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16 results on '"Quentin Giai Gianetto"'

1. Bacterial Membrane Vesicles as a Novel Strategy for Extrusion of Antimicrobial Bismuth Drug in Helicobacter pylori

Author

Sumith Kumar, Christine Schmitt, Olivier Gorgette, Martial Marbouty, Magalie Duchateau, Quentin Giai Gianetto, Mariette Matondo, Jean-Michel Guigner, and Hilde De Reuse

Subjects
Helicobacter pylori, bismuth, membrane vesicles, antibiotic resistance, polyphosphate granules, chromosome conformation capture, Microbiology, QR1-502
Abstract

ABSTRACT Bacterial antibiotic resistance is a major threat to human health. A combination of antibiotics with metals is among the proposed alternative treatments. Only one such combination is successfully used in clinics; it associates antibiotics with the metal bismuth to treat infections by Helicobacter pylori. This bacterial pathogen colonizes the human stomach and is associated with gastric cancer, killing 800,000 individuals yearly. The effect of bismuth in H. pylori treatment is not well understood in particular for sublethal doses such as those measured in the plasma of treated patients. We addressed this question and observed that bismuth induces the formation of homogeneously sized membrane vesicles (MVs) with unique protein cargo content enriched in bismuth-binding proteins, as shown by quantitative proteomics. Purified MVs of bismuth-exposed bacteria were strongly enriched in bismuth as measured by inductively coupled plasma optical emission spectrometry (ICP-OES), unlike bacterial cells from which they originate. Thus, our results revealed a novel function of MVs in bismuth detoxification, where secreted MVs act as tool to discard bismuth from the bacteria. Bismuth also induces the formation of intracellular polyphosphate granules that are associated with changes in nucleoid structure. Nucleoid compaction in response to bismuth was established by immunogold electron microscopy and refined by the first chromosome conformation capture (Hi-C) analysis of H. pylori. Our results reveal that even low doses of bismuth induce profound changes in H. pylori physiology and highlight a novel defense mechanism that involves MV-mediated bismuth extrusion from the bacteria and a probable local DNA protective response where polyphosphate granules are associated with nucleoid compaction. IMPORTANCE Bacterial resistance to antibiotics is a major threat to human health. Treatments combining antibiotics with metals were proposed to circumvent this hurdle. Only one such combination is successfully used in clinics associating antibiotics with the metal bismuth to treat infections by the human pathogen Helicobacter pylori. H. pylori causes 800,000 deaths by gastric cancer yearly. How bismuth impacts H. pylori and its response to this toxic metal were ill defined. We discovered that upon bismuth exposure, H. pylori secretes membrane vesicles that are enriched in bismuth. Bismuth also induces the formation of intracellular polyphosphate granules associated with compaction of the chromosome. Upon bismuth exposure, H. pylori displays both defense and protection mechanisms, with bismuth extrusion by vesicles and shielding of the chromosome.

Published
2022
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2. RNase E and HupB dynamics foster mycobacterial cell homeostasis and fitness

Author

Anna Griego, Thibaut Douché, Quentin Giai Gianetto, Mariette Matondo, and Giulia Manina

Subjects
Environmental science, Ecology, Biological sciences, Microbiology, Science
Abstract

Summary: RNA turnover is a primary source of gene expression variation, in turn promoting cellular adaptation. Mycobacteria leverage reversible mRNA stabilization to endure hostile conditions. Although RNase E is essential for RNA turnover in several species, its role in mycobacterial single-cell physiology and functional phenotypic diversification remains unexplored. Here, by integrating live-single-cell and quantitative-mass-spectrometry approaches, we show that RNase E forms dynamic foci, which are associated with cellular homeostasis and fate, and we discover a versatile molecular interactome. We show a likely interaction between RNase E and the nucleoid-associated protein HupB, which is particularly pronounced during drug treatment and infection, where phenotypic diversity increases. Disruption of RNase E expression affects HupB levels, impairing Mycobacterium tuberculosis growth homeostasis during treatment, intracellular replication, and host spread. Our work lays the foundation for targeting the RNase E and its partner HupB, aiming to undermine M. tuberculosis cellular balance, diversification capacity, and persistence.

Published
2022
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3. Ser/Thr Kinase-Dependent Phosphorylation of the Peptidoglycan Hydrolase CwlA Controls Its Export and Modulates Cell Division in Clostridioides difficile

Author

Transito Garcia-Garcia, Sandrine Poncet, Elodie Cuenot, Thibaut Douché, Quentin Giai Gianetto, Johann Peltier, Pascal Courtin, Marie-Pierre Chapot-Chartier, Mariette Matondo, Bruno Dupuy, Thomas Candela, and Isabelle Martin-Verstraete

Subjects
Microbiology, QR1-502
Abstract

Bacterial cells are encased in a PG exoskeleton that helps to maintain cell shape and confers physical protection. To allow bacterial growth and cell separation, PG needs to be continuously remodeled by hydrolytic enzymes that cleave PG at critical sites.

Published
2021
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4. Phosphorylation-Dependent Assembly of a 14-3-3 Mediated Signaling Complex during Red Blood Cell Invasion by Plasmodium falciparum Merozoites

Author

Kunal R. More, Inderjeet Kaur, Quentin Giai Gianetto, Brandon M. Invergo, Thibault Chaze, Ravi Jain, Christéle Huon, Petra Gutenbrunner, Hendrik Weisser, Mariette Matondo, Jyoti S. Choudhary, Gordon Langsley, Shailja Singh, and Chetan E. Chitnis

Subjects
host cell invasion, host-parasite interaction, malaria, signaling, Microbiology, QR1-502
Abstract

ABSTRACT Red blood cell (RBC) invasion by Plasmodium merozoites requires multiple steps that are regulated by signaling pathways. Exposure of P. falciparum merozoites to the physiological signal of low K+, as found in blood plasma, leads to a rise in cytosolic Ca2+, which mediates microneme secretion, motility, and invasion. We have used global phosphoproteomic analysis of merozoites to identify signaling pathways that are activated during invasion. Using quantitative phosphoproteomics, we found 394 protein phosphorylation site changes in merozoites subjected to different ionic environments (high K+/low K+), 143 of which were Ca2+ dependent. These included a number of signaling proteins such as catalytic and regulatory subunits of protein kinase A (PfPKAc and PfPKAr) and calcium-dependent protein kinase 1 (PfCDPK1). Proteins of the 14-3-3 family interact with phosphorylated target proteins to assemble signaling complexes. Here, using coimmunoprecipitation and gel filtration chromatography, we demonstrate that Pf14-3-3I binds phosphorylated PfPKAr and PfCDPK1 to mediate the assembly of a multiprotein complex in P. falciparum merozoites. A phospho-peptide, P1, based on the Ca2+-dependent phosphosites of PKAr, binds Pf14-3-3I and disrupts assembly of the Pf14-3-3I-mediated multiprotein complex. Disruption of the multiprotein complex with P1 inhibits microneme secretion and RBC invasion. This study thus identifies a novel signaling complex that plays a key role in merozoite invasion of RBCs. Disruption of this signaling complex could serve as a novel approach to inhibit blood-stage growth of malaria parasites. IMPORTANCE Invasion of red blood cells (RBCs) by Plasmodium falciparum merozoites is a complex process that is regulated by intricate signaling pathways. Here, we used phosphoproteomic profiling to identify the key proteins involved in signaling events during invasion. We found changes in the phosphorylation of various merozoite proteins, including multiple kinases previously implicated in the process of invasion. We also found that a phosphorylation-dependent multiprotein complex including signaling kinases assembles during the process of invasion. Disruption of this multiprotein complex impairs merozoite invasion of RBCs, providing a novel approach for the development of inhibitors to block the growth of blood-stage malaria parasites.

