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Phagosomal Rupture by Mycobacterium Tuberculosis Phagosomal Rupture by Mycobacterium tuberculosis Results in Toxicity and Host Cell Death Roxane Simeone, Alexandre Bobard, Juliane Lippmann, Wilbert Bitter, Laleh Majlessi, Roland Brosch, Jost Enninga To cite this version: Roxane Simeone, Alexandre Bobard, Juliane Lippmann, Wilbert Bitter, Laleh Majlessi, et al.. Phago- somal Rupture by Mycobacterium tuberculosis Results in Toxicity and Host Cell Death. PLoS Pathogens, Public Library of Science, 2012, 8 (2), pp.e1002507. 10.1371/journal.ppat.1002507. pasteur-01899479 HAL Id: pasteur-01899479 https://hal-pasteur.archives-ouvertes.fr/pasteur-01899479 Submitted on 19 Oct 2018 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Distributed under a Creative Commons Attribution| 4.0 International License Phagosomal Rupture by Mycobacterium tuberculosis Results in Toxicity and Host Cell Death Roxane Simeone1., Alexandre Bobard2., Juliane Lippmann2, Wilbert Bitter3, Laleh Majlessi4,5, Roland Brosch1"*, Jost Enninga2"* 1 Institut Pasteur, Unit for Integrated Mycobacterial Pathogenomics, Paris, France, 2 Institut Pasteur, Research Group ‘‘Dynamics of Host-Pathogen Interactions’’, Paris, France, 3 VU University, Molecular and Medical Microbiology, Amsterdam, The Netherlands, 4 Institut Pasteur, Unite´ de Re´gulation Immunitaire et Vaccinologie, Paris, France, 5 INSERM U1041, Paris, France Abstract Survival within macrophages is a central feature of Mycobacterium tuberculosis pathogenesis. Despite significant advances in identifying new immunological parameters associated with mycobacterial disease, some basic questions on the intracellular fate of the causative agent of human tuberculosis in antigen-presenting cells are still under debate. To get novel insights into this matter, we used a single-cell fluorescence resonance energy transfer (FRET)-based method to investigate the potential cytosolic access of M. tuberculosis and the resulting cellular consequences in an unbiased, quantitative way. Analysis of thousands of THP-1 macrophages infected with selected wild-type or mutant strains of the M. tuberculosis complex unambiguously showed that M. tuberculosis induced a change in the FRET signal after 3 to 4 days of infection, indicating phagolysosomal rupture and cytosolic access. These effects were not seen for the strains M. tuberculosisDRD1 or BCG, both lacking the ESX-1 secreted protein ESAT-6, which reportedly shows membrane-lysing properties. Complementation of these strains with the ESX-1 secretion system of M. tuberculosis restored the ability to cause phagolysosomal rupture. In addition, control experiments with the fish pathogen Mycobacterium marinum showed phagolysosomal translocation only for ESX-1 intact strains, further validating our experimental approach. Most importantly, for M. tuberculosis as well as for M. marinum we observed that phagolysosomal rupture was followed by necrotic cell death of the infected macrophages, whereas ESX-1 deletion- or truncation-mutants that remained enclosed within phagolysosomal compartments did not induce such cytotoxicity. Hence, we provide a novel mechanism how ESX-1 competent, virulent M. tuberculosis and M. marinum strains induce host cell death and thereby escape innate host defenses and favor their spread to new cells. In this respect, our results also open new research directions in relation with the extracellular localization of M. tuberculosis inside necrotic lesions that can now be tackled from a completely new perspective. Citation: Simeone R, Bobard A, Lippmann J, Bitter W, Majlessi L, et al. (2012) Phagosomal Rupture by Mycobacterium tuberculosis Results in Toxicity and Host Cell Death. PLoS Pathog 8(2): e1002507. doi:10.1371/journal.ppat.1002507 Editor: Sabine Ehrt, Weill Medical College of Cornell University, United States of America Received August 3, 2011; Accepted December 12, 2011; Published February 2, 2012 Copyright: ß 2012 Simeone et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the Institut Pasteur PTR372 (to R.B.), the European Community’s Seventh Framework Program under grant agreements nu201762 (to L.M., W.B. and R.B.) and nu241745 (to R.B.), a European Research Council Starting Grant Award nu 261166 (to J.E.), and a grant nu ANR-09-MIEN-020-01 from the Agence Nationale pour la Recherche (to J.E.