Search

Your search keyword '"European Project: 682809,EndoSubvert(2017)"' showing total 23 results
Start Over Author "European Project: 682809,EndoSubvert(2017)"
23 results on '"European Project: 682809,EndoSubvert(2017)"'

1. Actin subversion for productive Plasmodium hepatocyte invasion [Spotlight]

Author

Frischknecht, Freddy, Enninga, Jost, University of Heidelberg, Medical Faculty, Dynamique des Interactions Hôte-Pathogène - Dynamics of Host-Pathogen Interactions, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Our work is supported by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) – Projektnummer 240245660 – SFB 1129, SPP2225 and SPP2332 (F.F.), the ANR (program ‘PureMagRupture’ and ‘RabReprogram’, to J.E.) as well as the ERC (CoG ‘Endosubvert’ to J.E.). J.E. is member of the LabExes IBEID and Milieu Interieur., ANR-21-CE35-0007,PureMagRupture,Un flux de travail de la gMEP (Genetic magnetic-extraction-proteomics) pour comprendre le lien entre la rupture vacuolaire de Shigella et l'autophagie(2021), ANR-22-CE15-0042,RabReprogram,Reprogrammation localisée des Rab GTPases au niveau de la vacuole contenant Shigella pour déclencher la rupture vacuolaire par mécanotransduction.(2022), ANR-10-LABX-0062,IBEID,Integrative Biology of Emerging Infectious Diseases(2010), and European Project: 682809,EndoSubvert(2017)

Subjects
actin rearrangements, Plasmodium, bacterial pathogens, [SDV]Life Sciences [q-bio], host cell invasion
Abstract

International audience; Productive invasion of hepatocytes by Plasmodium sporozoites is a key step of infection. The parasites traverse hepatocytes before targeting one of them to form a parasitophorous vacuole for parasite expansion. Schepis et al. show the induction of membrane ruffling via host Rho GTPases by Plasmodium sporozoites facilitating productive invasion.

Published
2023
Full Text
View/download PDF

2. Modification of phosphoinositides by the Shigella effector IpgD during host cell infection

Author

Tran van Nhieu, Guy, Latour-Lambert, Patricia, Enninga, Jost, Institut de Biologie Intégrative de la Cellule (I2BC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Dynamique des Interactions Hôte-Pathogène - Dynamics of Host-Pathogen Interactions, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), JE and GTVN are funded by the Agence National pour la Recherche Projects 'PureMagRupture' (JE, GTVN) and 'RabReprogram' (JE). JE acknowledges support from the Institut Pasteur and the European Research Council (ERC-CoG 'Endosubvert'). JE is member of the LabEx IBEID and Milieu Interieur. GTVN is supported by the Inserm (Institut National de la Santé et de la Recherche Médicale) and the CNRS (Centre National de la Recherche Scientifique), ANR-21-CE35-0007,PureMagRupture,Un flux de travail de la gMEP (Genetic magnetic-extraction-proteomics) pour comprendre le lien entre la rupture vacuolaire de Shigella et l'autophagie(2021), ANR-10-LABX-0062,IBEID,Integrative Biology of Emerging Infectious Diseases(2010), ANR-10-LABX-0069,MILIEU INTERIEUR,GENETIC & ENVIRONMENTAL CONTROL OF IMMUNE PHENOTYPE VARIANCE: ESTABLISHING A PATH TOWARDS PERSONALIZED MEDICINE(2010), and European Project: 682809,EndoSubvert(2017)

Subjects
Microbiology (medical), [SDV]Life Sciences [q-bio], Immunology, Phosphatidylinositols, invasion, Microbiology, Phosphoric Monoester Hydrolases, dissemination, Shigella flexneri, phosphatase, Infectious Diseases, phosphatidyl-inositol polyphosphate, inflammation, Humans, IpgD, Shigella, phosphotransferase, Dysentery, Bacillary
Abstract

Shigella, the causative agent of bacillary dysentery, subvert cytoskeletal and trafficking processes to invade and replicate in epithelial cells using an arsenal of bacterial effectors translocated through a type III secretion system. Here, we review the various roles of the type III effector IpgD, initially characterized as phosphatidylinositol 4,5 bisphosphate (PI4,5P2) 4-phosphatase. By decreasing PI4,5P2 levels, IpgD triggers the disassembly of cortical actin filaments required for bacterial invasion and cell migration. PI5P produced by IpgD further stimulates signaling pathways regulating cell survival, macropinosome formation, endosomal trafficking and dampening of immune responses. Recently, IpgD was also found to exhibit phosphotransferase activity leading to PI3,4P2 synthesis adding a new flavor to this multipotent bacterial enzyme. The substrate of IpgD, PI4,5P2 is also the main substrate hydrolyzed by endogenous phospholipases C to produce inositoltriphosphate (InsP3), a major Ca2+ second messenger. Hence, beyond the repertoire of effects associated with the direct diversion of phoshoinositides, IpgD indirectly down-regulates InsP3-mediated Ca2+ release by limiting InsP3 production. Furthermore, IpgD controls the intracellular lifestyle of Shigella promoting Rab8/11 -dependent recruitment of the exocyst at macropinosomes to remove damaged vacuolar membrane remnants and promote bacterial cytosolic escape. IpgD thus emerges as a key bacterial effector for the remodeling of host cell membranes.

Published
2022
Full Text
View/download PDF

3. Fit to dwell in many places – The growing diversity of intracellular Salmonella niches

Author

Chak Hon Luk, Jost Enninga, Camila Valenzuela, Dynamique des Interactions Hôte-Pathogène - Dynamics of Host-Pathogen Interactions, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), The Francis Crick Institute [London], CHL is part of the Pasteur - Paris University (PPU) International PhD Program. This project has received funding from the European Union's Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement No 665807. CHL is a recipient of the Doctoral Fellowship from the Croucher Foundation and 4th year Doctoral Award from Fondation pour la Recherche Médicale. JE acknowledges the support from the European Union (ERC-CG-grant: EndoSubvert), and the ANR (program: HBPSensing and PureMagRupture). JE is part of the IBEID and the Milieu Interieur LabExes., ANR-17-CE15-0006,HBPsensing,Mécanisme de détection de l'heptose 1,7-bisphosphate lors d'infections bactériennes à Gram négatif(2017), ANR-21-CE35-0007,PureMagRupture,Un flux de travail de la gMEP (Genetic magnetic-extraction-proteomics) pour comprendre le lien entre la rupture vacuolaire de Shigella et l'autophagie(2021), ANR-10-LABX-0062,IBEID,Integrative Biology of Emerging Infectious Diseases(2010), European Project: 665807,H2020,H2020-MSCA-COFUND-2014,PASTEURDOC(2015), and European Project: 682809,EndoSubvert(2017)

Subjects
Microbiology (medical), host cell, Infectious Diseases, Salmonella, fibroblasts, [SDV]Life Sciences [q-bio], Immunology, host-pathogen interactions, Microbiology, epithelial cells, macrophages
Abstract

Salmonella enterica is capable of invading different host cell types including epithelial cells and M cells during local infection, and immune cells and fibroblasts during the subsequent systemic spread. The intracellular lifestyles of Salmonella inside different cell types are remarkable for their distinct residential niches, and their varying replication rates. To study this, researchers have employed different cell models, such as various epithelial cells, immune cells, and fibroblasts. In epithelial cells, S. Typhimurium dwells within modified endolysosomes or gains access to the host cytoplasm. In the cytoplasm, the pathogen is exposed to the host autophagy machinery or poised for rapid multiplication, whereas it grows at a slower rate or remains dormant within the endomembrane-bound compartments. The swift bimodal lifestyle is not observed in fibroblasts and immune cells, and it emerges that these cells handle intracellular S. Typhimurium through different clearance machineries. Moreover, in these cell types S. Typhimurium grows withing modified phagosomes of distinct functional composition by adopting targeted molecular countermeasures. The preference for one or the other intracellular niche and the diverse cell type-specific Salmonella lifestyles are determined by the complex interactions between a myriad of bacterial effectors and host factors. It is important to understand how this communication is differentially regulated dependent on the host cell type and on the distinct intracellular growth rate. To support the efforts in deciphering Salmonella invasion across the different infection models, we provide a systematic comparison of the findings yielded from cell culture models. We also outline the future directions towards a better understanding of these differential Salmonella intracellular lifestyles.

Published
2022
Full Text
View/download PDF

4. Time-Resolved Fluorescence Microscopy Screens on Host Protein Subversion During Bacterial Cell Invasion

Author

Lisa Sanchez, Yuen-Yan Chang, Nora Mellouk, Jost Enninga, Dynamique des Interactions Hôte-Pathogène - Dynamics of Host-Pathogen Interactions, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Université Paris Cité (UPCité), National Institute of Child Health and Human Development [Bethesda], National Institutes of Health, The DIHP unit is supported by an ERC grant (ERC-CoG-Endosubvert), and the ANR (StopBugEntry, HBPSensing), and it is part of the two LabExes IBEID and Milieu Interieur., ANR-15-CE15-0017,StopBugEntry,Identification des nouvelles molécules cellulaires cibles pour combattre les infections bactériennes(2015), ANR-17-CE15-0006,HBPsensing,Mécanisme de détection de l'heptose 1,7-bisphosphate lors d'infections bactériennes à Gram négatif(2017), ANR-10-LABX-0062,IBEID,Integrative Biology of Emerging Infectious Diseases(2010), ANR-10-LABX-0069,MILIEU INTERIEUR,GENETIC & ENVIRONMENTAL CONTROL OF IMMUNE PHENOTYPE VARIANCE: ESTABLISHING A PATH TOWARDS PERSONALIZED MEDICINE(2010), and European Project: 682809,EndoSubvert(2017)

Subjects
Intracellular bacterial pathogens, BAR domain-containing proteins, [SDV.BC.IC]Life Sciences [q-bio]/Cellular Biology/Cell Behavior [q-bio.CB], High-content screening, Rab GTPases, Membrane trafficking, Bacterial infection, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], Shigella flexneri
Abstract

International audience; Intracellular bacterial pathogens have evolved a plethora of strategies to invade eukaryotic cells. By manipulating host signaling pathways, in particular vesicular trafficking, these microbes subvert host functions to promote their internalization and to establish an intracellular niche. During these events, host endomembrane compartments are dynamically reorganized. Shigella flexneri, the causative agent of bacillary dysentery, recruits components of the host recycling pathway and the exocyst of non-phagocytic enterocytes in the vicinity of its entry site to facilitate its access to the host cytosol. These factors are either dynamically tethered to in situ formed macropinosomes or to the bacteria-containing vacuole itself. The underlying interactions cannot readily be monitored as individual bacterial infection events take place without synchronicity using cellular infection models. Therefore, time-resolved screens by fluorescence microscopy represent a powerful tool for the study of host subversion. Such screens can be performed with libraries of fluorescently tagged host factors. Using the cytosolic pathogenic agent Shigella flexneri as a model, we provide detailed protocols for such medium-to-high throughput multidimensional imaging screening of the dynamic host-pathogen cross talk. Our workflow is designed to be easily adapted for the study of different host factor libraries and different pathogen models.