Published
2020
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5. RadD Contributes to R-Loop Avoidance in Sub-MIC Tobramycin

Author

Veronica Negro, Evelyne Krin, Sebastian Aguilar Pierlé, Thibault Chaze, Quentin Giai Gianetto, Sean P. Kennedy, Mariette Matondo, Didier Mazel, and Zeynep Baharoglu

Subjects
DNA repair, R-loop, antibiotic resistance, Microbiology, QR1-502
Abstract

ABSTRACT We have previously identified Vibrio cholerae mutants in which the stress response to subinhibitory concentrations of aminoglycoside is altered. One gene identified, VC1636, encodes a putative DNA/RNA helicase, recently named RadD in Escherichia coli. Here we combined extensive genetic characterization and high-throughput approaches in order to identify partners and molecular mechanisms involving RadD. We show that double-strand DNA breaks (DSBs) are formed upon subinhibitory tobramycin treatment in the absence of radD and recBCD and that formation of these DSBs can be overcome by RNase H1 overexpression. Loss of RNase H1, or of the transcription-translation coupling factor EF-P, is lethal in the radD deletion mutant. We propose that R-loops are formed upon sublethal aminoglycoside treatment, leading to the formation of DSBs that can be repaired by the RecBCD homologous recombination pathway, and that RadD counteracts such R-loop accumulation. We discuss how R-loops that can occur upon translation-transcription uncoupling could be the link between tobramycin treatment and DNA break formation. IMPORTANCE Bacteria frequently encounter low concentrations of antibiotics. Active antibiotics are commonly detected in soil and water at concentrations much below lethal concentration. Although sub-MICs of antibiotics do not kill bacteria, they can have a major impact on bacterial populations by contributing to the development of antibiotic resistance through mutations in originally sensitive bacteria or acquisition of DNA from resistant bacteria. It was shown that concentrations as low as 100-fold below the MIC can actually lead to the selection of antibiotic-resistant cells. We seek to understand how bacterial cells react to such antibiotic concentrations using E. coli, the Gram-negative bacterial paradigm, and V. cholerae, the causative agent of cholera. Our findings shed light on the processes triggered at the DNA level by antibiotics targeting translation, how damage occurs, and what the bacterial strategies are to respond to such DNA damage.

Published
2019
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6. Biological differences between FIM2 and FIM3 fimbriae of Bordetella pertussis: not just the serotype

Author

Soraya Matczak, Valérie Bouchez, Pauline Leroux, Thibaut Douché, Nils Collinet, Annie Landier, Quentin Giai Gianetto, Sophie Guillot, Julia Chamot-Rooke, Milena Hasan, Mariette Matondo, Sylvain Brisse, Julie Toubiana, Biodiversité et Epidémiologie des Bactéries pathogènes - Biodiversity and Epidemiology of Bacterial Pathogens, Institut Pasteur [Paris] (IP)-Université Paris Cité (UPCité), 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), Centre national de Référence de la Coqueluche et autres Bordetelloses - National Reference Center for Whooping Cough and other Bordetella infections (CNR), Hub Bioinformatique et Biostatistique - Bioinformatics and Biostatistics HUB, 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), Cytometrie et Biomarqueurs – Cytometry and Biomarkers (UTechS CB), Département de Pédiatrie et maladies infectieuses [CHU Necker], CHU Necker - Enfants Malades [AP-HP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP), JT received funding from Ville de Paris as part of the EMERGENCE program. SM received funding from la Fondation pour la Recherche Médicale (FRM). SB received funding from INCEPTION program (ANR-16-CONV-0005)., and ANR-16-CONV-0005,INCEPTION,Institut Convergences pour l'étude de l'Emergence des Pathologies au Travers des Individus et des populatiONs(2016)

Subjects
Infectious Diseases, [SDV]Life Sciences [q-bio], Immunology, Microbiology, Microbes and Infection
Abstract

International audience; Introduction: Bordetella pertussis still circulates worldwide despite vaccination. Fimbriae are components of some acellular pertussis vaccines. Population fluctuations of B. pertussis fimbrial serotypes (FIM2 and FIM3) are observed, and fim3 alleles (fim3-1 [clade 1] and fim3-2 [clade 2]) mark a major phylogenetic subdivision of B. pertussis. Objectives: To compare microbiological characteristics and expressed protein profiles between fimbrial serotypes FIM2 and FIM3 and genomic clades. Methods: A total of 23 isolates were selected. Absolute protein abundance of the main virulence factors, autoagglutination and biofilm formation, bacterial survival in whole blood, induced blood cell cytokine secretion, and global proteome profiles were assessed. Results: Compared to FIM3, FIM2 isolates produced more fimbriae, less cellular pertussis toxin subunit 1 and more biofilm, but auto-agglutinated less. FIM2 isolates had a lower survival rate in cord blood, but induced higher levels of IL-4, IL-8 and IL-1 secretion. Global proteomecomparisons uncovered 15 differentially produced proteins between FIM2 and FIM3 isolates, involved in adhesion and metabolism of metals. FIM3 isolates of clade 2 produced more FIM3 and more biofilm compared to clade 1. Conclusion: FIM serotype and fim3 clades are associated with proteomic and other biological differences, which may have implications on pathogenesis and epidemiological emergence.

Published
2023

7. Listeriolysin S: A bacteriocin from

Author

Jazmín, Meza-Torres, Mickaël, Lelek, Juan J, Quereda, Martin, Sachse, Giulia, Manina, Dmitry, Ershov, Jean-Yves, Tinevez, Lilliana, Radoshevich, Claire, Maudet, Thibault, Chaze, Quentin, Giai Gianetto, Mariette, Matondo, Marc, Lecuit, Isabelle, Martin-Verstraete, Christophe, Zimmer, Hélène, Bierne, Olivier, Dussurget, Pascale, Cossart, and Javier, Pizarro-Cerdá

Subjects
Cytoplasm, Cell Membrane, Biological Sciences, microfluidic microscopy, Listeria monocytogenes, Microbiology, contact-dependent inhibition (CDI), Hemolysin Proteins, Adenosine Triphosphate, Bacteriocins, bacteriocin, Listeria monocytogenes (Lm), Listeriolysin S (LLS)
Abstract

