scholarly journals ESCRT and autophagy cooperate to repair ESX-1-dependent damage to the Mycobacterium-containing vacuole

2018 ◽  
Author(s):  
Ana T. López-Jiménez ◽  
Elena Cardenal-Muñoz ◽  
Florence Leuba ◽  
Lilli Gerstenmaier ◽  
Monica Hagedorn ◽  
...  
Keyword(s):  
Dictyostelium Discoideum ◽  
Membrane Damage ◽  
Mycobacterium Marinum ◽  
Eukaryotic Cells ◽  
Intracellular Pathogens ◽  
Membrane Repair ◽  
Endosomal Sorting

AbstractPhagocytes capture invader microbes within the bactericidal phagosome. Some pathogens subvert killing by damaging and escaping from this compartment. To prevent and fight bacterial escape, cells contain and repair the membrane damage, or finally eliminate the cytosolic escapees. All eukaryotic cells engage highly conserved mechanisms to ensure integrity of membranes in a multitude of physiological and pathological situations, including the Endosomal Sorting Complex Required for Transport (ESCRT) and autophagy machineries. In Dictyostelium discoideum, recruitment of the ESCRT-III protein Snf7/Chmp4/Vps32 and the ATPase Vps4 to sites of membrane repair relies on the ESCRT-I component Tsg101 and occurs in absence of Ca2+. The ESX-1 dependent membrane perforations produced by the pathogen Mycobacterium marinum separately engage both ESCRT and autophagy. In absence of Tsg101, M. marinum escapes earlier to the cytosol, where it is restricted by xenophagy. We propose that ESCRT has an evolutionary conserved function in containing intracellular pathogens in intact compartments.

scholarly journals The Dictyostelium discoideum E3 ubiquitin ligase TrafE coordinates endolysosomal damage response and cell-autonomous immunity to Mycobacterium marinum.

2021 ◽  
Author(s):  
Lyudmil Raykov ◽  
Manon Mottet ◽  
Jahn Nitschke ◽  
Thierry Soldati
Keyword(s):  
Cell Death ◽  
Dictyostelium Discoideum ◽  
Cellular Response ◽  
Membrane Damage ◽  
Mycobacterium Marinum ◽  
Chemical Components ◽  
Key Factors ◽  
Endosomal Sorting ◽  
Damage Response ◽  
Intracellular Compartments

Cells are perpetually challenged by pathogens, protein aggregates or chemicals, that induce plasma membrane or endolysosomal compartments damage. Endolysosomal perforations are recognised as severe stress, however the mechanisms of the cellular response that ensure quality control, repair and endolysosomal homeostasis are just beginning to be unravelled. The endosomal sorting complex required for transport (ESCRT) and the autophagy machinery are recruited to damaged membranes to either repair or to remove membrane remnants. Crucial element of the endolysosomal damage response (ELDR) are factors that sense damage, paralleled by extensive tagging of the damaged organelles with signals, such as ubiquitin, required for the recruitment of ELDR components. Unattended membrane damage leads to leakage of harmful components including protons and reactive oxygen species that cause cell death. To explore ELDR key factors responsible for detection and marking of damaged compartments we use the professional phagocyte Dictyostelium discoideum. We found an evolutionary conserved E3-ligase TrafE that is robustly recruited to intracellular compartments disrupted after infection with Mycobacterium marinum or after sterile damage caused by chemical components. Importantly, we show that the absence of TrafE severely compromises the xenophagy restriction of bacteria as well as autophagy-mediated and ESCRT-mediated ELDR, resulting in early cell death.

scholarly journals Mycobacterium tuberculosisType VII Secretion System Effectors Differentially Impact the ESCRT Endomembrane Damage Response

mBio ◽  
2018 ◽  
Vol 9 (6) ◽  
Author(s):  
Ekansh Mittal ◽  
Michael L. Skowyra ◽  
Grace Uwase ◽  
Emir Tinaztepe ◽  
Alka Mehra ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Recent Work ◽  
Host Response ◽  
Membrane Damage ◽  
Secretion System ◽  
Intracellular Pathogens ◽  
Dependent Manner ◽  
Membrane Repair ◽  
Content Type ◽  
Endosomal Sorting