Published
2022
Full Text
View/download PDF

5. Intracellular niche switching as host subversion strategy of bacterial pathogens

Author

Gutierrez, Maximiliano, Enninga, Jost, The Francis Crick Institute [London], Dynamique des Interactions Hôte-Pathogène - Dynamics of Host-Pathogen Interactions, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Work in the Host pathogen-interactions in tuberculosis lab is supported by the Francis Crick Institute (to MGG), which receives its core funding from Cancer Research UK (FC001092), the UK Medical Research Council (FC001092) and the Wellcome Trust (FC001092). This research was funded in whole, or in part, by the Wellcome Trust (FC001092). The Dynamics of Host Pathogen Interaction Unit acknowledges funding from the Institut Pasteur, the European Union (ERC-CoG 'Endosubvert'), and the Agence National pour la Recherche (Program 'HBP Sensing', 'PureMagRupture'). Furthermore, the JE team is supported by the LabExes 'Milieu Interieur' and 'IBEID'., ANR-17-CE15-0006,HBPsensing,Mécanisme de détection de l'heptose 1,7-bisphosphate lors d'infections bactériennes à Gram négatif(2017), ANR-21-CE35-0007,PureMagRupture,Un flux de travail de la gMEP (Genetic magnetic-extraction-proteomics) pour comprendre le lien entre la rupture vacuolaire de Shigella et l'autophagie(2021), ANR-10-LABX-0069,MILIEU INTERIEUR,GENETIC & ENVIRONMENTAL CONTROL OF IMMUNE PHENOTYPE VARIANCE: ESTABLISHING A PATH TOWARDS PERSONALIZED MEDICINE(2010), ANR-10-LABX-0062,IBEID,Integrative Biology of Emerging Infectious Diseases(2010), and European Project: 682809,EndoSubvert(2017)

Subjects
Model organisms, Cytosol, Bacteria, [SDV.BC.IC]Life Sciences [q-bio]/Cellular Biology/Cell Behavior [q-bio.CB], Host-Pathogen Interactions, Infectious Disease, Cell Biology, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM]
Abstract

International audience; Numerous bacterial pathogens “confine” themselves within host cells with an intracellular localization as main or exclusive niche. Many of them switch dynamically between a membrane-bound or cytosolic lifestyle. This requires either membrane damage and/or repair of the bacterial-containing compartment. Niche switching has profound consequences on how the host cell recognizes the pathogens in time and space for elimination. Moreover, niche switching impacts how bacteria communicate with host cells to obtain nutrients, and it affects the accessibility to antibiotics. Understanding the local environments and cellular phenotypes that lead to niche switching is critical for developing new host-targeted antimicrobial strategies, and has the potential to shed light into fundamental cellular processes.

Published
2021
Full Text
View/download PDF

6. Purification of infection-associated macropinosomes by magnetic isolation for proteomic characterization

Author

Quentin Giai Gianetto, Magalie Duchateau, Virginie Stévenin, Mariette Matondo, Jost Enninga, Yuen-Yan Chang, Dynamique des Interactions Hôte-Pathogène - Dynamics of Host-Pathogen Interactions, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Leiden University Medical Center (LUMC), Universiteit Leiden, Plateforme de Protéomique / Proteomics platform, Université Paris Cité (UPCité)-Spectrométrie de Masse pour la Biologie – Mass Spectrometry for Biology (UTechS MSBio), Institut Pasteur [Paris] (IP)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité)-Institut Pasteur [Paris] (IP)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Hub Bioinformatique et Biostatistique - Bioinformatics and Biostatistics HUB, Institut Pasteur [Paris] (IP)-Université Paris Cité (UPCité), National Institutes of Health [Bethesda] (NIH), Y-Y.C was supported by a fellowship from the Fondation pour la Recherche Médicale (FRM, SPF20160936275). V.S. was supported by a PhD fellowship from the University Paris Diderot (now Université de Paris) allocated by the ENS Paris-Saclay, a grant from the FRM (FRM, FDT20170436843), and an extension grant from the BCI Department of Institut Pasteur. J.E. is a member of the LabEx consortia IBEID and MilieuInterieur. J.E. also acknowledges support from the ANR (grant StopBugEntry and AutoHostPath) and the ERC (CoG EndoSubvert)., ANR-10-LABX-0062,IBEID,Integrative Biology of Emerging Infectious Diseases(2010), ANR-10-LABX-0069,MILIEU INTERIEUR,GENETIC & ENVIRONMENTAL CONTROL OF IMMUNE PHENOTYPE VARIANCE: ESTABLISHING A PATH TOWARDS PERSONALIZED MEDICINE(2010), ANR-15-CE15-0017,StopBugEntry,Identification des nouvelles molécules cellulaires cibles pour combattre les infections bactériennes(2015), ANR-15-CE15-0018,AutoHostPath,Rôles alternatifs pour les récepteurs de l'autophagie dans les interactions hôte-pathogène(2015), European Project: 682809,EndoSubvert(2017), Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Spectrométrie de Masse pour la Biologie – Mass Spectrometry for Biology (UTechS MSBio), and Institut Pasteur [Paris]-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur [Paris]-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)

Subjects
Proteomics, 0303 health sciences, Chemistry, Vesicle, Pinocytosis, Production cost, Magnetic Phenomena, Endocytic cycle, Cell migration, Composition analysis, General Biochemistry, Genetics and Molecular Biology, 3. Good health, Cell biology, 03 medical and health sciences, 0302 clinical medicine, Endocytic vesicle, Eukaryotic Cells, Magnetic isolation, Cell Movement, [SDV.BC.IC]Life Sciences [q-bio]/Cellular Biology/Cell Behavior [q-bio.CB], [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], 030217 neurology & neurosurgery, 030304 developmental biology
Abstract

International audience; Macropinocytosis refers to the nonselective uptake of extracellular molecules into many different types of eukaryotic cells within large fluid-filled vesicles named macropinosomes. Macropinosomes are relevant for a wide variety of cellular processes, such as antigen sampling in immune cells, homeostasis in the kidney, cell migration or pathogen uptake. Understanding the molecular composition of the different macropinosomes formed during these processes has helped to differentiate their regulations from other endocytic events. Here, we present a magnetic purification protocol that segregates scarce macropinosomes from other endocytic vesicles at a high purity and in a low-cost and unbiased manner. Our protocol takes advantage of moderate-sized magnetic beads of 100 nm in diameter coupled to mass-spectrometry-based proteomic analysis. Passing the cell lysate through a table-top magnet allows the quick retention of the bead-containing macropinosomes. Unlike other cell-fractionation-based methodologies, our protocol minimizes sample loss and production cost without prerequisite knowledge of the macropinosomes and with minimal laboratory experience. We describe a detailed procedure for the isolation of infection-associated macropinosomes during bacterial invasion and the optimization steps to readily adapt it to various studies. The protocol can be performed in 3 d to provide highly purified and enriched macropinosomes for qualitative proteomic composition analysis.

Published
2021
Full Text
View/download PDF

7. New methods to decrypt emerging macropinosome functions during the host–pathogen crosstalk

Author

Jost Enninga, Virginie Stévenin, Yuen-Yan Chang, National Institute of Child Health and Human Development [Bethesda], National Institutes of Health, Dynamique des Interactions Hôte-Pathogène - Dynamics of Host-Pathogen Interactions, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Leiden University Medical Center (LUMC), École Doctorale Bio Sorbonne Paris Cité [Paris] (ED BioSPC), Université Sorbonne Paris Cité (USPC)-Université de Paris (UP), Y-Y.C. and V.S. were supported by grants from the Fondation pour la Recherche Médicale (FRM, SPF20160936275 and FDT20170436843). J.E. is a member of the LabEx consortia IBEID and MilieuInterieur. J.E. also acknowledges support from the ANR (grant StopBugEntry, AutoHostPath and HBPSensing) and the ERC (CoG EndoSubvert)., ANR-10-LABX-0062,IBEID,Integrative Biology of Emerging Infectious Diseases(2010), ANR-10-LABX-0069,MILIEU INTERIEUR,GENETIC & ENVIRONMENTAL CONTROL OF IMMUNE PHENOTYPE VARIANCE: ESTABLISHING A PATH TOWARDS PERSONALIZED MEDICINE(2010), ANR-15-CE15-0017,StopBugEntry,Identification des nouvelles molécules cellulaires cibles pour combattre les infections bactériennes(2015), ANR-15-CE15-0018,AutoHostPath,Rôles alternatifs pour les récepteurs de l'autophagie dans les interactions hôte-pathogène(2015), ANR-17-CE15-0006,HBPsensing,Mécanisme de détection de l'heptose 1,7-bisphosphate lors d'infections bactériennes à Gram négatif(2017), European Project: 682809,EndoSubvert(2017), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Universiteit Leiden, École Doctorale Bio Sorbonne Paris Cité [Paris] (ED562 - BioSPC), and Université Sorbonne Paris Cité (USPC)-Université Paris Cité (UPCité)

Subjects
media_common.quotation_subject, Immunology, Cellular functions, Endosomes, Computational biology, Biology, Microbiology, 03 medical and health sciences, Salmonella, Virology, [SDV.BC.IC]Life Sciences [q-bio]/Cellular Biology/Cell Behavior [q-bio.CB], Humans, Compartment (development), [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], Macropinosome, Internalization, Dysentery, Bacillary, 030304 developmental biology, media_common, Microreviews, 0303 health sciences, Endocytic Process, 030306 microbiology, Intracellular parasite, Microreview, Crosstalk (biology), Host-Pathogen Interactions, Salmonella Infections, Shigella
Abstract

Large volumes of liquid and other materials from the extracellular environment are internalised by eukaryotic cells via an endocytic process called macropinocytosis. It is now recognised that this fundamental and evolutionarily conserved pathway is hijacked by numerous intracellular pathogens as an entry portal to the host cell interior. Yet, an increasing number of additional cellular functions of macropinosomes in pathologic processes have been reported beyond this role for fluid internalisation. It emerges that the identity of macropinosomes can vary hugely and change rapidly during their lifetime. A deeper understanding of this important multi‐faceted compartment is based on novel methods for their investigation. These methods are either imaging‐based for the tracking of macropinosome dynamics, or they provide the means to extract macropinosomes at high purity for comprehensive proteomic analyses. Here, we portray these new approaches for the investigation of macropinosomes. We document how these method developments have provided insights for a new understanding of the intracellular lifestyle of the bacterial pathogens Shigella and Salmonella. We suggest that a systematic complete characterisation of macropinosome subversion with these approaches during other infection processes and pathologies will be highly beneficial for our understanding of the underlying cellular and molecular processes., Viruses and intracellular bacteria commonly hijack macropinocytosis to invade host cell. Classically, subversion of macropinocytosis by intracellular pathogens was perceived as purely entry‐related. Yet, recent discoveries showed that pathogen‐induced ruffling leads to the formation of distinct class of macropinosomes termed infection‐associated macropinosomes (IAMs). Thanks to novel method developments, it has emerged that these non‐pathogen‐containing IAMs contribute to establish pathogen replicative niches; for instance by facilitating cytosolic escape or by promoting the maturation of a permissive pathogen‐containing compartment.

Published
2021
Full Text
View/download PDF

8. Salmonella enters a dormant state within human epithelial cells for persistent infection

Author

Perrine Bomme, Camila Valenzuela, Magdalena Gil, Yuen-Yan Chang, Chak Hon Luk, Jost Enninga, Adeline Mallet, Léa Swistak, Dynamique des Interactions Hôte-Pathogène - Dynamics of Host-Pathogen Interactions, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Université Paris Cité (UPCité), Plateforme BioImagerie Ultrastructurale – Ultrastructural BioImaging Platform (UTechS UBI), Institut Pasteur [Paris] (IP), This research was supported by fellowships from Croucher Foundation (HK) and Fondation pour la Recherche Médicale (FRM) to C.H.L. and Y.Y.C.. C.H.L. is part of the Pasteur - Paris University (PPU) International PhD Program. J.E. is supported by the ERC-CoG 'Endosubvert'. The Enninga lab is part of the LabEx IBEID and Milieu Interieure. AM and PB are supported for equipment from the French Government Programme Investissements d’Avenir France BioImaging (FBI, N° ANR-10-INSB-04-01) and are also members of the LabEx IBEID., ANR-10-LABX-0062,IBEID,Integrative Biology of Emerging Infectious Diseases(2010), ANR-10-LABX-0069,MILIEU INTERIEUR,GENETIC & ENVIRONMENTAL CONTROL OF IMMUNE PHENOTYPE VARIANCE: ESTABLISHING A PATH TOWARDS PERSONALIZED MEDICINE(2010), ANR-10-INBS-0004,France-BioImaging,Développment d'une infrastructure française distribuée coordonnée(2010), European Project: 682809,EndoSubvert(2017), Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Université de Paris (UP), and Institut Pasteur [Paris]

