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108 results on '"Microbiologie structurale - Structural Microbiology (Microb. Struc. (UMR_3528 / U-Pasteur_5))"'

1. 3D structures of the Plasmodium vivax subtilisin-like drug target SUB1 reveal conformational changes to accommodate a substrate-derived α-ketoamide inhibitor

Author: Martinez, Mariano, Batista, Fernando, Maurel, Manon, Bouillon, Anthony, Ortega Varga, Laura, Wehenkel, Anne Marie, Le Chevalier-Sontag, Lucile, Blondel, Arnaud, Haouz, Ahmed, Hernandez, Jean-François, Alzari, Pedro, Barale, Jean-Christophe, Microbiologie structurale - Structural Microbiology (Microb. Struc. (UMR_3528 / U-Pasteur_5)), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Institut des Biomolécules Max Mousseron [Pôle Chimie Balard] (IBMM), Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM)-Ecole Nationale Supérieure de Chimie de Montpellier (ENSCM), Université de Montpellier (UM), Bioinformatique structurale - Structural Bioinformatics, Cristallographie (Plateforme) - Crystallography (Platform), ANR, CNRS et Institut Pasteur, For this work, A. Bouillon and M. Martinez were supported by Agence Nationale pour la Recherche (ANR) grant ANR-11-RPIB-002 and FB and LO were supported by grant ANR-19-CE18-0010. LO was also supported by ANR-17-CE11-0030 and by the ‘URGENCE COVID-19’ fundraising campaign of Institut Pasteur. Part of this work and M. Maurel were supported by Carnot Chimie Balard Institute through ANR program 11 CARN 0001-01., ANR-11-RPIB-0002,MaPI,Développement d'un composé 'leader' à l'origine d'une nouvelle génération d'anti-paludiques: une approche multi-cibles(2011), ANR-19-CE18-0010,SPIM,Synthèse, biologie et structure pour guider l'optimisation d'Inhibiteurs de la peptidase SUB1 de Plasmodium, une cible potentielle pour le traitement du paludisme(2019), and ANR-17-CE11-0030,NICOFIVE,Découverte de modulateurs allostériques ciblant les récepteurs nicotiniques alpha5(2017)
Subjects: Plasmodium, malaria, [CHIM.CRIS]Chemical Sciences/Cristallography, therapeutic target, subtilisin, [SDV.MP.PAR]Life Sciences [q-bio]/Microbiology and Parasitology/Parasitology, [CHIM.THER]Chemical Sciences/Medicinal Chemistry, crystalography
Abstract: International audience; The constant selection and propagation of multi-resistant Plasmodium sp. parasites require the identification of new antimalarial candidates involved in as-yet untargeted metabolic pathways. Subtilisin-like protease 1 (SUB1) belongs to a new generation of drug targets because it plays a crucial role during egress of the parasite from infected host cells at different stages of its life cycle. SUB1 is characterized by an unusual pro-region that tightly interacts with its cognate catalytic domain, thus precluding 3D structural analysis of enzyme–inhibitor complexes. In the present study, to overcome this limitation, stringent ionic conditions and controlled proteolysis of recombinant full-length P. vivax SUB1 were used to obtain crystals of an active and stable catalytic domain (PvS1 Cat ) without a pro-region. High-resolution 3D structures of PvS1 Cat , alone and in complex with an α-ketoamide substrate-derived inhibitor (MAM-117), showed that, as expected, the catalytic serine of SUB1 formed a covalent bond with the α-keto group of the inhibitor. A network of hydrogen bonds and hydrophobic interactions stabilized the complex, including at the P1′ and P2′ positions of the inhibitor, although P′ residues are usually less important in defining the substrate specificity of subtilisins. Moreover, when associated with a substrate-derived peptidomimetic inhibitor, the catalytic groove of SUB1 underwent significant structural changes, particularly in its S4 pocket. These findings pave the way for future strategies for the design of optimized SUB1-specific inhibitors that may define a novel class of antimalarial candidates.
