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13 results on '"ANR-10-EQPX-0004,Imaginex BioMed,Plateau de microscopie de criblage à haut débit et d'analyse à très haute résolution(2010)"'

1. Optineurin links Hace1-dependent Rac ubiquitylation to integrin-mediated mechanotransduction to control bacterial invasion and cell division

Author

Laurent Gagnoux, Serena Petracchini, Benoit Ladoux, Amel Mettouchi, Martial Balland, Mads Daugaard, Mukund Gupta, Emmanuel Lemichez, Francis Impens, Poul H. Sorensen, Sébastien Janel, Elisa Vitiello, Anne Doye, Frank Lafont, Florian Fage, Atef Asnacios, Teresa Mendes Maia, Daniel Hamaoui, Jerome Gilleron, Centre méditerranéen de médecine moléculaire (C3M), Université Nice Sophia Antipolis (1965 - 2019) (UNS), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Côte d'Azur (UCA), Equipe labellisée Ligue contre le Cancer, Matière et Systèmes Complexes (MSC), Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Laboratoire Interdisciplinaire de Physique [Saint Martin d’Hères] (LIPhy ), Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes (UGA), Cellular Microbiology and Physics of Infection Group [Lille] (CMPI), Centre d’Infection et d’Immunité de Lille - INSERM U 1019 - UMR 9017 - UMR 8204 (CIIL), Institut Pasteur de Lille, Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Lille-Centre Hospitalier Régional Universitaire [Lille] (CHRU Lille)-Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur de Lille, Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Lille-Centre Hospitalier Régional Universitaire [Lille] (CHRU Lille)-Centre National de la Recherche Scientifique (CNRS), Institut Jacques Monod (IJM (UMR_7592)), Vlaams Instituut voor Biotechnologie [Ghent, Belgique] (VIB), Universiteit Gent = Ghent University (UGENT), Institut de Biologie Valrose (IBV), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Côte d'Azur (UCA), Vancouver Prostate Centre [Vancouver General Hospital] (VPC), Vancouver General Hospital, University of British Columbia (UBC), Toxines bactériennes - Bacterial Toxins, Université Paris Cité (UPCité)-Microbiologie Intégrative et Moléculaire (UMR6047), Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), This study was supported by institutional INSERM and Institut Pasteur fundings, 'Investments for the Future' LabEx SIGNALIFE ANR-11-LABX-0028-01 and grants from the Ligue Nationale contre le Cancer (LNCC labellisation and RS20/75-63), Association pour la Recherche contre le Cancer (ARC PJA20191209650), EPIC-XS, project number 823839, funded by the Horizon 2020 programme of the European Union (PRC-5074) and FRM-Piraud prize to E.L., Bio-SPC 'Contrat Doctoral' PhD fellowship to S.P., LNCC PhD fellowship to D.H., the Vancouver Prostate Centre to M.D., and Team Finn and The Ride to Concur Cancer to P.H.S., Fundings from ANR to A.A. ('ImmunoMeca' ANR-12-BSV5-0007-01, 'Initiatives d’excellence' Idex ANR-11-IDEX-0005-02, and 'Labex Who Am I?' ANR-11-LABX-0071)and to F.L. (ANR-10-EQPX-04-01), and from FEDER (12001407) to F.L. B.L. acknowledges financial supports from the Mechanobiology Institute, the European Research Council under the European Union's 7th Framework Programme (FP7/2007-2013) / ERC n° 617233 and NUS-USPC collaborative program., ANR-11-LABX-0028,SIGNALIFE,Réseau d'Innovation sur les Voies de Signalisation en Sciences de la Vie(2011), ANR-12-BSV5-0007,ImmunoMeca,Caractérisation du rôle de la mécanique dans l'immunité: vers un modèle intégré de l'activation des cellules T(2012), ANR-11-IDEX-0005,USPC,Université Sorbonne Paris Cité(2011), ANR-11-LABX-0071,WHO AM I,Determinants de l'Identité : de la molécule à l'individu(2011), ANR-10-EQPX-0004,Imaginex BioMed,Plateau de microscopie de criblage à haut débit et d'analyse à très haute résolution(2010), European Project: 617233,EC:FP7:ERC,ERC-2013-CoG,DURACELL(2014), Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS)-Université de Paris (UP), Université Nice Sophia Antipolis (... - 2019) (UNS), Matière et Systèmes Complexes (MSC (UMR_7057)), Centre National de la Recherche Scientifique (CNRS)-Université de Paris (UP), Centre National de la Recherche Scientifique (CNRS)-Centre Hospitalier Régional Universitaire [Lille] (CHRU Lille)-Université de Lille-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut Pasteur de Lille, Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP)-Centre National de la Recherche Scientifique (CNRS)-Centre Hospitalier Régional Universitaire [Lille] (CHRU Lille)-Université de Lille-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut Pasteur de Lille, Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP), National University of Singapore (NUS), Université de Paris (UP)-Centre National de la Recherche Scientifique (CNRS), Universiteit Gent = Ghent University [Belgium] (UGENT), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Côte d'Azur (UCA)-Centre National de la Recherche Scientifique (CNRS), and Institut Pasteur [Paris]-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité)

Subjects
rac1 GTP-Binding Protein, Integrins, Cell division, Mechanotransduction, Ubiquitin-Protein Ligases, [SDV]Life Sciences [q-bio], Integrin, General Physics and Astronomy, RAC1, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, Mechanotransduction, Cellular, General Biochemistry, Genetics and Molecular Biology, Extracellular matrix, Focal adhesion, Integrin signalling, 03 medical and health sciences, 0302 clinical medicine, Medicine and Health Sciences, Cyclin D1, 030304 developmental biology, Optineurin, 0303 health sciences, CNF1 toxin, Multidisciplinary, biology, Chemistry, Ubiquitination, Biology and Life Sciences, Cell migration, General Chemistry, Hostpathogen interaction, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, Cell biology, E3 ubiquitin ligase, biology.protein, Rac1 GTPase, Cell Division, 030217 neurology & neurosurgery, Tumour suppressor
Abstract

Extracellular matrix (ECM) elasticity is perceived by cells via focal adhesion structures, which transduce mechanical cues into chemical signalling to conform cell behavior. Although the contribution of ECM compliance to the control of cell migration or division is extensively studied, little is reported regarding infectious processes. We study this phenomenon with the extraintestinal Escherichia coli pathogen UTI89. We show that UTI89 takes advantage, via its CNF1 toxin, of integrin mechanoactivation to trigger its invasion into cells. We identify the HACE1 E3 ligase-interacting protein Optineurin (OPTN) as a protein regulated by ECM stiffness. Functional analysis establishes a role of OPTN in bacterial invasion and integrin mechanical coupling and for stimulation of HACE1 E3 ligase activity towards the Rac1 GTPase. Consistent with a role of OPTN in cell mechanics, OPTN knockdown cells display defective integrin-mediated traction force buildup, associated with limited cellular invasion by UTI89. Nevertheless, OPTN knockdown cells display strong mechanochemical adhesion signalling, enhanced Rac1 activation and increased cyclin D1 translation, together with enhanced cell proliferation independent of ECM stiffness. Together, our data ascribe a new function to OPTN in mechanobiology. Uropathogenic strains of Escherichia coli (UPEC) are a leading cause of urinary tract infections (UTIs) and invasion involves Rho GTPase members, notably Rac1, to drive actin cytoskeleton rearrangement leading to engulfment. Here, Petracchini et al. provide evidence of an ECM stiffnessmodulated role of Optineurin (OPTN), which regulates HACE1-dependant Rac1 activity and thus controls integrinmediated mechanotransduction and bacterial invasion.