Significance Listeria monocytogenes (Lm) is a bacterial pathogen that causes listeriosis, a foodborne disease characterized by gastroenteritis, meningitis, bacteremia, and abortions in pregnant women. The most severe human listeriosis outbreaks are associated with a subset of Lm hypervirulent clones that encode the bacteriocin Listeriolysin S (LLS), which modifies the gut microbiota and allows efficient Lm gut colonization and invasion of deeper organs. Our present work identifies the killing mechanism displayed by LLS to outcompete gut commensal bacteria, demonstrating that it induces membrane permeabilization and membrane depolarization of target bacteria. Moreover, we show that LLS is a thiazole/oxazole–modified microcin that displays a contact-dependent inhibition mechanism., Listeriolysin S (LLS) is a thiazole/oxazole–modified microcin (TOMM) produced by hypervirulent clones of Listeria monocytogenes. LLS targets specific gram-positive bacteria and modulates the host intestinal microbiota composition. To characterize the mechanism of LLS transfer to target bacteria and its bactericidal function, we first investigated its subcellular distribution in LLS-producer bacteria. Using subcellular fractionation assays, transmission electron microscopy, and single-molecule superresolution microscopy, we identified that LLS remains associated with the bacterial cell membrane and cytoplasm and is not secreted to the bacterial extracellular space. Only living LLS-producer bacteria (and not purified LLS-positive bacterial membranes) display bactericidal activity. Applying transwell coculture systems and microfluidic-coupled microscopy, we determined that LLS requires direct contact between LLS-producer and -target bacteria in order to display bactericidal activity, and thus behaves as a contact-dependent bacteriocin. Contact-dependent exposure to LLS leads to permeabilization/depolarization of the target bacterial cell membrane and adenosine triphosphate (ATP) release. Additionally, we show that lipoteichoic acids (LTAs) can interact with LLS and that LTA decorations influence bacterial susceptibility to LLS. Overall, our results suggest that LLS is a TOMM that displays a contact-dependent inhibition mechanism.

Published
2021

8. Experimental evolution links post-transcriptional regulation to Leishmania fitness gain

Author

Laura Piel, K. Shanmugha Rajan, Giovanni Bussotti, Hugo Varet, Rachel Legendre, Caroline Proux, Thibaut Douché, Quentin Giai-Gianetto, Thibault Chaze, Thomas Cokelaer, Barbora Vojtkova, Nadav Gordon-Bar, Tirza Doniger, Smadar Cohen-Chalamish, Praveenkumar Rengaraj, Céline Besse, Anne Boland, Jovana Sadlova, Jean-François Deleuze, Mariette Matondo, Ron Unger, Petr Volf, Shulamit Michaeli, Pascale Pescher, Gerald F. Späth, Parasitologie moléculaire et Signalisation / Molecular Parasitology and Signaling, Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris Cité (UPCité), The Everard and Mina Goodman Faculty of Life Sciences, Bar-Ilan University [Israël], Hub Bioinformatique et Biostatistique - Bioinformatics and Biostatistics HUB, Institut Pasteur [Paris] (IP)-Université Paris Cité (UPCité), Biomics (plateforme technologique), 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), Charles University [Prague] (CU), The Mina and Everard Goodman Faculty of Life Sciences, Centre National de Recherche en Génomique Humaine (CNRGH), Commissariat à l'énergie atomique et aux énergies alternatives (CEA), This work was supported by the Fondation de la Recherche Médicale contract FDT201805005619 (LP), the Agence Nationale pour la Recherche Labex ‘French Alliance for Parasitology and Health Care’ contract ANR-11-LABX-0024 (GFS, LP, PP, GB) and Labex ‘Integrative Biology of Emerging Infectious Diseases’ contract ANR-10-LABX-62-IBEID, the Campus France Franco-Israeli Programme Hubert Curien Maimonide 2018 (GFS and SM), the France Génomique National infrastructure, funded as part of the « Investissements d’Avenir » program managed by the Agence Nationale pour la Recherche contract ANR-10-INBS-09 (HV, RL, CP), the EU H2020 project LeiSHield-MATI - REP-778298-1 (GS), and the ERD Funds, project CePaViP (CZ.02.1.01/0.0/0.0/ 16_019/0000759) (BV, JS, PV)., We thank the CEA-CNRGH for its contribution to the sequencing costs and all the CEA-CNRGH staff who performed sample preparation and sequencing for their excellent technical assistance, ANR-11-LABX-0024,ParaFrap,Alliance française contre les maladies parasitaires(2011), ANR-10-LABX-0062,IBEID,Integrative Biology of Emerging Infectious Diseases(2010), ANR-10-INBS-0009,France-Génomique,Organisation et montée en puissance d'une Infrastructure Nationale de Génomique(2010), and European Project: 778298,H2020,H2020-MSCA-RISE-2017,LeiSHield-MATI(2018)

Subjects
Proteomics, Gene Expression Regulation, [SDV]Life Sciences [q-bio], Virology, Immunology, Genetics, Humans, Leishmaniasis, Visceral, Parasitology, Molecular Biology, Microbiology, Genomic Instability, Leishmania donovani
Abstract

The protozoan parasite Leishmania donovani causes fatal human visceral leishmaniasis in absence of treatment. Genome instability has been recognized as a driver in Leishmania fitness gain in response to environmental change or chemotherapy. How genome instability generates beneficial phenotypes despite potential deleterious gene dosage effects is unknown. Here we address this important open question applying experimental evolution and integrative systems approaches on parasites adapting to in vitro culture. Phenotypic analyses of parasites from early and late stages of culture adaptation revealed an important fitness tradeoff, with selection for accelerated growth in promastigote culture (fitness gain) impairing infectivity (fitness costs). Comparative genomics, transcriptomics and proteomics analyses revealed a complex regulatory network associated with parasite fitness gain, with genome instability causing highly reproducible, gene dosage-independent and -dependent changes. Reduction of flagellar transcripts and increase in coding and non-coding RNAs implicated in ribosomal biogenesis and protein translation were not correlated to dosage changes of the corresponding genes, revealing a gene dosage-independent, post-transcriptional mechanism of regulation. In contrast, abundance of gene products implicated in post-transcriptional regulation itself correlated to corresponding gene dosage changes. Thus, RNA abundance during parasite adaptation is controled by direct and indirect gene dosage changes. We correlated differential expression of small nucleolar RNAs (snoRNAs) with changes in rRNA modification, providing first evidence that Leishmania fitness gain in culture may be controlled by post-transcriptional and epitranscriptomic regulation. Our findings propose a novel model for Leishmania fitness gain in culture, where differential regulation of mRNA stability and the generation of modified ribosomes may potentially filter deleterious from beneficial gene dosage effects and provide proteomic robustness to genetically heterogenous, adapting parasite populations. This model challenges the current, genome-centric approach to Leishmania epidemiology and identifies the Leishmania transcriptome and non-coding small RNome as potential novel sources for the discovery of biomarkers that may be associated with parasite phenotypic adaptation in clinical settings.