Subjects
Bacterial Diseases, Salmonella typhimurium, Salmonella, THP-1 Cells, Host cells, Cultured tumor cells, Epithelial cells, Pathology and Laboratory Medicine, medicine.disease_cause, Epithelium, Mice, White Blood Cells, Medical Conditions, Spectrum Analysis Techniques, Animal Cells, Antibiotics, [SDV.BC.IC]Life Sciences [q-bio]/Cellular Biology/Cell Behavior [q-bio.CB], Medicine and Health Sciences, Flow cytometry, Biology (General), Pathogen, 0303 health sciences, Antimicrobials, Effector, Drugs, 3T3 Cells, Virus Latency, Bacterial Pathogens, Infectious Diseases, Medical Microbiology, Spectrophotometry, Salmonella Infections, Cell lines, Cytophotometry, Pathogens, Cellular Types, Anatomy, Biological cultures, Intracellular, Research Article, Genomic Islands, Virulence Factors, QH301-705.5, Immune Cells, Immunology, Virulence, Biology, Microbiology, 03 medical and health sciences, Enterobacteriaceae, Microbial Control, Virology, Genetics, medicine, Animals, Humans, Secretion, HeLa cells, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], Microbial Pathogens, Molecular Biology, 030304 developmental biology, Pharmacology, Intracellular pathogens, Blood Cells, Bacteria, 030306 microbiology, Intracellular parasite, Macrophages, Organisms, Biology and Life Sciences, Cell Biology, RC581-607, Cell cultures, Pathogenicity island, Research and analysis methods, Biological Tissue, Vacuoles, bacteria, Parasitology, Caco-2 Cells, Immunologic diseases. Allergy, Viral Transmission and Infection
Abstract

Salmonella Typhimurium (S. Typhimurium) is an enteric bacterium capable of invading a wide range of hosts, including rodents and humans. It targets different host cell types showing different intracellular lifestyles. S. Typhimurium colonizes different intracellular niches and is able to either actively divide at various rates or remain dormant to persist. A comprehensive tool to determine these distinct S. Typhimurium lifestyles remains lacking. Here we developed a novel fluorescent reporter, Salmonella INtracellular Analyzer (SINA), compatible for fluorescence microscopy and flow cytometry in single-bacterium level quantification. This identified a S. Typhimurium subpopulation in infected epithelial cells that exhibits a unique phenotype in comparison to the previously documented vacuolar or cytosolic S. Typhimurium. This subpopulation entered a dormant state in a vesicular compartment distinct from the conventional Salmonella-containing vacuoles (SCV) as well as the previously reported niche of dormant S. Typhimurium in macrophages. The dormant S. Typhimurium inside enterocytes were viable and expressed Salmonella Pathogenicity Island 2 (SPI-2) virulence factors at later time points. We found that the formation of these dormant S. Typhimurium is not triggered by the loss of SPI-2 effector secretion but it is regulated by (p)ppGpp-mediated stringent response through RelA and SpoT. We predict that intraepithelial dormant S. Typhimurium represents an important pathogen niche and provides an alternative strategy for S. Typhimurium pathogenicity and its persistence., Author summary Salmonella Typhimurium is a clinically relevant bacterial pathogen that causes Salmonellosis. It can actively or passively invade various host cell types and reside in a Salmonella-containing vacuole (SCV) within host cells. The SCV can be remodeled into a replicative niche with the aid of Salmonella Type III Secretion System 2 (T3SS2) effectors or else, the SCV is ruptured for the access of the nutrient-rich host cytosol. Depending on the infected host cell type, S. Typhimurium undertake different lifestyles that are distinct by their subcellular localization, replication rate and metabolic rate. We present here a novel fluorescent reporter system that rapidly detects S. Typhimurium lifestyles using fluorescence microscopy and flow cytometry. We identified a dormant S. Typhimurium population within enterocyte that displays capacities in host cell persistence, dormancy exit and antibiotic tolerance. We deciphered the (p)ppGpp stringent response pathway that suppresses S. Typhimurium dormancy in enterocytes while promoting dormancy in macrophages, pinpointing a divergent physiological consequence regulated by the same set of S. Typhimurium molecular mediators. Altogether, our work demonstrated the potential of fluorescent reporters in facile bacterial characterization, and revealed a dormant S. Typhimurium population in human enterocytes that are phenotypically distinct from that observed in macrophages and fibroblasts.

Published
2021
Full Text
View/download PDF

9. Micropatterning of cells on EM grids for efficient cryo-correlative light electron microscopy

Author

Matthijn Vos, Jost Enninga, Léa Swistak, Anna Sartori-Rupp, Dynamique des Interactions Hôte-Pathogène - Dynamics of Host-Pathogen Interactions, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Université de Paris (UP), Plateforme BioImagerie Ultrastructurale – Ultrastructural BioImaging Platform (UTechS UBI), Institut Pasteur [Paris], Plateforme technologique Nanoimagerie / Nanoimaging Technological Platform, This work was funded by the Domaine d’Intérêt Majeur ELICIT 2020 (DIM Elicit 2020) from the Région Ile-de-France. J.E. is supported by the ERC-CoG ‘Endosubvert’ and by the ANR grant HBPSensing. The Enninga lab is part of the LabEx IBEID and Milieu Interieur., Thanks to Dr. Kristine Schauer (Institut Curie) for sharing expertise on micropatterning., ANR-17-CE15-0006,HBPsensing,Mécanisme de détection de l'heptose 1,7-bisphosphate lors d'infections bactériennes à Gram négatif(2017), ANR-10-LABX-0062,IBEID,Integrative Biology of Emerging Infectious Diseases(2010), ANR-10-LABX-0069,MILIEU INTERIEUR,GENETIC & ENVIRONMENTAL CONTROL OF IMMUNE PHENOTYPE VARIANCE: ESTABLISHING A PATH TOWARDS PERSONALIZED MEDICINE(2010), European Project: 682809,EndoSubvert(2017), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Université Paris Cité (UPCité), and Institut Pasteur [Paris] (IP)

Subjects
Correlative, 0303 health sciences, Materials science, Ion beam, business.industry, 030302 biochemistry & molecular biology, law.invention, 03 medical and health sciences, Cryo-FIB/SEM, law, Correlative light electron microscopy (CLEM), Microscopy, Cryo-ET, [SDV.BC.IC]Life Sciences [q-bio]/Cellular Biology/Cell Behavior [q-bio.CB], Ultrastructure, Optoelectronics, Tomography, Electron microscope, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], business, EM grid micropatterning, 030304 developmental biology, Lamella (cell biology), Micropatterning, Cryo-EM
Abstract

International audience; Cryo-transmission electron microscopy (cryo-TEM) provides access to high resolution information of adherent cell ultrastructures in a close to native environment but only volumes of less than 500 nm can be imaged. Cryo-focused ion beam (FIB) milling overcomes this obstacle through the generation of thin lamella of less than 200 nm. These lamellas can be imaged by cryo-electron tomography (cryo-ET) giving access to ultrastructural data within the volume of the imaged cells. Nevertheless, a lack of control on the positioning of the samples on the electron microscopy (EM) grids drastically constrains its throughput. The use of custom-designed micropatterned EM grids bypasses these issues by accurately positioning cells in areas that allow FIB milling followed by cryo-ET. Combined with fluorescent light microscopy in correlative light electron microscopy (CLEM) pipelines to pinpoint specific events and automated FIB milling, micropatterning of cells on EM grids has the potential of dramatically accelerating the workflow of cryo-ET. Here, a detailed description is provided of the key steps necessary to implement photomicropatterning of EM grids for improved CLEM pipelines.

Published
2021
Full Text
View/download PDF

10. SopB- and SifA-dependent shaping of the Salmonella-containing vacuole proteome in the social amoeba Dictyostelium discoideum

Author

Carlos A. Santiviago, Magdalena Gil, Andrea Sabag, Ítalo M. Urrutia, Jost Enninga, Camila Valenzuela, Universidad de Chile = University of Chile [Santiago] (UCHILE), Dynamique des Interactions Hôte-Pathogène - Dynamics of Host-Pathogen Interactions, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), This work was supported by FONDECYT grants 1140754 and 1171844, to Carlos A. Santiviago. Camila Valenzuela and Ítalo M. Urrutia were supported by CONICYT fellowships 21140615 and 21150005, respectively. The team of Jost Enninga is supported by an ERC CoG grant (EndoSubvert), and acknowledges support from the ANR (StopBugEntry and AutoHostPath programs). Jost Enninga is a member of the LabExes IBEID and Milieu Interieur, ANR-15-CE15-0017,StopBugEntry,Identification des nouvelles molécules cellulaires cibles pour combattre les infections bactériennes(2015), ANR-15-CE15-0018,AutoHostPath,Rôles alternatifs pour les récepteurs de l'autophagie dans les interactions hôte-pathogène(2015), ANR-10-LABX-0062,IBEID,Integrative Biology of Emerging Infectious Diseases(2010), ANR-10-LABX-0069,MILIEU INTERIEUR,GENETIC & ENVIRONMENTAL CONTROL OF IMMUNE PHENOTYPE VARIANCE: ESTABLISHING A PATH TOWARDS PERSONALIZED MEDICINE(2010), European Project: 682809,EndoSubvert(2017), and Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)

Subjects
Proteomics, Salmonella typhimurium, Salmonella, food.ingredient, intracellular survival, proteome, Mutant, Immunology, Protozoan Proteins, Vacuole, macromolecular substances, medicine.disease_cause, Microbiology, Dictyostelium discoideum, Amoeba (genus), 03 medical and health sciences, food, Bacterial Proteins, Virology, [SDV.BC.IC]Life Sciences [q-bio]/Cellular Biology/Cell Behavior [q-bio.CB], medicine, Animals, Secretion, Dictyostelium, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], Amoeba, 030304 developmental biology, 0303 health sciences, biology, 030306 microbiology, Effector, biochemical phenomena, metabolism, and nutrition, biology.organism_classification, Cell biology, T3SS, Proteome, Host-Pathogen Interactions, Mutation, Salmonella Infections, Vacuoles, bacteria, SCV, Intracellular
Abstract

The ability ofSalmonellato survive and replicate within mammalian host cells involves the generation of a membranous compartment known as theSalmonella-containing vacuole (SCV).Salmonellaemploys a number of effector proteins that are injected into host cells for SCV formation using its type-three secretion systems encoded in SPI-1 and SPI-2 (T3SSSPI-1and T3SSSPI-2, respectively). Recently, we reported thatS. Typhimurium requires T3SSSPI-1and T3SSSPI-2to survive in the model amoebaDictyostelium discoideum. Despite these findings, the involved effector proteins have not been identified yet. Therefore, we evaluated the role of two majorS. Typhimurium effectors SopB and SifA duringD. discoideumintracellular niche formation. First, we established thatS. Typhimurium resides in a vacuolar compartment withinD. discoideum. Next, we isolated SCVs from amoebae infected with wild type or the ΔsopBand ΔsifAmutant strains ofS. Typhimurium, and we characterized the composition of this compartment by quantitative proteomics. This comparative analysis suggests thatS. Typhimurium requires SopB and SifA to modify the SCV proteome in order to generate a suitable intracellular niche inD. discoideum. Accordingly, we observed that SopB and SifA are needed for intracellular survival ofS. Typhimurium in this organism. Thus, our results provide insight into the mechanisms employed bySalmonellato survive intracellularly in phagocytic amoebae.ImportanceThe molecular mechanisms involved inSalmonellasurvival to predation by phagocytic amoebae, such asD. discoideum, remains poorly understood. Although we established thatS. Typhimurium requires two specialized type-three secretion systems to survive inD. discoideum, no effector protein has been implicated in this process so far. Here, we confirmed the presence of a membrane-bound compartment containingS. Typhimurium inD. discoideum, and purified theD. discoideumSCV to characterize the associated proteome. In doing so, we established a key role for effector proteins SopB and SifA in remodeling the protein content of the SCV that ultimately allow the intracellular survival ofS. Typhimurium inD. discoideum. We also discuss similarities and differences with the proteomes of the human SCV. These findings contribute to unravel the mechanisms used bySalmonellato survive in the environment exploiting phagocytic amoebae as a reservoir.