Published
2022
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2. Large scale screening discovers clofoctol as an inhibitor of SARS-CoV-2 replication that reduces COVID-19-like pathology

Author

Belouzard, Sandrine, Machelart, Arnaud, Sencio, Valentin, Vausselin, Thibaut, Hoffmann, Eik, Deboosere, Nathalie, Rouillé, Yves, Desmarets, Lowiese, Séron, Karin, Danneels, Adeline, Robil, Cyril, Belloy, Loïc, Moreau, Camille, Piveteau, Catherine, Biela, Alexandre, Vandeputte, Alexandre, Heumel, Séverine, Deruyter, Lucie, Dumont, Julie, Leroux, Florence, Engelmann, Ilka, Kazali Alidjinou, Enagnon, Hober, Didier, Brodin, Priscille, Beghyn, Terence, Trottein, François, Déprez, Benoît, Dubuisson, Jean, Centre d’Infection et d’Immunité de Lille - INSERM U 1019 - UMR 9017 - UMR 8204 (CIIL), Institut Pasteur de Lille, Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Lille-Centre Hospitalier Régional Universitaire [Lille] (CHRU Lille)-Centre National de la Recherche Scientifique (CNRS), Apteeus [Lille], Plateformes Lilloises en Biologie et Santé - UAR 2014 - US 41 (PLBS), Médicaments et molécules pour agir sur les Systèmes Vivants - U 1177 (M2SV), Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Lille, Pathogenèse virale du diabète de type 1 - ULR 3610 (Laboratoire de Virologie), Université de Lille-Centre Hospitalier Régional Universitaire [Lille] (CHRU Lille), This work was supported by the Institut Pasteur de Lille, the Fondation pour la Recherche Médicale (FRM) and the Agence Nationale de la Recherche (ANR) (Project FRM_ANR Flash 20 ANTICOV), the Centre National de la Recherche Scientifique (CNRS, COVIDprogram) and the I-Site Foundation (I-Site_Covid20_ANTI-SARS2).The platform used in this work was supported by the European Union (ERC-STG INTRACELLTB grant 260901), the ANR (ANR-10-EQPX-04-01), the 'Fonds Européen de Développement Régional' (Feder) (12001407 [D-AL] EquipEx ImagInEx BioMed), CPER-CTRL (Centre Transdisciplinaire de Recherche sur la Longévité) and the Région Nord-Pas-de-Calais (convention 12000080)., ANR-21-COVR-0022,ANTICOV,Valeur pronostique des auto-anticorps ANTi-Interféron de type I chez les patients pris en charge pour un syndrome de détresse respiratoire aiguë associé au COVID-19 : une étude de cohorte observationnelle multicentriq(2021), ANR-10-EQPX-0004,Imaginex BioMed,Plateau de microscopie de criblage à haut débit et d'analyse à très haute résolution(2010), European Project: 260901,EC:FP7:ERC,ERC-2010-StG_20091118,INTRACELLTB(2010), Centre National de la Recherche Scientifique (CNRS)-Centre Hospitalier Régional Universitaire [Lille] (CHRU Lille)-Université de Lille-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut Pasteur de Lille, Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP), Plateformes Lilloises en Biologie et Santé - UMS 2014 - US 41 (PLBS), Dubuisson, Jean, Valeur pronostique des auto-anticorps ANTi-Interféron de type I chez les patients pris en charge pour un syndrome de détresse respiratoire aiguë associé au COVID-19 : une étude de cohorte observationnelle multicentriq - - ANTICOV2021 - ANR-21-COVR-0022 - COVID-19 - VALID, Equipements d'excellence - Plateau de microscopie de criblage à haut débit et d'analyse à très haute résolution - - Imaginex BioMed2010 - ANR-10-EQPX-0004 - EQPX - VALID, and A Chemical Genomics Approach of Intracellular Mycobacterium tuberculosis Towards Defining Specific Host Pathogen Interactions - INTRACELLTB - - EC:FP7:ERC2010-12-01 - 2015-11-30 - 260901 - VALID

Subjects
[SDV.MP.VIR] Life Sciences [q-bio]/Microbiology and Parasitology/Virology, [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology
Abstract

The fastest way to implement a treatment against a new rapidly emerging viral disease consists in screening the potential antiviral activity of drugs approved for human use. This has the advantage of shortening regulatory preclinical development steps. Here, we screened a library of drug compounds, already registered in one or several geographical areas, to identify those exhibiting antiviral activity against SARS-CoV-2 with relevant potency. Of the 1,942 compounds tested, 21 exhibited a substantial antiviral activity in Vero-81 cells. Among them, clofoctol, an antibacterial drug used for the treatment of bacterial respiratory tract infections, was further investigated due to its favorable safety profile and its pharmacokinetic properties. Notably, the peak concentration of clofoctol that can be achieved in human lungs is more than 20 times higher than its IC 95 measured against SARS-CoV-2 in human pulmonary cells. Mechanistically, this compound inhibits SARS-CoV-2 at a post-entry step by specifically blocking translation initiation of viral RNA. Lastly, therapeutic treatment of human ACE2 receptor transgenic mice decreased viral load, reduced inflammatory gene expression and improved pulmonary pathology. Altogether, these data strongly support clofoctol as a therapeutic candidate for the treatment of COVID-19 patients.

Published
2021

3. The fate of Hepatitis E virus capsid protein is regulated by an Arginine-Rich Motif

Author

Hervouet, Kévin, Ferrié, Martin, Ankavay, Maliki, Montpellier, Claire, Camuzet, Charline, Alexandre, Virginie, Dembélé, Aïcha, Lecoeur, Cécile, Foe, Arnold Thomas, Bouquet, Peggy, Hot, David, Vausselin, Thibaut, Saliou, Jean-Michel, Salomé-Desnoulez, Sophie, Vandeputte, Alexandre, Marsollier, Laurent, Brodin, Priscille, Dreux, Marlène, Rouillé, Yves, Dubuisson, Jean, Aliouat-Denis, Cécile-Marie, Cocquerel, Laurence, Centre d’Infection et d’Immunité de Lille - INSERM U 1019 - UMR 9017 - UMR 8204 (CIIL), Institut Pasteur de Lille, Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Lille-Centre Hospitalier Régional Universitaire [Lille] (CHRU Lille)-Centre National de la Recherche Scientifique (CNRS), Plateformes Lilloises en Biologie et Santé - UMS 2014 - US 41 (PLBS), Centre de Recherche en Cancérologie et Immunologie Nantes-Angers (CRCINA), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Nantes - UFR de Médecine et des Techniques Médicales (UFR MEDECINE), Université de Nantes (UN)-Université de Nantes (UN)-Centre hospitalier universitaire de Nantes (CHU Nantes)-Centre National de la Recherche Scientifique (CNRS)-Université d'Angers (UA), 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), This work was supported by a grant from the Frenchagency ANRS-Maladies infectieuses émergentes. This work was also supported by the Pasteur Institute of Lille, Région Haut-de-France and Inserm-transfert. K.H., M.A. and T.V. were supported by a fellowship from the ANRS. M.F. was supported by a fellowship from the Pasteur Institute of Lille and Région Hauts-de-France. The PLBS platform used in this work was supported by the ANR (ANR-10-EQPX-04-01) and Feder (12001407 [D-AL] EquipEx ImagInEx BioMed)., ANR-10-EQPX-0004,Imaginex BioMed,Plateau de microscopie de criblage à haut débit et d'analyse à très haute résolution(2010), Plateformes Lilloises en Biologie et Santé - UAR 2014 - US 41 (PLBS), Université d'Angers (UA)-Université de Nantes (UN)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Centre hospitalier universitaire de Nantes (CHU Nantes), 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), Cocquerel, Laurence, and Equipements d'excellence - Plateau de microscopie de criblage à haut débit et d'analyse à très haute résolution - - Imaginex BioMed2010 - ANR-10-EQPX-0004 - EQPX - VALID

Subjects
[SDV] Life Sciences [q-bio], animal diseases, viruses, [SDV]Life Sciences [q-bio], virus diseases
Abstract

Posté sur bioRxiv le 2021-05-26; Producing multifunctional proteins is one of the major strategies developed by viruses to condense their genetic information. Here, we investigated the molecular determinants of the multifunctionality of hepatitis E virus (HEV) ORF2 capsid protein. We previously identified 3 isoforms of ORF2 which are partitioned in different subcellular compartments to perform distinct functions. Notably, the infectious ORF2 (ORF2i) protein is the structural component of the virion, whereas the genome-free secreted and glycosylated ORF2 proteins likely act as a humoral immune decoy. We identified a 5 amino acid Arginine-Rich Motif (ARM) located in the ORF2 N-terminal region as a central regulator of the subcellular localizations and functions of ORF2 isoforms. We showed that the ARM controls ORF2 nuclear translocation, promoting regulation of host antiviral responses. This motif also regulates the dual topology and functionality of ORF2 signal peptide, leading to the production of either cytosolic infectious ORF2i or reticular non-infectious glycosylated ORF2 forms. Furthermore, the ARM likely serves as a cleavage site of the glycosylated ORF2 protein. Finally, it promotes ORF2 membrane association that is likely essential for particle assembly. In conclusion, our observations highlight ORF2 ARM as a unique central regulator of ORF2 addressing that finely controls the HEV lifecycle.