Published
2022

9. Insights into amebiasis using a human 3D-intestinal model

Author

Nancy Guillén, Mariette Matondo, Julien Fernandes, Jean-Yves Coppée, Rachel Legendre, Silvia Castellanos-Castro, Hugo Varet, David Hardy, Odile Sismeiro, Arturo Aguilar-Rojas, Quentin Giai Gianetto, Jean-Christophe Olivo-Marin, Analyse d'images biologiques - Biological Image Analysis (BIA), Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Instituto Mexicano del Seguro Social [Mexico City, Mexico] (IMSS), Universidad Autónoma de la Ciudad de México (UACM), 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), Transcriptome et Epigénome (PF2), Institut Pasteur [Paris], Hub Bioinformatique et Biostatistique - Bioinformatics and Biostatistics HUB, BioImagerie Photonique – Photonic BioImaging (UTechS PBI), Neuropathologie expérimentale / Experimental neuropathology, Institut Pasteur [Paris]-Université de Paris (UP), The project has received funding from the European ERA‐NET Infect‐ERA program AMOEBAC (French National Agency for Research [ANR] grants ANR‐14‐IFEC‐0001‐01 and ANR‐14‐IFEC‐0001‐02). Biomics Platform (Institute Pasteur) is supported by France Génomique (ANR‐10‐INBS‐09‐09) and IBISA. AAR is a recipient of Research Career Development Awards from Fundación‐IMSS, México. SCC received a postdoctoral fellowship with number 173697 from the program S190‐Conacyt, 2017, Mexico., ANR-14-IFEC-0001,AMOEBAC,Entamoeba histolytica-bacterium interaction and the role of this interaction in intestinal pathogenesis.(2014), ANR-10-INBS-0009,France-Génomique,Organisation et montée en puissance d'une Infrastructure Nationale de Génomique(2010), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Institut Pasteur [Paris] (IP)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Institut Pasteur [Paris] (IP), and Institut Pasteur [Paris] (IP)-Université Paris Cité (UPCité)

Subjects
Models, Anatomic, Immunology, Virulence, Inflammation, Microbiology, Host-Parasite Interactions, Adherens junction, 03 medical and health sciences, Entamoeba histolytica, Immune system, Virology, medicine, Humans, [SDV.MP.PAR]Life Sciences [q-bio]/Microbiology and Parasitology/Parasitology, Fibroblast, 030304 developmental biology, 0303 health sciences, Microscopy, Confocal, biology, 030306 microbiology, Amebiasis, biology.organism_classification, In vitro, 3. Good health, Intestines, secretome, medicine.anatomical_structure, 3D-intestinal model, Infectious disease (medical specialty), inflammation, Dysentery, Amebic, medicine.symptom, transcriptome
Abstract

International audience; Entamoeba histolytica is the causative agent of amebiasis, an infectious disease targeting the intestine and the liver in humans. Two types of intestinal infection are caused by this parasite: silent infection, which occurs in the majority of cases, and invasive disease, which affects 10% of infected persons. To understand the intestinal pathogenic process, several in vitro models, such as cell cultures, human tissue explants or human intestine xenografts in mice, have been employed. Nevertheless, our knowledge on the early steps of amebic intestinal infection and the molecules involved during human-parasite interaction is scarce, in part due to limitations in the experimental settings. In the present work, we took advantage of tissue engineering approaches to build a three-dimensional (3D)-intestinal model that is able to replicate the general characteristics of the human colon. This system consists of an epithelial layer that develops tight and adherens junctions, a mucus layer and a lamina propria-like compartment made up of collagen containing macrophages and fibroblast. By means of microscopy imaging, omics assays and the evaluation of immune responses, we show a very dynamic interaction between E. histolytica and the 3D-intestinal model. Our data highlight the importance of several virulence markers occurring in patients or in experimental models, but they also demonstrate the involvement of under described molecules and regulatory factors in the amoebic invasive process.

Published
2020

10. Impact of the gut microbiota on the m 6 A epitranscriptome of mouse cecum and liver

Author

Pascale Cossart, Marie-Agnès Dillies, Sabrina Jabs, Amine Ghozlane, Quentin Giai Gianetto, Vincent Guérineau, Christophe Bécavin, David Touboul, Alessandro Pagliuso, Giulia Spanò, Mariette Matondo, Marie-Anne Nahori, Thibault Chaze, Anne Biton, Interactions Bactéries-Cellules (UIBC), Institut National de la Recherche Agronomique (INRA)-Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM), Hub Bioinformatique et Biostatistique - Bioinformatics and Biostatistics HUB, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Institut de Chimie des Substances Naturelles (ICSN), Institut de Chimie du CNRS (INC)-Université Paris-Saclay-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), We thank Marion Bérard and the Gnotobiology platform of the Institut Pasteur for support in conducting gnotobiology experiments, the Pasteur Biomics platforms for Transcriptomics and Epigenetics and Metagenomics for providing RNA sequencing and 16S rRNA sequencing services, Cédric Fund, Biomics Platform, C2RT, Institut Pasteur, Paris, France, supported by France Génomique (ANR-10-INBS-09-09) and IBISA, Carla Saleh and Olivier Dussurget for helpful discussions, Hugo Varet (Institut Pasteur Bionformatics and Statistics Hub) for advice concerning statistical analysis, Stevenn Volant for help with analysis of 16S sequencing data. This work was supported by grants to P.C.: European Research Council (ERC) Advanced Grant BacCellEpi (670823), ANR Investissement d’Avenir Programme (10-LABX-62-IBEID), ERANET-Infect-ERA PROANTILIS (ANR-13-IFEC-0004-02) and the Fondation Le Roch-Les Mousquetaires. S.J. was supported by a Danone Research fellowship and European Research Council (ERC) Advanced Grant BacCellEpi (670823)., ANR-10-INBS-0009,France-Génomique,Organisation et montée en puissance d'une Infrastructure Nationale de Génomique(2010), ANR-10-LABX-0062,IBEID,Integrative Biology of Emerging Infectious Diseases(2010), ANR-13-IFEC-0004,PROANTILIS,Subversive pro- and anti-inflammation trade-offs promote infection by Listeria monocytogenes(2013), European Project: 670823,H2020,ERC-2014-ADG,BacCellEpi(2015), Nahori, marie-anne, Organisation et montée en puissance d'une Infrastructure Nationale de Génomique - - France-Génomique2010 - ANR-10-INBS-0009 - INBS - VALID, Integrative Biology of Emerging Infectious Diseases - - IBEID2010 - ANR-10-LABX-0062 - LABX - VALID, ERA-NET Infect-ERA - Subversive pro- and anti-inflammation trade-offs promote infection by Listeria monocytogenes - - PROANTILIS2013 - ANR-13-IFEC-0004 - IFEC - VALID, Bacterial, cellular and epigenetic factors that control enteropathogenicity - BacCellEpi - - H20202015-10-01 - 2018-09-30 - 670823 - VALID, Institut National de la Recherche Agronomique (INRA)-Institut Pasteur [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), and Institut Pasteur [Paris]-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)

Subjects
0301 basic medicine, Adenosine, Methyltransferase, Science, [SDV]Life Sciences [q-bio], General Physics and Astronomy, Biology, Gut flora, Methylation, digestive system, Article, General Biochemistry, Genetics and Molecular Biology, Microbiology, Transcriptome, Mice, 03 medical and health sciences, Cecum, 0302 clinical medicine, fluids and secretions, Gene expression, medicine, Animals, RNA, Messenger, lcsh:Science, Multidisciplinary, Bacteria, Methyltransferases, General Chemistry, biology.organism_classification, Commensalism, Gastrointestinal Microbiome, Mice, Inbred C57BL, [SDV] Life Sciences [q-bio], stomatognathic diseases, 030104 developmental biology, medicine.anatomical_structure, Liver, Female, lcsh:Q, Microbiome, 030217 neurology & neurosurgery, Akkermansia muciniphila
Abstract