Published
2021
Full Text
View/download PDF

11. The histone demethylase KDM6B fine-tunes the host response to Streptococcus pneumoniae

Author

Emma Patey, Robert S. Heyderman, Caroline M. Weight, Laura Barrio, Michael G. Connor, Tiphaine M. N. Camarasa, Daniel P. Miller, Jost Enninga, Richard J. Lamont, Orhan Rasid, Melanie Hamon, Chromatine et Infection - Chromatin and Infection, Institut Pasteur [Paris], Université Paris Diderot, Sorbonne Paris Cité, Paris, France, Université Paris Diderot - Paris 7 (UPD7), Dynamique des Interactions Hôte-Pathogène - Dynamics of Host-Pathogen Interactions, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), University College of London [London] (UCL), Virginia Commonwealth University (VCU), University of Louisville, M.G.C. is supported by the Pasteur Foundation Fellowship. T.M.N.C. is supported by Foundation pour la Recherche Médicale, grant no. PMJ201810007628 (Prix MARIANE JOSSO), and a donation from Credit Agricole. M.A.H. is supported by the Institut Pasteur, and the Agence National de la Recherche (ANREpiBActIn), and is a member of the IBEID LabEx. C.M.W. was supported by the Medical Research Council (MR/T016329/1). We thank R. S. Heyderman (UCL) for in-depth discussion on the manuscript, and he is supported by the MRC (MR/T016329/1). J.E. and L.B., members of the Dynamics of Host–Pathogen interactions Unit (Institut Pasteur), are supported by the European Commission (ERC-CoG-Endosubvert) and the ANR-HBPsensing, and are members of the IBEID and Milieu Interieur LabExes. R.J.L. is funded by NIH/NIDCR nos. DE01111, DE012505, DE017921 and DE023193., ANR-17-CE12-0007,EpiBactIn,Modifications epigenomiques induites par l'interaction hote-bacteries(2017), ANR-10-LABX-0062,IBEID,Integrative Biology of Emerging Infectious Diseases(2010), ANR-17-CE15-0006,HBPsensing,Mécanisme de détection de l'heptose 1,7-bisphosphate lors d'infections bactériennes à Gram négatif(2017), ANR-10-LABX-0069,MILIEU INTERIEUR,GENETIC & ENVIRONMENTAL CONTROL OF IMMUNE PHENOTYPE VARIANCE: ESTABLISHING A PATH TOWARDS PERSONALIZED MEDICINE(2010), European Project: 682809,EndoSubvert(2017), Institut Pasteur [Paris] (IP), and Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)

Subjects
Microbiology (medical), Serotype, Jumonji Domain-Containing Histone Demethylases, Immunology, Lysine, [SDV.BC.BC]Life Sciences [q-bio]/Cellular Biology/Subcellular Processes [q-bio.SC], medicine.disease_cause, Applied Microbiology and Biotechnology, Microbiology, Pneumococcal Infections, Mice, 03 medical and health sciences, Histone H3, In vivo, [SDV.MHEP.MI]Life Sciences [q-bio]/Human health and pathology/Infectious diseases, Streptococcus pneumoniae, Genetics, medicine, Animals, Humans, Enzyme Inhibitors, Promoter Regions, Genetic, 030304 developmental biology, 0303 health sciences, biology, 030306 microbiology, NF-kappa B, Epithelial Cells, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, Cell Biology, Benzazepines, Chromatin Assembly and Disassembly, Interleukin-11, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, In vitro, 3. Good health, Cell biology, Mice, Inbred C57BL, Disease Models, Animal, Pyrimidines, Histone, Gene Expression Regulation, A549 Cells, Alveolar Epithelial Cells, Host-Pathogen Interactions, biology.protein, Demethylase
Abstract

Streptococcus pneumoniae is a natural colonizer of the human respiratory tract and an opportunistic pathogen. Although epithelial cells are among the first to encounter pneumococci, the cellular processes and contribution of epithelial cells to the host response are poorly understood. Here, we show that a S. pneumoniae serotype 6B ST90 strain, which does not cause disease in a murine infection model, induces a unique NF-κB signature response distinct from an invasive-disease-causing isolate of serotype 4 (TIGR4). This signature is characterized by activation of p65 and requires a histone demethylase KDM6B. We show, molecularly, that the interaction of the 6B strain with epithelial cells leads to chromatin remodelling within the IL-11 promoter in a KDM6B-dependent manner, where KDM6B specifically demethylates histone H3 lysine 27 dimethyl. Remodelling of the IL-11 locus facilitates p65 access to three NF-κB sites that are otherwise inaccessible when stimulated by IL-1β or TIGR4. Finally, we demonstrate through chemical inhibition of KDM6B with GSK-J4 inhibitor and through exogenous addition of IL-11 that the host responses to the 6B ST90 and TIGR4 strains can be interchanged both in vitro and in a murine model of infection in vivo. Our studies therefore reveal how a chromatin modifier governs cellular responses during infection. A non-invasive Streptococcus pneumoniae strain induces a unique NF-κB signature response in epithelial cells that requires the histone demethylase KDM6B. Modulation of KDM6B can interchange the host response to non-invasive and invasive pneumococcal strains, demonstrating the biological role of KDM6B in cellular responses during infection.

Published
2021
Full Text
View/download PDF

12. A Role for Taok2 in Listeria monocytogenes Vacuolar Escape

Author

Alain Charbit, Jost Enninga, Nathalie Aulner, Noelia Lopez-Montero, Jason Ziveri, Steven Rolland, Javier Pizarro-Cerdá, Pascale Cossart, Camille Morel, Juan J. Quereda, Théodore Grenier, Anne Danckaert, Interactions Bactéries-Cellules (UIBC), Institut Pasteur [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Universidad Cardenal Herrera-CEU (CEU-UCH), Dynamique des Interactions Hôte-Pathogène - Dynamics of Host-Pathogen Interactions, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Institut Necker Enfants-Malades (INEM - UM 111 (UMR 8253 / U1151)), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université de Paris (UP), Institut de Génomique Fonctionnelle de Lyon (IGFL), École normale supérieure - Lyon (ENS Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Institut Pasteur [Paris], Yersinia, Institut National de la Recherche Agronomique (INRA)-Institut Pasteur [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM), Université Paris Descartes, Sorbonne Paris Cité, Paris, France, Inflammasome NLRP3 – NLRP3 Inflammasome, Centre International de Recherche en Infectiologie - UMR (CIRI), Université de Lyon-Université de Lyon-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-École normale supérieure - Lyon (ENS Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), BioImagerie Photonique – Photonic BioImaging (UTechS PBI), This work was supported by Institut Pasteur, Université Paris Diderot, Agence Nationale de la Recherche (grant numbers ANR-15-CE15-0017 StopBugEntry to J. P. C., J. E., and A. C., and ANR-10-INSB-04-01, France-BioImaging to N. A.). J. J. Q. is supported by the Spanish Ministry of Science, Innovation and Universities (grant number RYC-2018-024985-I Ramón y Cajal contract). J. E. was supported by the European Research Council (consolidator grant EndoSubvert). P. B. I. is supported by the Région Ile de France (DIM1Health) and Groupement d’Intérêt Scientifique Infrastructures en Biologie Santé et Agronomie. The teams of P. C., J. P. C., N. A., and J. E. are members of Laboratory of Excellence Integrative Biology of Emerging Infectious Diseases., ANR-15-CE15-0017,StopBugEntry,Identification des nouvelles molécules cellulaires cibles pour combattre les infections bactériennes(2015), ANR-10-INBS-0004,France-BioImaging,Développment d'une infrastructure française distribuée coordonnée(2010), ANR-10-LABX-0062,IBEID,Integrative Biology of Emerging Infectious Diseases(2010), European Project: 682809,EndoSubvert(2017), Producción Científica UCH 2022, UCH. Departamento de Producción y Sanidad Animal, Salud Pública Veterinaria y Ciencia y Tecnología de los Alimentos, Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Charbit, Alain, Identification des nouvelles molécules cellulaires cibles pour combattre les infections bactériennes - - StopBugEntry2015 - ANR-15-CE15-0017 - AAPG2015 - VALID, Développment d'une infrastructure française distribuée coordonnée - - France-BioImaging2010 - ANR-10-INBS-0004 - INBS - VALID, Integrative Biology of Emerging Infectious Diseases - - IBEID2010 - ANR-10-LABX-0062 - LABX - VALID, Common mechanisms of host membrane trafficking subversion by intracellular pathogens to rupture bacterial containing vacuoles - EndoSubvert - 2017-01-01 - 2021-12-31 - 682809 - VALID, Institut National de la Recherche Agronomique (INRA)-Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Institut Pasteur [Paris] (IP), Centre International de Recherche en Infectiologie (CIRI), École normale supérieure de Lyon (ENS de Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Université Jean Monnet - Saint-Étienne (UJM)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-École normale supérieure de Lyon (ENS de Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Université Jean Monnet - Saint-Étienne (UJM)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), and Producción Científica UCH 2020

Subjects
0301 basic medicine, Cytoplasm, media_common.quotation_subject, Citoplasma, 030106 microbiology, Vacuole, Biology, medicine.disease_cause, Pathogenic bacteria, 03 medical and health sciences, Hemolysin Proteins, Cytosol, Listeria monocytogenes, Bacterial Proteins, [SDV.MHEP.MI]Life Sciences [q-bio]/Human health and pathology/Infectious diseases, medicine, Immunology and Allergy, Humans, Células epiteliales, vacuolar escape, Listeriosis, Kinase activity, Internalization, Pathogen, ComputingMilieux_MISCELLANEOUS, media_common, Epithelial cells, siRNA screen, Kinase, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, Cell biology, STE1-like kinase, 030104 developmental biology, Infectious Diseases, [SDV.MP]Life Sciences [q-bio]/Microbiology and Parasitology, Bacterias patógenas, Vacuoles, [SDV.MHEP.MI] Life Sciences [q-bio]/Human health and pathology/Infectious diseases, [SDV.MP.BAC] Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, Taok2
Abstract

Este artículo se encuentra disponible en la siguiente URL: https://academic.oup.com/jid/advance-article/doi/10.1093/infdis/jiaa367/5862450 En esta investigación también participan: Nathalie Aulner, Anne Danckaert, Alain Charbit, Jost Enninga, Pascale Cossart y Javier Pizarro Cerdá. The bacterial pathogen Listeria monocytogenes invades host cells, ruptures the internalization vacuole, and reaches the cytosol for replication. A high-content small interfering RNA (siRNA) microscopy screen allowed us to identify epithelial cell factors involved in L. monocytogenes vacuolar rupture, including the serine/threonine kinase Taok2. Kinase activity inhibition using a specific drug validated a role for Taok2 in favoring L. monocytogenes cytoplasmic access. Furthermore, we showed that Taok2 recruitment to L. monocytogenes vacuoles requires the presence of pore-forming toxin listeriolysin O. Overall, our study identified the first set of host factors modulating L. monocytogenes vacuolar rupture and cytoplasmic access in epithelial cells.