Published
2021

4. The sps Genes Encode an Original Legionaminic Acid Pathway Required for Crust Assembly in Bacillus subtilis

Author

Thomas Dubois, Frederic Krzewinski, Nao Yamakawa, Christelle Lemy, Audrey Hamiot, Loïc Brunet, Anne-Sophie Lacoste, Yuryi Knirel, Yann Guerardel, Christine Faille, DUBOIS, Thomas, APPEL À PROJETS GÉNÉRIQUE 2018 - La Mécanique des Fluides au Service de la Sécurité Alimentaire - - FEFS2018 - ANR-18-CE21-0010 - AAPG2018 - VALID, Equipements d'excellence - Plateau de microscopie de criblage à haut débit et d'analyse à très haute résolution - - Imaginex BioMed2010 - ANR-10-EQPX-0004 - EQPX - VALID, Unité Matériaux et Transformations - UMR 8207 (UMET), Centrale Lille-Institut de Chimie du CNRS (INC)-Université de Lille-Centre National de la Recherche Scientifique (CNRS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Unité de Glycobiologie Structurale et Fonctionnelle - UMR 8576 (UGSF), Université de Lille-Centre National de la Recherche Scientifique (CNRS), Plateformes Lilloises en Biologie et Santé - UAR 2014 - US 41 (PLBS), Institut Pasteur de Lille, Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Lille-Centre Hospitalier Régional Universitaire [Lille] (CHRU Lille)-Centre National de la Recherche Scientifique (CNRS), ND Zelinsky Institute of Organic Chemistry [Moscow, Russia], Region Hauts-de-FranceGoToS3NuTeX, ANR-18-CE21-0010,FEFS,La Mécanique des Fluides au Service de la Sécurité Alimentaire(2018), ANR-10-EQPX-0004,Imaginex BioMed,Plateau de microscopie de criblage à haut débit et d'analyse à très haute résolution(2010), Institut de Chimie du CNRS (INC)-Université de Lille-Centre National de la Recherche Scientifique (CNRS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Centrale Lille Institut (CLIL), Unité de Glycobiologie Structurale et Fonctionnelle (UGSF), Université de Lille-Centre National de la Recherche Scientifique (CNRS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Plateformes Lilloises en Biologie et Santé - UMS 2014 - US 41 (PLBS), Université de Lille, CNRS, Unité Matériaux et Transformations - UMR 8207 [UMET], Unité de Glycobiologie Structurale et Fonctionnelle (UGSF) - UMR 8576, and Plateformes Lilloises en Biologie et Santé - UAR 2014 - US 41 [PLBS]

Subjects
Bacillus subtilis, bacterial adhesion, crust, legionaminic acid, nonulosonic acid, spores, fungi, [SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Microbiology, QR1-502
Abstract

International audience; The crust is the outermost spore layer of most Bacillus strains devoid of an exosporium. This outermost layer, composed of both proteins and carbohydrates, plays a major role in the adhesion and spreading of spores into the environment. Recent studies have identified several crust proteins and have provided insights about their organization at the spore surface. However, although carbohydrates are known to participate in adhesion, little is known about their composition, structure, and localization. In this study, we showed that the spore surface of Bacillus subtilis is covered with legionaminic acid (Leg), a nine-carbon backbone nonulosonic acid known to decorate the flagellin of the human pathogens Helicobacter pylori and Campylobacter jejuni We demonstrated that the spsC, spsD, spsE, spsG, and spsM genes of Bacillus subtilis are required for Leg biosynthesis during sporulation, while the spsF gene is required for Leg transfer from the mother cell to the surface of the forespore. We also characterized the activity of SpsM and highlighted an original Leg biosynthesis pathway in B. subtilis Finally, we demonstrated that Leg is required for the assembly of the crust around the spores, and we showed that in the absence of Leg, spores were more adherent to stainless steel probably because of their reduced hydrophilicity and charge.IMPORTANCE Bacillus species are a major economic and food safety concern of the food industry because of their food spoilage-causing capability and persistence. Their persistence is mainly due to their ability to form highly resistant spores adhering to the surfaces of industrial equipment. Spores of the Bacillus subtilis group are surrounded by the crust, a superficial layer which plays a key role in their adhesion properties. However, knowledge of the composition and structure of this layer remains incomplete. Here, for the first time, we identified a nonulosonic acid (Leg) at the surfaces of bacterial spores (B. subtilis). We uncovered a novel Leg biosynthesis pathway, and we demonstrated that Leg is required for proper crust assembly. This work contributes to the description of the structure and composition of Bacillus spores which has been under way for decades, and it provides keys to understanding the importance of carbohydrates in Bacillus adhesion and persistence in the food industry.

Published
2020

5. Intracellular and in vivo evaluation of imidazo[2,1-b]thiazole-5-carboxamide anti-tuberculosis compounds

Author

Garrett C. Moraski, Nathalie Deboosere, Anne J. Lenaerts, Courtney Hastings, Alexandre Vandeputte, Priscille Brodin, Heath A. Weaver, Lisa K. Woolhiser, Marvin J. Miller, Kate L. Marshall, Department of Chemistry and Biochemistry [Bozeman, MT, États-Unis], Montana State University (MSU), Centre d’Infection et d’Immunité de Lille - INSERM U 1019 - UMR 9017 - UMR 8204 (CIIL), Institut Pasteur de Lille, Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Lille-Centre Hospitalier Régional Universitaire [Lille] (CHRU Lille)-Centre National de la Recherche Scientifique (CNRS), Mycobacteria Research Laboratories [Fort Collins, CO, États-Unis], Department of Microbiology, Immunology and Pathology [Fort Collins, CO, États-Unis], Colorado State University [Fort Collins] (CSU)-Colorado State University [Fort Collins] (CSU), Department of Chemistry and Biochemistry [Notre Dame, IN, États-Unis], University of Notre Dame [Indiana] (UND), This study was financially supported in part by the National Institutes of Health by grant R37AI054193 (M.J.M., G.C.M) and the National Institute of Allergy and Infectious Diseases, Contract No. HHSN272201100009I. Funding for Proteomics, Metabolomics and Mass Spectrometry Facility (at MSU) was made possible by the MJ Murdock Charitable Trust and NIH P20GM103474. Funding for the 500 NMR (at MSU) was provided by the NSF-Major Research Instrumentation program (NSF-MRI: DBI-1532078), the Murdock Charitable Trust Foundation (015066:MNL), and support from the MSU Vice-President for Research and Economic Development office. Work in Priscille Brodin’s lab was also supported from the European Community (ERC-STG INTRACELLTB n˚ 260901, MM4TB n˚260872), the Agence Nationale de la Recherche (ANR-10-EQPX-04-01, ANR-14-CE08-0017, ANR-16-CE35-0009), and the Feder (12001407 (D-AL) Equipex Imaginex BioMed) and the Région Nord Pas de Calais (convention n˚12000080)., ANR-14-CE08-0017,ANTI-TB-NANO,Une galénique ' verte ' à base de nanoparticules de cyclodextrines pour un traitement plus efficace de la tuberculose(2014), ANR-16-CE35-0009,TBemerg,Naissance d'un tueur: facteurs génétiques et adaptations métaboliques impliquées dans l'émergence des bacilles tuberculeux épidémiques(2016), ANR-10-EQPX-0004,Imaginex BioMed,Plateau de microscopie de criblage à haut débit et d'analyse à très haute résolution(2010), European Project: 260901,EC:FP7:ERC,ERC-2010-StG_20091118,INTRACELLTB(2010), European Project: 260872,EC:FP7:HEALTH,FP7-HEALTH-2010-single-stage,MM4TB(2011), Bodescot, Myriam, Appel à projets générique - Une galénique ' verte ' à base de nanoparticules de cyclodextrines pour un traitement plus efficace de la tuberculose - - ANTI-TB-NANO2014 - ANR-14-CE08-0017 - Appel à projets générique - VALID, Naissance d'un tueur: facteurs génétiques et adaptations métaboliques impliquées dans l'émergence des bacilles tuberculeux épidémiques - - TBemerg2016 - ANR-16-CE35-0009 - AAPG2016 - VALID, Equipements d'excellence - Plateau de microscopie de criblage à haut débit et d'analyse à très haute résolution - - Imaginex BioMed2010 - ANR-10-EQPX-0004 - EQPX - VALID, A Chemical Genomics Approach of Intracellular Mycobacterium tuberculosis Towards Defining Specific Host Pathogen Interactions - INTRACELLTB - - EC:FP7:ERC2010-12-01 - 2015-11-30 - 260901 - VALID, More Medicines for Tuberculosis - MM4TB - - EC:FP7:HEALTH2011-02-01 - 2016-01-31 - 260872 - VALID, Centre d’Infection et d’Immunité de Lille (CIIL) - U1019 - UMR 8204 (CIIL), Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Lille-Centre National de la Recherche Scientifique (CNRS), ANR-14-CE08-0017,ANTI-TB-NANO,Une galénique « verte » à base de nanoparticules de cyclodextrines pour un traitement plus efficace de la tuberculose(2014), ANR-10-EQPX-0004/10-EQPX-0004,Imaginex BioMed,Plateau de microscopie de criblage à haut débit et d'analyse à très haute résolution(2010), Centre National de la Recherche Scientifique (CNRS)-Centre Hospitalier Régional Universitaire [Lille] (CHRU Lille)-Université de Lille-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut Pasteur de Lille, and Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP)