The intestinal microbiota modulates host physiology and gene expression via mechanisms that are not fully understood. Here we examine whether host epitranscriptomic marks are affected by the gut microbiota. We use methylated RNA-immunoprecipitation and sequencing (MeRIP-seq) to identify N6-methyladenosine (m6A) modifications in mRNA of mice carrying conventional, modified, or no microbiota. We find that variations in the gut microbiota correlate with m6A modifications in the cecum, and to a lesser extent in the liver, affecting pathways related to metabolism, inflammation and antimicrobial responses. We analyze expression levels of several known writer and eraser enzymes, and find that the methyltransferase Mettl16 is downregulated in absence of a microbiota, and one of its target mRNAs, encoding S-adenosylmethionine synthase Mat2a, is less methylated. We furthermore show that Akkermansia muciniphila and Lactobacillus plantarum affect specific m6A modifications in mono-associated mice. Our results highlight epitranscriptomic modifications as an additional level of interaction between commensal bacteria and their host., The intestinal microbiota modulates host physiology and gene expression via unclear mechanisms. Here, Jabs et al. show that variations in the gut microbiota correlate with N6-methyladenosine modifications of host mRNAs in the cecum and liver of mice.

Published
2020

11. Phosphorylation-Dependent Assembly of a 14-3-3 Mediated Signaling Complex During Red Blood Cell Invasion by Plasmodium falciparum Merozoites

Author

Jyoti S. Choudhary, Brandon M. Invergo, Christèle Huon, Petra Gutenbrunner, Mariette Matondo, Inderjeet Kaur, Kunal R. More, Chetan E. Chitnis, Shailja Singh, Ravi Jain, Hendrik Weisser, Thibault Chaze, Gordon Langsley, Quentin Giai Gianetto, Biologie de Plasmodium et Vaccins - Malaria Parasite Biology and Vaccines, Institut Pasteur [Paris], International Centre for Genetic Engineering and Biotechnology [New Delhi] (ICGEB), 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, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), European Bioinformatics Institute [Hinxton] (EMBL-EBI), EMBL Heidelberg, The Wellcome Trust Sanger Institute [Cambridge], Jawaharlal Nehru University (JNU), Biologie cellulaire comparative des Apicomplexes, [Institut Cochin] Departement Infection, immunité, inflammation, Institut Cochin (IC UM3 (UMR 8104 / U1016)), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université de Paris (UP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université de Paris (UP)-Institut Cochin (IC UM3 (UMR 8104 / U1016)), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université de Paris (UP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université de Paris (UP), This work was supported by a MolSigMal grant (ANR-17-CE15-0010) from Agence Nationale de la Recherche (ANR) to C.E.C. and internal funds from the Institut Pasteur. G.L. acknowledges support from the Labex ParaFrap (ANR-11-LABX-0024). K.R.M. received a short-term fellowship from the European Molecular Biology Organization (EMBO)., ANR-17-CE15-0010,MolSigMal,Evènements moléculaires de la signalisation induite par l'invasion des globules rouges par les parasites paludéens(2017), ANR-11-LABX-0024,ParaFrap,Alliance française contre les maladies parasitaires(2011), Institut Pasteur [Paris] (IP), Institut Pasteur [Paris] (IP)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), 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 National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité)-Institut Cochin (IC UM3 (UMR 8104 / U1016)), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), PORTELETTE, Virginie, Evènements moléculaires de la signalisation induite par l'invasion des globules rouges par les parasites paludéens - - MolSigMal2017 - ANR-17-CE15-0010 - AAPG2017 - VALID, and Laboratoires d'excellence - Alliance française contre les maladies parasitaires - - ParaFrap2011 - ANR-11-LABX-0024 - LABX - VALID

Subjects
Proteomics, Erythrocytes, Multiprotein complex, [SDV.BBM.BS] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Plasmodium falciparum, Protozoan Proteins, malaria, Microbiology, Host-Microbe Biology, Microneme, 03 medical and health sciences, 0302 clinical medicine, hemic and lymphatic diseases, Virology, parasitic diseases, Humans, Protein phosphorylation, Phosphorylation, Protein kinase A, host cell invasion, 030304 developmental biology, 0303 health sciences, biology, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Merozoites, Kinase, Chemistry, Phosphoproteomics, hemic and immune systems, biology.organism_classification, host-parasite interaction, QR1-502, 3. Good health, Cell biology, 14-3-3 Proteins, Signal transduction, signaling, 030217 neurology & neurosurgery, circulatory and respiratory physiology, Signal Transduction, Research Article
Abstract

Invasion of red blood cells (RBCs) by Plasmodium falciparum merozoites is a complex process that is regulated by intricate signaling pathways. Here, we used phosphoproteomic profiling to identify the key proteins involved in signaling events during invasion. We found changes in the phosphorylation of various merozoite proteins, including multiple kinases previously implicated in the process of invasion. We also found that a phosphorylation-dependent multiprotein complex including signaling kinases assembles during the process of invasion. Disruption of this multiprotein complex impairs merozoite invasion of RBCs, providing a novel approach for the development of inhibitors to block the growth of blood-stage malaria parasites., Red blood cell (RBC) invasion by Plasmodium merozoites requires multiple steps that are regulated by signaling pathways. Exposure of P. falciparum merozoites to the physiological signal of low K+, as found in blood plasma, leads to a rise in cytosolic Ca2+, which mediates microneme secretion, motility, and invasion. We have used global phosphoproteomic analysis of merozoites to identify signaling pathways that are activated during invasion. Using quantitative phosphoproteomics, we found 394 protein phosphorylation site changes in merozoites subjected to different ionic environments (high K+/low K+), 143 of which were Ca2+ dependent. These included a number of signaling proteins such as catalytic and regulatory subunits of protein kinase A (PfPKAc and PfPKAr) and calcium-dependent protein kinase 1 (PfCDPK1). Proteins of the 14-3-3 family interact with phosphorylated target proteins to assemble signaling complexes. Here, using coimmunoprecipitation and gel filtration chromatography, we demonstrate that Pf14-3-3I binds phosphorylated PfPKAr and PfCDPK1 to mediate the assembly of a multiprotein complex in P. falciparum merozoites. A phospho-peptide, P1, based on the Ca2+-dependent phosphosites of PKAr, binds Pf14-3-3I and disrupts assembly of the Pf14-3-3I-mediated multiprotein complex. Disruption of the multiprotein complex with P1 inhibits microneme secretion and RBC invasion. This study thus identifies a novel signaling complex that plays a key role in merozoite invasion of RBCs. Disruption of this signaling complex could serve as a novel approach to inhibit blood-stage growth of malaria parasites.