Published
2021
Full Text
View/download PDF

13. The actin comet guides the way: How<scp>Listeria</scp>actin subversion has impacted cell biology, infection biology and structural biology

Author

Jost Enninga, Sonja Kühn, Dynamique des Interactions Hôte-Pathogène - Dynamics of Host-Pathogen Interactions, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), This work has been supported by a postdoctoral fellowship from the Institut Pasteur to S.K. (Bourse Roux‐Cantarini) and by a grant from the ERC (CoG Endosubvert) to J.E. The Enninga team is supported by the LabEx IBEID and by the LabEx Milieu Interieur., ANR-10-LABX-0062,IBEID,Integrative Biology of Emerging Infectious Diseases(2010), ANR-10-LABX-0069,MILIEU INTERIEUR,GENETIC & ENVIRONMENTAL CONTROL OF IMMUNE PHENOTYPE VARIANCE: ESTABLISHING A PATH TOWARDS PERSONALIZED MEDICINE(2010), European Project: 682809,EndoSubvert(2017), and Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS)

Subjects
Movement, Immunology, Cellular microbiology, macromolecular substances, Biology, medicine.disease_cause, Microbiology, Polymerization, actin dynamics, 03 medical and health sciences, Infection biology, Cytosol, Bacterial Proteins, Listeria monocytogenes, Virology, [SDV.BC.IC]Life Sciences [q-bio]/Cellular Biology/Cell Behavior [q-bio.CB], Actin dynamics, medicine, Humans, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], nucleation promoting factor, Actin, Pascale Cossart, 030304 developmental biology, 0303 health sciences, Binding Sites, 030306 microbiology, Membrane Proteins, ActA, biology.organism_classification, Actins, Cell biology, Cytoskeletal Proteins, Structural biology, Listeria
Abstract

International audience; The discovery of the role of ActA to polymerise actin at one pole of Listeria monocytogenes represents a key event in the field of cellular microbiology. It uncovered much more than the molecular principle behind actin-based motility of Listeria within the cytosol of infected cells, and it changed the way how actin dynamics could be studied and eventually understood. The ActA discovery took place at a time when cell biology, biochemistry and microbiology came together in a very fruitful fashion. Here, we provide an overview of the science that took place around this event. Then, we outline the wide array of research fields that have been impacted by this finding. This ranges from structural and biophysical investigations on actin and its dynamics, the role of actin polymerisation during infection with different pathogens, to actin-dynamics during various pathologies. Like a comet in the sky, Pascale Cossart's work on ActA has inspired and will inspire generations of (life) scientists.

Published
2020
Full Text
View/download PDF

14. Shigella hijacks the exocyst to cluster macropinosomes for efficient vacuolar escape

Author

Jost Enninga, Magalie Duchateau, Quentin Giai Gianetto, Véronique Hourdel, Norbert Reiling, Cristina Rodrigues, Mariette Matondo, Virginie Stévenin, Yuen-Yan Chang, Dynamique des Interactions Hôte-Pathogène - Dynamics of Host-Pathogen Interactions, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Spectrométrie de Masse pour la Biologie – Mass Spectrometry for Biology (UTechS MSBio), Institut Pasteur [Paris]-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Hub Bioinformatique et Biostatistique - Bioinformatics and Biostatistics HUB, Research Center Borstel - Leibniz Lung Center [Germany], This work is supported by a fellowship from the Fondation pour la Recherche Medicale (FRM-SPF20160936275) to Y-Y.C. and by grants from the ERC (EndoSubvert) and the ANR (StopBugEntry and AutoHostPath), We acknowledge the helpful discussions with all lab members of the DIHP unit, in particular Camille Rey, Magdalena Gil Taran and Laura Barrio Cano for their feedback on the manuscript. We thank Aleix Boquet-Pujadas for his technical support in image analysis and software Icy, ANR-15-CE15-0017,StopBugEntry,Identification des nouvelles molécules cellulaires cibles pour combattre les infections bactériennes(2015), ANR-15-CE15-0018,AutoHostPath,Rôles alternatifs pour les récepteurs de l'autophagie dans les interactions hôte-pathogène(2015), European Project: 682809,EndoSubvert(2017), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), and Institut Pasteur [Paris] (IP)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)

Subjects
Cultured tumor cells, Glycobiology, Priming (immunology), Vacuole, Pathology and Laboratory Medicine, Biochemistry, Shigella flexneri, Contractile Proteins, Medicine and Health Sciences, Biology (General), Glucans, 0303 health sciences, biology, Effector, 030302 biochemistry & molecular biology, Small interfering RNA, Cell biology, Nucleic acids, Medical Microbiology, Cell lines, Pathogens, Cellular Structures and Organelles, Biological cultures, Signal Transduction, Research Article, Virulence Factors, QH301-705.5, Immunology, Exocyst, Microbiology, 03 medical and health sciences, Bacterial Proteins, Polysaccharides, Virology, Genetics, Humans, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, HeLa cells, Non-coding RNA, Microbial Pathogens, Dextran, Molecular Biology, Actin, Dysentery, Bacillary, 030304 developmental biology, Intracellular pathogens, Bacteria, Intracellular parasite, Organisms, Biology and Life Sciences, Proteins, Cell Biology, RC581-607, Cell cultures, biology.organism_classification, Actins, Gene regulation, Research and analysis methods, Cytoskeletal Proteins, Cytosol, rab GTP-Binding Proteins, Vacuoles, RNA, Bacterial pathogens, Parasitology, Shigella, Gene expression, Immunologic diseases. Allergy
Abstract

Shigella flexneri invades host cells by entering within a bacteria-containing vacuole (BCV). In order to establish its niche in the host cytosol, the bacterium ruptures its BCV. Contacts between S. flexneri BCV and infection-associated macropinosomes (IAMs) formed in situ have been reported to enhance BCV disintegration. The mechanism underlying S. flexneri vacuolar escape remains however obscure. To decipher the molecular mechanism priming the communication between the IAMs and S. flexneri BCV, we performed mass spectrometry-based analysis of the magnetically purified IAMs from S. flexneri-infected cells. While proteins involved in host recycling and exocytic pathways were significantly enriched at the IAMs, we demonstrate more precisely that the S. flexneri type III effector protein IpgD mediates the recruitment of the exocyst to the IAMs through the Rab8/Rab11 pathway. This recruitment results in IAM clustering around S. flexneri BCV. More importantly, we reveal that IAM clustering subsequently facilitates an IAM-mediated unwrapping of the ruptured vacuole membranes from S. flexneri, enabling the naked bacterium to be ready for intercellular spread via actin-based motility. Taken together, our work untangles the molecular cascade of S. flexneri-driven host trafficking subversion at IAMs to develop its cytosolic lifestyle, a crucial step en route for infection progression at cellular and tissue level., Author summary Shigella flexneri is a clinically relevant bacterial pathogen that causes bacillary dysentery. It invades the host cell by injecting a repository of bacterial effectors through its type III secretion system. Upon its entry into host cell, S. flexneri resides shortly in a bacteria-containing vacuole (BCV), which is rapidly ruptured for the cytosolic propagation and infection progression of the pathogen. Infection-associated macropinosomes (IAMs) formed in situ during S. flexneri entry are found to promote the efficient vacuolar escape of S. flexneri with an unclear mechanism. We present here the molecular players involving in the BCV-IAM interactions obtained by proteomic analysis of magnetically-purified IAMs. We decipher the successive steps of S. flexneri BCV escape, pinpointing the bacterial effector-mediated hijacking of host trafficking pathways to promote the BCV disintegration and displacement of BCV membranes from the bacteria. This study sheds light on the mechanism by which bacterial pathogens modulate BCV vacuolar integrity through interaction with infection-induced vesicle compartments for the establishment of their intracellular replicative niches and pathogenicities.

Published
2020
Full Text
View/download PDF

15. Imaging macropinosomes during Shigella infections

Author

Anna Sartori-Rupp, Yuen-Yan Chang, Noelia Lopez-Montero, Sonja Kühn, Jost Enninga, Dynamique des Interactions Hôte-Pathogène - Dynamics of Host-Pathogen Interactions, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), This work was supported by the Institut Pasteur, the Agence National pour la Recherche (ANR) through the grants AutoHostPath and StopBugEntry. We thank Nora Mellouk for providing the data shown in the upper row of Fig. 2B and Allon Weiner for providing the original data for Fig. 5. J.E. acknowledges grants from the European Commission (ERC Consolidator Grant 'EndoSubvert') and the ANR (StopBugEntry and AutoHostPath), S.K. is recipient of a Pasteur-Roux (Institut Pasteur) and Y.Y.C. of a FRM postdoctoral fellowship, respectively. The DIHP research unit is member of the LabEx 'IBEID' and 'Milieu Interieur'., ANR-15-CE15-0018,AutoHostPath,Rôles alternatifs pour les récepteurs de l'autophagie dans les interactions hôte-pathogène(2015), ANR-15-CE15-0017,StopBugEntry,Identification des nouvelles molécules cellulaires cibles pour combattre les infections bactériennes(2015), European Project: 682809,EndoSubvert(2017), and Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)

Subjects
0301 basic medicine, Fluorescence-lifetime imaging microscopy, MESH: Dysentery, Bacillary, Endosome, Endosomes, MESH: Microscopy, Electron, MESH: Bacteriological Techniques, medicine.disease_cause, Endocytosis, MESH: Shigella, Fluorescence imaging, General Biochemistry, Genetics and Molecular Biology, Microbiology, 03 medical and health sciences, [SDV.BC.IC]Life Sciences [q-bio]/Cellular Biology/Cell Behavior [q-bio.CB], medicine, Humans, Shigella, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], Macropinosome, Molecular Biology, Dysentery, Bacillary, Intracellular pathogens, Macropinocytosis, Bacteriological Techniques, MESH: Humans, biology, Pinocytosis, Intracellular parasite, MESH: Host-Pathogen Interactions, biology.organism_classification, MESH: Pinocytosis, Cell biology, Microscopy, Electron, 030104 developmental biology, MESH: Endosomes, Host-Pathogen Interactions, MESH: Biomarkers, Correlative light electron microscopy, Biomarkers, Bacteria
Abstract

International audience; Macropinocytosis is the uptake of extracellular fluid within vesicles of varying size that takes place during numerous cellular processes in a large variety of cells. A growing number of pathogens, including viruses, parasites, and bacteria are known to induce macropinocytosis during their entry into targeted host cells. We have recently discovered that the human enteroinvasive, bacterial pathogen Shigella causes in situ macropinosome formation during its entry into epithelial cells. These infection-associated macropinosomes are not generated to ingest the bacteria, but are instead involved in Shigella's intracellular niche formation. They make contacts with the phagocytosed shigellae to promote vacuolar membrane rupture and their cytosolic release. Here, we provide an overview of the different imaging approaches that are currently used to analyze macropinocytosis during infectious processes with a focus on Shigella entry. We detail the advantages and disadvantages of genetically encoded reporters as well as chemical probes to trace fluid phase uptake. In addition, we report how such reporters can be combined with ultrastructural approaches for correlative light electron microscopy either in thin sections or within large volumes. The combined imaging techniques introduced here provide a detailed characterization of macropinosomes during bacterial entry, which, apart from Shigella, are relevant for numerous other ones, including Salmonella, Brucella or Mycobacteria.

Published
2017
Full Text
View/download PDF

16. Tracing a fat or sweet lifestyle - New insights on catabolic paths of intracellular Salmonella

Author

Jost Enninga, Magdalena Gil, Dynamique des Interactions Hôte-Pathogène - Dynamics of Host-Pathogen Interactions, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), This work was funded by the ANR (LabEx IBEID, projectStopBugEntry and project AutoHostPath) and the EuropeanCommission (Consolidator Grant EndoSubvert)., ANR-15-CE15-0017,StopBugEntry,Identification des nouvelles molécules cellulaires cibles pour combattre les infections bactériennes(2015), ANR-15-CE15-0018,AutoHostPath,Rôles alternatifs pour les récepteurs de l'autophagie dans les interactions hôte-pathogène(2015), European Project: 682809,EndoSubvert(2017), and Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS)

Subjects
Salmonella typhimurium, 0301 basic medicine, Microbiology (medical), Cytoplasm, Salmonella, Immunology, Biology, medicine.disease_cause, Microbiology, Mice, 03 medical and health sciences, Bacterial Proteins, [SDV.BC.IC]Life Sciences [q-bio]/Cellular Biology/Cell Behavior [q-bio.CB], medicine, Animals, Humans, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], Promoter Regions, Genetic, Life Style, ComputingMilieux_MISCELLANEOUS, 2. Zero hunger, Virulence, Catabolism, Life style, Macrophages, Gene Expression Regulation, Bacterial, Flow Cytometry, Adaptation, Physiological, Editorial, 030104 developmental biology, Infectious Diseases, Biochemistry, Vacuoles, Parasitology, Metabolic Networks and Pathways, Intracellular, Research Paper
Abstract

Salmonella enterica serovar Typhimurium (S. Typhimurium) is a Gram-negative pathogen that causes various host-specific diseases. During their life cycle, Salmonellae survive frequent exposures to a variety of environmental stresses, e.g. carbon-source starvation. The virulence of this pathogen relies on its ability to establish a replicative niche, named Salmonella-containing vacuole, inside host cells. However, the microenvironment of the SCV and the bacterial metabolic pathways required during infection are largely undefined. In this work we developed different biological probes whose expression is modulated by the environment and the physiological state of the bacterium. We constructed transcriptional reporters by fusing promoter regions to the gfpmut3a gene to monitor the expression profile of genes involved in glucose utilization and lipid catabolism. The induction of these probes by a specific metabolic change was first tested in vitro, and then during different conditions of infection in macrophages. We were able to determine that Entner-Doudoroff is the main metabolic pathway utilized by Salmonella during infection in mouse macrophages. Furthermore, we found sub-populations of bacteria expressing genes involved in pathways for the utilization of different sources of carbon. These populations are modified in presence of different metabolizable substrates, suggesting the coexistence of Salmonella with diverse metabolic states during the infection.