Subjects
Bacterial Diseases, Physiology, Antitubercular Agents, Carboxamide, Pharmacology, chemistry.chemical_compound, Mice, White Blood Cells, Animal Cells, [SDV.MHEP.MI]Life Sciences [q-bio]/Human health and pathology/Infectious diseases, Immune Physiology, Chlorocebus aethiops, Medicine and Health Sciences, Cytochrome P-450 Enzyme Inhibitors, ADME, Liquid Chromatography, 0303 health sciences, Multidisciplinary, Chromatographic Techniques, Imidazoles, Animal Models, 3. Good health, Body Fluids, Actinobacteria, Infectious Diseases, Blood, Experimental Organism Systems, [SDV.SP.PHARMA] Life Sciences [q-bio]/Pharmaceutical sciences/Pharmacology, [SDV.MHEP.MI] Life Sciences [q-bio]/Human health and pathology/Infectious diseases, Medicine, medicine.symptom, Cellular Types, Anatomy, Research Article, medicine.drug_class, Immune Cells, Science, Immunology, Mouse Models, Microbial Sensitivity Tests, Research and Analysis Methods, Microbiology, Blood Plasma, 03 medical and health sciences, Model Organisms, In vivo, medicine, Potency, Animals, Humans, Tuberculosis, Animal Models of Disease, Thiazole, Vero Cells, 030304 developmental biology, Blood Cells, Bacteria, 030306 microbiology, Macrophages, Organisms, Biology and Life Sciences, Mycobacterium tuberculosis, Cell Biology, Tropical Diseases, In vitro, High Performance Liquid Chromatography, Thiazoles, Animal Models of Infection, RAW 264.7 Cells, Mechanism of action, chemistry, Microsome, Animal Studies, [SDV.SP.PHARMA]Life Sciences [q-bio]/Pharmaceutical sciences/Pharmacology, Caco-2 Cells, Spleen
Abstract

International audience; The imidazo[2,1-b]thiazole-5-carboxamides (ITAs) are a promising class of anti-tuberculosis agents shown to have potent activity in vitro and to target QcrB, a key component of the mycobacterial cytochrome bcc-aa3 super complex critical for the electron transport chain. Herein we report the intracellular macrophage potency of nine diverse ITA analogs with MIC values ranging from 0.0625-2.5 μM and mono-drug resistant potency ranging from 0.0017 to 7 μM. The in vitro ADME properties (protein binding, CaCo-2, human microsomal stability and CYP450 inhibition) were determined for an outstanding compound of the series, ND-11543. ND-11543 was tolerable at >500 mg/kg in mice and at a dose of 200 mg/kg displayed good drug exposure in mice with an AUC(0-24h) >11,700 ng·hr/mL and a >24 hr half-life. Consistent with the phenotype observed with other QcrB inhibitors, compound ND-11543 showed efficacy in a chronic murine TB infection model when dosed at 200 mg/kg for 4 weeks. The efficacy was not dependent upon exposure, as pre-treatment with a known CYP450-inhibitor did not substantially improve efficacy. The ITAs are an interesting scaffold for the development of new anti-TB drugs especially in combination therapy based on their favorable properties and novel mechanism of action.

Published
2020

6. Strength of Neisseria meningitidis binding to endothelial cells requires highly-ordered CD147/β2-adrenoceptor clusters assembled by alpha-actinin-4

Author

Maïssa, Nawal, Covarelli, Valentina, Janel, Sébastien, Durel, Beatrice, Simpson, Nandi, Bernard, Sandra, Pardo-Lopez, Liliana, Bouzinba-Ségard, Haniaa, Faure, Camille, Scott, Mark G.H., Coureuil, Mathieu, Morand, Philippe, Lafont, Frank, Nassif, Xavier, Marullo, Stefano, Bourdoulous, Sandrine, Institut Cochin (IC UM3 (UMR 8104 / U1016)), Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Centre d’Infection et d’Immunité de Lille - INSERM U 1019 - UMR 9017 - UMR 8204 (CIIL), Institut Pasteur de Lille, Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Lille-Centre Hospitalier Régional Universitaire [Lille] (CHRU Lille)-Centre National de la Recherche Scientifique (CNRS), Institut Necker Enfants-Malades (INEM - UM 111 (UMR 8253 / U1151)), CHU Necker - Enfants Malades [AP-HP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP), N.M. was supported by a doctoral fellowship from Université Paris Descartes and the Fondation pour la Recherche Médicale of France. V.C. was supported by a postdoctoral fellowship from the Fondation pour la Recherche Médicale. S.C.B. was supported by a doctoral fellowship from Université Paris Diderot and Association pour la Recherche sur le Cancer. This work was supported by collaborative research grants the Agence Nationale de la Recherche of France (ANR-14-CE14-0010-02) and the Université Paris Descartes (AAP-2011) to S.B., X.N. and S.M., from the Fondation pour la Recherche Médicale to X.N. and S.M. and from the ANR (ANR-10-EQPX-04-01) to F.L., ANR-14-CE14-0010,NEMBRAIN,La barrière hémato-encéphalique: de la méningite bactérienne à la délivrance de médicaments dans le cerveau(2014), ANR-10-EQPX-0004,Imaginex BioMed,Plateau de microscopie de criblage à haut débit et d'analyse à très haute résolution(2010), Centre National de la Recherche Scientifique (CNRS)-Centre Hospitalier Régional Universitaire [Lille] (CHRU Lille)-Université de Lille-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut Pasteur de Lille, Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP), Faure, Camille, Appel à projets générique - La barrière hémato-encéphalique: de la méningite bactérienne à la délivrance de médicaments dans le cerveau - - NEMBRAIN2014 - ANR-14-CE14-0010 - Appel à projets générique - VALID, and Equipements d'excellence - Plateau de microscopie de criblage à haut débit et d'analyse à très haute résolution - - Imaginex BioMed2010 - ANR-10-EQPX-0004 - EQPX - VALID

Subjects
[SDV] Life Sciences [q-bio], [SDV]Life Sciences [q-bio], Science
Abstract

International audience; Neisseria meningitidis (meningococcus) is an invasive bacterial pathogen that colonizes human vessels, causing thrombotic lesions and meningitis. Establishment of tight interactions with endothelial cells is crucial for meningococci to resist haemodynamic forces. Two endothelial receptors, CD147 and the β2-adrenergic receptor (β2AR), are sequentially engaged by meningococci to adhere and promote signalling events leading to vascular colonization, but their spatiotemporal coordination is unknown. Here we report that CD147 and β2AR form constitutive hetero-oligomeric complexes. The scaffolding protein α-actinin-4 directly binds to the cytosolic tail of CD147 and governs the assembly of CD147–β2AR complexes in highly ordered clusters at bacterial adhesion sites. This multimolecular assembly process increases the binding strength of meningococci to endothelial cells under shear stress, and creates molecular platforms for the elongation of membrane protrusions surrounding adherent bacteria. Thus, the specific organization of cellular receptors has major impacts on host–pathogen interaction.

