zheng Zhou. "The small GTPase Rab2 functions in the removal of apoptotic cells in Caenorhabditis elegans." Journal of Cell Biology 180, no. 2 (January 28, 2008): 357–73. http://dx.doi.org/10.1083/jcb.200708130.
Full textAbstract:
We identify here a novel class of loss-of-function alleles of uncoordinated locomotion(unc)-108, which encodes the Caenorhabditis elegans homologue of the mammalian small guanosine triphosphatase Rab2. Like the previously isolated dominant-negative mutants, unc-108 loss-of-function mutant animals are defective in locomotion. In addition, they display unique defects in the removal of apoptotic cells, revealing a previously uncharacterized function for Rab2. unc-108 acts in neurons and engulfing cells to control locomotion and cell corpse removal, respectively, indicating that unc-108 has distinct functions in different cell types. Using time-lapse microscopy, we find that unc-108 promotes the degradation of engulfed cell corpses. It is required for the efficient recruitment and fusion of lysosomes to phagosomes and the acidification of the phagosomal lumen. In engulfing cells, UNC-108 is enriched on the surface of phagosomes. We propose that UNC-108 acts on phagosomal surfaces to promote phagosome maturation and suggest that mammalian Rab2 may have a similar function in the degradation of apoptotic cells.
11
Grinstein, S., and W. Furuya. "Assessment of Na+-H+ exchange activity in phagosomal membranes of human neutrophils." American Journal of Physiology-Cell Physiology 254, no. 2 (February 1, 1988): C272—C285. http://dx.doi.org/10.1152/ajpcell.1988.254.2.c272.
Full textAbstract:
To assess the presence of Na+-H+ exchange in internalized membranes, the phagosomal pH was monitored in suspensions of intact human neutrophils by measuring the fluorescence of ingested bacteria (Micrococcus lysodeikticus) prelabeled with a pH-sensitive dye. Uptake of fluoresceinated bacteria was confirmed by flow cytometry and by phase-contrast and electron microscopy. Manipulation of the cytoplasmic ion content was accomplished by permeabilization of the plasma membrane with nystatin, which did not alter phagosomal permeability. At 37 degrees C, the phagosomal interior acidified at a maximal rate of 0.135 +/- 0.003 pH units/min (n = 10). The endogenous Na+-H+ exchanger does not affect phagosomal acidification, since the rate and extent of the pH change were not altered by 1) omission of intraphagosomal Na+ and 2) addition of the permeant inhibitor methylisobutylamiloride or by trapping amiloride in the phagosome during bacterial ingestion. Moreover, amiloride-sensitive Na+-H+ exchange was not detectable when Na+ or H+ gradients were imposed across the phagosomal membrane. Under comparable conditions, Na+-H+ exchange could be readily detected across the surface membrane. These data imply that the Na+-H+ antiporters are either inactivated in the phagosome or are segregated and not internalized into the phagosomal membrane.
12
Roux, Anne-Laure, Albertus Viljoen, Aïcha Bah, Roxane Simeone, Audrey Bernut, Laura Laencina, Therese Deramaudt, et al. "The distinct fate of smooth and rough Mycobacterium abscessus variants inside macrophages." Open Biology 6, no. 11 (November 2016): 160185. http://dx.doi.org/10.1098/rsob.160185.
Full textAbstract:
Mycobacterium abscessus is a pathogenic, rapidly growing mycobacterium responsible for pulmonary and cutaneous infections in immunocompetent patients and in patients with Mendelian disorders, such as cystic fibrosis (CF). Mycobacterium abscessus is known to transition from a smooth (S) morphotype with cell surface-associated glycopeptidolipids (GPL) to a rough (R) morphotype lacking GPL. Herein, we show that M. abscessus S and R variants are able to grow inside macrophages and are present in morphologically distinct phagosomes. The S forms are found mostly as single bacteria within phagosomes characterized by a tightly apposed phagosomal membrane and the presence of an electron translucent zone (ETZ) surrounding the bacilli. By contrast, infection with the R form leads to phagosomes often containing more than two bacilli, surrounded by a loose phagosomal membrane and lacking the ETZ. In contrast to the R variant, the S variant is capable of restricting intraphagosomal acidification and induces less apoptosis and autophagy. Importantly, the membrane of phagosomes enclosing the S forms showed signs of alteration, such as breaks or partial degradation. Although not frequently encountered, these events suggest that the S form is capable of provoking phagosome–cytosol communication. In conclusion, M. abscessus S exhibits traits inside macrophages that are reminiscent of slow-growing mycobacterial species.
13
Mantegazza, Adriana R., Ariel Savina, Mónica Vermeulen, Laura Pérez, Jorge Geffner, Olivier Hermine, Sergio D. Rosenzweig, Florence Faure, and Sebastián Amigorena. "NADPH oxidase controls phagosomal pH and antigen cross-presentation in human dendritic cells." Blood 112, no. 12 (December 1, 2008): 4712–22. http://dx.doi.org/10.1182/blood-2008-01-134791.
Full textAbstract:
Abstract The phagocyte NADPH oxidase (NOX2) is critical for the bactericidal activity of phagocytic cells and plays a major role in innate immunity. We showed recently that NOX2 activity in mouse dendritic cells (DCs) prevents acidification of phagosomes, promoting antigen cross-presentation. Inorder to investigate the role of NOX2 in the regulation of the phagosomal pH in human DCs, we analyzed the production of reactive oxygen species (ROS) and the phagosomal/endosomal pH in monocyte-derived DCs and macrophages (MØs) from healthy donors or patients with chronic granulomatous disease (CGD). As expected, we found that human MØs acidify their phagosomes more efficiently than human DCs. Accordingly, the expression of the vacuolar proton ATPase (V-H+-ATPase) was higher in MØs than in DCs. Phagosomal ROS production, however, was also higher in MØs than in DCs, due to higher levels of gp91phox expression and recruitment to phagosomes. In contrast, in the absence of active NOX2, the phagosomal and endosomal pH decreased. Both in the presence of a NOX2 inhibitor and in DCs derived from patients with CGD, the cross-presentation of 2 model tumor antigens was impaired. We conclude that NOX2 activity participates in the regulation of the phagosomal and endosomal pH in human DCs, and is required for efficient antigen cross-presentation.
14
Vieira, Otilia V., Rene E. Harrison, Cameron C. Scott, Harald Stenmark, David Alexander, Jun Liu, Jean Gruenberg, Alan D. Schreiber, and Sergio Grinstein. "Acquisition of Hrs, an Essential Component of Phagosomal Maturation, Is Impaired by Mycobacteria." Molecular and Cellular Biology 24, no. 10 (May 15, 2004): 4593–604. http://dx.doi.org/10.1128/mcb.24.10.4593-4604.2004.
Full textAbstract:
ABSTRACT Pathogenic mycobacteria survive within macrophages by precluding the fusion of phagosomes with late endosomes or lysosomes. Because the molecular determinants of normal phagolysosome formation are poorly understood, the sites targeted by mycobacteria remain unidentified. We found that Hrs, an adaptor molecule involved in protein sorting, associates with phagosomes prior to their fusion with late endosomes or lysosomes. Recruitment of Hrs required the interaction of its FYVE domain with phagosomal phosphatidylinositol 3-phosphate, but two other attachment sites were additionally involved. Depletion of Hrs by use of small interfering RNA impaired phagosomal maturation, preventing the acquisition of lysobisphosphatidic acid and reducing luminal acidification. As a result, the maturation of phagosomes formed in Hrs-depleted cells was arrested at an early stage, characterized by the acquisition and retention of sorting endosomal markers. This phenotype is strikingly similar to that reported to occur in phagosomes of cells infected by mycobacteria. We therefore tested whether Hrs is recruited to phagosomes containing mycobacteria. Hrs associated readily with phagosomes containing inert particles but poorly with mycobacterial phagosomes. Moreover, Hrs was found more frequently in phagosomes containing avirulent Mycobacterium smegmatis than in phagosomes with the more virulent Mycobacterium marinum. These findings suggest that the inability to recruit Hrs contributes to the arrest of phagosomal maturation induced by pathogenic mycobacteria.