Published
2020
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12. Differentiation-dependent susceptibility of human muscle cells to Zika virus infection

Author

Gillian Butler-Browne, Jim Zoladek, Ingo Riederer, Quentin Giai Gianetto, Philippe Roingeard, Patricia Jeannin, Julien Burlaud-Gaillard, Pierre-Emmanuel Ceccaldi, Mariette Matondo, Valérie Choumet, Thibault Chaze, Vincent Legros, Vincent Mouly, Philippe V. Afonso, Antoine Gessain, Mariela-Natacha Gonzàlez, Epidémiologie et Physiopathologie des Virus Oncogènes (EPVO (UMR_3569 / U-Pasteur_3)), Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS)-Université de Paris (UP), Morphogénèse et antigénicité du VIH et du virus des Hépatites (MAVIVH - U1259 Inserm - CHRU Tours), Université de Tours-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre Hospitalier Régional Universitaire de Tours (CHRU TOURS), Plateforme IBISA de Microscopie Electronique [CHRU de Tours] (UNIV Tours), Centre Hospitalier Régional Universitaire de Tours (CHRU TOURS)-Université de Tours, Spectrométrie de Masse pour la Biologie – Mass Spectrometry for Biology (UTechS MSBio), Centre National de la Recherche Scientifique (CNRS)-Centre de Ressources et de Recherche Technologique - Center for Technological Resources and Research (C2RT), Institut Pasteur [Paris]-Institut Pasteur [Paris], Hub Bioinformatique et Biostatistique - Bioinformatics and Biostatistics HUB, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Institut de Myologie, Centre National de la Recherche Scientifique (CNRS)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Association française contre les myopathies (AFM-Téléthon)-Sorbonne Université (SU), Centre de recherche en myologie, Association française contre les myopathies (AFM-Téléthon)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), Fundação Oswaldo Cruz (FIOCRUZ), Réseau International des Instituts Pasteur (RIIP), Environnement et Risques infectieux - Environment and Infectious Risks (ERI), Institut Pasteur [Paris], This work was supported by a Pasteur-Fiocruz grant. VL was supported by a fellowship from the 'Fondation pour la Recherche Médicale' (FRM), IR and MG by CNPq/Ciências sem Fronteiras program, CNPq/Brazilian National Institute of Science and Technology on Neuroimmunomodulation (INCT-NIM). FOCEM - Mercosur Structural Convergence Fund 03/11. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript., Centre International de Recherche en Infectiologie - UMR (CIRI), 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), Epidémiologie et Physiopathologie des Virus Oncogènes / Oncogenic Virus Epidemiology and Pathophysiology (EPVO (UMR_3569 / U-Pasteur_3)), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Centre Hospitalier Régional Universitaire de Tours (CHRU Tours)-Université de Tours (UT), Institut Pasteur [Paris] (IP)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Centre de recherche en Myologie – U974 SU-INSERM, Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU), Institut Pasteur [Paris] (IP), Matondo, Mariette, Institut Pasteur [Paris]-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Association française contre les myopathies (AFM-Téléthon)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), UMRS974, Université Pierre et Marie Curie - Paris 6 (UPMC), Centre de Recherche en Myologie, 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), and Fundação Oswaldo Cruz / Oswaldo Cruz Foundation (FIOCRUZ)

Subjects
Proteomics, Pulmonology, RC955-962, Apoptosis, Pathology and Laboratory Medicine, Zika virus, 0302 clinical medicine, Medical Conditions, Animal Cells, [SDV.MHEP.MI]Life Sciences [q-bio]/Human health and pathology/Infectious diseases, Arctic medicine. Tropical medicine, MESH: Proteins, Chikungunya, ComputingMilieux_MISCELLANEOUS, [SDV.MHEP.ME] Life Sciences [q-bio]/Human health and pathology/Emerging diseases, [SDV.MHEP.ME]Life Sciences [q-bio]/Human health and pathology/Emerging diseases, Zika Virus Infection, Stem Cells, MESH: Proteomics, 3. Good health, Medical Microbiology, Arboviral Infections, Viral Pathogens, Interferon Type I, [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology, Public aspects of medicine, Cellular Types, MESH: Zika Virus, Microbiology, 03 medical and health sciences, Respiratory Disorders, MESH: Zika Virus Infection, Humans, Microbial Pathogens, MESH: Humans, Flaviviruses, MESH: Host-Pathogen Interactions, Public Health, Environmental and Occupational Health, Organisms, Biology and Life Sciences, Chikungunya Infection, Proteins, medicine.disease, Tropical Diseases, Virology, MESH: Cell Line, 030104 developmental biology, Biological Tissue, Interferon type I, Developmental Biology, 0301 basic medicine, RNA viruses, MESH: Cell Death, MESH: Interferon Type I, MESH: Muscle Cells, Viral Diseases, [SDV]Life Sciences [q-bio], Muscle Fibers, Skeletal, medicine.disease_cause, Virus Replication, Dengue fever, Myoblasts, Cytopathogenic Effect, Viral, Medicine and Health Sciences, Morphogenesis, Myocyte, Cytopathic effect, [SDV.MP.VIR] Life Sciences [q-bio]/Microbiology and Parasitology/Virology, MESH: Muscle Fibers, Skeletal, Cell Death, Myogenesis, Cell Differentiation, Muscle Differentiation, Infectious Diseases, Cell Processes, Viruses, Host-Pathogen Interactions, [SDV.MHEP.MI] Life Sciences [q-bio]/Human health and pathology/Infectious diseases, Disease Susceptibility, Stem cell, RA1-1270, Pathogens, Anatomy, medicine.drug, Research Article, Neglected Tropical Diseases, MESH: Cell Differentiation, 030231 tropical medicine, Muscle Tissue, MESH: Disease Susceptibility, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, MESH: Stem Cells, Biology, Cell Line, medicine, MESH: Myoblasts, Muscle Cells, MESH: Virus Replication, Cell Biology, Zika Virus, biology.organism_classification, MESH: Cytopathogenic Effect, Viral, Respiratory Infections
Abstract

Muscle cells are potential targets of many arboviruses, such as Ross River, Dengue, Sindbis, and chikungunya viruses, that may be involved in the physiopathological course of the infection. During the recent outbreak of Zika virus (ZIKV), myalgia was one of the most frequently reported symptoms. We investigated the susceptibility of human muscle cells to ZIKV infection. Using an in vitro model of human primary myoblasts that can be differentiated into myotubes, we found that myoblasts can be productively infected by ZIKV. In contrast, myotubes were shown to be resistant to ZIKV infection, suggesting a differentiation-dependent susceptibility. Infection was accompanied by a caspase-independent cytopathic effect, associated with paraptosis-like cytoplasmic vacuolization. Proteomic profiling was performed 24h and 48h post-infection in cells infected with two different isolates. Proteome changes indicate that ZIKV infection induces an upregulation of proteins involved in the activation of the Interferon type I pathway, and a downregulation of protein synthesis. This work constitutes the first observation of primary human muscle cells susceptibility to ZIKV infection, and differentiation-dependent restriction of infection from myoblasts to myotubes. Since myoblasts constitute the reservoir of stem cells involved in reparation/regeneration in muscle tissue, the infection of muscle cells and the viral-induced alterations observed here could have consequences in ZIKV infection pathogenesis., Author summary Muscle cells are potential targets of many arboviruses, such as Ross River, Dengue, Sindbis, and chikungunya viruses, and may be involved in the disease manifestation. During the recent outbreak of Zika virus (ZIKV), myalgia was one of the most frequently reported symptoms. We investigated the susceptibility of human muscle cells to ZIKV infection. Using an in vitro model of human muscle stem cells (myoblasts) that can be differentiated into differentiated muscle cells (myotubes), we found that myoblasts can be infected by ZIKV. In contrast, myotubes were shown to be resistant to ZIKV infection. Infection induced the death of infected cells. Protein levels 24h and 48h post-infection indicate that ZIKV infection induces an upregulation of proteins involved in the activation of the Interferon type I pathway, and a downregulation of protein synthesis. This work constitutes the first observation of primary human muscle cells susceptibility to ZIKV infection, muscle stem cells being susceptible while differentiated muscle cells are resistant. Since myoblasts constitute the reservoir of stem cells involved in reparation/regeneration in muscle tissue, the infection of muscle cells and the viral-induced alterations observed here could have consequences during ZIKV infection.