Published
2017

7. Reversion of antibiotic resistance in Mycobacterium tuberculosis by spiroisoxazoline SMARt-420

Author

Alexandre Wohlkonig, David Hot, Sebastien Gagneux, Marc Gitzinger, Julia Feldmann, Mireia Coscolla, Marion Flipo, Nicolas Willand, Martin Moune, Kamel Djaout, Mehdi Kiass, Priscille Brodin, Vincent Delorme, Vanessa Mathys, Rosangela Frita, Karine Soetaert, Benoit Deprez, Ludovic Huot, Nicolas Blondiaux, René Wintjens, Christian Kemmer, Alain R. Baulard, Vincent Trebosc, Camille Locht, Matthieu Desroses, Rudy Antoine, Willand, Nicolas, Appel à projets générique - Reprogrammation de la bioactivation des thioamides comme alternative antituberculeuse - - Tea-4-Two2014 - ANR-14-CE14-0027 - Appel à projets générique - VALID, Equipements d'excellence - Plateau de microscopie de criblage à haut débit et d'analyse à très haute résolution - - Imaginex BioMed2010 - ANR-10-EQPX-0004 - EQPX - VALID, EGID Diabetes Pole - - EGID2010 - ANR-10-LABX-0046 - LABX - VALID, Plate forme Lilloise de séquençage du génome humain pour une médecine personnalisée - - LIGAN PM2010 - ANR-10-EQPX-0007 - EQPX - VALID, A Chemical Genomics Approach of Intracellular Mycobacterium tuberculosis Towards Defining Specific Host Pathogen Interactions - INTRACELLTB - - EC:FP7:ERC2010-12-01 - 2015-11-30 - 260901 - VALID, Compensatory Evolution and Epistasis in Multidrug-resistant Mycobacterium tuberculosis - EVODRTB - - EC:FP7:ERC2013-04-01 - 2018-03-31 - 309540 - VALID, More Medicines for Tuberculosis - MM4TB - - EC:FP7:HEALTH2011-02-01 - 2016-01-31 - 260872 - VALID, Centre d’Infection et d’Immunité de Lille - INSERM U 1019 - UMR 9017 - UMR 8204 (CIIL), Institut Pasteur de Lille, Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Lille-Centre Hospitalier Régional Universitaire [Lille] (CHRU Lille)-Centre National de la Recherche Scientifique (CNRS), Médicaments et molécules pour agir sur les Systèmes Vivants - U 1177 (M2SV), Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Lille, Karolinska Institutet [Stockholm], Institut Scientifique de Santé Publique [Belgique] - Scientific Institute of Public Health [Belgium] (WIV-ISP), Réseau International des Instituts Pasteur (RIIP), Tuberculosis Lab [Korea], Institut Pasteur Korea - Institut Pasteur de Corée, Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP), Bioversys AG [Basel], University of Basel (Unibas), Université libre de Bruxelles (ULB), VIB-VUB Center for Structural Biology [Bruxelles], VIB [Belgium], Vrije Universiteit Brussel [Bruxelles] (VUB), Swiss Tropical and Public Health Institute [Basel], This work was supported by l’Agence Nationale de la Recherche (ANR), France (Tea-4-Two, ANR-14-CE14-0027-01) (ANR-10-EQPX-04-01), by EU grants ERC-STG INTRACELLTB no 260901, the Feder (12001407 (D-AL), PRIM (NewBio4Tb), European Research Council (grant 309540-EVODRTB), SystemsX.ch, Institut National de la Santé et de la Recherche Médicale, Université de Lille, Institut Pasteur de Lille, Centre National de la Recherche Scientifique, the Région Hauts-de-France (convention no. 12000080), and Société d’Accélération du Transfert de Technologie Nord. R.W. is Research Associate at the National Fund for Scientific Research (FNRS-FRS) (Belgium). M.M. was supported by PRIM (NewBio4Tb), V.D. was supported by EU grant 260872, and V.T. was suppported by the Marie Curie Initial Training Network (ITN-2013-607694-Translocation). The authors also thank the Unité Mixte de Recherche UMR 8199, Lille Integrated Genomics Network for Advanced Personalized Medicine (LIGAN-PM) Genomics platform (Lille, France), which belongs to the Federation de Recherche 3508 Labex EGID (European Genomics Institute for Diabetes, ANR-10-LABX-46) and was supported by the Agence Nationale de la Recherche (ANR) Equipex 2010 session (ANR-10-EQPX-07-01, LIGAN-PM). The LIGAN-PM Genomics platform is supported by the Fonds Européen de Développement Régional and the Region Hauts-de-France., ANR-14-CE14-0027,Tea-4-Two,Reprogrammation de la bioactivation des thioamides comme alternative antituberculeuse(2014), ANR-10-EQPX-0004,Imaginex BioMed,Plateau de microscopie de criblage à haut débit et d'analyse à très haute résolution(2010), ANR-10-LABX-0046,EGID,EGID Diabetes Pole(2010), ANR-10-EQPX-0007,LIGAN PM,Plate forme Lilloise de séquençage du génome humain pour une médecine personnalisée(2010), European Project: 260901,EC:FP7:ERC,ERC-2010-StG_20091118,INTRACELLTB(2010), European Project: 309540,EC:FP7:ERC,ERC-2012-StG_20111109,EVODRTB(2013), European Project: 260872,EC:FP7:HEALTH,FP7-HEALTH-2010-single-stage,MM4TB(2011), and Centre National de la Recherche Scientifique (CNRS)-Centre Hospitalier Régional Universitaire [Lille] (CHRU Lille)-Université de Lille-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut Pasteur de Lille

Subjects
0301 basic medicine, Tuberculosis, medicine.drug_class, [CHIM.THER] Chemical Sciences/Medicinal Chemistry, 030106 microbiology, Antibiotics, Reversion, [CHIM.THER]Chemical Sciences/Medicinal Chemistry, Microbiology, Mycobacterium tuberculosis, 03 medical and health sciences, Antibiotic resistance, [CHIM] Chemical Sciences, medicine, [CHIM]Chemical Sciences, chemistry.chemical_classification, Multidisciplinary, biology, medicine.disease, biology.organism_classification, 3. Good health, 030104 developmental biology, Enzyme, chemistry, Ethionamide, Bacteria, medicine.drug
Abstract

Countering TB prodrug resistance The arsenal of antibiotics for treating tuberculosis (TB) contains many prodrugs, such as ethionamide, which need activation by normal metabolism to release their toxic effects. Ethionamide is potentiated by small molecules. Blondiaux et al. screened for more potent analogs and identified a lead compound called SMARt-420. This small molecule inactivates a TetR-like repressor, EthR2, and boosts ethionamide activation. SMARt-420 successfully promoted clearance of multidrug-resistant strains of Mycobacterium tuberculosis from the lungs of mice. Science , this issue p. 1206

Published
2017

8. Targeting Type IV pili as an antivirulence strategy against invasive meningococcal disease

Author

Jean-Philippe Barnier, Haniaa Bouzinba-Segard, Anne Gouge, Anne Jamet, Mathieu Coureuil, Philippe Morand, Xavier Nassif, Philippe Pelissier, Frank Lafont, Béatrice Durel, Marion Le Bris, Sandrine Bourdoulous, Olivier Join-Lambert, Nicolas Barois, Daniel Euphrasie, Kevin Denis, Loic Le Guennec, Camille Faure, Institut Cochin (IC UM3 (UMR 8104 / U1016)), Centre National de la Recherche Scientifique (CNRS)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM), 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), CHU Necker - Enfants Malades [AP-HP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP), Centre d’Infection et d’Immunité de Lille - INSERM U 1019 - UMR 9017 - UMR 8204 (CIIL), Centre National de la Recherche Scientifique (CNRS)-Centre Hospitalier Régional Universitaire [Lille] (CHRU Lille)-Université de Lille-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut Pasteur de Lille, Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP), Centre hospitalier Saint-Joseph [Paris], K.D. and L.LG. were supported by a doctoral fellowship from la Region Ile de France and the Fondation pour la Recherche Médicale, respectively. This work was supported by collaborative research grants from the Agence Nationale de la Recherche of France (grant no. ANR-14-IFEC14-0006) to S.B. and X.N. in the framework of the Infect-ERA joint transnational call (European funding for infectious diseases research), by the Société d’Accélération du Transfert de Technologie (grant no. ANR-10-SATT-05-01) to S.B. and by grant no. ANR-10-EQPX-04-01 to F.L., We thank M. Robert-Genthon and F. Chretien for providing the P. aeruginosa PAO1 strain and brain tissues, M. Favier of the histology facility, A. Schmitt and T. Guilbert of the imaging facility, and K. Bailly and M. Andrieu of the cytometry facility of the Institut Cochin for their expert technical help., ANR-10-SATT-0005,SATT Erganeo,ERGANEO(2010), ANR-10-EQPX-0004,Imaginex BioMed,Plateau de microscopie de criblage à haut débit et d'analyse à très haute résolution(2010), Institut Pasteur de Lille, Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Lille-Centre Hospitalier Régional Universitaire [Lille] (CHRU Lille)-Centre National de la Recherche Scientifique (CNRS), ANR-10-SATT-0005,SATT ERGANEO,SATT ERGANEO (EX IDF INNOV)(2010), Centre d’Infection et d’Immunité de Lille (CIIL) - U1019 - UMR 8204 (CIIL), Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Lille-Centre National de la Recherche Scientifique (CNRS), Groupe hospitalier Paris Saint-Joseph - Hôpital, ANR: 10-SATT-0005,SATT IDF INNOV,IDF INNOV(2010), and ANR-10-EQPX-0004/10-EQPX-0004,Imaginex BioMed,Plateau de microscopie de criblage à haut débit et d'analyse à très haute résolution(2010)

Subjects
Microbiology (medical), Gram-negative bacteria, Immunology, Fimbria, Inflammation, medicine.disease_cause, Applied Microbiology and Biotechnology, Microbiology, [SDV.MP.PRO]Life Sciences [q-bio]/Microbiology and Parasitology/Protistology, Virulence factor, Pilus, Bacterial genetics, 03 medical and health sciences, Genetics, medicine, [SDV.MP.MYC]Life Sciences [q-bio]/Microbiology and Parasitology/Mycology, 030304 developmental biology, 0303 health sciences, biology, 030306 microbiology, Neisseria meningitidis, Cell Biology, biology.organism_classification, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, 3. Good health, [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology, medicine.symptom, Bacteria
Abstract

Bacterial virulence factors are attractive targets for the development of therapeutics. Type IV pili, which are associated with a remarkable array of properties including motility, the interaction between bacteria and attachment to biotic and abiotic surfaces, represent particularly appealing virulence factor targets. Type IV pili are present in numerous bacterial species and are critical for their pathogenesis. In this study, we report that trifluoperazine and related phenothiazines block functions associated with Type IV pili in different bacterial pathogens, by affecting piliation within minutes. Using Neisseria meningitidis as a paradigm of Gram-negative bacterial pathogens that require Type IV pili for pathogenesis, we show that piliation is sensitive to altered activity of the Na+ pumping NADH–ubiquinone oxidoreductase (Na+−NQR) complex and that these compounds probably altered the establishment of the sodium gradient. In vivo, these compounds exert a strong protective effect. They reduce meningococcal colonization of the human vessels and prevent subsequent vascular dysfunctions, intravascular coagulation and overwhelming inflammation, the hallmarks of invasive meningococcal infections. Finally, they reduce lethality. This work provides a proof of concept that compounds with activity against bacterial Type IV pili could beneficially participate in the treatment of infections caused by Type IV pilus-expressing bacteria. Phenothiazines inhibit Type IV pili function in Neisseria meningitidis and protect against bacterial colonization of blood vessels and lethality during infection.