Published
2017
Full Text
View/download PDF

17. Actin Assembly around the Shigella-Containing Vacuole Promotes Successful Infection

Author

Chiara Zurzolo, Stéphanie Lebreton, Camila Valenzuela, John Bergqvist, Jost Enninga, Laura Barrio, Magdalena Gil, Sonja Kühn, Dynamique des Interactions Hôte-Pathogène - Dynamics of Host-Pathogen Interactions, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Trafic membranaire et Pathogénèse, Institut Pasteur [Paris], This work was supported by grants from the European Research Council (ERC) (EndoSubvert) to J.E., Agence Nationale de la Recherche (ANR) to J.E. (StopBugEntry) and to C.Z. (ANR-16-CE16-0019-01, NEUROTUNN). J.E. is a member of the LabEx consortium IBEID and MilieuInterieur. S.K. is a recipient of a Pasteur-Roux fellowship., ANR-15-CE15-0017,StopBugEntry,Identification des nouvelles molécules cellulaires cibles pour combattre les infections bactériennes(2015), ANR-16-CE16-0019,Neurotunn,Role des nanotubes membranaires dans la propagation d'agrégats protéiques impliqués dans les maladie neurodégénératives(2016), European Project: 682809,EndoSubvert(2017), Kuhn, S., Bergqvist, J., Gil, M., Valenzuela, C., Barrio, L., Lebreton, S., Zurzolo, C., Enninga, J., Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), and Institut Pasteur [Paris] (IP)

Subjects
0301 basic medicine, ε, fatty acylation, actin cytoskeleton, Coronin, effector proteins, Arp2/3 complex, vacuolar rupture, Nε fatty acylation, macromolecular substances, Vacuole, host-pathogen interaction, Article, General Biochemistry, Genetics and Molecular Biology, Shigella flexneri, 03 medical and health sciences, 0302 clinical medicine, [SDV.BC.IC]Life Sciences [q-bio]/Cellular Biology/Cell Behavior [q-bio.CB], Animals, host-pathogen interactions, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], N-WASP, effector protein, Actin, biology, Chemistry, intracellular pathogen, CDC42, Cofilin, biology.organism_classification, Actin cytoskeleton, Actins, 3. Good health, Cell biology, 030104 developmental biology, intracellular pathogens, Vacuoles, biology.protein, N(ε) fatty acylation, 030217 neurology & neurosurgery, Cortactin
Abstract

Summary The enteroinvasive bacterium Shigella flexneri forces its uptake into non-phagocytic host cells through the translocation of T3SS effectors that subvert the actin cytoskeleton. Here, we report de novo actin polymerization after cellular entry around the bacterium-containing vacuole (BCV) leading to the formation of a dynamic actin cocoon. This cocoon is thicker than any described cellular actin structure and functions as a gatekeeper for the cytosolic access of the pathogen. Host CDC42, TOCA-1, N-WASP, WIP, the Arp2/3 complex, cortactin, coronin, and cofilin are recruited to the actin cocoon. They are subverted by T3SS effectors, such as IpgD, IpgB1, and IcsB. IcsB immobilizes components of the actin polymerization machinery at the BCV dependent on its fatty acyltransferase activity. This represents a unique microbial subversion strategy through localized entrapment of host actin regulators causing massive actin assembly. We propose that the cocoon promotes subsequent invasion steps for successful Shigella infection., Graphical Abstract, Highlights • A thick actin cocoon forms de novo around the Shigella-containing vacuole upon entry • The effector IcsB entraps host actin regulators at the vacuole by lipidation • Cdc42, N-WASP, and the Arp2/3 complex are major actin cocoon regulators • Cocoon formation promotes subsequent Shigella niche formation and dissemination, Kühn et al. present the formation of a massive actin cocoon structure around the Shigella-containing vacuole upon bacterial entry in enterocytes. This cocoon is formed by the pathogen-induced localization of host actin regulators at the phagosome. It favors efficient cytosolic access and spread for successful infection.

Published
2020
Full Text
View/download PDF

18. Transcytosis subversion by M cell-to-enterocyte spread promotes Shigella flexneri and Listeria monocytogenes intracellular bacterial dissemination

Author

Yuen-Yan Chang, Jost Enninga, Patricia Latour-Lambert, Hugo Varet, Camille Rey, Caroline Proux, Rachel Legendre, Jean-Yves Coppée, Dynamique des Interactions Hôte-Pathogène - Dynamics of Host-Pathogen Interactions, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Hub Bioinformatique et Biostatistique - Bioinformatics and Biostatistics HUB, Transcriptome et Epigénome (PF2), Institut Pasteur [Paris] (IP), CR acknowledges a grant from Danone Research. YYC is supported through a postdoctoral fellowship from the Fondation pour la Recherche Médicale (FRM). JE acknowledges grant support from Institut Pasteur (GPF 'MCellHTLV'), the European Union (ERC CoG 'EndoSubvert'), and the ANR (Grants 'StopBugEntry' and 'AutoHostPath'). The DIHP unit is member of the IBEID and MI LabExes., We thank Felix A. Rey and John Rohde for critical comments of the manuscript. We thank Philippe Sansonetti, Javier Pizarro-Cerda and Pascale Cossart for bacterial strains. We thank Marie-Agnes Dillies for advice on single cell transcriptomic statistical analysis, Valentina Libri and the Center for Translational Science (CRT) / Cytometry and Biomarkers Unit of Technology and Service (CB UTechS) at Institut Pasteur for support in conducting this study, and Jean-Yves Tinevez and the Imagopole France–BioImaging infrastructure, supported by the French National Research Agency (ANR 10-INSB-04-01, Investments for the Future), for advice and access to microscopes. Finally, we thank Gianfranco Grompone and Muriel Derrien for enabling this study., ANR-15-CE15-0017,StopBugEntry,Identification des nouvelles molécules cellulaires cibles pour combattre les infections bactériennes(2015), ANR-15-CE15-0018,AutoHostPath,Rôles alternatifs pour les récepteurs de l'autophagie dans les interactions hôte-pathogène(2015), European Project: 682809,EndoSubvert(2017), Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), and Institut Pasteur [Paris]

Subjects
Cell Lines, Pathology and Laboratory Medicine, Shigella flexneri, White Blood Cells, Gastrointestinal tract, Animal Cells, [SDV.BC.IC]Life Sciences [q-bio]/Cellular Biology/Cell Behavior [q-bio.CB], Medicine and Health Sciences, Listeriosis, Pathogen motility, Biology (General), Caco-2 cells, Microfold cell, Fluorescence microscopy, Microscopy, 0303 health sciences, 030302 biochemistry & molecular biology, Light Microscopy, Acquired immune system, Bacterial Pathogens, 3. Good health, Cell biology, Transcytosis, Medical Microbiology, Biological Cultures, Pathogens, Cellular Types, Anatomy, Intracellular, Research Article, QH301-705.5, Virulence Factors, Immune Cells, Immunology, Biology, Research and Analysis Methods, Microbiology, 03 medical and health sciences, Immune system, Virology, Genetics, Humans, Antibody-Producing Cells, Microbial Pathogens, Molecular Biology, Tropism, Dysentery, Bacillary, 030304 developmental biology, Intracellular pathogens, B cells, Blood Cells, Bacteria, Intracellular parasite, Organisms, Biology and Life Sciences, Epithelial Cells, Cell Biology, RC581-607, biology.organism_classification, Listeria monocytogenes, Enterocytes, Parasitology, Shigella, Immunologic diseases. Allergy, Digestive System
Abstract

Microfold (M) cell host-pathogen interaction studies would benefit from the visual analysis of dynamic cellular and microbial interplays. We adapted a human in vitro M cell model to physiological bacterial infections, expression of fluorescent localization reporters and long-term three-dimensional time-lapse microscopy. This approach allows following key steps of M cell infection dynamics at subcellular resolution, from the apical onset to basolateral epithelial dissemination. We focused on the intracellular pathogen Shigella flexneri, classically reported to transcytose through M cells to initiate bacillary dysentery in humans, while eliciting poorly protective immune responses. Our workflow was critical to reveal that S. flexneri develops a bimodal lifestyle within M cells leading to rapid transcytosis or delayed vacuolar rupture, followed by direct actin motility-based propagation to neighboring enterocytes. Moreover, we show that Listeria monocytogenes, another intracellular pathogen sharing a tropism for M cells, disseminates in a similar manner and evades M cell transcytosis completely. We established that actin-based M cell-to-enterocyte spread is the major dissemination pathway for both pathogens and avoids their exposure to basolateral compartments in our system. Our results challenge the notion that intracellular pathogens are readily transcytosed by M cells to inductive immune compartments in vivo, providing a potential mechanism for their ability to evade adaptive immunity., Author summary Microfold (M) epithelial cells are important for the onset of infections and induction of immune responses in many mucosal diseases. We extended a human in vitro M cell model to apical infections, expression of fluorescent host and microbial reporters and real-time fluorescence microscopy. Focusing on the human intracellular pathogen S. flexneri, responsible for bacillary dysentery, this workflow allowed us to uncover that the bacterium can subvert the immunological sampling function of M cells by promoting a cytosolic lifestyle and spreading directly to neighboring enterocytes. This mechanism was shared with the etiologic agent of listeriosis, the intracellular pathogen L. monocytogenes and allowed both pathogens to avoid exposure to underlying immune compartments. These results may provide a mechanism for the ability of intracellular pathogens to evade adaptive immunity in vivo, emphasizing the importance of advanced studies of M cell host-pathogen interactions to understand early steps of mucosal invasion and their consequences on immunity.