15
Jabado, Nada, Andrzej Jankowski, Samuel Dougaparsad, Virginie Picard, Sergio Grinstein, and Philippe Gros. "Natural Resistance to Intracellular Infections." Journal of Experimental Medicine 192, no. 9 (October 30, 2000): 1237–48. http://dx.doi.org/10.1084/jem.192.9.1237.
Full textAbstract:
Mutations at the natural resistance–associated macrophage protein 1 (Nramp1) locus cause susceptibility to infection with antigenically unrelated intracellular pathogens. Nramp1 codes for an integral membrane protein expressed in the lysosomal compartment of macrophages, and is recruited to the membrane of phagosomes soon after the completion of phagocytosis. To define whether Nramp1 functions as a transporter at the phagosomal membrane, a divalent cation-sensitive fluorescent probe was designed and covalently attached to a porous particle. The resulting conjugate, zymosan–FF6, was ingested by macrophages and its fluorescence emission was recorded in situ after phagocytosis, using digital imaging. Quenching of the probe by Mn2+ was used to monitor the flux of divalent cations across the phagosomal membrane in peritoneal macrophages obtained from Nramp1-expressing (+/+) and Nramp1-deficient (−/−) macrophages. Phagosomes from Nramp1+/+ mice extrude Mn2+ faster than their Nramp−/− counterparts. The difference in the rate of transport is eliminated when acidification of the phagosomal lumen is dissipated, suggesting that divalent metal transport through Nramp1 is H+ dependent. These studies suggest that Nramp1 contributes to defense against infection by extrusion of divalent cations from the phagosomal space. Such cations are likely essential for microbial function and their removal from the phagosomal microenvironment impairs pathogenesis, resulting in enhanced bacteriostasis or bactericidal activity.
16
Clemens, Daniel L., Bai-Yu Lee, and Marcus A. Horwitz. "Virulent and Avirulent Strains of Francisella tularensis Prevent Acidification and Maturation of Their Phagosomes and Escape into the Cytoplasm in Human Macrophages." Infection and Immunity 72, no. 6 (June 2004): 3204–17. http://dx.doi.org/10.1128/iai.72.6.3204-3217.2004.
Full textAbstract:
ABSTRACT Francisella tularensis, the agent of tularemia, is an intracellular pathogen, but little is known about the compartment in which it resides in human macrophages. We have examined the interaction of a recent virulent clinical isolate of F. tularensis subsp. tularensis and the live vaccine strain with human macrophages by immunoelectron and confocal immunofluorescence microscopy. We assessed the maturation of the F. tularensis phagosome by examining its acquisition of the lysosome-associated membrane glycoproteins (LAMPs) CD63 and LAMP1 and the acid hydrolase cathepsin D. Two to four hours after infection, vacuoles containing live F. tularensis cells acquired abundant staining for LAMPs but little or no staining for cathepsin D. However, after 4 h, the colocalization of LAMPs with live F. tularensis organisms declined dramatically. In contrast, vacuoles containing formalin-killed bacteria exhibited intense staining for all of these late endosomal/lysosomal markers at all time points examined (1 to 16 h). We examined the pH of the vacuoles 3 to 4 h after infection by quantitative immunogold staining and by fluorescence staining for lysosomotropic agents. Whereas phagosomes containing killed bacteria stained intensely for these agents, indicating a marked acidification of the phagosomes (pH 5.5), phagosomes containing live F. tularensis did not concentrate these markers and thus were not appreciably acidified (pH 6.7). An ultrastructural analysis of the F. tularensis compartment revealed that during the first 4 h after uptake, the majority of F. tularensis bacteria reside within phagosomes with identifiable membranes. The cytoplasmic side of the membranes of ∼50% of these phagosomes was coated with densely staining fibrils of ∼30 nm in length. In many cases, these coated phagosomal membranes appeared to bud, vesiculate, and fragment. By 8 h after infection, the majority of live F. tularensis bacteria lacked any ultrastructurally discernible membrane separating them from the host cell cytoplasm. These results indicate that F. tularensis initially enters a nonacidified phagosome with LAMPs but without cathepsin D and that the phagosomal membrane subsequently becomes morphologically disrupted, allowing the bacteria to gain direct access to the macrophagic cytoplasm. The capacity of F. tularensis to alter the maturation of its phagosome and to enter the cytoplasm is likely an important element of its capacity to parasitize macrophages and has major implications for vaccine development.
17
Yin, Jianhua, Yaling Huang, Pengfei Guo, Siqi Hu, Sawako Yoshina, Nan Xuan, Qiwen Gan, Shohei Mitani, Chonglin Yang, and Xiaochen Wang. "GOP-1 promotes apoptotic cell degradation by activating the small GTPase Rab2 in C. elegans." Journal of Cell Biology 216, no. 6 (April 19, 2017): 1775–94. http://dx.doi.org/10.1083/jcb.201610001.
Full textAbstract:
Apoptotic cells generated by programmed cell death are engulfed by phagocytes and enclosed within plasma membrane–derived phagosomes. Maturation of phagosomes involves a series of membrane-remodeling events that are governed by the sequential actions of Rab GTPases and lead to formation of phagolysosomes, where cell corpses are degraded. Here we identified gop-1 as a novel regulator of apoptotic cell clearance in Caenorhabditis elegans. Loss of gop-1 affects phagosome maturation through the RAB-5–positive stage, causing defects in phagosome acidification and phagolysosome formation, phenotypes identical to and unaffected by loss of unc-108, the C. elegans Rab2. GOP-1 transiently associates with cell corpse–containing phagosomes, and loss of its function abrogates phagosomal association of UNC-108. GOP-1 interacts with GDP-bound and nucleotide-free UNC-108/Rab2, disrupts GDI-UNC-108 complexes, and promotes activation and membrane recruitment of UNC-108/Rab2 in vitro. Loss of gop-1 also abolishes association of UNC-108 with endosomes, causing defects in endosome and dense core vesicle maturation. Thus, GOP-1 is an activator of UNC-108/Rab2 in multiple processes.
18
Belhaouane, Imène, Amine Pochet, Jonathan Chatagnon, Eik Hoffmann, Christophe J. Queval, Nathalie Deboosère, Céline Boidin-Wichlacz, et al. "Tirap controls Mycobacterium tuberculosis phagosomal acidification." PLOS Pathogens 19, no. 3 (March 8, 2023): e1011192. http://dx.doi.org/10.1371/journal.ppat.1011192.
Full textAbstract:
Progression of tuberculosis is tightly linked to a disordered immune balance, resulting in inability of the host to restrict intracellular bacterial replication and its subsequent dissemination. The immune response is mainly characterized by an orchestrated recruitment of inflammatory cells secreting cytokines. This response results from the activation of innate immunity receptors that trigger downstream intracellular signaling pathways involving adaptor proteins such as the TIR-containing adaptor protein (Tirap). In humans, resistance to tuberculosis is associated with a loss-of-function in Tirap. Here, we explore how genetic deficiency in Tirap impacts resistance to Mycobacterium tuberculosis (Mtb) infection in a mouse model and ex vivo. Interestingly, compared to wild type littermates, Tirap heterozygous mice were more resistant to Mtb infection. Upon investigation at the cellular level, we observed that mycobacteria were not able to replicate in Tirap-deficient macrophages compared to wild type counterparts. We next showed that Mtb infection induced Tirap expression which prevented phagosomal acidification and rupture. We further demonstrate that the Tirap-mediated anti-tuberculosis effect occurs through a Cish-dependent signaling pathway. Our findings provide new molecular evidence about how Mtb manipulates innate immune signaling to enable intracellular replication and survival of the pathogen, thus paving the way for host-directed approaches to treat tuberculosis.