Published
2019

9. Stiffness tomography of eukaryotic intracellular compartments by atomic force microscopy

Author

Michka Popoff, Séverine Divoux, Frank Lafont, Elisabeth Werkmeister, Sébastien Janel, Franck Perez, Nicolas Barois, Centre d’Infection et d’Immunité de Lille - INSERM U 1019 - UMR 9017 - UMR 8204 (CIIL), Institut Pasteur de Lille, Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Lille-Centre Hospitalier Régional Universitaire [Lille] (CHRU Lille)-Centre National de la Recherche Scientifique (CNRS), Institut d’Électronique, de Microélectronique et de Nanotechnologie - UMR 8520 (IEMN), Centrale Lille-Université de Lille-Centre National de la Recherche Scientifique (CNRS)-Université Polytechnique Hauts-de-France (UPHF)-JUNIA (JUNIA), Université catholique de Lille (UCL)-Université catholique de Lille (UCL), Plateforme BioImaging Center Lille (BICeL), Plateformes Lilloises en Biologie et Santé - UAR 2014 - US 41 (PLBS), Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Lille-Centre Hospitalier Régional Universitaire [Lille] (CHRU Lille)-Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur de Lille, Compartimentation et dynamique cellulaires (CDC), Institut Curie [Paris]-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), We thank A. Bongiovanni and J. Warein for expert technical assistance. Thanks to Y. Ciczora and S. Bovio for valuable discussions and L. Redondo-Morata for reading the manuscript. The work was funded by grants from the University Lille 1 (MP), ANR-10-EQPX-04-01, and FEDER 12001407 (FL)., ANR-10-EQPX-0004,Imaginex BioMed,Plateau de microscopie de criblage à haut débit et d'analyse à très haute résolution(2010), Centre National de la Recherche Scientifique (CNRS)-Centre Hospitalier Régional Universitaire [Lille] (CHRU Lille)-Université de Lille-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut Pasteur de Lille, Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP), Centrale Lille-Institut supérieur de l'électronique et du numérique (ISEN)-Université de Valenciennes et du Hainaut-Cambrésis (UVHC)-Université de Lille-Centre National de la Recherche Scientifique (CNRS)-Université Polytechnique Hauts-de-France (UPHF), Plateformes Lilloises en Biologie et Santé - UMS 2014 - US 41 (PLBS), LAFONT, Frank, and Equipements d'excellence - Plateau de microscopie de criblage à haut débit et d'analyse à très haute résolution - - Imaginex BioMed2010 - ANR-10-EQPX-0004 - EQPX - VALID

Subjects
Cytoplasm, [SDV.IB.IMA]Life Sciences [q-bio]/Bioengineering/Imaging, [SPI.NANO] Engineering Sciences [physics]/Micro and nanotechnologies/Microelectronics, Cell, 02 engineering and technology, Microscopy, Atomic Force, 010402 general chemistry, Microtubules, 01 natural sciences, law.invention, law, Microscopy, Organelle, medicine, Humans, General Materials Science, [SPI.NANO]Engineering Sciences [physics]/Micro and nanotechnologies/Microelectronics, Cytoskeleton, ComputingMilieux_MISCELLANEOUS, Cell Nucleus, Viscosity, Chemistry, Cell Membrane, 021001 nanoscience & nanotechnology, Elasticity, 0104 chemical sciences, Characterization (materials science), medicine.anatomical_structure, [SDV.IB.IMA] Life Sciences [q-bio]/Bioengineering/Imaging, Biophysics, Electron microscope, 0210 nano-technology, Intracellular, HeLa Cells
Abstract

International audience; Precise localization and biophysical characterization of cellular structures is a key to the understanding of biological processes happening both inside the cell and at the cell surface. Atomic force microscopy is a powerful tool to study the cell surface – topography, elasticity, viscosity, interactions – and also the viscoelastic behavior of the underlying cytoplasm, cytoskeleton or the nucleus. Here, we demonstrate the ability of atomic force microscopy to also map and characterize organelles and microorganisms inside cells, at the nanoscale, by combining stiffness tomography with super-resolution fluorescence and electron microscopy. By using this correlative approach, we could both identify and characterize intracellular compartments. The validation of this approach was performed by monitoring the stiffening effect according to the metabolic status of the mitochondria in living cells in real-time.

Published
2019

10. Mitochondrial Dynamics and Activity in Legionella-Infected Cells

Author

Carmen Buchrieser, Ok-Ryul Song, Pedro Escoll, Priscille Brodin, Centre d’Infection et d’Immunité de Lille - INSERM U 1019 - UMR 9017 - UMR 8204 (CIIL), Centre National de la Recherche Scientifique (CNRS)-Centre Hospitalier Régional Universitaire [Lille] (CHRU Lille)-Université de Lille-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut Pasteur de Lille, Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP), Biologie des Bactéries intracellulaires - Biology of Intracellular Bacteria, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Work in C.B. laboratory is financed by Institut Pasteur and ANR (grant no. ANR-10-LABX-62-IBEID). P.E. is funded by the Fondation pour la Recherche Médicale (FRM) project DEQ20120323697., P.B. and O.R.S. received financial support from the European Community (ERC-STG INTRACELLTBno. 260901, MM4TB no. 260872), the ANR (grant no. ANR-10-EQPX-04-01), and the Région Nord Pas de Calais (convention no. 12000080)., Carmen Buchrieser, Hubert Hilbi, We thank Nathalie Aulner and the Imagopole-CiTech (part of France-BioImaging supported by ANR grant no. ANR-10-INSB-04-01, Conseil de la Région Ile-de-France, FRM), and the Technology Core of the Center for Translational Science (CRT) at Institut Pasteur, for technical support during the setup of the methods presented here., ANR-10-LABX-0062,IBEID,Integrative Biology of Emerging Infectious Diseases(2010), ANR-10-EQPX-0004,Imaginex BioMed,Plateau de microscopie de criblage à haut débit et d'analyse à très haute résolution(2010), ANR-10-INBS-0004,France-BioImaging,Développment d'une infrastructure française distribuée coordonnée(2010), European Project: 260901,EC:FP7:ERC,ERC-2010-StG_20091118,INTRACELLTB(2010), European Project: 260872,EC:FP7:HEALTH,FP7-HEALTH-2010-single-stage,MM4TB(2011), Institut Pasteur de Lille, Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Lille-Centre Hospitalier Régional Universitaire [Lille] (CHRU Lille)-Centre National de la Recherche Scientifique (CNRS), and Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)

Subjects
Host mitochondria, Bioenergetics, Legionella, Mitochondrial respiration assay, MESH: Host-Pathogen Interactions, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, MESH: Mitochondria / metabolism, Legionella pneumophila, Microbiology, 03 medical and health sciences, Living-cell assays, MESH: Data Analysis, MESH: Macrophages / metabolism, MitoTracker dyes, MESH: Macrophages / microbiology, 030304 developmental biology, MESH: Legionellosis / microbiology, MESH: Mitochondrial Dynamics, 0303 health sciences, Mitochondrial morphology, MESH: Humans, biology, 030306 microbiology, Mitochondrial fission, MESH: Molecular Imaging / methods, biology.organism_classification, Seahorse, Mitochondrial respiration, MESH: Image Processing, Computer-Assisted, MESH: Legionellosis / metabolism, MESH: Legionella / physiology, High-content microscopy, Mitochondrial dynamics, MESH: Cell Respiration, MESH: Cells, Cultured
Abstract

International audience; The study of Legionella pneumophila interactions with host mitochondria during infection has been historically limited by the techniques available to analyze and quantify mitochondrial dynamics and activity in living cells. Recently, new, powerful techniques such as high-content microscopy or mitochondrial respiration assays (Seahorse) have been developed to quantitatively analyze mitochondrial parameters. Here we present state-of-the-art methods adapted to analyze mitochondrial dynamics and activity during Legionella infection of living human primary macrophages.

