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Autophagy 2018M. tuberculosis causes an enormous worldwide burden of disease. Its success depends upon subverting the antimicrobial capacity of macrophages. We have known for decades...
M. tuberculosis causes an enormous worldwide burden of disease. Its success depends upon subverting the antimicrobial capacity of macrophages. We have known for decades that M. tuberculosis impairs phagosomal trafficking to avoid lysosomal degradation, but the mechanism is unclear. Recent work has described a phagolysosomal pathway called LC3-associated phagocytosis (LAP), in which LC3 associates with microbe-containing phagosomes. Macrophage pathogen recognition receptors (PRRs) initiate LAP, and NADPH oxidase and RUBCN/RUBICON are required for LAP. We discovered that CpsA, an exported M. tuberculosis virulence factor, blocks LAP by interfering with recruitment of CYBB/NOX2 (cytochrome b-245, beta polypeptide) to the mycobacterial phagosome. In macrophages and in mice, M. tuberculosis mutants lacking cpsA are successfully cleared by NADPH oxidase and the ensuing LC3-associated lysosomal trafficking pathway. CpsA belongs to the LytR-CpsA-Psr family, which is found widely in Gram-positive bacilli. This family is known for its enzymatic role in cell wall assembly. However, our data suggest that CpsA inhibits CYBB oxidase independently of a cell wall function. Thus, CpsA may have evolved from an enzyme involved in cell wall integrity to an indispensable virulence factor that M. tuberculosis uses to evade the innate immune response.
Topics: Animals; Autophagy; Bacterial Proteins; Host-Pathogen Interactions; Intracellular Signaling Peptides and Proteins; Macrophages; Mice; Mycobacterium tuberculosis; Phagocytosis; Phagosomes
PubMed: 29313424
DOI: 10.1080/15548627.2018.1425054 -
The Pediatric Infectious Disease Journal Nov 2010Autophagy is a well-known survival mechanism of the cell. Autophagosomes remove excessive proteins and thereby maintain homeostasis within the cell. Autophagy is now... (Review)
Review
Autophagy is a well-known survival mechanism of the cell. Autophagosomes remove excessive proteins and thereby maintain homeostasis within the cell. Autophagy is now recognized as a component of both innate and adaptive immune responses to bacterial and viral pathogens common to children. These pathogens include Streptococcus, tuberculosis, as well as hepatitis and herpes viruses. Varicella-zoster virus infection provides an excellent example of autophagy in humans, because abundant autophagosomes are easily detected in the skin vesicles of both varicella and zoster. Engineered herpes viruses, which elicit autophagy responses, are being used currently in clinical therapeutic trials against brain cancer. Furthermore, defective autophagy of bacteria may explain in part the pathogenesis of Crohn disease. However, at present, there is no single screening diagnostic assay by which to measure autophagy, as a means to investigate an etiologic role in children with an as yet undefined immunodeficiency. Instead, translational researchers are measuring individual components of the cellular autophagy pathway in both humans and animal models, to correlate autophagy responses with severity of infection. Autophagy certainly will remain a subject of immunology investigations in children in the coming decade.
Topics: Animals; Autophagy; Bacterial Infections; Child; Host-Pathogen Interactions; Humans; Phagosomes; Virus Diseases
PubMed: 20562672
DOI: 10.1097/INF.0b013e3181e77f43 -
The Journal of Clinical Investigation Jan 2015Life and health span can be prolonged by calorie limitation or by pharmacologic agents that mimic the effects of caloric restriction. Both starvation and the genetic... (Review)
Review
Life and health span can be prolonged by calorie limitation or by pharmacologic agents that mimic the effects of caloric restriction. Both starvation and the genetic inactivation of nutrient signaling converge on the induction of autophagy, a cytoplasmic recycling process that counteracts the age-associated accumulation of damaged organelles and proteins as it improves the metabolic fitness of cells. Here we review experimental findings indicating that inhibition of the major nutrient and growth-related signaling pathways as well as the upregulation of anti-aging pathways mediate life span extension via the induction of autophagy. Furthermore, we discuss mounting evidence suggesting that autophagy is not only necessary but, at least in some cases, also sufficient for increasing longevity.
Topics: Acetylation; Animals; Autophagy; Humans; Longevity; Paracrine Communication; Phagosomes; Protein Processing, Post-Translational
PubMed: 25654554
DOI: 10.1172/JCI73946 -
FEBS Letters Jun 2013Autophagy is a highly conserved intracytoplasmic degradation pathway for proteins, oligomers, organelles and pathogens. It initiates with the formation of a cup-shaped... (Review)
Review
Autophagy is a highly conserved intracytoplasmic degradation pathway for proteins, oligomers, organelles and pathogens. It initiates with the formation of a cup-shaped double membrane structure called the phagophore. The membrane origin for autophagosomes has been a key question for the field. ATG9 and ATG16L1, or their yeast orthologues, are key proteins that regulate autophagosome biogenesis, and may be associated with distinct membrane sources. Here we review the biology of autophagy with a focus on ATG16L1 and ATG9, and we summarise the current knowledge of their trafficking in relation to autophagic stimuli and autophagosome formation.