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: [email protected] (RB); [email protected] (JE) . These authors contributed equally to this work. " These authors are joint senior authors on this work. Introduction particularity is thought to be linked to the capacity of the bacterium to persist and replicate within macrophages [4,5]. However, as has The intracellular localization of bacterial pathogens has impor- been recently reported by cryo-immunogold electron microscopy, tant consequences for the sensing by the host and the induced host other cellular mechanisms that involve translocation of M. immune responses. Hence, numerous studies have investigated the tuberculosis from the phagosome into the cytosol of the infected host intracellular niche of the microbes. Particularly, phagolysosome cell might play crucial roles at later time points of the infection fusion is a central cellular mechanism used by host cells to cope with process [6]. In the same study it was shown that the attenuated infection. Intracellular pathogens like Mycobacterium tuberculosis are Mycobacterium bovis Bacille de Calmette et Gue´rin vaccine (BCG), known to avoid lysosomal fusion through the manipulation of host lacking the 6kD Early Secretory Antigenic Target ESAT-6 (EsxA) signal transduction pathways [1]. Following phagocytosis by a host and its 10kD Culture Filtrate Protein partner CFP-10 (EsxB) due to macrophage and/or dendritic cell, M. tuberculosis typically resides in the deletion of the region of difference RD1 [7,8] remained within a phagosomal compartment that maintains many characteristics of the phagolysosomal compartment, similar to an M. tuberculosis CFP- an early endosome. The maturation towards an acidified phago- 10 transposon mutant, which was not detected within the host lysosome is blocked or retarded by M. tuberculosis [2,3]. This cytoplasm [6]. PLoS Pathogens | www.plospathogens.org 1 February 2012 | Volume 8 | Issue 2 | e1002507 Mtb Phagosomal Rupture Induces Host Cell Death Author Summary Results Mycobacterium tuberculosis is one of the most life- Mycobacterial strains express and display beta-lactamase threatening pathogens of all time. Despite the develop- activity ment of vaccines and antibiotics, this pathogen is still a The aim of our study was to analyze the capacity of selected major public health problem. Also the HIV epidemic has an mycobacteria to reach the cytosol during macrophage infection via important impact on the rise of M. tuberculosis infections a robust, sensitive and quantitative approach at the single cell since immunodeficient people are highly susceptible. level. For this, we had to adapt our FRET based reporter that was Commonly, M. tuberculosis has been thought to reside in previously used to monitor the intracellular localization of a membrane-bound compartment within its host cells Salmonella and Shigella species [11] (Figure S1) to the slowly during the entire infection cycle from invasion to cell death. Using a fluorescence-based method, we provide growing mycobacteria. In the host cell, membrane permeable evidence that M. tuberculosis is able to rupture its CCF-4-AM molecules diffuse freely across the cellular plasma membrane-bound compartment and gain access to the membrane, are subsequently trapped in the cytosol and excluded host cytosol, where it can elicit cell death. Furthermore, we from endosomes and other organelles by anion conversion into show that this effect is dependent on a functional type VII CCF-4 upon cytosolic esterase action. The use of this cytosolic secretion system named ESX-1. Most importantly, we were probe relies on the bacterial expression and exposure of beta- able to track the dynamics of infection to understand the lactamase. Cleavage of CCF-4 by beta-lactamase-expressing consequences of M. tuberculosis phagosomal rupture. This bacteria leads to a switch of the FRET signal from 535 nm revealed that phagosomal rupture results in cell toxicity (green) to 450 nm (blue) upon 405 nm excitation. As mycobacteria and host cell death involving necrosis. Together, our data are naturally resistant to beta-lactam antibiotics, such as provide a new angle in the worldwide fight against M. ampicillin, due to the constitutive expression of endogenous tuberculosis and could lead to new approaches in the beta-lactamases [21], we assumed that this activity could be development of
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