Published
2019

11. Standardized Nanomechanical Atomic Force Microscopy Procedure (SNAP) for Measuring Soft and Biological Samples

Author

Schillers, Hermann, Rianna, Carmela, Schäpe, Jens, Luque, Tomas, Doschke, Holger, Wälte, Mike, Uriarte, Juan José, Campillo, Noelia, Michanetzis, Georgios P. A., Bobrowska, Justyna, Dumitru, Andra, Herruzo, Elena T., Bovio, Simone, Parot, Pierre, Galluzzi, Massimiliano, Podestà, Alessandro, Puricelli, Luca, Scheuring, Simon, Missirlis, Yannis, Garcia, Ricardo, Odorico, Michael, Teulon, Jean-Marie, Lafont, Frank, Lekka, Malgorzata, Rico, Felix, Rigato, Annafrancesca, Pellequer, Jean-Luc, Oberleithner, Hans, Navajas, Daniel, Radmacher, Manfred, Westfälische Wilhelms-Universität Münster ( WWU ), University of Bremen, University of Barcelona, University of Patras [Patras], Polish Academy of Sciences, Instituto de Ciencia de Materiales de Madrid [Madrid] ( ICMM ), Institut Pasteur de Lille, Réseau International des Instituts Pasteur ( RIIP ), Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ), Università degli studi di Milano [Milano], Department Radiation Oncology (Weill Cornell Medicine), Weill Medical College of Cornell University [New York], Institut de Chimie Séparative de Marcoule ( ICSM - UMR 5257 ), Université Montpellier 2 - Sciences et Techniques ( UM2 ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Ecole Nationale Supérieure de Chimie de Montpellier ( ENSCM ) -Université de Montpellier ( UM ) -Centre National de la Recherche Scientifique ( CNRS ), Institut de biologie structurale ( IBS - UMR 5075 ), Université Joseph Fourier - Grenoble 1 ( UJF ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Centre National de la Recherche Scientifique ( CNRS ) -Université Grenoble Alpes ( UGA ), BIO-AFM-LAB Bio Atomic Force Microscopy Laboratory ( Bio-AFM-Lab ), Aix Marseille Université ( AMU ) -Institut National de la Santé et de la Recherche Médicale ( INSERM ), ANR-10-EQPX-0004/10-EQPX-0004,Imaginex BioMed,Plateau de microscopie de criblage à haut débit et d'analyse à très haute résolution ( 2010 ), European Project : 340177,EC:FP7:ERC,ERC-2013-ADG,3DNANOMECH ( 2014 ), Westfälische Wilhelms-Universität Münster (WWU), Instituto de Ciencia de Materiales de Madrid (ICMM), Consejo Superior de Investigaciones Científicas [Madrid] (CSIC), Réseau International des Instituts Pasteur (RIIP), Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Università degli Studi di Milano [Milano] (UNIMI), Etude de la Matière en Mode Environnemental (L2ME), Institut de Chimie Séparative de Marcoule (ICSM - UMR 5257), Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC)-Université de Montpellier (UM)-Ecole Nationale Supérieure de Chimie de Montpellier (ENSCM)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC)-Université de Montpellier (UM)-Ecole Nationale Supérieure de Chimie de Montpellier (ENSCM)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Institut de biologie structurale (IBS - UMR 5075 ), Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), BIO-AFM-LAB Bio Atomic Force Microscopy Laboratory (Bio-AFM-Lab), Aix Marseille Université (AMU)-Institut National de la Santé et de la Recherche Médicale (INSERM), ANR-10-EQPX-0004,Imaginex BioMed,Plateau de microscopie de criblage à haut débit et d'analyse à très haute résolution(2010), European Project: 340177,EC:FP7:ERC,ERC-2013-ADG,3DNANOMECH(2014), Universitat de Barcelona, Ministerio de Ciencia e Innovación (España), International AFMBioMed Conferences Association, Università degli Studi di Milano, Ministerio de Economía y Competitividad (España), German Research Foundation, European Research Council, Agence Nationale de la Recherche (France), National Science Centre (Poland), Westfälische Wilhelms-Universität Münster = University of Münster (WWU), University of Patras, Polska Akademia Nauk = Polish Academy of Sciences (PAN), Università degli Studi di Milano = University of Milan (UNIMI), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Ecole Nationale Supérieure de Chimie de Montpellier (ENSCM)-Institut de Chimie du CNRS (INC)-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Ecole Nationale Supérieure de Chimie de Montpellier (ENSCM)-Institut de Chimie du CNRS (INC)-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)

Subjects
[SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Nanotecnologia, Science, technology, industry, and agriculture, Reproducibility of Results, Biomecànica, Hydrogels, Microscòpia de força atòmica, Microscopy, Atomic Force, Article, Madin Darby Canine Kidney Cells, Atomic force microscopy, Dogs, Elastic Modulus, Medicine, Animals, Nanotechnology, Biomechanics, Stress, Mechanical, [ SDV.BBM.BS ] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM]
Abstract

We present a procedure that allows a reliable determination of the elastic (Young's) modulus of soft samples, including living cells, by atomic force microscopy (AFM). The standardized nanomechanical AFM procedure (SNAP) ensures the precise adjustment of the AFM optical lever system, a prerequisite for all kinds of force spectroscopy methods, to obtain reliable values independent of the instrument, laboratory and operator. Measurements of soft hydrogel samples with a well-defined elastic modulus using different AFMs revealed that the uncertainties in the determination of the deflection sensitivity and subsequently cantilever's spring constant were the main sources of error. SNAP eliminates those errors by calculating the correct deflection sensitivity based on spring constants determined with a vibrometer. The procedure was validated within a large network of European laboratories by measuring the elastic properties of gels and living cells, showing that its application reduces the variability in elastic moduli of hydrogels down to 1%, and increased the consistency of living cells elasticity measurements by a factor of two. The high reproducibility of elasticity measurements provided by SNAP could improve significantly the applicability of cell mechanics as a quantitative marker to discriminate between cell types and conditions., This work was supported by grants to D.N., T.L., N.C., and J.J.U. (Spanish Ministry of Economy and Competitiveness FIS-PI14/00280), M.L. and J.B. (National Science Center NCN DEC-2011/01/M/ST3/00711), H.O. (Deutsche Forschungsgemeinschaft, Koselleck 68/1 and Centre of Excellence, Cells in Motion (CIM), University of Münster, Germany), S.B. and F.L. (ANR-10-EQPX-04-01) and A.D., E.T.H., R.G. (CSD2010-00024, ERC-AdG-340177). A.P. thanks the Dept. of Physics of the University of Milano for financial support under the project “Piano di Sviluppo dell’Ateneo per la Ricerca 2014”, actions: “A. Upgrade of instrumentation”, and “B. Support to young researchers”. We acknowledge the support of the International AFMBioMed Conference Association.

Published
2017

12. Legionella pneumophila Modulates Mitochondrial Dynamics to Trigger Metabolic Repurposing of Infected Macrophages

Author

Carmen Buchrieser, Bernhard Steiner, Priscille Brodin, Pedro Escoll, Ok-Ryul Song, Flávia Viana, Francis Impens, Jean-Christophe Olivo-Marin, Hubert Hilbi, Thibault Lagache, HOFFMANN, Isabelle, Integrative Biology of Emerging Infectious Diseases - - IBEID2010 - ANR-10-LABX-0062 - LABX - VALID, ERA-NET Infect-ERA - Eukaryotic genes in vacuolar pathogens and symbionts - Implications for virulence, metabolism and ecology - - EUGENPATH2013 - ANR-13-IFEC-0003 - IFEC - VALID, Développment d'une infrastructure française distribuée coordonnée - - France-BioImaging2010 - ANR-10-INBS-0004 - INBS - VALID, Equipements d'excellence - Plateau de microscopie de criblage à haut débit et d'analyse à très haute résolution - - Imaginex BioMed2010 - ANR-10-EQPX-0004 - EQPX - VALID, A Chemical Genomics Approach of Intracellular Mycobacterium tuberculosis Towards Defining Specific Host Pathogen Interactions - INTRACELLTB - - EC:FP7:ERC2010-12-01 - 2015-11-30 - 260901 - VALID, More Medicines for Tuberculosis - MM4TB - - EC:FP7:HEALTH2011-02-01 - 2016-01-31 - 260872 - VALID, Biologie des Bactéries intracellulaires - Biology of Intracellular Bacteria, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Centre d’Infection et d’Immunité de Lille - INSERM U 1019 - UMR 9017 - UMR 8204 (CIIL), Institut Pasteur de Lille, Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Lille-Centre Hospitalier Régional Universitaire [Lille] (CHRU Lille)-Centre National de la Recherche Scientifique (CNRS), Institute of Medical Microbiology [Zurich], Universität Zürich [Zürich] = University of Zurich (UZH), Analyse d'images biologiques - Biological Image Analysis (BIA), Universiteit Gent = Ghent University (UGENT), Work in CB laboratory is financed by Institut Pasteur and ANR-10-LABX-62-IBEID. F.V. was funded by ANR-13-IFEC-0003-02 (Infect-ERA project EUGENPATH), P.E. by the Fondation pour la Recherche Médicale (FRM) project DEQ20120323697., Work in JCOM laboratory was partially funded by a Roux Fellowship (Institut Pasteur) to TL and ANR-10-INBS-04-06 FranceBioImaging and ANR-10-LABX-62-IBEID. HH was supported by the Institute of Medical Microbiology, University of Zürich, the Swiss National Science Foundation (SNF, 31003A_153200), and the German Bundesministerium für Bildung und Forschung (BMBF, 031A410A, Infect-ERA project EUGENPATH). FI is supported by VIB and Odysseus grant G0F8616N from the Research Foundation Flanders (FWO). PB and ORS received financial support from the European Community (ERC-STG INTRACELLTB n° 260901, MM4TB n°260872), the ANR-10-EQPX-04-01 and the Région Nord Pas de Calais (convention n° 12000080)., We thank N Aulner, JY Tinevez and the Imagopole-CiTech (part of FranceBioImaging supported by ANR-10-INSB-04-01, Conseil de la Région Ile-de-France, FRM) and the Technology Core of the Center for Translational Science (CRT) at Institut Pasteur, for support in conducting this study., ANR-10-LABX-0062,IBEID,Integrative Biology of Emerging Infectious Diseases(2010), ANR-13-IFEC-0003,EUGENPATH,Eukaryotic genes in vacuolar pathogens and symbionts - Implications for virulence, metabolism and ecology(2013), ANR-10-INBS-0004,France-BioImaging,Développment d'une infrastructure française distribuée coordonnée(2010), ANR-10-EQPX-0004,Imaginex BioMed,Plateau de microscopie de criblage à haut débit et d'analyse à très haute résolution(2010), European Project: 260901,EC:FP7:ERC,ERC-2010-StG_20091118,INTRACELLTB(2010), European Project: 260872,EC:FP7:HEALTH,FP7-HEALTH-2010-single-stage,MM4TB(2011), Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS)-Centre Hospitalier Régional Universitaire [Lille] (CHRU Lille)-Université de Lille-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut Pasteur de Lille, Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP), Universiteit Gent = Ghent University [Belgium] (UGENT), and University of Zurich