Topics: Animals; Autophagy; Autophagy-Related Proteins; Carrier Proteins; Humans; Membrane Proteins; Phagosomes; Protein Transport; Vesicular Transport Proteins
PubMed: 23669359
DOI: 10.1016/j.febslet.2013.04.025 -
Nature Communications Oct 2018Selective types of autophagy mediate the clearance of specific cellular components and are essential to maintain cellular homeostasis. However, tools to directly induce...
Selective types of autophagy mediate the clearance of specific cellular components and are essential to maintain cellular homeostasis. However, tools to directly induce and monitor such pathways are limited. Here we introduce the PIM (particles induced by multimerization) assay as a tool for the study of aggrephagy, the autophagic clearance of aggregates. The assay uses an inducible multimerization module to assemble protein clusters, which upon induction recruit ubiquitin, p62, and LC3 before being delivered to lysosomes. Moreover, use of a dual fluorescent tag allows for the direct observation of cluster delivery to the lysosome. Using flow cytometry and fluorescence microscopy, we show that delivery to the lysosome is partially dependent on p62 and ATG7. This assay will help in elucidating the spatiotemporal dynamics and control mechanisms underlying aggregate clearance by the autophagy-lysosomal system.
Topics: Autophagy; Flow Cytometry; Fluorescence Recovery After Photobleaching; HEK293 Cells; HeLa Cells; Humans; Lysosomes; Microscopy, Fluorescence; Phagosomes; Protein Aggregates; Ubiquitin
PubMed: 30315152
DOI: 10.1038/s41467-018-06674-4 -
Investigative Ophthalmology & Visual... Feb 2017The eye is an immune-privileged microenvironment that has adapted several mechanisms of immune regulation to prevent inflammation. One of these potential mechanisms is...
PURPOSE
The eye is an immune-privileged microenvironment that has adapted several mechanisms of immune regulation to prevent inflammation. One of these potential mechanisms is retinal pigment epithelial cells (RPE) altering phagocytosis in macrophages.
METHODS
The conditioned media of RPE eyecups from eyes of healthy mice and mice with experimental autoimmune uveitis (EAU) were used to treat primary macrophage phagocytizing pHrodo bacterial bioparticles. In addition, the neuropeptides were depleted from the conditioned media of healthy RPE eyecups and used to treat phagocytizing macrophages. The conditioned media from healthy and EAU RPE eyecups were assayed for IL-6, and IL-6 was added to the healthy conditioned media, and neutralized in the EAU conditioned media. The macrophages were treated with the conditioned media and assayed for fluorescence. The macrophages were imaged, and the fluorescence intensity, relative to active phagolysosomes, was measured. Also, the macrophages were assayed using fluorescent viability dye staining.
RESULTS
The conditioned media from healthy, but not from EAU RPE eyecups suppressed phagolysosome activation. Depletion of the neuropeptides alpha-melanocyte-stimulating hormone and neuropeptide Y from the healthy RPE eyecup conditioned media resulted in macrophage death. In the EAU RPE eyecup conditioned media was 0.96 ± 0.18 ng/mL of IL-6, and when neutralized the conditioned media suppressed phagolysosome activation.
CONCLUSIONS
The healthy RPE through soluble molecules, including alpha-melanocyte-stimulating hormone and neuropeptide Y, suppresses the activation of the phagolysosome in macrophages. In EAU, the IL-6 produced by the RPE promotes the activation of phagolysosomes in macrophages. These results demonstrate that under healthy conditions, RPE promotes an altered pathway of phagocytized material in macrophages with implications on antigen processing and clearance.
Topics: Animals; Cells, Cultured; Culture Media, Conditioned; Disease Models, Animal; Epithelial Cells; Interleukin-6; Macrophage Activation; Macrophages; Mice; Phagosomes; Retinal Pigment Epithelium; Uveitis
PubMed: 28241314
DOI: 10.1167/iovs.16-21082 -
Traffic (Copenhagen, Denmark) Nov 2013The production of reactive oxygen species (ROS) within immune cell phagosomes is critical for antimicrobial activity and for correct antigen processing, and influences... (Review)
Review
The production of reactive oxygen species (ROS) within immune cell phagosomes is critical for antimicrobial activity and for correct antigen processing, and influences signaling pathways that direct host responses to infection and inflammation. Because excess oxidants can cause tissue damage and oxidative stress, phagocytes must precisely control both the location and timing of NADPH oxidase activity. How differential regulation is achieved at phagosomes is not well understood. Recent studies have revealed that the PI(3)P phosphoinositide plays an important role in locally boosting phagosomal NADPH oxidase activity through its binding to the p40(phox) NADPH oxidase subunit. Furthermore, phox subunit dynamics at phagosomes may regulate the timing of the oxidative burst. Novel elements regulating catalytic core trafficking include Rab27 and SNAP-23. In addition to trafficking events, the activity of the electrogenic oxidase is also governed by ionic fluxes, which are constrained at phagosomes owing to low intraphagosomal volume and dynamic display of channels, transporters, and pumps. New insights on the interdependence of phagosomal pH and ROS have been recently elucidated, and chloride channels important for microbicidal functions, including CFTR, and CLIC family channels, have been identified. Finally, periphagosomal calcium microdomains and calcium-dependent S100A8/9 protein recruitment may help fine-tune spatiotemporal regulation of NADPH oxidase activation for an effective immune response.