Published: 2023
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2. Phages against Noncapsulated Klebsiella pneumoniae: Broader Host range, Slower Resistance

Author: Lourenço, Marta, Osbelt, Lisa, Passet, Virginie, Gravey, François, Megrian, Daniela, Strowig, Till, Rodrigues, Carla, Brisse, Sylvain, Biodiversité et Epidémiologie des Bactéries pathogènes - Biodiversity and Epidemiology of Bacterial Pathogens, Institut Pasteur [Paris] (IP)-Université Paris Cité (UPCité), Helmholtz Centre for Infection Research (HZI), German Center for Infection Research - partner site Hannover-Braunschweig (DZIF), Dynamique Microbienne associée aux Infections Urinaires et Respiratoires (DYNAMICURE), Université de Caen Normandie (UNICAEN), Normandie Université (NU)-Normandie Université (NU)-Université de Rouen Normandie (UNIROUEN), Normandie Université (NU)-Institut National de la Santé et de la Recherche Médicale (INSERM), Microbiologie structurale - Structural Microbiology (Microb. Struc. (UMR_3528 / U-Pasteur_5)), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), This work was mainly supported by the Joint Programming Initiative on Antimicrobial Resistance (JPIAMR) project CRISPR-ATTACK (Advancing CRISPR antimicrobials to combat the bacterial pathogen Klebsiella pneumoniae) under the French Agence Nationale de la Recherche grant ANR-18-JAM2-0004-04, S.B. and 01KI1824 to T.S. C.R. was also supported financially by a Pasteur-Roux fellowship by the Institut Pasteur. The BEBP laboratory is supported by the French Government Investissement d’Avenir Program Laboratoire d’Excellence, Integrative Biology of Emerging Infectious Diseases (ANR10LABX62IBEID). T.S. was also supported by the Federal Ministry of Science under the project DF-AMR2:DECOLONIZE (01KI2131), JPI-AMR Germany (01KI1824) as well as by the German Center for Infection Research (DZIF, TTU 06.826)., We thank Olaya Rendueles Garcia and Eduardo Rocha for sharing the mutant strains that were used for the anti-Kd phage isolation. We thank the Biomics Platform, C2RT, Institut Pasteur, Paris, France, supported by France Génomique (ANR-10-INBS-09-09) and IBISA, especially Marc Monot, Elodie Turc, Laure Lemée and Georges Haustant, for the sequencing project management, the preparation of the genomic libraries, and the sequencing. We thank Melanie Hennart for the bioinformatics methodological input and Anne-Marie Wehenkel for the help with the protein analyses. We are grateful to Jin-Town Wang for sharing the strain NTUH-K2044. We thank Quentin Lamy-Besnier, Chiara Crestani, and Olaya Rendueles Garcia for the critical reading of the manuscript., ANR-18-JAM2-0004,CRISPRattacK(2018), and ANR-10-LABX-0062,IBEID,Integrative Biology of Emerging Infectious Diseases(2010)
Subjects: Klebsiella pneumoniae, in vivo, bacteriophages, phage therapy, bacteriophage-bacteria interactions, phage resistance, [SDV.MHEP.MI]Life Sciences [q-bio]/Human health and pathology/Infectious diseases, genomics, host range, bacteriophage therapy, antimicrobial resistance, noncapsulated mutants, phage-bacteria interactions
Abstract: International audience; Klebsiella pneumoniae (Kp), a human gut colonizer and opportunistic pathogen, is a major contributor to the global burden of antimicrobial resistance. Virulent bacteriophages represent promising agents for decolonization and therapy. However, the majority of anti-Kp phages that have been isolated thus far are highly specific to unique capsular types (anti-K phages), which is a major limitation to phage therapy prospects due to the highly polymorphic capsule of Kp. Here, we report on an original anti-Kp phage isolation strategy, using capsule-deficient Kp mutants as hosts (anti-Kd phages). We show that anti-Kd phages have a broad host range, as the majority are able to infect noncapsulated mutants of multiple genetic sublineages and O-types. Additionally, anti-Kd phages induce a lower rate of resistance emergence in vitro and provide increased killing efficiency when in combination with anti-K phages. In vivo, anti-Kd phages are able to replicate in mouse guts colonized with a capsulated Kp strain, suggesting the presence of noncapsulated Kp subpopulations. The original strategy proposed here represents a promising avenue that circumvents the Kp capsule host restriction barrier, offering promise for therapeutic development.IMPORTANCE Klebsiella pneumoniae (Kp) is an ecologically generalist bacterium as well as an opportunistic pathogen that is responsible for hospital-acquired infections and a major contributor to the global burden of antimicrobial resistance. In the last decades, limited advances have been made in the use of virulent phages as alternatives or complements to antibiotics that are used to treat Kp infections. This work demonstrates the potential value of an anti-Klebsiella phage isolation strategy that addresses the issue of the narrow host range of anti-K phages. Anti-Kd phages may be active in infection sites in which capsule expression is intermittent or repressed or in combination with anti-K phages, which often induce the loss of capsule in escape mutants.
Published: 2023
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3. Legionella para-effectors target chromatin and promote bacterial replication

Author: Daniel Schator, Sonia Mondino, Jérémy Berthelet, Cristina Di Silvestre, Mathilde Ben Assaya, Christophe Rusniok, Fernando Rodrigues-Lima, Annemarie Wehenkel, Carmen Buchrieser, Monica Rolando, Biologie des Bactéries intracellulaires - Biology of Intracellular Bacteria, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Unité de Biologie Fonctionnelle et Adaptative (BFA (UMR_8251 / U1133)), Université Paris Diderot - Paris 7 (UPD7)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Microbiologie structurale - Structural Microbiology (Microb. Struc. (UMR_3528 / U-Pasteur_5)), Institut Pasteur [Paris] (IP)-Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), Génomique des Microorganismes Pathogènes, Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Toxines bactériennes dans la relation hôtes-pathogènes, Université Nice Sophia Antipolis (1965 - 2019) (UNS), and COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-IFR50-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Côte d'Azur (UCA)
Subjects: Multidisciplinary, [SDV]Life Sciences [q-bio], General Physics and Astronomy, General Chemistry, General Biochemistry, Genetics and Molecular Biology
Abstract: Legionella pneumophila replicates intracellularly by secreting effectors via a type IV secretion system. One of these effectors is a eukaryotic methyltransferase (RomA) that methylates K14 of histone H3 (H3K14me3) to counteract host immune responses. However, it is not known how L. pneumophila infection catalyses H3K14 methylation as this residue is usually acetylated. Here we show that L. pneumophila secretes a eukaryotic-like histone deacetylase (LphD) that specifically targets H3K14ac and works in synergy with RomA. Both effectors target host chromatin and bind the HBO1 histone acetyltransferase complex that acetylates H3K14. Full activity of RomA is dependent on the presence of LphD as H3K14 methylation levels are significantly decreased in a ∆lphD mutant. The dependency of these two chromatin-modifying effectors on each other is further substantiated by mutational and virulence assays revealing that the presence of only one of these two effectors impairs intracellular replication, while a double knockout (∆lphD∆romA) can restore intracellular replication. Uniquely, we present evidence for “para-effectors”, an effector pair, that actively and coordinately modify host histones to hijack the host response. The identification of epigenetic marks modulated by pathogens has the potential to lead to the development of innovative therapeutic strategies to counteract bacterial infection and strengthening host defences.