19
Lâm, Thiên-Trí, Bernd Giese, Deepak Chikkaballi, Anika Kühn, Wanja Wolber, Jan Pané-Farré, Daniel Schäfer, Susanne Engelmann, Martin Fraunholz, and Bhanu Sinha. "Phagolysosomal Integrity Is Generally Maintained after Staphylococcus aureus Invasion of Nonprofessional Phagocytes but Is Modulated by Strain 6850." Infection and Immunity 78, no. 8 (June 7, 2010): 3392–403. http://dx.doi.org/10.1128/iai.00012-10.
Full textAbstract:
ABSTRACT Staphylococcus aureus is a major cause of a variety of both local and systemic infections. It can invade human host cells, a process that may account for disseminated and recurrent infections. S. aureus postinvasion events in nonprofessional phagocytes are only partially understood. While morphological data suggest a phagosomal escape, there is a lack of corroborating functional data. Using a combination of pH determination and morphological techniques, we have tested the integrity of Staphylococcus-containing phagosomes in 293 (HEK-293), HeLa, and EA.hy926 cells over time. Rapid acidification of S. aureus-containing phagosomes occurred and was sustained for up to 24 h. All S. aureus strains tested displayed equally sustained intraphagosomal pH levels without exhibiting any correlation with pH level and hemolytic activity. The membrane morphology of the phagosomal compartment was heterogeneous, even under conditions where acidic pH was fully maintained, an observation incompatible with phagolysosomal membrane destruction. As an exception, S. aureus strain 6850 showed a reduced phagosomal acidification signal 6 h after invasion. Additionally, only strain 6850 failed to localize to LAMP-1-positive vesicles in HeLa cells, although this was observed only rarely. Several other strongly beta-hemolytic strains did not modulate phagolysosomal pH, suggesting that S. aureus α-toxin and β-toxin are not sufficient for this process. Taken together, our data suggest that S. aureus-containing phagolysosomes generally remain functionally intact in nonprofessional phagocytes, thereby contrasting with transmission electron micrographic results.
20
Sullivan, Jonathan Tabb, Ellen F. Young, Jessica R. McCann, and Miriam Braunstein. "The Mycobacterium tuberculosis SecA2 System Subverts Phagosome Maturation To Promote Growth in Macrophages." Infection and Immunity 80, no. 3 (January 3, 2012): 996–1006. http://dx.doi.org/10.1128/iai.05987-11.
Full textAbstract:
The ability ofMycobacterium tuberculosisto grow in macrophages is critical to the virulence of this important pathogen. One wayM. tuberculosisis thought to maintain a hospitable niche in macrophages is by arresting the normal process of phagosomes maturing into acidified phagolysosomes. The process of phagosome maturation arrest byM. tuberculosisis not fully understood, and there has remained a need to firmly establish a requirement for phagosome maturation arrest forM. tuberculosisgrowth in macrophages. Other intracellular pathogens that control the phagosomal environment use specialized protein export systems to deliver effectors of phagosome trafficking to the host cell. InM. tuberculosis, the accessory SecA2 system is a specialized protein export system that is required for intracellular growth in macrophages. In studying the importance of the SecA2 system in macrophages, we discovered that SecA2 is required for phagosome maturation arrest. Shortly after infection, phagosomes containing a ΔsecA2mutant ofM. tuberculosiswere more acidified and showed greater association with markers of late endosomes than phagosomes containing wild-typeM. tuberculosis. We further showed that inhibitors of phagosome acidification rescued the intracellular growth defect of the ΔsecA2mutant, which demonstrated that the phagosome maturation arrest defect of the ΔsecA2mutant is responsible for the intracellular growth defect. This study demonstrates the importance of phagosome maturation arrest forM. tuberculosisgrowth in macrophages, and it suggests there are effectors of phagosome maturation that are exported into the host environment by the accessory SecA2 system.
21
Xu, Meng, Yubing Liu, Liyuan Zhao, Qiwen Gan, Xiaochen Wang, and Chonglin Yang. "The lysosomal cathepsin protease CPL-1 plays a leading role in phagosomal degradation of apoptotic cells in Caenorhabditis elegans." Molecular Biology of the Cell 25, no. 13 (July 2014): 2071–83. http://dx.doi.org/10.1091/mbc.e14-01-0015.
Full textAbstract:
During programmed cell death, the clearance of apoptotic cells is achieved by their phagocytosis and delivery to lysosomes for destruction in engulfing cells. However, the role of lysosomal proteases in cell corpse destruction is not understood. Here we report the identification of the lysosomal cathepsin CPL-1 as an indispensable protease for apoptotic cell removal in Caenorhabditis elegans. We find that loss of cpl-1 function leads to strong accumulation of germ cell corpses, which results from a failure in degradation rather than engulfment. CPL-1 is expressed in a variety of cell types, including engulfment cells, and its mutation does not affect the maturation of cell corpse–containing phagosomes, including phagosomal recruitment of maturation effectors and phagosome acidification. Of importance, we find that phagosomal recruitment and incorporation of CPL-1 occurs before digestion of cell corpses, which depends on factors required for phagolysosome formation. Using RNA interference, we further examine the role of other candidate lysosomal proteases in cell corpse clearance but find that they do not obviously affect this process. Collectively, these findings establish CPL-1 as the leading lysosomal protease required for elimination of apoptotic cells in C. elegans.
22
Dragotakes, Quigly, Ella Jacobs, Lia Sanchez Ramirez, Olivia Insun Yoon, Caitlin Perez-Stable, Hope Eden, Jenlu Pagnotta, et al. "Bet-hedging antimicrobial strategies in macrophage phagosome acidification drive the dynamics of Cryptococcus neoformans intracellular escape mechanisms." PLOS Pathogens 18, no. 7 (July 11, 2022): e1010697. http://dx.doi.org/10.1371/journal.ppat.1010697.
Full textAbstract:
The fungus Cryptococcus neoformans is a major human pathogen with a remarkable intracellular survival strategy that includes exiting macrophages through non-lytic exocytosis (Vomocytosis) and transferring between macrophages (Dragotcytosis) by a mechanism that involves sequential events of non-lytic exocytosis and phagocytosis. Vomocytosis and Dragotcytosis are fungal driven processes, but their triggers are not understood. We hypothesized that the dynamics of Dragotcytosis could inherit the stochasticity of phagolysosome acidification and that Dragotcytosis was triggered by fungal cell stress. Consistent with this view, fungal cells involved in Dragotcytosis reside in phagolysosomes characterized by low pH and/or high oxidative stress. Using fluorescent microscopy, qPCR, live cell video microscopy, and fungal growth assays we found that the that mitigating pH or oxidative stress reduced Dragotcytosis frequency, whereas ROS susceptible mutants of C. neoformans underwent Dragotcytosis more frequently. Dragotcytosis initiation was linked to phagolysosomal pH, oxidative stresses, and macrophage polarization state. Dragotcytosis manifested stochastic dynamics thus paralleling the dynamics of phagosomal acidification, which correlated with the inhospitality of phagolysosomes in differently polarized macrophages. Hence, randomness in phagosomal acidification randomly created a population of inhospitable phagosomes where fungal cell stress triggered stochastic C. neoformans non-lytic exocytosis dynamics to escape a non-permissive intracellular macrophage environment.
23
Sasaki, Ayaka, Isei Nakae, Maya Nagasawa, Keisuke Hashimoto, Fumiko Abe, Kota Saito, Masamitsu Fukuyama, et al. "Arl8/ARL-8 functions in apoptotic cell removal by mediating phagolysosome formation inCaenorhabditis elegans." Molecular Biology of the Cell 24, no. 10 (May 15, 2013): 1584–92. http://dx.doi.org/10.1091/mbc.e12-08-0628.