Published
2020

13. 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
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14. RadD Contributes to R-Loop Avoidance in Sub-MIC Tobramycin

Author

Quentin Giai Gianetto, Didier Mazel, Sean Kennedy, Mariette Matondo, Thibault Chaze, Zeynep Baharoglu, Evelyne Krin, Sebastian Aguilar Pierlé, Veronica Negro, Plasticité du Génome Bactérien - Bacterial Genome Plasticity (PGB), Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Collège doctoral [Sorbonne universités], Sorbonne Université (SU), 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, Centre de Bioinformatique, Biostatistique et Biologie Intégrative (C3BI), Work in the Mazel lab is funded by the Institut Pasteur, the Centre National de la Recherche Scientifique (CNRS-UMR3525), the French National Research Agency (ANR Unibac ANR-17-CE13-0010-01), and the French Government’s Investissement d’Avenir program, Laboratoire d’Excellence 'Integrative Biology of Emerging Infectious Diseases'(grant no. ANR-10-LABX-62-IBEID). V.N. was funded by fellowships from DIM Malinf 140051 (Conseil régional d’Île-de-France) and FRM grant DBF20160635736. S.A.P. was supported by a postdoctoral fellowship from the Roux Foundation (Institut Pasteur), the FRM grant DBF20160635736, and the ANR Unibac ANR-17-CE13-0010-01, We thank Christophe Thomas for his help with protein purification, Melis Asal and Ricardo Albino Camacho for their participation in strain constructions, and Julie Lambert for UV tests. We thank Micheline Fromont for the yeast strains and plasmids and for her help with the two-hybrid experiment. We are grateful to Bénédicte Michel for the gift of several E. coli strains. We thank Melanie Blokesch for the Vibrio cholerae WT strain used in this study. We thank Sébastien Fleurier for advice for TUNEL experiments., ANR-17-CE13-0010,UniBac,Etude de l'émergence de la résistance aux antibiotiques et de la diversification génétique chez les bactéries à l'échelle de cellules uniques.(2017), ANR-10-LABX-0062,IBEID,Integrative Biology of Emerging Infectious Diseases(2010), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Collège Doctoral, and Institut Pasteur [Paris] (IP)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)

Subjects
DNA, Bacterial, Molecular Biology and Physiology, antibiotic resistance, DNA repair, DNA damage, [SDV]Life Sciences [q-bio], Microbial Sensitivity Tests, medicine.disease_cause, Microbiology, 03 medical and health sciences, [SDV.BBM.GTP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN], Virology, Escherichia coli, medicine, DNA Breaks, Double-Stranded, Vibrio cholerae, 030304 developmental biology, Adenosine Triphosphatases, RecBCD, 0303 health sciences, Microbial Viability, biology, 030306 microbiology, Chemistry, Escherichia coli Proteins, Aminoglycoside, biology.organism_classification, QR1-502, Anti-Bacterial Agents, 3. Good health, Homologous Recombination Pathway, Tobramycin, R-loop, R-Loop Structures, Bacteria, Research Article
Abstract

Bacteria frequently encounter low concentrations of antibiotics. Active antibiotics are commonly detected in soil and water at concentrations much below lethal concentration. Although sub-MICs of antibiotics do not kill bacteria, they can have a major impact on bacterial populations by contributing to the development of antibiotic resistance through mutations in originally sensitive bacteria or acquisition of DNA from resistant bacteria. It was shown that concentrations as low as 100-fold below the MIC can actually lead to the selection of antibiotic-resistant cells. We seek to understand how bacterial cells react to such antibiotic concentrations using E. coli, the Gram-negative bacterial paradigm, and V. cholerae, the causative agent of cholera. Our findings shed light on the processes triggered at the DNA level by antibiotics targeting translation, how damage occurs, and what the bacterial strategies are to respond to such DNA damage., We have previously identified Vibrio cholerae mutants in which the stress response to subinhibitory concentrations of aminoglycoside is altered. One gene identified, VC1636, encodes a putative DNA/RNA helicase, recently named RadD in Escherichia coli. Here we combined extensive genetic characterization and high-throughput approaches in order to identify partners and molecular mechanisms involving RadD. We show that double-strand DNA breaks (DSBs) are formed upon subinhibitory tobramycin treatment in the absence of radD and recBCD and that formation of these DSBs can be overcome by RNase H1 overexpression. Loss of RNase H1, or of the transcription-translation coupling factor EF-P, is lethal in the radD deletion mutant. We propose that R-loops are formed upon sublethal aminoglycoside treatment, leading to the formation of DSBs that can be repaired by the RecBCD homologous recombination pathway, and that RadD counteracts such R-loop accumulation. We discuss how R-loops that can occur upon translation-transcription uncoupling could be the link between tobramycin treatment and DNA break formation.

Published
2019

15. Assembly and disassembly of Aspergillus fumigatus conidial rodlets

Author

Isabel Valsecchi, Jennifer I. Lai, Emmanuel Stephen-Victor, Ariane Pillé, Audrey Beaussart, Victor Lo, Chi L.L. Pham, Vishukumar Aimanianda, Ann H. Kwan, Magalie Duchateau, Quentin Giai Gianetto, Mariette Matondo, Melanie Lehoux, Donald C. Sheppard, Yves F. Dufrene, Jagadeesh Bayry, J. Iñaki Guijarro, Margaret Sunde, Jean-Paul Latgé, Aspergillus, Institut Pasteur [Paris] (IP), Plateforme technologique de RMN biologique - Biological NMR Technological Platform, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), The University of Sydney, Centre de Recherche des Cordeliers (CRC (UMR_S_1138 / U1138)), École Pratique des Hautes Études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Université Paris Diderot - Paris 7 (UPD7)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU), Université Catholique de Louvain = Catholic University of Louvain (UCL), Laboratoire Interdisciplinaire des Environnements Continentaux (LIEC), Institut Ecologie et Environnement (INEE), Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)-Institut national des sciences de l'Univers (INSU - CNRS)-Observatoire Terre et Environnement de Lorraine (OTELo), Institut national des sciences de l'Univers (INSU - CNRS)-Université de Lorraine (UL)-Centre National de la Recherche Scientifique (CNRS)-Institut national des sciences de l'Univers (INSU - CNRS)-Université de Lorraine (UL)-Centre National de la Recherche Scientifique (CNRS), Plateforme de Protéomique / Proteomics platform, 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)-Institut Pasteur [Paris] (IP)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Centre de Bioinformatique, Biostatistique et Biologie Intégrative (C3BI), McGill University = Université McGill [Montréal, Canada], This work was supported partially by the grant Aspergillus by Aviesan, the grant DEQ20150331722 LATGE Equipe FRM 2015 and the French Australian FAST project FR110012. This work was additionally funded by the French ANR (HYDROPHOBIN ANR-10-BLAN-1309 and FUNHYDRO ANR-16-CE110020-01) and the Institut Pasteur PTR 529. AP was a recipient of an Université Pierre et Marie Curie (UPMC) fellowship. The NMR spectrometer was partially supported by the Région Ile de France (SESAME grant)., ANR-10-BLAN-1309,HYDROPHOBIN,HYDROPHOBINE DES CONIDIES d'Aspergillus fumigatus : ANALYSE STRUCTURALE ET REPONSE IMMUNE(2010), ANR-16-CE11-0020,FUNHYDRO,Amyloïdes fonctionnels formés par les hydrophobines du pathogène fongique Aspergillus fumigatus(2016), Institut Pasteur [Paris], Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), École pratique des hautes études (EPHE), 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), UCL - SST/LIBST - Louvain Institute of Biomolecular Science and Technology, École pratique des hautes études (EPHE)-Université Paris Diderot - Paris 7 (UPD7)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU), Université Catholique de Louvain (UCL), Centre National de la Recherche Scientifique (CNRS)-Centre de Ressources et de Recherche Technologique - Center for Technological Resources and Research (C2RT), Institut Pasteur [Paris]-Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS)-Centre de Ressources et de Recherche Technologique - Center for Technological Resources and Research (C2RT), Institut Pasteur [Paris]-Institut Pasteur [Paris], and McGill University