Published: 2023
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4. A novel nuclease is the executing part of a bacterial plasmid defense system

Author: Manuela Weiß, Giacomo Giacomelli, Mathilde Ben Assaya, Finja Grundt, Ahmed Haouz, Feng Peng, Stéphanie Petrella, Anne Marie Wehenkel, Marc Bramkamp, Christian-Albrechts University of Kiel, Ludwig-Maximilians-Universität München (LMU), Institut für Allgemeine Mikrobiologie, Christian-Albrechts-Universität zu Kiel (CAU), Microbiologie structurale - Structural Microbiology (Microb. Struc. (UMR_3528 / U-Pasteur_5)), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Cristallogenèse et Diffraction des Rayons X (Plate-forme/PF6), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), University of Vienna [Vienna], Ludwig-Maximilians University [Munich] (LMU), and Kiel University
Subjects: [SDV]Life Sciences [q-bio]
Abstract: Cells are continuously facing the risk of taking up foreign DNA that can compromise genomic and cellular integrity. Therefore, bacteria are in a constant arms race with mobile genetic elements such as phages, transposons and plasmids. They have developed several active strategies against invading DNA molecules that can be seen as a bacterial ‘innate immune system’. Anti-phage systems are usually organized in ‘defense islands’ and can consist of restriction-modification (R-M) systems, CRISPR-Cas, and abortive infection (Abi) systems. Despite recent advances in the field, much less is known about plasmid defense systems. We have recently identified the MksBEFG system in Corynebacterium glutamicum as a novel plasmid defense system which comprises homologues of the condensin system MukFEB. Here, we investigated the molecular arrangement of the MksBEFG complex. Importantly, we identified MksG as a novel nuclease that degrades plasmid DNA and is, thus, the executing part of the system. The crystal structure of MksG revealed a dimeric assembly through its DUF2220 C-terminal domains. This domain is homologous to the TOPRIM domain of the topoisomerase II family of enzymes and contains the corresponding divalent ion binding site that is essential for DNA cleavage in topoisomerases, explaining the in vitro nuclease activity of MksG. We further show that the MksBEF subunits exhibit an ATPase cycle similar to MukBEF in vitro and we reason that this reaction cycle, in combination with the nuclease activity provided by MksG, allows for processive degradation of invading plasmids. Super-resolution localization microscopy revealed that the Mks system is spatially regulated via to the polar scaffold protein DivIVA. Introduction of plasmids increases the diffusion rate and alters the localization of MksG, indicating an activation of the system in vivo.
Published: 2022

5. Characteristics of the GlnH and GlnX Signal Transduction Proteins Controlling PknG-Mediated Phosphorylation of OdhI and 2-Oxoglutarate Dehydrogenase Activity in Corynebacterium glutamicum

Author: Lea Sundermeyer, Graziella Bosco, Srushti Gujar, Melanie Brocker, Meike Baumgart, Dieter Willbold, Oliver H. Weiergräber, Marco Bellinzoni, Michael Bott, Forschungszentrum Jülich GmbH, Heinrich Heine Universität Düsseldorf = Heinrich Heine University [Düsseldorf], Microbiologie structurale - Structural Microbiology (Microb. Struc. (UMR_3528 / U-Pasteur_5)), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), This work was financially supported by the DFG-ANR project MetActino (DFG BO 903/4-1, ANR-18-CE92-0003) and by the Heinrich Heine University Düsseldorf within the framework of the Bio24 graduate school., and ANR-18-CE92-0003,METACTINO,Déchiffrer la plasticité métabolique des Actinobactéries(2018)
Subjects: Microbiology (medical), 2-oxoglutarate dehydrogenase, General Immunology and Microbiology, Ecology, Physiology, [SDV]Life Sciences [q-bio], bacterial signaling, Cell Biology, forkhead-associated domain, protein phosphorylation, Corynebacterium glutamicum, Actinobacteria, Infectious Diseases, four-helix bundle, ddc:570, Genetics, periplasmic binding protein, serine/threonine kinases, signal transduction
Abstract: International audience; In Corynebacterium glutamicum the protein kinase PknG phosphorylates OdhI and thereby abolishes the inhibition of 2-oxoglutarate dehydrogenase activity by unphosphorylated OdhI. Our previous studies suggested that PknG activity is controlled by the periplasmic binding protein GlnH and the transmembrane protein GlnX, because ΔglnH and ΔglnX mutants showed a growth defect on glutamine similar to that of a ΔpknG mutant. We have now confirmed the involvement of GlnH and GlnX in the control of OdhI phosphorylation by analyzing the OdhI phosphorylation status and glutamate secretion in ΔglnH and ΔglnX mutants and by characterizing ΔglnX suppressor mutants. We provide evidence for GlnH being a lipoprotein and show by isothermal titration calorimetry that it binds l-aspartate and l-glutamate with moderate to low affinity, but not l-glutamine, l-asparagine, or 2-oxoglutarate. Based on a structural comparison with GlnH of Mycobacterium tuberculosis, two residues critical for the binding affinity were identified and verified. The predicted GlnX topology with four transmembrane segments and two periplasmic domains was confirmed by PhoA and LacZ fusions. A structural model of