Full textAbstract:
Efficient clearance of apoptotic cells by phagocytes is important for development, tissue homeostasis, and the prevention of autoimmune responses. Phagosomes containing apoptotic cells undergo acidification and mature from Rab5-positive early to Rab7-positive late stages. Phagosomes finally fuse with lysosomes to form phagolysosomes, which degrade apoptotic cells; however, the molecular mechanism underlying phagosome–lysosome fusion is not fully understood. Here we show that the Caenorhabditis elegans Arf-like small GTPase Arl8 (ARL-8) is involved in phagolysosome formation and is required for the efficient removal of apoptotic cells. Loss of function of arl-8 results in the accumulation of apoptotic germ cells. Both the engulfment of the apoptotic cells by surrounding somatic sheath cells and the phagosomal maturation from RAB-5- to RAB-7-positive stages occur in arl-8 mutants. However, the phagosomes fail to fuse with lysosomes in the arl-8 mutants, leading to the accumulation of RAB-7-positive phagosomes and the delayed degradation of apoptotic cells. ARL-8 localizes primarily to lysosomes and physically interacts with the homotypic fusion and protein sorting complex component VPS-41. Collectively our findings reveal that ARL-8 facilitates apoptotic cell removal in vivo by mediating phagosome–lysosome fusion during phagocytosis.
24
Morris, Paul E. R., Stephen Renshaw, Simon J. Foster, Andrew Peden, and David Dockrell. "2601. Identification of Staphylococcus aureus Genetic Factors Associatiated with the Subversion of Macrophage Phagosomal Acidification." Open Forum Infectious Diseases 6, Supplement_2 (October 2019): S904. http://dx.doi.org/10.1093/ofid/ofz360.2279.
Full textAbstract:
Abstract Background S. aureus is a major medical pathogen contributing to healthcare-associated costs and mortality. Metastatic S. aureus infection is commonly associated with skin and soft-tissue infections. Macrophages as resident tissue phagocytes are essential for bacterial clearance and effectively phagocytose and kill S. aureus at low inocula. Formation of an intracellular reservoir develops when the capacity for macrophages to eradicate S. aureus is overwhelmed at increased inocula. Phagosomal maturation after bacterial ingestion involves sequential fusion with endosomes and lysosomes, reducing luminal pH, facilitating bacterial degradation. In the context of intracellular S. aureus, incomplete phagosomal maturation is demonstrable with impaired acidification and failure of lysosome fusion. Inhibition of phagosomal maturation is dependent upon bacterial factors as it is reversed in heat-killed bacteria. Intracellular survival of S. aureus is associated with reduced antimicrobial effectivity and increased complications. Within the intracellular environment, persistence of S. aureus is associated with adaptation of gene expression which confers greater resistance to antimicrobial effector mechanisms. Methods An ordered mutant library of S. aureus provides the opportunity to give a comprehensive evaluation of gene function. The Nebraska transposon (Tn) mutant library contains 1952 sequence-defined Tn insertion mutants derivative of USA300 LAC S. aureus, each with a single non-essential gene deletion. The mutants were labeled with a fluorescent stain activated at pH < 6 and challenged against differentiated human monocyte-derived macrophages for 4 hours. A high-content microscopy screen was developed to identify the bacterial genes associated with impairment of phagosomal acidification. Results The results of the high-throughput screen indicate the global regulators agr and saeR, hemolysin A and catalase are associated with the inhibition of phagosomal acidification. Conclusion The burden of S. aureus bacteremia and metastatic disease makes the targeting of intracellular S. aureus essential. Identification of bacterial factors associated with impaired phagosomal acidification and maturation offers targets to limit S. aureus infections. Disclosures All authors: No reported disclosures.
25
Despras, Guillaume, Alsu I. Zamaleeva, Lucie Dardevet, Céline Tisseyre, Joao Gamelas Magalhaes, Charlotte Garner, Michel De Waard, et al. "H-Rubies, a new family of red emitting fluorescent pH sensors for living cells." Chemical Science 6, no. 10 (2015): 5928–37. http://dx.doi.org/10.1039/c5sc01113b.
Full textAbstract:
H-Rubies is a family of pH probes that display a bright red fluorescence upon acidification. They have been used as molecular form to monitor mitochondrial acidification and as functionalised forms to provide ratiometric systems to measure phagosomal and endosomal pH in macrophages.
26
Ramachandra, Lakshmi, Jamie L. Smialek, Sam S. Shank, Marilyn Convery, W. Henry Boom, and Clifford V. Harding. "Phagosomal Processing of Mycobacterium tuberculosis Antigen 85B Is Modulated Independently of Mycobacterial Viability and Phagosome Maturation." Infection and Immunity 73, no. 2 (February 2005): 1097–105. http://dx.doi.org/10.1128/iai.73.2.1097-1105.2005.
Full textAbstract:
ABSTRACT Control of Mycobacterium tuberculosis infection requires CD4 T-cell responses and major histocompatibility complex class II (MHC-II) processing of M. tuberculosis antigens (Ags). We have previously demonstrated that macrophages process heat-killed (HK) M. tuberculosis more efficiently than live M. tuberculosis. These observations suggested that live M. tuberculosis may inhibit Ag processing by inhibiting phagosome maturation or that HK M. tuberculosis may be less resistant to Ag processing. In the present study we examined the correlation between M. tuberculosis viability and phagosome maturation and efficiency of Ag processing. Since heat treatment could render M. tuberculosis Ags more accessible to proteolysis, M. tuberculosis was additionally killed by antibiotic treatment and radiation. Processing of HK, live, radiation-killed (RadK), or rifampin-killed (RifK) M. tuberculosis in activated murine bone marrow macrophages was examined by using an I-Ab-restricted T-cell hybridoma cell line (BB7) that recognizes an epitope derived from Ag 85B. Macrophages processed HK M. tuberculosis more rapidly and efficiently than they processed live, RadK, or RifK M. tuberculosis. Live, RadK, and RifK M. tuberculosis cells were processed with similar efficiencies for presentation to BB7 T hybridoma cells. Furthermore, phagosomes containing live or RadK M. tuberculosis expressed fewer M. tuberculosis peptide-MHC-II complexes than phagosomes containing HK M. tuberculosis expressed. Since only live M. tuberculosis was able to prevent acidification of the phagosome, our results suggest that regulation of phagosome maturation does not explain the differences in processing of different forms of M. tuberculosis. These findings suggest that the mechanisms used by M. tuberculosis to inhibit phagosomal maturation differ from the mechanisms involved in modulating phagosome Ag processing.
27
Stewart, Graham R., Janisha Patel, Brian D. Robertson, Aaron Rae, and Douglas B. Young. "Mycobacterial Mutants with Defective Control of Phagosomal Acidification." PLoS Pathogens 1, no. 3 (November 25, 2005): e33. http://dx.doi.org/10.1371/journal.ppat.0010033.
Full text
28
Bah, Aïcha, Merlin Sanicas, Jérôme Nigou, Christophe Guilhot, Catherine Astarie-Dequeker, and Isabelle Vergne. "The Lipid Virulence Factors of Mycobacterium tuberculosis Exert Multilayered Control over Autophagy-Related Pathways in Infected Human Macrophages." Cells 9, no. 3 (March 9, 2020): 666. http://dx.doi.org/10.3390/cells9030666.