Subjects
0301 basic medicine, Dynamins, DNM1L, [SDV]Life Sciences [q-bio], 030106 microbiology, 2405 Parasitology, 610 Medicine & health, Vacuole, Mitochondrion, bioenergetics, [SDV.BBM.BM] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, Microbiology, Legionella pneumophila, GTP Phosphohydrolases, Mitochondrial Proteins, Type IV Secretion Systems, 03 medical and health sciences, Mice, Bacterial Proteins, Virology, Animals, Humans, host-pathogen interactions, Secretion, Cells, Cultured, biology, 10179 Institute of Medical Microbiology, Effector, Intracellular parasite, Macrophages, 2404 Microbiology, Autophagy, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, biology.organism_classification, mitochondrial dynamics, Cell biology, virulence, [SDV] Life Sciences [q-bio], live cell imaging, 030104 developmental biology, RAW 264.7 Cells, 2406 Virology, 570 Life sciences, Parasitology, Legionnaires' Disease, Microtubule-Associated Proteins
Abstract

International audience; The intracellular bacteria Legionella pneumophila encodes a type IV secretion system (T4SS) that injects effector proteins into macrophages in order to establish and replicate within the Legionella-containing vacuole (LCV). Once generated, the LCV interacts with mitochondria through unclear mechanisms. We show that Legionella uses both T4SS-independent and T4SS-dependent mechanisms to respectively interact with mitochondria and induce mitochondrial fragmentation that ultimately alters mitochondrial metabolism. The T4SS effector MitF, a Ran GTPase activator, is required for fission of the mitochondrial network. These effects of MitF occur through accumulation of mitochondrial DNM1L, a GTPase critical for fission. Furthermore mitochondrial respiration is abruptly halted in a T4SS-dependent manner, while T4SS-independent upregulation of cellular glycolysis remains elevated. Collectively, these alterations in mitochondrial dynamics promote a Warburg-like phenotype in macrophages that favors bacterial replication. Hence the rewiring of cellular bioenergetics to create a replication permissive niche in host cells is a virulence strategy of L. pneumophila.

Published
2017

13. Mycobacterium tuberculosis LppM Displays an Original Structure and Domain Composition Linked to a Dual Localization

Author

Romain Veyron-Churlet, Angelique De Visch, Philippe Barthe, Jérôme Nigou, Priscille Brodin, Jean-Michel Saliou, Martine Gilleron, Stanislas Tomavo, Martin Cohen-Gonsaud, GILLERON, Martine, Equipements d'excellence - Plateau de microscopie de criblage à haut débit et d'analyse à très haute résolution - - Imaginex BioMed2010 - ANR-10-EQPX-0004 - EQPX - VALID, Laboratoires d'excellence - Alliance française contre les maladies parasitaires - - ParaFrap2011 - ANR-11-LABX-0024 - LABX - VALID, Infrastructure Française pour la Biologie Structurale Intégrée - - FRISBI2010 - ANR-10-INBS-0005 - INBS - VALID, A Chemical Genomics Approach of Intracellular Mycobacterium tuberculosis Towards Defining Specific Host Pathogen Interactions - INTRACELLTB - - EC:FP7:ERC2010-12-01 - 2015-11-30 - 260901 - VALID, More Medicines for Tuberculosis - MM4TB - - EC:FP7:HEALTH2011-02-01 - 2016-01-31 - 260872 - VALID, Centre de Biochimie Structurale [Montpellier] (CBS), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), Centre d’Infection et d’Immunité de Lille - INSERM U 1019 - UMR 9017 - UMR 8204 (CIIL), Institut Pasteur de Lille, Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Lille-Centre Hospitalier Régional Universitaire [Lille] (CHRU Lille)-Centre National de la Recherche Scientifique (CNRS), Institut de pharmacologie et de biologie structurale (IPBS), Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS), R.V.C. acknowledges support from the Région Nord-Pas-de-Calais. Financial support for this work was provided by the European Community (ERC-STG INTRACELLTB grant no. 260901, MM4TB grant no. 260872), the Agence Nationale de la Recherche (ANR-10-EQPX-04-01 and ANR-11-LABX-0024), the Fonds Européen de Développement Economique Régionale (12001407 [D-AL] Equipex Imaginex BioMed and 13003300-42405 Labex Parafrap), the Métropole Européene de Lille (MEL) and the Region Nord Pas de Calais (2011_10248), and French Infrastructure for Integrated Structural Biology (FRISBI) ANR-10-INSB-05-01., ANR-10-EQPX-0004,Imaginex BioMed,Plateau de microscopie de criblage à haut débit et d'analyse à très haute résolution(2010), ANR-11-LABX-0024,ParaFrap,Alliance française contre les maladies parasitaires(2011), ANR-10-INBS-0005,FRISBI,Infrastructure Française pour la Biologie Structurale Intégrée(2010), European Project: 260901,EC:FP7:ERC,ERC-2010-StG_20091118,INTRACELLTB(2010), European Project: 260872,EC:FP7:HEALTH,FP7-HEALTH-2010-single-stage,MM4TB(2011), Université de Toulouse (UT)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM)-Institut National de la Santé et de la Recherche Médicale (INSERM), Centre National de la Recherche Scientifique (CNRS)-Centre Hospitalier Régional Universitaire [Lille] (CHRU Lille)-Université de Lille-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut Pasteur de Lille, Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP), Centre National de la Recherche Scientifique (CNRS)-Université Toulouse III - Paul Sabatier (UT3), and Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées

Subjects
0301 basic medicine, Models, Molecular, Phagocytosis, Lipoproteins, 030106 microbiology, Plasma protein binding, Phosphatidylinositols, Mass Spectrometry, Protein Structure, Secondary, Microbiology, Mycobacterium tuberculosis, 03 medical and health sciences, chemistry.chemical_compound, Immune system, Bacterial Proteins, Protein Domains, Structural Biology, Cell Wall, [SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology, Macrophage, Protein Isoforms, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Phosphatidylinositol, Molecular Biology, Binding Sites, biology, Effector, Gene Expression Regulation, Bacterial, biology.organism_classification, 3. Good health, Culture Media, Secretory protein, chemistry
Abstract

International audience; Mycobacterium tuberculosis (Mtb) encodes several bacterial effectors impacting the colonization of phagocytes. LppM (Rv2171) is both implicated in phagocytosis and able to efficiently block phagosomal acidification in the macrophage, two key processes contributing to Mtb persistence. We show that LppM is anchored to the mycobacterial cell wall by a C-terminal membrane domain. However, the protein also exists as a truncated protein secreted into the culture medium. The LppM solution structure we solve here displays no similarity with other Mtb lipoproteins also involved in phagosomal maturation (i.e., LprG). In addition, we demonstrate that the protein may be able to bind rare molecular species of phosphatidylinositol mannosides, bacterial compounds known to affect the host immune response. Thus, our data demonstrate a dual localization of LppM and provide a unique perspective on the regulation of protein secretion and localization in Mtb.

Published
2016

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