ABSTRACTIntracellular pathogens have varied strategies to breach the endolysosomal barrier so that they can deliver effectors to the host cytosol, access nutrients, replicate in the cytoplasm, and avoid degradation in the lysosome. In the case ofMycobacterium tuberculosis, the bacterium perforates the phagosomal membrane shortly after being taken up by macrophages. Phagosomal damage depends upon the mycobacterial ESX-1 type VII secretion system (T7SS). Sterile insults, such as silica crystals or membranolytic peptides, can also disrupt phagosomal and endolysosomal membranes. Recent work revealed that the host endosomal sorting complex required for transport (ESCRT) machinery rapidly responds to sterile endolysosomal damage and promotes membrane repair. We hypothesized that ESCRTs might also respond to pathogen-induced phagosomal damage and thatM. tuberculosiscould impair this host response. Indeed, we found that ESCRT-III proteins were recruited toM. tuberculosisphagosomes in anESX-1-dependent manner. We previously demonstrated that the mycobacterial effectors EsxG/TB9.8 and EsxH/TB10.4, both secreted by the ESX-3 T7SS, can inhibit ESCRT-dependent trafficking of receptors to the lysosome. Here, we additionally show that ESCRT-III recruitment to sites of endolysosomal damage is antagonized by EsxG and EsxH, both within the context ofM. tuberculosisinfection and sterile injury. Moreover, EsxG and EsxH themselves respond within minutes to membrane damage in a manner that is independent of calcium and ESCRT-III recruitment. Thus, our study reveals that T7SS effectors and ESCRT participate in a series of measures and countermeasures for control of phagosome integrity.IMPORTANCEMycobacterium tuberculosiscauses tuberculosis, which kills more people than any other infection.M. tuberculosisgrows in macrophages, cells that specialize in engulfing and degrading microorganisms. Like many intracellular pathogens, in order to cause disease,M. tuberculosisdamages the membrane-bound compartment (phagosome) in which it is enclosed after macrophage uptake. Recent work showed that when chemicals damage this type of intracellular compartment, cells rapidly detect and repair the damage, using machinery called the endosomal sorting complex required for transport (ESCRT). Therefore, we hypothesized that ESCRT might also respond to pathogen-induced damage. At the same time, our previous work showed that the EsxG-EsxH heterodimer ofM. tuberculosiscan inhibit ESCRT, raising the possibility thatM. tuberculosisimpairs this host response. Here, we show that ESCRT is recruited to damagedM. tuberculosisphagosomes and that EsxG-EsxH undermines ESCRT-mediated endomembrane repair. Thus, our studies demonstrate a battle between host and pathogen over endomembrane integrity.

scholarly journals Disruption of vacuolin microdomains in the host Dictyostelium discoideum increases resistance to Mycobacterium marinum-induced membrane damage and infection

2021 ◽  
Author(s):  
Cristina Bosmani ◽  
Angélique Perret ◽  
Florence Leuba ◽  
Aurélie Guého ◽  
Nabil Hanna ◽  
...  
Keyword(s):  
Dictyostelium Discoideum ◽  
Membrane Damage ◽  
Mycobacterium Marinum ◽  
Vesicular Trafficking ◽  
Membrane Microdomains ◽  
Membrane Association ◽  
Phagosome Maturation ◽  
Closely Related Species ◽  
Important Host ◽  
And Function

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, manipulates the host phagosome maturation pathway to replicate intracellularly. Mycobacterium marinum, a closely-related species, and Dictyostelium discoideum, a social amoeba and alternative phagocytic host, have been used as models to study host-pathogen interactions occurring during mycobacterial infections. Vacuolins, functional homologues of the mammalian flotillins, organize membrane microdomains and play a role in vesicular trafficking. Various pathogens have been reported to manipulate their membrane association and function. During infection of D. discoideum with M. marinum, Vacuolin C was specifically and highly induced and all three vacuolin isoforms were enriched at the mycobacteria-containing-vacuole (MCV). In addition, absence of vacuolins reduced escape from the MCV and conferred resistance to M. marinum infection. Moreover, ESAT-6, the membrane-disrupting virulence factor of M. marinum, was less associated with membranes when vacuolins were absent. Together, these results suggest that vacuolins are important host factors that are manipulated by mycobacteria to inflict membrane damage and escape from their compartment.

scholarly journals Time-resolved RNA-seq profiling of the infection of Dictyostelium discoideum by Mycobacterium marinum reveals an integrated host response to damage and stress