GlnX suggested that, with the exception of a poorly ordered N-terminal region, the entire protein is composed of α-helices and small loops or linkers, and it revealed similarities to other bacterial transmembrane receptors. Our results suggest that the GlnH-GlnX-PknG-OdhI-OdhA signal transduction cascade serves to adapt the flux of 2-oxoglutarate between ammonium assimilation via glutamate dehydrogenase and energy generation via the tricarboxylic acid (TCA) cycle to the availability of the amino group donors l-glutamate and l-aspartate in the environment.IMPORTANCE Actinobacteria comprise a large number of species playing important roles in biotechnology and medicine, such as Corynebacterium glutamicum, the major industrial amino acid producer, and Mycobacterium tuberculosis, the pathogen causing tuberculosis. Many actinobacteria use a signal transduction process in which the phosphorylation status of OdhI (corynebacteria) or GarA (mycobacteria) regulates the carbon flux at the 2-oxoglutarate node. Inhibition of 2-oxoglutarate dehydrogenase by unphosphorylated OdhI shifts the flux of 2-oxoglutarate from the TCA cycle toward glutamate formation and, thus, ammonium assimilation. Phosphorylation of OdhI/GarA is catalyzed by the protein kinase PknG, whose activity was proposed to be controlled by the periplasmic binding protein GlnH and the transmembrane protein GlnX. In this study, we combined genetic, biochemical, and structural modeling approaches to characterize GlnH and GlnX of C. glutamicum and confirm their roles in the GlnH-GlnX-PknG-OdhI-OdhA signal transduction cascade. These findings are relevant also to other Actinobacteria employing a similar control process.
Published: 2022
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6. FtsEX-independent control of RipA-mediated cell separation in Corynebacteriales

Author: Quentin Gaday, Daniela Megrian, Giacomo Carloni, Mariano Martinez, Bohdana Sokolova, Mathilde Ben Assaya, Pierre Legrand, Sebastien Brûlé, Ahmed Haouz, Anne Marie Wehenkel, Pedro M. Alzari, Microbiologie structurale - Structural Microbiology (Microb. Struc. (UMR_3528 / U-Pasteur_5)), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Synchrotron SOLEIL (SSOLEIL), Centre National de la Recherche Scientifique (CNRS), Cristallographie (Plateforme) - Crystallography (Platform), This work was funded by the Agence Nationale de la Recherche (ANR) through the projects PhoCellDiv (ANR-18-CE11-0017) granted to P.M.A. and MecaDiv (ANR-21-CE11-0003) granted to A.M.W., and by institutional grants from the Institut Pasteur, the CNRS, and Université Paris Cité. Molecular graphics were done with ChimeraX, developed at UCSF with support from NIH (R01-GM129325) and NIAID. We gratefully acknowledge the core facilities at the Institut Pasteur C2RT (Centre for Technological Resources and Research), in particular B. Raynal, P. England (PFBMI), and P. Weber, C. Pissis, A. Mechaly (PFC). We also acknowledge the synchrotron source Soleil (Saint-Aubin, France) for granting access to the facility and the staff of Proxima 1 and Proxima 2A beamlines for helpful assistance during X-ray data collection. G.C. was funded by the Unipharma Erasmus exchange program. Q.G. was funded by MTCI PhD school (ED 563)., ANR-18-CE11-0017,PhoCellDiv,Mécanismes moléculaires phospho-dépendants de l'assemblage et de la régulation du divisome bactérien(2018), and ANR-21-CE11-0003,MecaDiv,Caractérisation mécanistique de sous-complexes du divisome bactérien reconstitués in vitro(2021)
Subjects: Multidisciplinary, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM]
Abstract: The bacterial cell wall is a multi-layered mesh, whose major component is peptidoglycan (PG), a sugar polymer cross-linked by short peptide stems. During cell division, a careful balance of PG synthesis and degradation, precisely coordinated both in time and space, is necessary to prevent uncontrolled destruction of the cell wall. In Corynebacteriales, the D,L endopeptidase RipA has emerged as a major PG hydrolase for cell separation, and RipA defaults have major implications for virulence of the human pathogens Mycobacterium tuberculosis and Corynebacterium diphtheriae . However, the precise mechanisms by which RipA mediates cell separation remain elusive. Here we report phylogenetic, biochemical, and structural analysis of the Corynebacterium glutamicum homologue of RipA, Cg1735. The crystal structures of full-length Cg1735 in two different crystal forms revealed the C-terminal NlpC/P60 catalytic domain obtruded by its N-terminal conserved coiled-coil domain, which locks the enzyme in an autoinhibited state. We show that this autoinhibition is relieved by the extracellular core domain of the transmembrane septal protein Cg1604. The crystal structure of Cg1604 revealed a (β/α) protein with an overall topology similar to that of receiver domains from response regulator proteins. The atomic model of the Cg1735–Cg1604 complex, based on bioinformatical and mutational analysis, indicates that a conserved, distal-membrane helical insertion in Cg1604 is responsible for Cg1735 activation. The reported data provide important insights into how intracellular cell division signal(s), yet to be identified, control PG hydrolysis during RipA-mediated cell separation in Corynebacteriales .