Full textAbstract:
Autophagy is an important innate immune defense mechanism that controls Mycobacterium tuberculosis (Mtb) growth inside macrophages. Autophagy machinery targets Mtb-containing phagosomes via xenophagy after damage to the phagosomal membrane due to the Type VII secretion system Esx-1 or via LC3-associated phagocytosis without phagosomal damage. Conversely, Mtb restricts autophagy-related pathways via the production of various bacterial protein factors. Although bacterial lipids are known to play strategic functions in Mtb pathogenesis, their role in autophagy manipulation remains largely unexplored. Here, we report that the lipid virulence factors sulfoglycolipids (SLs) and phthiocerol dimycocerosates (DIMs) control autophagy-related pathways through distinct mechanisms in human macrophages. Using knock-out and knock-in mutants of Mtb and Mycobacterium bovis BCG (Bacille Calmette Guerin) and purified lipids, we found that (i) Mtb mutants with DIM and SL deficiencies promoted functional autophagy via an MyD88-dependent and phagosomal damage-independent pathway in human macrophages; (ii) SLs limited this pathway by acting as TLR2 antagonists; (iii) DIMs prevented phagosomal damage-independent autophagy while promoting Esx-1-dependent xenophagy; (iv) and DIMs, but not SLs, limited the acidification of LC3-positive Mtb compartments. In total, our study reveals an unexpected and intricate role for Mtb lipid virulence factors in controlling autophagy-related pathways in human macrophages, thus providing further insight into the autophagy manipulation tactics deployed by intracellular bacterial pathogens.
29
Prajsnar, Tomasz K., Bartosz J. Michno, Niedharsan Pooranachandran, Andrew K. Fenton, Tim J. Mitchell, David H. Dockrell, and Stephen A. Renshaw. "Phagosomal Acidification Is Required to Kill Streptococcus pneumoniae in a Zebrafish Model." Cellular Microbiology 2022 (June 9, 2022): 1–13. http://dx.doi.org/10.1155/2022/9429516.
Full textAbstract:
Streptococcus pneumoniae (the pneumococcus) is a major human pathogen causing invasive disease, including community-acquired bacteraemia, and remains a leading cause of global mortality. Understanding the role of phagocytes in killing bacteria is still limited, especially in vivo. In this study, we established a zebrafish model to study the interaction between intravenously administered pneumococci and professional phagocytes such as macrophages and neutrophils, to unravel bacterial killing mechanisms employed by these immune cells. Our model confirmed the key role of polysaccharide capsule in promoting pneumococcal virulence through inhibition of phagocytosis. Conversely, we show pneumococci lacking a capsule are rapidly internalised by macrophages. Low doses of encapsulated S. pneumoniae cause near 100% mortality within 48 hours postinfection (hpi), while 50 times higher doses of unencapsulated pneumococci are easily cleared. Time course analysis of in vivo bacterial numbers reveals that while encapsulated pneumococcus proliferates to levels exceeding 105 CFU at the time of host death, unencapsulated bacteria are unable to grow and are cleared within 20 hpi. Using genetically induced macrophage depletion, we confirmed an essential role for macrophages in bacterial clearance. Additionally, we show that upon phagocytosis by macrophages, phagosomes undergo rapid acidification. Genetic and chemical inhibition of vacuolar ATPase (v-ATPase) prevents intracellular bacterial killing and induces host death indicating a key role of phagosomal acidification in immunity to invading pneumococci. We also show that our model can be used to study the efficacy of antimicrobials against pneumococci in vivo. Collectively, our data confirm that larval zebrafish can be used to dissect killing mechanisms during pneumococcal infection in vivo and highlight key roles for phagosomal acidification in macrophages for pathogen clearance.
30
Leliefeld, Pieter H. C., Janesh Pillay, Nienke Vrisekoop, Marjolein Heeres, Tamar Tak, Matthijs Kox, Suzan H. M. Rooijakkers, et al. "Differential antibacterial control by neutrophil subsets." Blood Advances 2, no. 11 (June 12, 2018): 1344–55. http://dx.doi.org/10.1182/bloodadvances.2017015578.
Full textAbstract:
Key Points Neutrophil subsets circulating during acute inflammation are characterized by differential bacterial containment capacity. Adequate antimicrobial containment is associated with profound phagosomal acidification yet independent of reactive oxygen species.
31
Vergne, Isabelle, Rutilio A. Fratti, Preston J. Hill, Jennifer Chua, John Belisle, and Vojo Deretic. "Mycobacterium tuberculosisPhagosome Maturation Arrest: Mycobacterial Phosphatidylinositol Analog Phosphatidylinositol Mannoside Stimulates Early Endosomal Fusion." Molecular Biology of the Cell 15, no. 2 (February 2004): 751–60. http://dx.doi.org/10.1091/mbc.e03-05-0307.
Full textAbstract:
Mycobacterium tuberculosis is a facultative intracellular pathogen that parasitizes macrophages by modulating properties of the Mycobacterium-containing phagosome. Mycobacterial phagosomes do not fuse with late endosomal/lysosomal organelles but retain access to early endosomal contents by an unknown mechanism. We have previously reported that mycobacterial phosphatidylinositol analog lipoarabinomannan (LAM) blocks a trans-Golgi network-to-phagosome phosphatidylinositol 3-kinase-dependent pathway. In this work, we extend our investigations of the effects of mycobacterial phosphoinositides on host membrane trafficking. We present data demonstrating that phosphatidylinositol mannoside (PIM) specifically stimulated homotypic fusion of early endosomes in an ATP-, cytosol-, and N-ethylmaleimide sensitive factor-dependent manner. The fusion showed absolute requirement for small Rab GTPases, and the stimulatory effect of PIM increased upon partial depletion of membrane Rabs with RabGDI. We found that stimulation of early endosomal fusion by PIM was higher when phosphatidylinositol 3-kinase was inhibited by wortmannin. PIM also stimulated in vitro fusion between model phagosomes and early endosomes. Finally, PIM displayed in vivo effects in macrophages by increasing accumulation of plasma membrane-endosomal syntaxin 4 and transferrin receptor on PIM-coated latex bead phagosomes. In addition, inhibition of phagosomal acidification was detected with PIM-coated beads. The effects of PIM, along with the previously reported action of LAM, suggest that M. tuberculosis has evolved a two-prong strategy to modify its intracellular niche: its products block acquisition of late endosomal/lysosomal constituents, while facilitating fusion with early endosomal compartments.
32
Jung, Joo-Yong, and Cory M. Robinson. "Interleukin-27 inhibits phagosomal acidification by blocking vacuolar ATPases." Cytokine 62, no. 2 (May 2013): 202–5. http://dx.doi.org/10.1016/j.cyto.2013.03.010.
Full text
33
Monteith, Andrew J., Heather Vincent, Sunah Kang, Patrick Li, Tauris Claiborne, Nathaniel Moorman, and Barbara Vilen. "Chronic mTOR activity impaires lysosome maturation in lupus." Journal of Immunology 198, no. 1_Supplement (May 1, 2017): 217.7. http://dx.doi.org/10.4049/jimmunol.198.supp.217.7.
Full textAbstract:
Abstract Multiple genetic and environmental factors activate lymphocytes and myeloid cells to promote systemic lupus erythematosus (SLE). This complexity has made it difficult to define underlying mechanisms. We previously demonstrated that hematopoietic cells from lupus-prone mice accumulate nuclear antigens on their cell surface due to an inability to mature the lysosome. On myeloid cells, the accumulation of nuclear antigens reflects IgG-immune complexes (IgG-ICs) bound to FcγRs. Further, lupus-prone mice lacking FcγRI fail to develop myeloid and lymphoid-associated pathologies suggesting a critical role for FcγRI in SLE. How FcγRI signaling influences lysosomal maturation is unclear. Herein, we show that lysosome acidification, a key step in the maturation process, relies on the strict regulation of mTORC2, activation of caspase-1, and cleavage of Rab39a. In macrophages from MRL/lpr mice, we find that chronic mTORC2 activation disrupts lysosomal maturation by inducing constitutive phosphorylation of cofilin. This prevents the association of cofilin with phagosomes containing IgG-ICs. As a result, inflammasome-independent activation of caspase-1 in proximity to the phagosome does not occur. Consequently, Rab39a remains intact on the phagosomal membrane, and the lysosome does not acidity. These findings reveal unique roles for mTORC2, cofilin, and pyroptototic caspases critical to an unappreciated signaling pathway promoting lysosome acidification. Further, this signaling cascade becomes disrupted in SLE, preventing the lysosome-mediated degradation of apoptotic debris upstream of multiple lupus-associated pathologies.