Subjects
hydrophobins, [SDV.IMM.II]Life Sciences [q-bio]/Immunology/Innate immunity, Applied Microbiology and Biotechnology, Microbiology, Article, 03 medical and health sciences, [SDV.MHEP.MI]Life Sciences [q-bio]/Human health and pathology/Infectious diseases, Amyloids, Hydrophobins, Rodlets, lcsh:QH573-671, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], [SDU.ENVI]Sciences of the Universe [physics]/Continental interfaces, environment, skin and connective tissue diseases, Molecular Biology, ComputingMilieux_MISCELLANEOUS, [SDV.MP.MYC]Life Sciences [q-bio]/Microbiology and Parasitology/Mycology, 030304 developmental biology, rodlets, 0303 health sciences, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], rodA, lcsh:Cytology, Aspergillus fumigatus, 030302 biochemistry & molecular biology, fungi, aspergillus fumigatus, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, Cell Biology, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], [SDV.BBM.BP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biophysics, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], RodA
Abstract

The rodlet structure present on the Aspergillus fumigatus conidial surface hides conidia from immune recognition. In spite of the essential biological role of the rodlets, the molecular basis for their self-assembly and disaggregation is not known. Analysis of the soluble forms of conidia-extracted and recombinant RodA by NMR spectroscopy has indicated the importance of disulfide bonds and identified two dynamic regions as likely candidates for conformational change and intermolecular interactions during conversion of RodA into the amyloid rodlet structure. Point mutations introduced into the RODA sequence confirmed that (1) mutation of a single cysteine was sufficient to block rodlet formation on the conidial surface and (2) both presumed amyloidogenic regions were needed for proper rodlet assembly. Mutations in the two putative amyloidogenic regions retarded and disturbed, but did not completely inhibit, the formation of the rodlets in vitro and on the conidial surface. Even in a disturbed form, the presence of rodlets on the surface of the conidia was sufficient to immunosilence the conidium. However, in contrast to the parental conidia, long exposure of mutant conidia lacking disulfide bridges within RodA or expressing RodA carrying the double (I115S/I146G) mutation activated dendritic cells with the subsequent secretion of proinflammatory cytokines. The immune reactivity of the RodA mutant conidia was not due to a modification in the RodA structure, but to the exposure of different pathogen-associated molecular patterns on the surface as a result of the modification of the rodlet surface layer. The full degradation of the rodlet layer, which occurs during early germination, is due to a complex array of cell wall bound proteases. As reported earlier, this loss of the rodlet layer lead to a strong anti-fumigatus host immune response in mouse lungs. Keywords: Aspergillus fumigatus, Hydrophobins, Rodlets, RodA, Amyloids

Published
2019

16. CotL, a new morphogenetic spore coat protein of Clostridium difficile

Author

Bruno Dupuy, Carolina Alves Feliciano, Mariette Matondo, Isabelle Martin-Verstraete, Thibaut Douché, Quentin Giai Gianetto, Pathogénèse des Bactéries Anaérobies / Pathogenesis of Bacterial Anaerobes (PBA (U-Pasteur_6)), Institut Pasteur [Paris]-Université Paris Diderot - Paris 7 (UPD7), 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, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Research in this study for the CAF fellowship was funded by the Institut Pasteur and ITN Marie Curie, Clospore (H2020‐MSCA‐ITN‐2014 642068)., The authors thank Maryse Moya‐Nilges for performing transmission electron microscopy, Jost Enniga and Giulia Manina for access to the fluorescence microscope, Adriano Henriques for providing the plasmids carrying the SNAPCd reporter gene and the antibody raised against CdeM, Aimee Shen for helpful discussion and providing the antibodies, Nigel Minton for the ClosTron system and Julian Garneau for the bioinformatics analysis., Centre National de la Recherche Scientifique (CNRS)-Centre de Ressources et de Recherche Technologique - Center for Technological Resources and Research (C2RT), Institut Pasteur [Paris]-Institut Pasteur [Paris], Institut Pasteur [Paris] (IP)-Université Paris Diderot - Paris 7 (UPD7), Institut Pasteur [Paris] (IP)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), and Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)

Subjects
Coat, [SDV]Life Sciences [q-bio], Mutant, Sigma Factor, Biology, Microbiology, 03 medical and health sciences, chemistry.chemical_compound, Bacterial Proteins, Sigma factor, Cell Wall, RNA polymerase, Gene, Ecology, Evolution, Behavior and Systematics, 030304 developmental biology, Spores, Bacterial, 0303 health sciences, 030306 microbiology, Clostridioides difficile, fungi, Exosporium, Clostridium difficile, 3. Good health, Spore, [SDV.MP]Life Sciences [q-bio]/Microbiology and Parasitology, chemistry, Muramidase
Abstract

International audience; The strict anaerobe Clostridium difficile is the most common cause of antibiotic-associated diarrhoea. The oxygen-resistant C. difficile spores play a central role in the infectious cycle, contributing to transmission, infection and recurrence. The spore surface layers, the coat and exosporium, enable the spores to resist physical and chemical stress. However, little is known about the mechanisms of their assembly. In this study, we characterized a new spore protein, CotL, which is required for the assembly of the spore coat. The cotL gene was expressed in the mother cell compartment under the dual control of the RNA polymerase sigma factors, σE and σK . CotL was localized in the spore coat, and the spores of the cotL mutant had a major morphologic defect at the level of the coat/exosporium layers. Therefore, the mutant spores contained a reduced amount of several coat/exosporium proteins and a defect in their localization in sporulating cells. Finally, cotL mutant spores were more sensitive to lysozyme and were impaired in germination, a phenotype likely to be associated with the structurally altered coat. Collectively, these results strongly suggest that CotL is a morphogenetic protein essential for the assembly of the spore coat in C. difficile.

Published
2018

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