Published: 2022
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7. 5-(4-Nitrophenyl)furan-2-carboxylic Acid

Author: Matteo Mori, Andrea Tresoldi, Stefania Villa, Giulia Cazzaniga, Marco Bellinzoni, Fiorella Meneghetti, Università degli Studi di Milano = University of Milan (UNIMI), Microbiologie structurale - Structural Microbiology (Microb. Struc. (UMR_3528 / U-Pasteur_5)), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), and This work was funded by the University of Milan, 'Linea B'.
Subjects: furan, MbtI, Organic Chemistry, SC-XRD, Mycobacterium tuberculosis, Settore CHIM/06 - Chimica Organica, Biochemistry, Settore CHIM/08 - Chimica Farmaceutica, synchrotron, antitubercular agent, [CHIM]Chemical Sciences, Physical and Theoretical Chemistry, iron acquisition
Abstract: International audience; The ever-evolving research in the field of antitubercular agents has led to the identification of several new potential drug classes. Among them, 5-phenyl-furan-2-carboxylic acids have emerged as innovative potential therapeutics, targeting iron acquisition in mycobacterial species. In our efforts to characterize the molecular interactions between these compounds and their protein target (MbtI from M. tuberculosis) by means of co-crystallization experiments, we unexpectedly obtained the structure of 5-(4-nitrophenyl)furan-2-carboxylic acid (1). Herein, we describe the preparation of the compound and its analysis by 1H NMR, 13C NMR, HRMS, and SC-XRD.
Published: 2022
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8. Complete Genome Sequence of Mesorhizobium ciceri Strain R30, a Rhizobium Used as a Commercial Inoculant for Chickpea in Argentina

Author: Emiliano Foresto, Santiago Revale, Emiliano Primo, Fiorela Nievas, Evangelina Carezzano, Mariana Puente, Pedro Alzari, Mariano Martínez, Mathilde Ben-Assaya, Damien Mornico, Maricel Santoro, Francisco Martínez-Abarca, Walter Giordano, Pablo Bogino, National University of Río Cuarto = Universidad Nacional de Río Cuarto (UNRC), The Wellcome Trust Centre for Human Genetics [Oxford], University of Oxford, Instituto Nacional de Tecnología Agropecuaria, Universidad Nacional de la Patagonia Austral (UNPA), Microbiologie structurale - Structural Microbiology (Microb. Struc. (UMR_3528 / U-Pasteur_5)), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Hub Bioinformatique et Biostatistique - Bioinformatics and Biostatistics HUB, Institut Pasteur [Paris] (IP)-Université Paris Cité (UPCité), Max Planck Institute for Chemical Ecology, Max-Planck-Gesellschaft, Grupo de Ecología Genética, Estación Experimental del Zaidín, Granada, Consejo Superior de Investigaciones Científicas [Madrid] (CSIC), This work was supported by grants from the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT) (PID2020-113207GBI00 funded by MCIN/AEI/10.13039/501100011033), by 'ERDF: A Way of Making Europe' (P20_0047), funded by the Junta de Andalucía PAIDI/FEDER/EU, and by the Biotechnology and Biosciences Research Council (BBSRC)., We are grateful to Plateforme de Microbiologie Mutualisée (P2M) and the Pasteur International Bioresources network (PIBnet) and to Institut Pasteur Paris for providing the resources for Illumina sequencing. We thank O.G.C. at the Wellcome Centre for Human Genetics for the sequencing data and B.M.R.C. for processing (supported by Wellcome Trust Core Award grant 203141/Z/16/Z and the NIHR Oxford BRC). We are also grateful to Vincent Enouf from Unité de Génétique Moléculaire des Virus à ARN-UMR3569 CNRS, Université de Paris, Centre National de Référence Virus des Infections Respiratoires (dont la grippe) and to F. Sgarlatta for proofreading the manuscript., Consejo Nacional de Investigaciones Científicas y Técnicas (Argentina), Agencia Nacional de Promoción Científica y Tecnológica (Argentina), Ministerio de Ciencia e Innovación (España), Junta de Andalucía, and European Commission
Subjects: Immunology and Microbiology (miscellaneous), Genetics, [SDE.BE]Environmental Sciences/Biodiversity and Ecology, Molecular Biology
Abstract: We report the complete genome sequence of Mesorhizobium ciceri strain R30, a rhizobium strain recommended and used as a commercial inoculant for chickpea in Argentina. The genome consists of almost 7 Mb, distributed into two circular replicons: a chromosome of 6.49 Mb and a plasmid of 0.46 Mb., This work was supported by grants from the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT) (PID2020-113207GBI00 funded by MCIN/AEI/10.13039/501100011033); by “ERDF: A Way of Making Europe” (P20_0047), funded by the Junta de Andalucía PAIDI/FEDER/EU; and by the Biotechnology and Biosciences Research Council (BBSRC). We are grateful to Plateforme de Microbiologie Mutualisée (P2M) and the Pasteur International Bioresources network (PIBnet) and to Institut Pasteur Paris for providing the resources for Illumina sequencing. We thank O.G.C. at the Wellcome Centre for Human Genetics for the sequencing data and B.M.R.C. for processing (supported by Wellcome Trust Core Award grant 203141/Z/16/Z and the NIHR Oxford BRC). We are also grateful to Vincent Enouf from Unité de Génétique Moléculaire des Virus à ARN-UMR3569 CNRS, Université de Paris, Centre National de Référence Virus des Infections Respiratoires (dont la grippe) and to F. Sgarlatta for proofreading the manuscript.