2019 ◽  
Author(s):  
N. Hanna ◽  
F. Burdet ◽  
A. Melotti ◽  
C. Bosmani ◽  
S. Kicka ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dictyostelium Discoideum ◽  
Host Response ◽  
Mycobacterium Marinum ◽  
Infection Model ◽  
Multiple Drug ◽  
Late Time ◽  
Membrane Repair ◽  
Time Resolved ◽  
Recent Emergence

ABSTRACTTuberculosis remains the most pervasive infectious disease and the recent emergence of multiple drug-resistant strains emphasizes the need for more efficient drug treatments. The experimentally versatile Dictyostelium discoideum – Mycobacterium marinum infection model provides a powerful system to study mycobacteria pathogenicity and host response. In this study, a time-resolved transcriptomic analysis of the amoeba D. discoideum was performed to decipher the different host pathways impacted during infection. We investigated how D. discoideum fine-tunes its gene expression in response to M. marinum infection by assessing the transcriptomic profile covering the critical stages of entry, establishment of a permissive niche, proliferation and dissemination (1, 3, 6, 12, 24 and 48 hours post infection). Differential gene expression provided a fingerprint of the transcriptome of the host cell in the presence of mycobacteria, and helped identify specific markers and molecular signatures of infection. Enrichment pathway analysis showed that most of the Biological Processes (BP) of upregulated genes at early time point of infection hinted towards damage response and cellular defence, especially in specific pathways involved in membrane repair (ESCRT) and bacteria elimination (autophagy). Whereas at late time points of infection, BP related to starvation were upregulated. Some other signatures were more unexpected, such as cell cycle (downregulation of cytosolic large & small ribosomal subunits) and upregulation of metabolic adaptations (lipids transport).

scholarly journals A cryo–electron tomography workflow reveals protrusion-mediated shedding on injured plasma membrane

2021 ◽  
Vol 7 (13) ◽  
pp. eabc6345
Author(s):  
Shrawan Kumar Mageswaran ◽  
Wei Yuan Yang ◽  
Yogaditya Chakrabarty ◽  
Catherine M. Oikonomou ◽  
Grant J. Jensen
Keyword(s):  
Plasma Membrane ◽  
Molecular Mechanisms ◽  
Electron Tomography ◽  
Membrane Damage ◽  
Light And Electron Microscopy ◽  
Actin Dynamics ◽  
Endosomal Sorting ◽  
Plasma Membrane Damage ◽  
Cryo Electron Tomography ◽  
Vacuolar Protein

Cryo–electron tomography (cryo-ET) provides structural context to molecular mechanisms underlying biological processes. Although straightforward to implement for studying stable macromolecular complexes, using it to locate short-lived structures and events can be impractical. A combination of live-cell microscopy, correlative light and electron microscopy, and cryo-ET will alleviate this issue. We developed a workflow combining the three to study the ubiquitous and dynamic process of shedding in response to plasma membrane damage in HeLa cells. We found filopodia-like protrusions enriched at damage sites and acting as scaffolds for shedding, which involves F-actin dynamics, myosin-1a, and vacuolar protein sorting 4B (a component of the ‘endosomal sorting complex required for transport’ machinery). Overall, shedding is more complex than current models of vesiculation from flat membranes. Its similarities to constitutive shedding in enterocytes argue for a conserved mechanism. Our workflow can also be adapted to study other damage response pathways and dynamic cellular events.

scholarly journals Listeriolysin O: from bazooka to Swiss army knife

2017 ◽  
Vol 372 (1726) ◽  
pp. 20160222 ◽  
Author(s):  
Suzanne E. Osborne ◽  
John H. Brumell
Keyword(s):  
Systemic Disease ◽  
Membrane Damage ◽  
Host Cells ◽  
Listeriolysin O ◽  
Tissue Destruction ◽  
Membrane Repair ◽  
Membrane Pores ◽  
Bacterial Dissemination ◽  
Versatile Tool ◽  
Cell Spread

Listeria monocytogenes ( Lm ) is a Gram-positive facultative intracellular pathogen. Infections in humans can lead to listeriosis, a systemic disease with a high mortality rate. One important mechanism of Lm dissemination involves cell-to-cell spread after bacteria have entered the cytosol of host cells. Listeriolysin O (LLO; encoded by the hly gene) is a virulence factor present in Lm that plays a central role in the cell-to-cell spread process. LLO is a member of the cholesterol-dependent cytolysin (CDC) family of toxins that were initially thought to promote disease largely by inducing cell death and tissue destruction—essentially acting like a ‘bazooka’. This view was supported by structural studies showing CDCs can form large pores in membranes. However, it is now appreciated that LLO has many subtle activities during Lm infection of host cells, and many of these likely do not involve large pores, but rather small membrane perforations. It is also appreciated that membrane repair pathways of host cells play a major role in limiting membrane damage by LLO and other toxins. LLO is now thought to represent a ‘Swiss army knife’, a versatile tool that allows Lm to induce many membrane alterations and cellular responses that promote bacterial dissemination during infection. This article is part of the themed issue ‘Membrane pores: from structure and assembly, to medicine and technology’.