Topics: Animals; Humans; Ion Transport; NADPH Oxidases; Phagocytosis; Phagosomes; Protein Transport; Reactive Oxygen Species
PubMed: 23980663
DOI: 10.1111/tra.12115 -
FEBS Letters Jun 2015Despite the availability of a large pool of experimental approaches and hypothetical considerations, the hunt for the enigmatic membrane origin of autophagosomes is... (Review)
Review
Despite the availability of a large pool of experimental approaches and hypothetical considerations, the hunt for the enigmatic membrane origin of autophagosomes is still on. In mammalian cells proposed scenarios for the formation of the autophagosomal membrane include both de novo assembly, and rearrangements plus maturation of pre-existing membrane sections from the endoplasmic reticulum (ER), plasma membrane, Golgi or mitochondria. Earlier, we identified the human WD-repeat protein interacting with phosphoinositides (WIPI) family and showed that WIPI proteins function as essential phosphatidylinositol 3-phosphate (PtdIns3P) effectors at the nascent autophagosome. Interestingly, WIPI proteins localize to both pre-existing endomembranes and nascent autophagosomes. In this context, and on the basis of historical records on the formation of autophagosomes, we discuss with appropriate modesty an alternative perspective on the membrane origin of autophagosomes.
Topics: Autophagy; Humans; Intracellular Membranes; Membrane Proteins; Phagosomes
PubMed: 25980605
DOI: 10.1016/j.febslet.2015.05.008 -
BioFactors (Oxford, England) 2014Macrophages are among the first cellular actors facing the invasion of microorganisms. These cells are able to internalize pathogens and destroy them by means of toxic... (Review)
Review
Macrophages are among the first cellular actors facing the invasion of microorganisms. These cells are able to internalize pathogens and destroy them by means of toxic mediators, many of which are produced enzymatically and have strong oxidizing capacity. Indeed, macrophages count on the NADPH oxidase complex activity, which is triggered during pathogen invasion and leads to the production of superoxide radical inside the phagosome. At the same time, the induction of nitric oxide synthase results in the production of nitric oxide in the cytosol which is able to readily diffuse to the phagocytic vacuole. Superoxide radical and nitric oxide react at diffusion controlled rates with each other inside the phagosome to yield peroxynitrite, a powerful oxidant capable to kill micro-organisms. Peroxynitrite toxicity resides on oxidations and nitrations of biomolecules in the target cell. The central role of peroxynitrite as a key effector molecule in the control of infections has been proven in a wide number of models. However, some microorganisms and virulent strains adapt to survive inside the potentially hostile oxidizing microenvironment of the phagosome by either impeding peroxynitrite formation or rapidly detoxifying it once formed. In this context, the outcome of the infection process is a result of the interplay between the macrophage-derived oxidizing cytotoxins such as peroxynitrite and the antioxidant defense machinery of the invading pathogens.
Topics: Animals; Host-Pathogen Interactions; Humans; Immunity, Innate; Macrophages; Oxidation-Reduction; Peroxynitrous Acid; Phagocytosis; Phagosomes
PubMed: 24281946
DOI: 10.1002/biof.1150 -
Traffic (Copenhagen, Denmark) Sep 2003The ability of Mycobacterium tuberculosis to enter host macrophages, and reside in a phagosome, which does not mature into a phagolysosome, is central to the spread of... (Review)
Review
The ability of Mycobacterium tuberculosis to enter host macrophages, and reside in a phagosome, which does not mature into a phagolysosome, is central to the spread of tuberculosis and the associated pandemic involving billions of people worldwide. Tuberculosis can be viewed as a disease with a significant intracellular trafficking and organellar biogenesis component. Current understanding of the block in M. tuberculosis phagosome maturation also sheds light on fundamental aspects of phagolysosome biogenesis. The maturation block involves interference with the recruitment and function of rabs, rab effectors (phosphatidylinositol 3-kinases and tethering molecules such as EEA1), SNAREs (Syntaxin 6 and cellubrevin) and Ca2+/calmodulin signaling. M. tuberculosis analogs of mammalian phosphatidylinositols interfere with these systems and associated processes.
Topics: Animals; Biological Transport; Biomarkers; Calcium; Calmodulin; Cell Membrane; Humans; Mycobacterium tuberculosis; Phagosomes; Phosphatidylinositol Phosphates
PubMed: 12911814
DOI: 10.1034/j.1600-0854.2003.00120.x