Published: 2022
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9. Efficacy of dihydroartemisinin/piperaquine in patients with non-complicated Plasmodium falciparum malaria in Yaoundé, Cameroon

Author: Xavier Iriart, Benoit Witkowski, Melissa Mairet-Khedim, Lawrence Ayong, Laure Otam, Sandrine E. Nsango, Christelle Ngou, Antoine Berry, Peggy Gandia, Isabelle Morlais, Nimol Khim, Camille Roesch, Sandie Menard, Sreynet Srun, Thomas Lanot, Francis Abega, Malaria Molecular Epidemiology, Institut Pasteur du Cambodge, Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP), Institut Toulousain des Maladies Infectieuses et Inflammatoires (Infinity), Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Microbiologie structurale - Structural Microbiology (Microb. Struc. (UMR_3528 / U-Pasteur_5)), Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS)-Université de Paris (UP), Université de Douala, Centre Pasteur du Cameroun, Réseau International des Instituts Pasteur (RIIP), Maladies infectieuses et vecteurs : écologie, génétique, évolution et contrôle (MIVEGEC), Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche pour le Développement (IRD [France-Sud]), Hôpital Purpan [Toulouse], CHU Toulouse [Toulouse], Université de Ngaoundéré/University of Ngaoundéré [Cameroun] (UN), Innovations Thérapeutiques et Résistances (InTheRes), Institut National de la Recherche Agronomique (INRA)-Ecole Nationale Vétérinaire de Toulouse (ENVT), Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées, This work was sponsored in part by the Institut National de la Santée de la Recherche Médicale, the ParaFrap Consortium and core funding from the Institut de Recherche pour le Développement & Pasteur Institute of Cambodia. S.N. was supported by an Institut Pasteur International Network Calmette & Yersin mobility grant, We would like to thank all the technical teams from the Malaria Research Unit of the Centre Pasteur of Cameroon, the Malaria Molecular Epidemiology Unit of the Pasteur Institute of Cambodia and the Eukaryotic Intracellular Parasites: Immunity and Chemoresistance Unit of the Centre for Physiopathology of Toulouse Purpan., Malaria Molecular Epidemiology (MMEU), Université de Toulouse (UT)-Université de Toulouse (UT)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Centre Hospitalier Universitaire de Toulouse (CHU Toulouse), Dispensaire Nkol Eton, Yaounde, Cameroon, Transmission-Interactions-Adaptations hôtes/vecteurs/pathogènes (MIVEGEC-TRIAD), Evolution des Systèmes Vectoriels (ESV), Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche pour le Développement (IRD [France-Sud])-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche pour le Développement (IRD [France-Sud])-Maladies infectieuses et vecteurs : écologie, génétique, évolution et contrôle (MIVEGEC), Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche pour le Développement (IRD [France-Sud])-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche pour le Développement (IRD [France-Sud]), Université de Toulouse (UT)-Université de Toulouse (UT)-Institut National Polytechnique (Toulouse) (Toulouse INP), and Université de Toulouse (UT)-Université de Toulouse (UT)
Subjects: Microbiology (medical), medicine.medical_treatment, Plasmodium falciparum, MESH: Malaria, 030231 tropical medicine, Dihydroartemisinin, Pharmacology, Antimalarials, 03 medical and health sciences, 0302 clinical medicine, Dihydroartemisinin/piperaquine, Piperaquine, MESH: Artemisinins, parasitic diseases, medicine, Humans, [SDV.MP.PAR]Life Sciences [q-bio]/Microbiology and Parasitology/Parasitology, Pharmacology (medical), In patient, Cameroon, Clinical efficacy, Malaria, Falciparum, Artemisinin, MESH: Plasmodium falciparum, 030304 developmental biology, 0303 health sciences, MESH: Humans, biology, business.industry, MESH: Malaria, Falciparum, MESH: Cameroon, medicine.disease, biology.organism_classification, MESH: Antimalarials, Artemisinins, Malaria, 3. Good health, Infectious Diseases, Quinolines, business, MESH: Quinolines, medicine.drug
Abstract: Background Dihydroartemisinin/piperaquine is increasingly used for the treatment of uncomplicated Plasmodium falciparum malaria in Africa. The efficacy of this combination in Cameroon is poorly documented, while resistance to dihydroartemisinin/piperaquine readily spreads in Southeast Asia. Objectives This study evaluated the clinical efficacy of dihydroartemisinin/piperaquine in Cameroon, as well as the molecular profile and phenotypic susceptibility of collected isolates to dihydroartemisinin and piperaquine. Patients and methods Dihydroartemisinin/piperaquine efficacy in 42 days was followed-up for 138 patients presenting non-complicated falciparum malaria. Piperaquine concentration was determined at day 7 for 124 patients. kelch13 gene polymorphisms (n = 150) and plasmepsin2 gene amplification (n = 148) were determined as molecular markers of resistance to dihydroartemisinin and piperaquine, respectively. Parasite susceptibility to dihydroartemisinin and piperaquine was determined using validated in vitro survival assays. Results The efficacy of dihydroartemisinin/piperaquine treatment was 100% after PCR correction. The reinfections were not associated with a variation of piperaquine concentration at day 7. Ninety-six percent (144/150) of the samples presented a WT allele of the kelch13 gene. Two percent (3/150) presented the non-synonymous mutation A578S, which is not associated with resistance to dihydroartemisinin. No duplication of the plasmepsin2 gene was observed (0/148). All the samples tested in vitro by survival assays (n = 87) were susceptible to dihydroartemisinin and piperaquine. Conclusions Dihydroartemisinin/piperaquine has demonstrated excellent therapeutic efficacy with no evidence of emerging artemisinin or piperaquine resistance in Yaoundé, Cameroon. This observation suggests that dihydroartemisinin/piperaquine could be a sustainable therapeutic solution for P. falciparum malaria if implemented in areas previously free of artemisinin- and piperaquine-resistant parasites, unlike Southeast Asia.