34
Rittig, Michael G., Maria-Teresa Alvarez-Martinez, Françoise Porte, Jean-Pierre Liautard, and Bruno Rouot. "Intracellular Survival of Brucellaspp. in Human Monocytes Involves Conventional Uptake but Special Phagosomes." Infection and Immunity 69, no. 6 (June 1, 2001): 3995–4006. http://dx.doi.org/10.1128/iai.69.6.3995-4006.2001.
Full textAbstract:
ABSTRACT Brucella spp. are facultative intracellular parasites of various mammals, including humans, typically infecting lymphoid as well as reproductive organs. We have investigated howB. suis and B. melitensis enter human monocytes and in which compartment they survive. Peripheral blood monocytes readily internalized nonopsonized brucellae and killed most of them within 12 to 18 h. The presence ofBrucella-specific antibodies (but not complement) increased the uptake of bacteria without increasing their intracellular survival, whereas adherence of the monocytes or incubation in Ca2+- and Mg2+-free medium reduced the uptake. Engulfment of all Brucella organisms (regardless of bacterial viability or virulence) initially resulted in phagosomes with tightly apposed walls (TP). Most TP were fully fusiogenic and matured to spacious phagolysosomes containing degraded bacteria, whereas some TP (more in monocyte-derived macrophages, HeLa cells, and CHO cells than in monocytes) remained tightly apposed to intact bacteria. Immediate treatment of infected host cells with the lysosomotropic base ammonium chloride caused a swelling of all phagosomes and a rise in the intraphagosomal pH, abolishing the intracellular survival of Brucella. These results indicate that (i) human monocytes readily internalizeBrucella in a conventional way using various phagocytosis-promoting receptors, (ii) the maturation of someBrucella phagosomes is passively arrested between the steps of acidification and phagosome-lysosome fusion, (iii) brucellae are killed in maturing but not in arrested phagosomes, and (iv) survival of internalized Brucella depends on an acidic intraphagosomal pH and/or close contact with the phagosomal wall.
35
Govoni, Gregory, François Canonne-Hergaux, Cheryl G. Pfeifer, Sandra L. Marcus, Scott D. Mills, David J. Hackam, Sergio Grinstein, Danielle Malo, B. Brett Finlay, and Philippe Gros. "Functional Expression of Nramp1 In Vitro in the Murine Macrophage Line RAW264.7." Infection and Immunity 67, no. 5 (May 1, 1999): 2225–32. http://dx.doi.org/10.1128/iai.67.5.2225-2232.1999.
Full textAbstract:
ABSTRACT Mutations at the Nramp1 locus in vivo cause susceptibility to infection by unrelated intracellular microbes.Nramp1 encodes an integral membrane protein abundantly expressed in the endosomal-lysosomal compartment of macrophages and is recruited to the phagosomal membrane following phagocytosis. The mechanism by which Nramp1 affects the biochemical properties of the phagosome to control microbial replication is unknown. To devise an in vitro assay for Nramp1 function, we introduced a wild-typeNramp1G169 cDNA into RAW 264.7 macrophages (which bear a homozygous mutant Nramp1D169 allele and thus are permissive to replication of specific intracellular parasites). Recombinant Nramp1 was expressed in a membranous compartment in RAW264.7 cells and was recruited to the membrane ofSalmonella typhimurium and Yersinia enterocolitica containing phagosomes. Evaluation of the antibacterial activity of RAW264.7 transfectants showed that expression of the recombinant Nramp1 protein abrogated intracellular replication of S. typhimurium. Studies with a replication-defectiveS. typhimurium mutant suggest that this occurs through an enhanced bacteriostatic activity. The effect of Nramp1 expression was specific, since (i) it was not seen in RAW264.7 transfectants overexpressing the closely related Nramp2 protein, and (ii) control RAW264.7 cells, Nramp1, and Nramp2 transfectants could all efficiently kill a temperature-sensitive, replication-defective mutant of S. typhimurium. Finally, increased antibacterial activity of the Nramp1 RAW264.7 transfectants was linked to increased phagosomal acidification, a distinguishing feature of primary macrophages expressing a wild-type Nramp1 allele. Together, these results indicate that transfection of Nramp1 cDNAs in the RAW264.7 macrophage cell line can be used as a direct assay to study both Nramp1 function and mechanism of action as well as to identify structure-function relationships in this protein.
36
Teresa Guereno, M., M. Rosario Silaf, A. Javier Bava, Ricardo Negroni, and Roberto A. Diez. "Decreased monocytic phagosomal acidification among chronic paracoccidioidomycosis patients. Herabgesetzte Ansauerung in Monozyten-Phagosomen bei Paracoccidioidomykose-Patienten." Mycoses 46, no. 9-10 (October 2003): 397–401. http://dx.doi.org/10.1046/j.0933-7407.2003.00916.x.
Full text
37
Sun-Wada, G. H., H. Tabata, N. Kawamura, M. Aoyama, and Y. Wada. "Direct recruitment of H+-ATPase from lysosomes for phagosomal acidification." Journal of Cell Science 122, no. 14 (June 23, 2009): 2504–13. http://dx.doi.org/10.1242/jcs.050443.
Full text
38
Haggie, Peter M., and A. S. Verkman. "Cystic Fibrosis Transmembrane Conductance Regulator-independent Phagosomal Acidification in Macrophages." Journal of Biological Chemistry 282, no. 43 (August 27, 2007): 31422–28. http://dx.doi.org/10.1074/jbc.m705296200.
Full text
39
Queval, Christophe J., Ok-Ryul Song, Jean-Philippe Carralot, Jean-Michel Saliou, Antonino Bongiovanni, Gaspard Deloison, Nathalie Deboosère, et al. "Mycobacterium tuberculosis Controls Phagosomal Acidification by Targeting CISH-Mediated Signaling." Cell Reports 20, no. 13 (September 2017): 3188–98. http://dx.doi.org/10.1016/j.celrep.2017.08.101.
Full text
40
Mishra, Richa, Sakshi Kohli, Nitish Malhotra, Parijat Bandyopadhyay, Mansi Mehta, MohamedHusen Munshi, Vasista Adiga, et al. "Targeting redox heterogeneity to counteract drug tolerance in replicating Mycobacterium tuberculosis." Science Translational Medicine 11, no. 518 (November 13, 2019): eaaw6635. http://dx.doi.org/10.1126/scitranslmed.aaw6635.
Full textAbstract:
The capacity of Mycobacterium tuberculosis (Mtb) to tolerate multiple antibiotics represents a major problem in tuberculosis (TB) management. Heterogeneity in Mtb populations is one of the factors that drives antibiotic tolerance during infection. However, the mechanisms underpinning this variation in bacterial population remain poorly understood. Here, we show that phagosomal acidification alters the redox physiology of Mtb to generate a population of replicating bacteria that display drug tolerance during infection. RNA sequencing of this redox-altered population revealed the involvement of iron-sulfur (Fe-S) cluster biogenesis, hydrogen sulfide (H2S) gas, and drug efflux pumps in antibiotic tolerance. The fraction of the pH- and redox-dependent tolerant population increased when Mtb infected macrophages with actively replicating HIV-1, suggesting that redox heterogeneity could contribute to high rates of TB therapy failure during HIV-TB coinfection. Pharmacological inhibition of phagosomal acidification by the antimalarial drug chloroquine (CQ) eradicated drug-tolerant Mtb, ameliorated lung pathology, and reduced postchemotherapeutic relapse in in vivo models. The pharmacological profile of CQ (Cmax and AUClast) exhibited no major drug-drug interaction when coadministered with first line anti-TB drugs in mice. Our data establish a link between phagosomal pH, redox metabolism, and drug tolerance in replicating Mtb and suggest repositioning of CQ to shorten TB therapy and achieve a relapse-free cure.