Published
2020
Full Text
View/download PDF

19. Mitochondrial reactive oxygen species regulate the induction of CD8+ T cells by plasmacytoid dendritic cells

Author

Claude Leclerc, Camille Guillerey, Catherine Fayolle, Jost Enninga, Gilles Dadaglio, Marine Oberkampf, Ariel Savina, Alexandre Bobard, Sebastian Amigorena, Juliette Mouriès, Eric Ogier-Denis, Pierre Rosenbaum, Régulation Immunitaire et Vaccinologie, Institut Pasteur [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris-Est Créteil Val-de-Marne - Paris 12 (UPEC UP12), Dynamique des Interactions Hôte-Pathogène - Dynamics of Host-Pathogen Interactions, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Immunité et cancer (U932), Université Paris Descartes - Paris 5 (UPD5)-Institut Curie [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM), Laboratoire Roche [Boulogne-Billancourt], Faculté de Médecine Xavier Bichat, Centre de recherche biomédicale Bichat-Beaujon (CRB3), Université Paris Diderot - Paris 7 (UPD7)-Institut National de la Santé et de la Recherche Médicale (INSERM), This work was supported by the Ligue Nationale Contre le Cancer (Equipe Labellisée 2017). J.E. acknowledges funding by the ERC (Consolidator grant EndoSubvert) and the ANR (program StopBugEntry and AutoHostPath)., ANR-15-CE15-0017,StopBugEntry,Identification des nouvelles molécules cellulaires cibles pour combattre les infections bactériennes(2015), ANR-15-CE15-0018,AutoHostPath,Rôles alternatifs pour les récepteurs de l'autophagie dans les interactions hôte-pathogène(2015), European Project: 682809,EndoSubvert(2017), Dynamique des Interactions Hôte-Pathogène, Institut Pasteur [Paris], Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut Curie-Université Paris Descartes - Paris 5 (UPD5), Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris-Est Créteil Val-de-Marne - Paris 12 (UPEC UP12), and Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)

Subjects
0301 basic medicine, Mitochondrial ROS, Mice, 129 Strain, Science, General Physics and Astronomy, Priming (immunology), Mice, Transgenic, macromolecular substances, Mitochondrion, CD8-Positive T-Lymphocytes, Major histocompatibility complex, General Biochemistry, Genetics and Molecular Biology, Article, 03 medical and health sciences, Immune system, Cross-Priming, Antigen, [SDV.BC.IC]Life Sciences [q-bio]/Cellular Biology/Cell Behavior [q-bio.CB], Cytotoxic T cell, Animals, Antigens, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], lcsh:Science, Cells, Cultured, Mice, Knockout, Antigen Presentation, Mice, Inbred BALB C, Multidisciplinary, biology, Chemistry, Histocompatibility Antigens Class I, hemic and immune systems, General Chemistry, Dendritic Cells, 3. Good health, Cell biology, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], Mitochondria, Mice, Inbred C57BL, 030104 developmental biology, biology.protein, lcsh:Q, Reactive Oxygen Species, CD8
Abstract

Cross-presentation allows exogenous antigen presentation in association with major histocompatibility complex class I molecules, a process crucial for the priming of CD8+ T-cell responses against viruses and tumors. By contrast to conventional dendritic cells (cDC), which cross-present antigens in the steady state, plasmacytoid dendritic cells (pDC) acquire this ability only after stimulation by Toll-like receptor (TLR) ligands. The intracellular pathways accounting for this functional difference are still unknown. Here we show that the induction of cross-presentation by pDCs is regulated by mitochondria through a reactive oxygen species (ROS)-dependent mechanism, involving pH alkalization and antigen protection. The reduction of mitochondrial ROS production dramatically decreases the cross-presentation capacity of pDCs, leading to a strong reduction of their capacity to trigger CD8+ T-cell responses. Our results demonstrate the importance of mitochondrial metabolism in pDC biology, particularly for the induction of adaptive immune responses., Cross-presentation allows exogenous antigens to be presented by the major histocompatibility I pathway. Here the authors show that the inducible cross-presentation by plasmacytoid dendritic cells is modulated by mitochondria-originated reactive oxygen species.

Published
2018
Full Text
View/download PDF

20. Identification of Parameters of Host Cell Vulnerability during Salmonella Infection by Quantitative Image Analysis and Modeling

Author

Christophe Gardella, Virginie Stévenin, Jost Enninga, Jakub Voznica, Alexandre Dufour, Ilia Belotserkovsky, Dynamique des Interactions Hôte-Pathogène - Dynamics of Host-Pathogen Interactions, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), École normale supérieure - Cachan (ENS Cachan), Université Paris-Sud - Paris 11 (UP11), Analyse d'images biologiques - Biological Image Analysis (BIA), Institut de la Vision, Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), Laboratoire de Physique Statistique de l'ENS (LPS), Fédération de recherche du Département de physique de l'Ecole Normale Supérieure - ENS Paris (FRDPENS), École normale supérieure - Paris (ENS-PSL), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Centre National de la Recherche Scientifique (CNRS)-École normale supérieure - Paris (ENS-PSL), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Diderot - Paris 7 (UPD7)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), Pathogénie microbienne moléculaire, Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM), V.S. was supported by a Ph.D. fellowship from the University Paris Diderot allocated by the ENS Cachan, Université Paris-Saclay. J.E. is a member of the LabEx consortia IBEID and MilieuInterieur. J.E. also acknowledges support from the ANR (grant StopBugEntry and AutoHostPath) and the ERC (CoG EndoSubvert)., ANR-15-CE15-0017,StopBugEntry,Identification des nouvelles molécules cellulaires cibles pour combattre les infections bactériennes(2015), ANR-15-CE15-0018,AutoHostPath,Rôles alternatifs pour les récepteurs de l'autophagie dans les interactions hôte-pathogène(2015), European Project: 682809,EndoSubvert(2017), Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS)-Sorbonne Université (SU)-Institut National de la Santé et de la Recherche Médicale (INSERM), École normale supérieure - Paris (ENS Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Centre National de la Recherche Scientifique (CNRS)-École normale supérieure - Paris (ENS Paris), Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut Pasteur [Paris], Langlais, Laurence, Identification des nouvelles molécules cellulaires cibles pour combattre les infections bactériennes - - StopBugEntry2015 - ANR-15-CE15-0017 - AAPG2015 - VALID, Rôles alternatifs pour les récepteurs de l'autophagie dans les interactions hôte-pathogène - - AutoHostPath2015 - ANR-15-CE15-0018 - AAPG2015 - VALID, and Common mechanisms of host membrane trafficking subversion by intracellular pathogens to rupture bacterial containing vacuoles - EndoSubvert - 2017-01-01 - 2021-12-31 - 682809 - VALID

Subjects
0301 basic medicine, Salmonella typhimurium, Salmonella, Cell Survival, Immunology, Cell, Population, Vulnerability, Salmonella infection, [SDV.BC.IC] Life Sciences [q-bio]/Cellular Biology/Cell Behavior [q-bio.CB], Biology, medicine.disease_cause, Microbiology, Models, Biological, 03 medical and health sciences, [SDV.BC.IC]Life Sciences [q-bio]/Cellular Biology/Cell Behavior [q-bio.CB], medicine, Humans, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], Permissive, education, [SDV.BBM.BC] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], ComputingMilieux_MISCELLANEOUS, education.field_of_study, Microscopy, Cellular Microbiology: Pathogen-Host Cell Molecular Interactions, Host (biology), medicine.disease, Virology, 3. Good health, 030104 developmental biology, Infectious Diseases, medicine.anatomical_structure, Cholesterol, Parasitology, Identification (biology), Caco-2 Cells, HeLa Cells
Abstract

Salmonella targets and enters epithelial cells at permissive entry sites: some cells are more likely to be infected than others. However, the parameters that lead to host cell heterogeneity are not known. Here, we quantitatively characterized host cell vulnerability to Salmonella infection based on imaged parameters. We performed successive infections of the same host cell population followed by automated high-throughput microscopy and observed that infected cells have a higher probability of being reinfected. Establishing a predictive model, we identified two combined origins of host cell vulnerability: pathogen-induced cellular vulnerability emerging from Salmonella uptake and persisting at later stages of the infection and host cell-inherent vulnerability. We linked the host cell-inherent vulnerability with its morphological attributes, such as local cell crowding, and with host cell cholesterol content. This showed that the probability of Salmonella infection success can be forecast from morphological or molecular host cell parameters.

Published
2017
Full Text
View/download PDF

21. The Shigella type III effector IpgD recodes Ca 2+ signals during invasion of epithelial cells

Author

Ceren Simsek, Christophe Erneux, Benjamin Wacquier, Alexandre Lehman, Daniel I Aguilar, Philippe J. Sansonetti, Laurence Arbibe, Sylviane Boucherie, Kevin Denis, Guy Tran Van Nhieu, Chun Hui Sun, Geneviève Dupont, Cesar Valencia-Gallardo, Laurent Combettes, Jost Enninga, Nathalie Carayol, Centre interdisciplinaire de recherche en biologie (CIRB), Labex MemoLife, École normale supérieure - Paris (ENS Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Ecole Superieure de Physique et de Chimie Industrielles de la Ville de Paris (ESPCI Paris), Université Paris sciences et lettres (PSL)-Collège de France (CdF (institution))-École normale supérieure - Paris (ENS Paris), Université Paris sciences et lettres (PSL)-Collège de France (CdF (institution))-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM), Intéractions cellulaires et physiopathologie hépathique (Orsay, Essonne) UMRS 1174 (ICPH ), Institut National de la Santé et de la Recherche Médicale (INSERM), Unité de Chronobiologie Théorique [Brussels, Belgium], Université libre de Bruxelles (ULB), Interdisciplinary Research Institute [Brussels, Belgium] (IRIBHM), Dynamique des Interactions Hôte-Pathogène - Dynamics of Host-Pathogen Interactions, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Institut Necker Enfants-Malades (INEM - UM 111 (UMR 8253 / U1151)), Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Université Paris Descartes - Paris 5 (UPD5), Pathogénie microbienne moléculaire, Institut Pasteur [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM), Chaire Microbiologie et Maladies infectieuses, Collège de France (CdF (institution)), Université Paris sciences et lettres (PSL)-Collège de France (CdF (institution)), The work was supported by the ANR grants MITOPATHO and PATHIMMUN, grant from Tournesol program No. 31268YG, and grants from the Labex Memolife and PSL IDEX Shigaforce. CHS is a recipient of a PhD grant from the China Scholarship Council. DA is a recipient of a CONACYT grant and Labex Memolife. CV‐G was a recipient of the PSL IDEX grant. NC is a recipient of a Marie‐Curie IRG grant. BW and GD are Research Fellow and Research Director at the Belgian F.R.S.‐F.N.R.S., respectively. This work was supported by the Fonds de la Recherche Scientifique‐FNRS under Grant No. J0043.14 and by the Fonds David and Alice Van Buuren. JE was supported by an ERC Starting ('Rupteffects' No. 261166) and a Consolidator Grant ('Endosubvert' No. 682809). LC, GD, and GTVN are recipients of a WBI‐France Exchange Program (Wallonie‐Bruxelles International, Fonds de la Recherche Scientifique, Ministère Français des Affaires étrangères et européennes, Ministère de l'Enseignement supérieur et de la Recherche dans le cadre des Partenariats Hubert Curien)., ANR-12-BSV3-0017,MITOPATHO,Rôle des mitochondries durant l'invasion des cellules par les bactéries Listeria et Shigella(2012), ANR-10-MIDI-0007,PATHIMMUN,Les pathogènes nous enseignent de nouvelles stratégies antiinflammatoires(2010), ANR-10-IDEX-0001,PSL,Paris Sciences et Lettres(2010), European Project: 261166,EC:FP7:ERC,ERC-2010-StG_20091118,RUPTEFFECTS(2011), European Project: 682809,EndoSubvert(2017), Langlais, Laurence, BLANC - Rôle des mitochondries durant l'invasion des cellules par les bactéries Listeria et Shigella - - MITOPATHO2012 - ANR-12-BSV3-0017 - BLANC - VALID, MECANISMES INTEGRES DE L'INFLAMMATION - Les pathogènes nous enseignent de nouvelles stratégies antiinflammatoires - - PATHIMMUN2010 - ANR-10-MIDI-0007 - MI2 - VALID, Initiative d'excellence - Paris Sciences et Lettres - - PSL2010 - ANR-10-IDEX-0001 - IDEX - VALID, Revealing the mechanism of host membrane rupture by invasive pathogens and its role in triggering the immune response - RUPTEFFECTS - - EC:FP7:ERC2011-03-01 - 2016-02-29 - 261166 - VALID, Common mechanisms of host membrane trafficking subversion by intracellular pathogens to rupture bacterial containing vacuoles - EndoSubvert - 2017-01-01 - 2021-12-31 - 682809 - VALID, École normale supérieure - Paris (ENS-PSL), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Collège de France (CdF (institution))-Ecole Superieure de Physique et de Chimie Industrielles de la Ville de Paris (ESPCI Paris), Université Paris sciences et lettres (PSL)-École normale supérieure - Paris (ENS-PSL), Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM), Collège de France - Chaire Microbiologie et Maladies infectieuses, and Université Paris sciences et lettres (PSL)