Published: 2021
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10. The Antibacterial Type VII Secretion System of Bacillus subtilis: Structure and Interactions of the Pseudokinase YukC/EssB

Author: Matteo Tassinari, Thierry Doan, Marco Bellinzoni, Maïalene Chabalier, Mathilde Ben-Assaya, Mariano Martinez, Quentin Gaday, Pedro M. Alzari, Eric Cascales, Rémi Fronzes, Francesca Gubellini, Microbiologie structurale - Structural Microbiology (Microb. Struc. (UMR_3528 / U-Pasteur_5)), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Institut Européen de Chimie et Biologie (IECB), Université de Bordeaux (UB)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Collège Doctoral, Sorbonne Université (SU), Laboratoire d'ingénierie des systèmes macromoléculaires (LISM), Aix Marseille Université (AMU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Microbiologie Fondamentale et Pathogénicité (MFP), Université Bordeaux Segalen - Bordeaux 2-Centre National de la Recherche Scientifique (CNRS), This work received financial support from the Institut Pasteur (Paris), the Centre National pour la Recherche Scientifique (CNRS), and Aix-Marseille Université. T.D. and E.C. acknowledge the Fondation Bettencourt-Schueller for the acquisition of the epifluorescence microscope. M.T. was supported by a scholarship from the Pasteur-Paris University (PPU) International PhD Program., We thank A. Haouz and the Crystallography Facility at the Institut Pasteur (Paris) for invaluable support in obtaining YukC crystals. We acknowledge the European Synchrotron Radiation Facility for provision of synchrotron radiation facilities, and we thank Gordon Leonard for assistance at beamline ID30A-3. We also thank the Synchrotron SOLEIL and its staff for initial tests. We are grateful to P. Tavares (I2BC, Université Paris-Sud, Université Paris-Saclay) and D. Rudner (Harvard University, USA) for the kind gifts of purified SPP1 phage and pDR111 plasmid, respectively. We acknowledge the Institut Pasteur platforms of Proteomics and Molecular Biophysics (S. Brûlé and B. Raynal) for the YukE MS analysis and nanoDSF experiments, respectively. We are very grateful to A. M. Wehenkel, O. Francetic, and S. Dramsi (Institut Pasteur, Paris) for helpful discussions and to M. Ricchetti (Institut Pasteur, Paris) for her support of M. Tassinari during his PhD work. We thank G. Pehau-Arnaudet (Unité Technologie et Service Bioimagerie Ultrastructurale, Institut Pasteur, Paris) for his help with initial electron microscopy investigation on YukC. We also thank L. Catoire and M. Casiraghi (IBPC, Paris) for the insertion of YukC in nanodiscs, even if the results could unfortunately not be inserted in the current study. A warm thanks to N. Minc (IJM, Paris) for helping with data analysis and for proofreading the manuscript. We acknowledge the contribution of Danguolė Norkūnaitė in inserting the yukC-P231A mutation on the plasmid used for the stabilisation tests., and martinez, mariano
Subjects: protein complexes, pseudokinases, interaction network, [SDV]Life Sciences [q-bio], pseudokinase, bacterial two-hybrid assay, Microbiology, secretion systems, Anti-Bacterial Agents, [SDV] Life Sciences [q-bio], Bacterial Proteins, Virology, Type VII Secretion Systems, type VIIb secretion system, structural biology, bacterial competition, crystallographic structure, Bacillus subtilis
Abstract: International audience; Type VIIb secretion systems (T7SSb) were recently proposed to mediate different aspects of Firmicutes physiology, including bacterial pathogenicity and competition. However, their architecture and mechanism of action remain largely obscure. Here, we present a detailed analysis of the T7SSb-mediated bacterial competition in Bacillus subtilis, using the effector YxiD as a model for the LXG secreted toxins. By systematically investigating protein-protein interactions, we reveal that the membrane subunit YukC contacts all T7SSb components, including the WXG100 substrate YukE and the LXG effector YxiD. YukC's crystal structure shows unique features, suggesting an intrinsic flexibility that is required for T7SSb antibacterial activity. Overall, our results shed light on the role and molecular organization of the T7SSb and demonstrate the potential of B. subtilis as a model system for extensive structure-function studies of these secretion machineries. IMPORTANCE Type VII secretion systems mediate protein extrusion from Gram-positive bacteria and are classified as T7SSa and T7SSb in Actinobacteria and in Firmicutes, respectively. Despite the genetic divergence of T7SSa and T7SSb, the high degree of structural similarity of their WXG100 substrates suggests similar secretion mechanisms. Recent advances revealed the structures of several T7SSa cytoplasmic membrane complexes, but the molecular mechanism of secretion and the T7SSb architecture remain obscure. Here, we provide hints on the organization of T7SSb in B. subtilis and a highresolution structure of its central pseudokinase subunit, opening new perspectives for the understanding of the T7SSb secretion mechanism by using B. subtilis as an amenable bacterial model.