41
Bönquist, Linda, Helena Lindgren, Igor Golovliov, Tina Guina, and Anders Sjöstedt. "MglA and Igl Proteins Contribute to the Modulation of Francisella tularensis Live Vaccine Strain-Containing Phagosomes in Murine Macrophages." Infection and Immunity 76, no. 8 (May 12, 2008): 3502–10. http://dx.doi.org/10.1128/iai.00226-08.
Full textAbstract:
ABSTRACT The Francisella tularensis live vaccine strain (LVS), in contrast to its iglC mutant, replicates in the cytoplasm of macrophages. We studied the outcome of infection of the murine macrophagelike cell line J774A.1 with LVS and with iglC, iglD, and mglA mutants, the latter of which is deficient in a global regulator. Compared to LVS, all of the mutants showed impaired intracellular replication up to 72 h, and the number of the mglA mutant bacteria even decreased. Colocalization with LAMP-1 was significantly increased for all mutants compared to LVS, indicating an impaired ability to escape into the cytoplasm. A lysosomal acidity-dependent dye accumulated in approximately 40% of the vacuoles containing mutant bacteria but not at all in vacuoles containing LVS. Preactivation of the macrophages with gamma interferon inhibited the intracellular growth of all strains and significantly increased acidification of phagosomes containing the mutants, but it only slightly increased the LAMP-1 colocalization. The intracellular replication and phagosomal escape of the iglC and iglD mutants were restored by complementation in trans. In conclusion, the IglC, IglD, and MglA proteins each directly or indirectly critically contribute to the virulence of F. tularensis LVS, including its intracellular replication, cytoplasmic escape, and inhibition of acidification of the phagosomes.
42
Schneider, Boris, Roy Gross, and Albert Haas. "Phagosome Acidification Has Opposite Effects on Intracellular Survival of Bordetella pertussis andB. bronchiseptica." Infection and Immunity 68, no. 12 (December 1, 2000): 7039–48. http://dx.doi.org/10.1128/iai.68.12.7039-7048.2000.
Full textAbstract:
ABSTRACT Bordetella pertussis is readily killed after uptake by professional phagocytes, whereas its close relative Bordetella bronchiseptica is not and can persist intracellularly for days. Phagocytosis of members of either species by a mouse macrophage cell line results in transport of the bacteria to a phagosomal compartment positive for the lysosome-associated membrane protein 1, the protease cathepsin D, and the late endosomal vacuolar proton-pumping ATPase but negative for the early endosome antigen 1 and the early endosomal transferrin receptor. In addition, we demonstrate thatBordetella-containing phagosomes rapidly acidify to pH 4.5 to 5.0. Taken together, these data demonstrate thatBordetella-containing phagosomes rapidly mature to an acidic late endosomal/lysosomal compartment. Following up on this observation, we determined that B. pertussis does not survive in bacterial growth media adjusted to a pH of 4.5, whereas this pH has only minor effects on the growth of B. bronchiseptica. Raising the intracellular pH in infected macrophages by the addition of bafilomycin A1, ammonium chloride, or monensin increases the survival of acid-sensitive B. pertussis but, surprisingly, decreases that of acid-tolerantB. bronchiseptica. In summary, we hypothesize that the differential survival of B. pertussis and B. bronchiseptica in macrophages is, at least in part, due to the differences in their acid tolerance.
43
Martínez, Alejandra, Carolina Prolo, Damián Estrada, Natalia Rios, María Noel Alvarez, María Dolores Piñeyro, Carlos Robello, Rafael Radi, and Lucía Piacenza. "Cytosolic Fe-superoxide dismutase safeguardsTrypanosoma cruzifrom macrophage-derived superoxide radical." Proceedings of the National Academy of Sciences 116, no. 18 (April 12, 2019): 8879–88. http://dx.doi.org/10.1073/pnas.1821487116.
Full textAbstract:
Trypanosoma cruzi, the causative agent of Chagas disease (CD), contains exclusively Fe-dependent superoxide dismutases (Fe-SODs). DuringT. cruziinvasion to macrophages, superoxide radical (O2•−) is produced at the phagosomal compartment toward the internalized parasite via NOX-2 (gp91-phox) activation. In this work,T. cruzicytosolic Fe-SODB overexpressers (pRIBOTEX–Fe-SODB) exhibited higher resistance to macrophage-dependent killing and enhanced intracellular proliferation compared with wild-type (WT) parasites. The higher infectivity of Fe-SODB overexpressers compared with WT parasites was lost in gp91-phox−/−macrophages, underscoring the role of O2•−in parasite killing. Herein, we studied the entrance of O2•−and its protonated form, perhydroxyl radical [(HO2•); pKa= 4.8], toT. cruziat the phagosome compartment. At the acidic pH values of the phagosome lumen (pH 5.3 ± 0.1), high steady-state concentrations of O2•−and HO2•were estimated (∼28 and 8 µM, respectively). Phagosomal acidification was crucial for O2•−permeation, because inhibition of the macrophage H+-ATPase proton pump significantly decreased O2•−detection in the internalized parasite. Importantly, O2•−detection, aconitase inactivation, and peroxynitrite generation were lower in Fe-SODB than in WT parasites exposed to external fluxes of O2•−or during macrophage infections. Other mechanisms of O2•−entrance participate at neutral pH values, because the anion channel inhibitor 5-nitro-2-(3-phenylpropylamino) benzoic acid decreased O2•−detection. Finally, parasitemia and tissue parasite burden in mice were higher in Fe-SODB–overexpressing parasites, supporting the role of the cytosolic O2•−-catabolizing enzyme as a virulence factor for CD.
44
Porte, Françoise, Jean-Pierre Liautard, and Stephan Köhler. "Early Acidification of Phagosomes ContainingBrucella suis Is Essential for Intracellular Survival in Murine Macrophages." Infection and Immunity 67, no. 8 (August 1, 1999): 4041–47. http://dx.doi.org/10.1128/iai.67.8.4041-4047.1999.
Full textAbstract:
ABSTRACT Brucella suis is a facultative intracellular pathogen of mammals, residing in macrophage vacuoles. In this work, we studied the phagosomal environment of these bacteria in order to better understand the mechanisms allowing survival and multiplication ofB. suis. Intraphagosomal pH in murine J774 cells was determined by measuring the fluorescence intensity of opsonized, carboxyfluorescein-rhodamine- and Oregon Green 488-rhodamine-labeled bacteria. Compartments containing live B. suis acidified to a pH of about 4.0 to 4.5 within 60 min. Acidification of B. suis-containing phagosomes in the early phase of infection was abolished by treatment of host cells with 100 nM bafilomycin A1, a specific inhibitor of vacuolar proton-ATPases. This neutralization at 1 h postinfection resulted in a 2- to 34-fold reduction of opsonized and nonopsonized viable intracellular bacteria at 4 and 6 h postinfection, respectively. Ammonium chloride and monensin, other pH-neutralizing reagents, led to comparable loss of intracellular viability. Addition of ammonium chloride at 7 h after the beginning of infection, however, did not affect intracellular multiplication of B. suis, in contrast to treatment at 1 h postinfection, where bacteria were completely eradicated within 48 h. Thus, we conclude that phagosomes with B. suis acidify rapidly after infection, and that this early acidification is essential for replication of the bacteria within the macrophage.
45
Via, L. E., R. A. Fratti, M. McFalone, E. Pagan-Ramos, D. Deretic, and V. Deretic. "Effects of cytokines on mycobacterial phagosome maturation." Journal of Cell Science 111, no. 7 (April 1, 1998): 897–905. http://dx.doi.org/10.1242/jcs.111.7.897.
Full textAbstract:
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.