scholarly journals Investigation of the host transcriptional response to intracellular bacterial infection using Dictyostelium discoideum as a host model

BMC Genomics ◽  
2019 ◽  
Vol 20 (1) ◽  
Author(s):  
Jonas Kjellin ◽  
Maria Pränting ◽  
Frauke Bach ◽  
Roshan Vaid ◽  
Bart Edelbroek ◽  
...  
Keyword(s):  
Dictyostelium Discoideum ◽  
High Throughput ◽  
Legionella Pneumophila ◽  
Transcriptional Response ◽  
Infection Model ◽  
Intracellular Pathogens ◽  
Host Pathogen Interactions ◽  
Human Macrophages ◽  
Wide Range ◽  
Host Pathogen

Abstract Background During infection by intracellular pathogens, a highly complex interplay occurs between the infected cell trying to degrade the invader and the pathogen which actively manipulates the host cell to enable survival and proliferation. Many intracellular pathogens pose important threats to human health and major efforts have been undertaken to better understand the host-pathogen interactions that eventually determine the outcome of the infection. Over the last decades, the unicellular eukaryote Dictyostelium discoideum has become an established infection model, serving as a surrogate macrophage that can be infected with a wide range of intracellular pathogens. In this study, we use high-throughput RNA-sequencing to analyze the transcriptional response of D. discoideum when infected with Mycobacterium marinum and Legionella pneumophila. The results were compared to available data from human macrophages. Results The majority of the transcriptional regulation triggered by the two pathogens was found to be unique for each bacterial challenge. Hallmark transcriptional signatures were identified for each infection, e.g. induction of endosomal sorting complexes required for transport (ESCRT) and autophagy genes in response to M. marinum and inhibition of genes associated with the translation machinery and energy metabolism in response to L. pneumophila. However, a common response to the pathogenic bacteria was also identified, which was not induced by non-pathogenic food bacteria. Finally, comparison with available data sets of regulation in human monocyte derived macrophages shows that the elicited response in D. discoideum is in many aspects similar to what has been observed in human immune cells in response to Mycobacterium tuberculosis and L. pneumophila. Conclusions Our study presents high-throughput characterization of D. discoideum transcriptional response to intracellular pathogens using RNA-seq. We demonstrate that the transcriptional response is in essence distinct to each pathogen and that in many cases, the corresponding regulation is recapitulated in human macrophages after infection by mycobacteria and L. pneumophila. This indicates that host-pathogen interactions are evolutionary conserved, derived from the early interactions between free-living phagocytic cells and bacteria. Taken together, our results strengthen the use of D. discoideum as a general infection model.

scholarly journals Membrane Repair: Mechanisms and Pathophysiology

2015 ◽  
Vol 95 (4) ◽  
pp. 1205-1240 ◽  
Author(s):  
Sandra T. Cooper ◽  
Paul L. McNeil
Keyword(s):  
Molecular Mechanisms ◽  
Eukaryotic Cells ◽  
Membrane Repair ◽  
Membrane Pore ◽  
Rapid Repair ◽  
Repair Mechanisms ◽  
Molecular Machinery ◽  
Repair Pathways ◽  
Different Tissues ◽  
Ischemic Stress

Eukaryotic cells have been confronted throughout their evolution with potentially lethal plasma membrane injuries, including those caused by osmotic stress, by infection from bacterial toxins and parasites, and by mechanical and ischemic stress. The wounded cell can survive if a rapid repair response is mounted that restores boundary integrity. Calcium has been identified as the key trigger to activate an effective membrane repair response that utilizes exocytosis and endocytosis to repair a membrane tear, or remove a membrane pore. We here review what is known about the cellular and molecular mechanisms of membrane repair, with particular emphasis on the relevance of repair as it relates to disease pathologies. Collective evidence reveals membrane repair employs primitive yet robust molecular machinery, such as vesicle fusion and contractile rings, processes evolutionarily honed for simplicity and success. Yet to be fully understood is whether core membrane repair machinery exists in all cells, or whether evolutionary adaptation has resulted in multiple compensatory repair pathways that specialize in different tissues and cells within our body.