Subjects
0301 basic medicine, MESH: Signal Transduction, MESH: Shigella flexneri, MESH: Calpain, Cell, Regulator, [SDV.BC.IC] Life Sciences [q-bio]/Cellular Biology/Cell Behavior [q-bio.CB], Shigella flexneri, [SDV.BC.IC]Life Sciences [q-bio]/Cellular Biology/Cell Behavior [q-bio.CB], [SDV.BBM.BC] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], MESH: Bacterial Proteins, biology, Effector, General Neuroscience, Calpain, Articles, Cell biology, Phosphotransferases (Alcohol Group Acceptor), medicine.anatomical_structure, MESH: Calcium, Host-Pathogen Interactions, Second messenger system, MESH: Phosphoric Monoester Hydrolases, IpgD, medicine.symptom, calpain, Signal Transduction, Programmed cell death, MESH: Dysentery, Bacillary, Inflammation, General Biochemistry, Genetics and Molecular Biology, MESH: Cell Adhesion, 03 medical and health sciences, Bacterial Proteins, Cell Adhesion, medicine, Humans, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], Cell adhesion, Molecular Biology, Dysentery, Bacillary, calcium, MESH: Humans, General Immunology and Microbiology, talin, MESH: Host-Pathogen Interactions, MESH: Phosphotransferases (Alcohol Group Acceptor), Phosphoric Monoester Hydrolases, 030104 developmental biology, MESH: HeLa Cells, biology.protein, Shigella, HeLa Cells
Abstract

The role of second messengers in the diversion of cellular processes by pathogens remains poorly studied despite their importance. Among these, Ca 2+ virtually regulates all known cell processes, including cytoskeletal reorganization, inflammation, or cell death pathways. Under physiological conditions, cytosolic Ca 2+ increases are transient and oscillatory, defining the so‐called Ca 2+ code that links cell responses to specific Ca 2+ oscillatory patterns. During cell invasion, Shigella induces atypical local and global Ca 2+ signals. Here, we show that by hydrolyzing phosphatidylinositol‐(4,5)bisphosphate, the Shigella type III effector IpgD dampens inositol‐(1,4,5)trisphosphate (InsP 3 ) levels. By modifying InsP 3 dynamics and diffusion, IpgD favors the elicitation of long‐lasting local Ca 2+ signals at Shigella invasion sites and converts Shigella ‐induced global oscillatory responses into erratic responses with atypical dynamics and amplitude. Furthermore, IpgD eventually inhibits InsP 3 ‐dependent responses during prolonged infection kinetics. IpgD thus acts as a pathogen regulator of the Ca 2+ code implicated in a versatility of cell functions. Consistent with this function, IpgD prevents the Ca 2+ ‐dependent activation of calpain, thereby preserving the integrity of cell adhesion structures during the early stages of infection.

Published
2017
Full Text
View/download PDF

22. Dynamic Growth and Shrinkage of the Salmonella-Containing Vacuole Determines the Intracellular Pathogen Niche

Author

Quentin Giai Gianetto, Yoann Le Toquin, Virginie Stévenin, Norbert Reiling, Magalie Duchateau, Chak Hon Luk, Audrey Salles, Mariette Matondo, Yuen-Yan Chang, Jost Enninga, Victoria Sohst, Dynamique des Interactions Hôte-Pathogène - Dynamics of Host-Pathogen Interactions, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Spectrométrie de Masse pour la Biologie – Mass Spectrometry for Biology (UTechS MSBio), Institut Pasteur [Paris] (IP)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Hub Bioinformatique et Biostatistique - Bioinformatics and Biostatistics HUB, BioImagerie Photonique – Photonic BioImaging (UTechS PBI), Institut Pasteur [Paris] (IP), Research group Microbial Interface Biology [Borstel Center, Germany] (RG-MIB), Research Center Borstel - Leibniz Lung Center [Germany], German Center for Infection Research - Partner Site Hamburg-Lübeck-Borstel-Riems, German Centre for Infection Research (DZIF), V. Stévenin was supported by a PhD fellowship from the University Paris Diderot allocated by the ENS Paris-Saclay, a grant from the Fondation pour la Recherche Médicale (FRM, FDT20170436843), and an extension grant from the BCI Department of Institut Pasteur. V. Sohst and N.R. were supported by a grant of the Deutsche Forschungsgemeinschaft (DFG) Excellence Cluster 306 'Inflammation at Interfaces' (MTP2). J.E. is a member of the LabEx consortia IBEID and MilieuInterieur. J.E. also acknowledges support from the ANR (grant StopBugEntry and AutoHostPath) and the ERC (CoG EndoSubvert). N.R. is very grateful for funding within the DFG priority program (SPP1580) (NR: Re1228 5-1, Re1228 5-2) and also thanks the German Center for Infection (DZIF) for financial support. UTechS PBI is part of the France–BioImaging infrastructure network (FBI) supported by the Agence Nationale pour la Recherche (ANR-10–INSB–04, Investments for the Future), and acknowledges support from ANR/FBI and the Région Ile-de-France (program 'Domaine d’Intérêt Majeur-Malinf') for the use of the Zeiss LSM 780 Elyra PS1 microscope., The authors acknowledge T. Galli, E. Boucrot, L. Johannes, and F. Garcia-del Portillo for providing tools and scientific feedback. The authors are deeply grateful to Sjaak Neefjes and his group for providing equipment, reagents, and scientific feedback to V. Stévenin during the revision period., ANR-15-CE15-0017,StopBugEntry,Identification des nouvelles molécules cellulaires cibles pour combattre les infections bactériennes(2015), ANR-15-CE15-0018,AutoHostPath,Rôles alternatifs pour les récepteurs de l'autophagie dans les interactions hôte-pathogène(2015), ANR-10-INBS-0004,France-BioImaging,Développment d'une infrastructure française distribuée coordonnée(2010), European Project: 682809,EndoSubvert(2017), Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Institut Pasteur [Paris]-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), and Institut Pasteur [Paris]

Subjects
Salmonella typhimurium, magnetic extraction, 0301 basic medicine, Salmonella, Vesicle fusion, Synaptosomal-Associated Protein 25, spacious vacuole-associated tubules (SVAT), [SDV]Life Sciences [q-bio], Salmonella enterica serovar Typhimurium, Vacuole, Biology, medicine.disease_cause, Article, General Biochemistry, Genetics and Molecular Biology, 03 medical and health sciences, Cytosol, proteomics, 0302 clinical medicine, medicine, Humans, Macropinosome, lcsh:QH301-705.5, Pathogen, membrane rupture, Qa-SNARE Proteins, membrane trafficking, vesicle fusion, biology.organism_classification, Cell biology, Salmonella-containing vacuole, 030104 developmental biology, macropinosome, lcsh:Biology (General), SNARE, Salmonella Infections, Vacuoles, Caco-2 Cells, compartmental size control, 030217 neurology & neurosurgery, Intracellular, Bacteria, HeLa Cells
Abstract

Summary Salmonella is a human and animal pathogen that causes gastro-enteric diseases. The key to Salmonella infection is its entry into intestinal epithelial cells, where the bacterium resides within a Salmonella-containing vacuole (SCV). Salmonella entry also induces the formation of empty macropinosomes, distinct from the SCV, in the vicinity of the entering bacteria. A few minutes after its formation, the SCV increases in size through fusions with the surrounding macropinosomes. Salmonella also induces membrane tubules that emanate from the SCV and lead to SCV shrinkage. Here, we show that these antipodal events are utilized by Salmonella to either establish a vacuolar niche or to be released into the cytosol by SCV rupture. We identify the molecular machinery underlying dynamic SCV growth and shrinkage. In particular, the SNARE proteins SNAP25 and STX4 participate in SCV inflation by fusion with macropinosomes. Thus, host compartment size control emerges as a pathogen strategy for intracellular niche regulation., Graphical Abstract, Highlights • The early SCV simultaneously grows and shrinks through fusion and tubule formation • SCV shrinkage promotes vacuolar rupture and cytosolic release • IAMs are enriched in the host SNAREs SNAP25 and STX4, enabling IAM-SCV fusion • Promoting SNX1-mediated tubule formation, SopB fosters SCV ruptures, When infecting epithelial cells, Salmonella can reside and replicate within vacuolar or cytosolic niches. These co-existing lifestyles provide alternative survival strategies for the bacteria. Stévenin et al. show that the balance between dynamic growth and shrinkage of the Salmonella-containing vacuole determines vacuolar maintenance or rupture and controls the pathogen niche.

Published
2019
Full Text
View/download PDF

23. The entry of Salmonella in a distinct tight compartment revealed at high temporal and ultrastructural resolution

Author

Michael Elbaum, Virginie Stévenin, Jacomine Krijnse-Locker, Katya Rechav, Jennifer Fredlund, Patricia Latour-Lambert, Jost Enninga, Adeline Mallet, Allon Weiner, José Carlos Santos, Dynamique des Interactions Hôte-Pathogène - Dynamics of Host-Pathogen Interactions, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Department of Materials and Interfaces [Rehovot, Israël], Weizmann Institute of Science [Rehovot, Israël], Microscopie ultrastructurale - Ultrapole (CITECH), Institut Pasteur [Paris], This work was funded by grants from the Pasteur‐Weizmann Foundation to J. E., by a fellowship from the Pasteur Foundation to J. F. by the FCT (SFRH/BD/51006/2010), and an Institut Pasteur PTR grant to J. C. S. by fellowships from the Pasteur‐Weizmann Foundation and the FRM to A. W., by a fellowship from the University Paris Diderot allocated by the ENS Cachan, Université Paris‐Saclay, and a grant from the FRM to V.S., and by grants from the ANR (StopBugEntry), and the European Research Council (Starting grant 'Rupteffects' and Consolidator grant 'EndoSubvert') to J. E., ANR-15-CE15-0017,StopBugEntry,Identification des nouvelles molécules cellulaires cibles pour combattre les infections bactériennes(2015), European Project: 261166,EC:FP7:ERC,ERC-2010-StG_20091118,RUPTEFFECTS(2011), European Project: 682809,EndoSubvert(2017), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), and Institut Pasteur [Paris] (IP)

Subjects
0301 basic medicine, Salmonella, Membrane ruffling, intracellular trafficking, 030106 microbiology, Immunology, Vacuole, medicine.disease_cause, Microbiology, 03 medical and health sciences, Cytosol, Virology, [SDV.BC.IC]Life Sciences [q-bio]/Cellular Biology/Cell Behavior [q-bio.CB], medicine, Humans, Compartment (development), Secretion, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], host cell invasion, biology, Salmonella enterica, Epithelial Cells, biology.organism_classification, Cell biology, correlative light electron mciroscopy, Host-Pathogen Interactions, Salmonella Infections, Ultrastructure, macropinosomes, Intracellular, HeLa Cells
Abstract

International audience; Salmonella enterica induces membrane ruffling and genesis of macropinosomes during its interactions with epithelial cells. This is achieved through the type three secretion system-1, which first mediates bacterial attachment to host cells and then injects bacterial effector proteins to alter host behaviour. Next, Salmonella enters into the targeted cell within an early membrane-bound compartment that matures into a slow growing, replicative niche called the Salmonella Containing Vacuole (SCV). Alternatively, the pathogen disrupts the membrane of the early compartment and replicate at high rate in the cytosol. Here, we show that the in situ formed macropinosomes, which have been previously postulated to be relevant for the step of Salmonella entry, are key contributors for the formation of the mature intracellular niche of Salmonella. We first clarify the primary mode of type three secretion system-1 induced Salmonella entry into epithelial cells by combining classical fluorescent microscopy with cutting edge large volume electron microscopy. We observed that Salmonella, similarly to Shigella, enters epithelial cells inside tight vacuoles rather than in large macropinosomes. We next apply this technology to visualise rupturing Salmonella containing compartments, and we use extended time-lapse microscopy to establish early markers that define which Salmonella will eventually hyper replicate. We show that at later infection stages, SCVs harbouring replicating Salmonella have previously fused with the in situ formed macropinosomes. In contrast, such fusion events could not be observed for hyper-replicating Salmonella, suggesting that fusion of the Salmonella entry compartment with macropinosomes is the first committed step of SCV formation.

Full Text
View/download PDF

Catalog

Books, media, physical & digital resources
See catalog results