46
Rabani, Razieh, Allen Volchuk, Mirjana Jerkic, Lindsay Ormesher, Linda Garces-Ramirez, Johnathan Canton, Claire Masterson, et al. "Mesenchymal stem cells enhance NOX2-dependent reactive oxygen species production and bacterial killing in macrophages during sepsis." European Respiratory Journal 51, no. 4 (March 8, 2018): 1702021. http://dx.doi.org/10.1183/13993003.02021-2017.
Full textAbstract:
Human mesenchymal stem/stromal cells (MSCs) have been reported to produce an M2-like, alternatively activated phenotype in macrophages. In addition, MSCs mediate effective bacterial clearance in pre-clinical sepsis models. Thus, MSCs have a paradoxical antimicrobial and anti-inflammatory response that is not understood.Here, we studied the phenotypic and functional response of monocyte-derived human macrophages to MSC exposure in vitro.MSCs induced two distinct, coexistent phenotypes: M2-like macrophages (generally elongated morphology, CD163+, acute phagosomal acidification, low NOX2 expression and limited phagosomal superoxide production) and M1-like macrophages characterised by high levels of phagosomal superoxide production. Enhanced phagosomal reactive oxygen species production was also observed in alveolar macrophages from a rodent model of pneumonia-induced sepsis. The production of M1-like macrophages was dependent on prostaglandin E2 and phosphatidylinositol 3-kinase. MSCs enhanced human macrophage phagocytosis of unopsonised bacteria and enhanced bacterial killing compared with untreated macrophages. Bacterial killing was significantly reduced by blockade of NOX2 using diphenyleneiodonium, suggesting that M1-like cells are primarily responsible for this effect. MSCs also enhanced phagocytosis and polarisation of M1-like macrophages derived from patients with severe sepsis.The enhanced antimicrobial capacity (M1-like) and inflammation resolving phenotype (M2-like) may account for the paradoxical effect of these cells in sepsis in vivo.
47
Toyooka, Kiminori, Shinji Takai, and Teruo Kirikae. "Rhodococcus equi can survive a phagolysosomal environment in macrophages by suppressing acidification of the phagolysosome." Journal of Medical Microbiology 54, no. 11 (November 1, 2005): 1007–15. http://dx.doi.org/10.1099/jmm.0.46086-0.
Full textAbstract:
Rhodococcus equi is one of the most important causes of pneumonia in foals and has emerged as a significant opportunistic pathogen of immunosuppressed hosts such as human immunodeficiency virus-infected patients. Virulent R. equi harbouring an 85 kb plasmid, but not the avirulent form lacking the plasmid, has the ability to survive in macrophages. However, the survival mechanism is not known. In the present study, morphological interactions were observed between virulent or plasmid-cured avirulent R. equi and phagolysosomes in murine macrophage-like J774.1 cells by immunocytological methods. The J774.1 cells phagocytosed virulent and avirulent bacteria to a similar extent, and both bacteria replicated in single membrane vacuoles at similar rates up to 6 h after infection. Thereafter, the virulent bacteria continued to grow, whereas the avirulent bacteria stopped growing. When the infected cells were stained with phagosomal and lysosomal markers and observed with a confocal fluorescence microscope, the majority of phagosomes containing these bacteria were fused with lysosomes. Neither R. equi organism has the ability to hinder phagosome-lysosome fusion. The acidity in phagolysosomes containing R. equi was examined by staining with LysoTracker Red DND-99, an acidotropic probe. The phagolysosomes containing virulent organisms were not acidic as compared with avirulent organisms. Over 90 % of the phagolysosomes containing avirulent R. equi were stained with LysoTracker 6 h after infection, whereas less than 50 % of those containing virulent R. equi were stained. Furthermore, when the supernatant obtained from a virulent R. equi culture was added to the cell cultures, the acidity of acidic compartments in macrophages was reduced. The authors conclude that some substance(s) produced by virulent R. equi suppress acidification in phagolysosomes, and help R. equi survival and replication in the bactericidal environment.
48
Kailasan Vanaja, Sivapriya, Vijay Rathinam, Parisa Kalantari, Katherine Fitzgerald, and John Leong. "Essential role of NLRP3 and AIM2 inflammasomes in IL-1β production induced by the extracellular pathogen, enterohemorrhagic Escherichia coli (157.1)." Journal of Immunology 186, no. 1_Supplement (April 1, 2011): 157.1. http://dx.doi.org/10.4049/jimmunol.186.supp.157.1.
Full textAbstract:
Abstract Enterohemorrhagic Escherichia coli (EHEC) induces elevated levels of IL-1β in infected patients. However, the molecular mechanisms involved in the activation of IL-1β by EHEC are unknown. Inflammasomes are multiprotein complexes in the cytosol that process proIL-1β to bioactive IL-1β. To identify the inflammasomes involved in IL-1β induction by EHEC, we infected macrophages derived from wild type and mice deficient in inflammasome components with EHEC and analyzed the processing and release of mature IL-1β. EHEC-induced IL-1β production was dependent on caspase-1 and the adaptor ASC. Notably, both NLRP3, a cytosolic sensor for microbial and endogenous products, and AIM2, a cytosolic sensor for DNA, were absolutely essential for EHEC-induced IL-1β production. Though EHEC is a non-cytosolic pathogen, bacterial products such as DNA were observed in the cytosol of infected cells, providing a potential means by which cytosolic inflammasomes might be activated. This also indicated that phagosomal degradation of bacteria is a prerequisite for inflammasome activation by EHEC. Supporting this hypothesis, blocking lysosomal acidification and type I interferon signaling that are essential for bacterial lysis in phagosome, led to a marked decrease in EHEC-induced IL-1β secretion. Thus, our results demonstrate that EHEC potently activates multiple inflammasomes as a result of release of bacterial products from phagosomes into the cytosol.
49
Peña-Ramos, Omar, and Zheng Zhou. "Measuring the acidification of the phagosomal lumen in live C. elegans embryos." STAR Protocols 4, no. 2 (June 2023): 102332. http://dx.doi.org/10.1016/j.xpro.2023.102332.
Full text
50
Barriere, Herve, Miklos Bagdany, Florian Bossard, Tsukasa Okiyoneda, Gabriella Wojewodka, Dieter Gruenert, Danuta Radzioch, and Gergely L. Lukacs. "Revisiting the Role of Cystic Fibrosis Transmembrane Conductance Regulator and Counterion Permeability in the pH Regulation of Endocytic Organelles." Molecular Biology of the Cell 20, no. 13 (July 2009): 3125–41. http://dx.doi.org/10.1091/mbc.e09-01-0061.
Full textAbstract:
Organellar acidification by the electrogenic vacuolar proton-ATPase is coupled to anion uptake and cation efflux to preserve electroneutrality. The defective organellar pH regulation, caused by impaired counterion conductance of the mutant cystic fibrosis transmembrane conductance regulator (CFTR), remains highly controversial in epithelia and macrophages. Restricting the pH-sensitive probe to CFTR-containing vesicles, the counterion and proton permeability, and the luminal pH of endosomes were measured in various cells, including genetically matched CF and non-CF human respiratory epithelia, as well as cftr+/+and cftr−/−mouse alveolar macrophages. Passive proton and relative counterion permeabilities, determinants of endosomal, lysosomal, and phagosomal pH-regulation, were probed with FITC-conjugated transferrin, dextran, and Pseudomonas aeruginosa, respectively. Although CFTR function could be documented in recycling endosomes and immature phagosomes, neither channel activation nor inhibition influenced the pH in any of these organelles. CFTR heterologous overexpression also failed to alter endocytic organellar pH. We propose that the relatively large CFTR-independent counterion and small passive proton permeability ensure efficient shunting of the proton-ATPase–generated membrane potential. These results have implications in the regulation of organelle acidification in general and demonstrate that perturbations of the endolysosomal organelles pH homeostasis cannot be linked to the etiology of the CF lung disease.