scholarly journals An Autocrine Proliferation Repressor Regulates Dictyostelium discoideum Proliferation and Chemorepulsion Using the G Protein-Coupled Receptor GrlH

mBio ◽  
2018 ◽  
Vol 9 (1) ◽  
Author(s):  
Yu Tang ◽  
Yuantai Wu ◽  
Sarah E. Herlihy ◽  
Francisco J. Brito-Aleman ◽  
Jose H. Ting ◽  
...  
Keyword(s):  
G Protein ◽  
Dictyostelium Discoideum ◽  
Protein A ◽  
G Protein Coupled Receptors ◽  
Eukaryotic Cells ◽  
Putative Receptor ◽  
Large Numbers ◽  
Eukaryotic Microbes ◽  
G Protein Coupled ◽  
Eukaryotic Microbe

ABSTRACT In eukaryotic microbes, little is known about signals that inhibit the proliferation of the cells that secrete the signal, and little is known about signals (chemorepellents) that cause cells to move away from the source of the signal. Autocrine proliferation repressor protein A (AprA) is a protein secreted by the eukaryotic microbe Dictyostelium discoideum. AprA is a chemorepellent for and inhibits the proliferation of D. discoideum. We previously found that cells sense AprA using G proteins, suggesting the existence of a G protein-coupled AprA receptor. To identify the AprA receptor, we screened mutants lacking putative G protein-coupled receptors. We found that, compared to the wild-type strain, cells lacking putative receptor GrlH (grlH¯ cells) show rapid proliferation, do not have large numbers of cells moving away from the edges of colonies, are insensitive to AprA-induced proliferation inhibition and chemorepulsion, and have decreased AprA binding. Expression of GrlH in grlH¯ cells (grlH¯/grlH OE ) rescues the phenotypes described above. These data indicate that AprA signaling may be mediated by GrlH in D. discoideum. IMPORTANCE Little is known about how eukaryotic cells can count themselves and thus regulate the size of a tissue or density of cells. In addition, little is known about how eukaryotic cells can sense a repellant signal and move away from the source of the repellant, for instance, to organize the movement of cells in a developing embryo or to move immune cells out of a tissue. In this study, we found that a eukaryotic microbe uses G protein-coupled receptors to mediate both cell density sensing and chemorepulsion.

scholarly journals Genome-Wide Transposon Mutagenesis Indicates that Mycobacterium marinum Customizes Its Virulence Mechanisms for Survival and Replication in Different Hosts

2015 ◽  
Vol 83 (5) ◽  
pp. 1778-1788 ◽  
Author(s):  
Eveline M. Weerdenburg ◽  
Abdallah M. Abdallah ◽  
Farania Rangkuti ◽  
Moataz Abd El Ghany ◽  
Thomas D. Otto ◽  
...  
Keyword(s):  
Insertion Site ◽  
Mycobacterium Marinum ◽  
Host Cells ◽  
Intracellular Pathogens ◽  
Virulence Determinants ◽  
Phagocytic Cells ◽  
General Hypothesis ◽  
Content Type ◽  
Genome Wide ◽  
A Genome

The interaction of environmental bacteria with unicellular eukaryotes is generally considered a major driving force for the evolution of intracellular pathogens, allowing them to survive and replicate in phagocytic cells of vertebrate hosts. To test this hypothesis on a genome-wide level, we determined for the intracellular pathogenMycobacterium marinumwhether it uses conserved strategies to exploit host cells from both protozoan and vertebrate origin. Using transposon-directed insertion site sequencing (TraDIS), we determined differences in genetic requirements for survival and replication in phagocytic cells of organisms from different kingdoms. In line with the general hypothesis, we identified a number of general virulence mechanisms, including the type VII protein secretion system ESX-1, biosynthesis of polyketide lipids, and utilization of sterols. However, we were also able to show thatM. marinumcontains an even larger set of host-specific virulence determinants, including proteins involved in the modification of surface glycolipids and, surprisingly, the auxiliary proteins of the ESX-1 system. Several of these factors were in fact counterproductive in other hosts. Therefore,M. marinumcontains different sets of virulence factors that are tailored for specific hosts. Our data imply that although amoebae could function as a training ground for intracellular pathogens, they do not fully prepare pathogens for crossing species barriers.

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