Published: 2022
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11. Structural Basis for the Binding of Allosteric Activators Leucine and ADP to Mammalian Glutamate Dehydrogenase

Author: Vasily A. Aleshin, Victoria I. Bunik, Eduardo M. Bruch, Marco Bellinzoni, Lomonosov Moscow State University (MSU), Sechenov First Moscow State Medical University, Microbiologie structurale - Structural Microbiology (Microb. Struc. (UMR_3528 / U-Pasteur_5)), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), and This research was supported by an Ostrogradski fellowship from the French Embassy in Moscow for a PhD international mobility to V.A.A., institutional funding from the Institut Pasteur and the CNRS, and an RSF grant to V.I.B. (grant no. 18-14-00116).
Subjects: ADP, Nitrogen, Glutamic Acid, Catalysis, Inorganic Chemistry, thiamine triphosphate, Animals, Physical and Theoretical Chemistry, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], Molecular Biology, Spectroscopy, acetylation, Mammals, molecular_biology, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], potassium, Organic Chemistry, glutamate dehydrogenase, General Medicine, Carbon, allosteric regulation, Computer Science Applications, Adenosine Diphosphate, Cattle, leucine
Abstract: International audience; Glutamate dehydrogenase (GDH) plays a key role in the metabolism of glutamate, an important compound at a cross-road of carbon and nitrogen metabolism and a relevant neurotransmitter. Despite being one of the first discovered allosteric enzymes, GDH still poses challenges for structural characterization of its allosteric sites. Only the structures with ADP, and at low (3.5 Å) resolution, are available for mammalian GDH complexes with allosteric activators. Here, we aim at deciphering a structural basis for the GDH allosteric activation using bovine GDH as a model. For the first time, we report a mammalian GDH structure in a ternary complex with the activators leucine and ADP, co-crystallized with potassium ion, resolved to 2.45 Å. An improved 2.4-angstrom resolution of the GDH complex with ADP is also presented. The ternary complex with leucine and ADP differs from the binary complex with ADP by the conformation of GDH C-terminus, involved in the leucine binding and subunit interactions. The potassium site, identified in this work, may mediate interactions between the leucine and ADP binding sites. Our data provide novel insights into the mechanisms of GDH activation by leucine and ADP, linked to the enzyme regulation by (de)acetylation.
Published: 2022
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12. Complete Genome Sequence of Bradyrhizobium sp. Strain C-145, a Nitrogen-Fixing Rhizobacterium Used as a Peanut Inoculant in Argentina

Author: Fiorela Nievas, Santiago Revale, Emiliano Foresto, Sacha Cossovich, Mariana Puente, Pedro Alzari, Mariano Martínez, Mathilde Ben-Assaya, Damien Mornico, Maricel Santoro, Francisco Martínez-Abarca, Walter Giordano, Pablo Bogino, Consejo Nacional de Investigaciones Científicas y Técnicas (Argentina), Agencia Nacional de Promoción Científica y Tecnológica (Argentina), Ministerio de Ciencia e Innovación (España), European Commission, Biotechnology and Biological Sciences Research Council (UK), Université de Paris, Institut Pasteur, National University of Río Cuarto = Universidad Nacional de Río Cuarto (UNRC), The Wellcome Trust Centre for Human Genetics [Oxford], University of Oxford, Instituto Nacional de Tecnología Agropecuaria, Universidad Nacional de la Patagonia Austral (UNPA), Microbiologie structurale - Structural Microbiology (Microb. Struc. (UMR_3528 / U-Pasteur_5)), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Hub Bioinformatique et Biostatistique - Bioinformatics and Biostatistics HUB, Institut Pasteur [Paris] (IP)-Université Paris Cité (UPCité), Max Planck Institute for Chemical Ecology, Max-Planck-Gesellschaft, Estacion Experimental del Zaidin-CSIC, and This work was supported by grants from the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT), the Spanish Ministerio de Ciencia e Innovación (research grant PID2020-113207GB-I00), ERDF (European Regional Development Funds), and the Biotechnology and Biosciences Research Council (BBSRC). It was also supported by Wellcome Trust Core Award grant number 203141/Z/16/Z and the NIHR Oxford BRC. We thank the Oxford Genomics Centre at the Wellcome Centre for Human Genetics (funded by Wellcome Trust grant reference 203141/Z/16/Z) for the sequencing data.
Subjects: [SDV.MP]Life Sciences [q-bio]/Microbiology and Parasitology, Immunology and Microbiology (miscellaneous), Genetics, Molecular Biology
Abstract: We present the complete genome sequence of Bradyrhizobium sp. strain C-145, one of the most widely used nitrogen-fixing rhizobacteria for inoculating peanut crops in Argentina. The genome consists of 9.53 Mbp in a single circular chromosome and was determined using a hybrid long- and short-read assembly approach., This work was supported by grants from the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT), the Spanish Ministerio de Ciencia e Innovación (research grant PID2020-113207GB-I00), ERDF (European Regional Development Funds), and the Biotechnology and Biosciences Research Council (BBSRC). It was also supported by Wellcome Trust Core Award grant number 203141/Z/16/Z and the NIHR Oxford BRC. We thank the Oxford Genomics Centre at the Wellcome Centre for Human Genetics (funded by Wellcome Trust grant reference 203141/Z/16/Z) for the sequencing data. We are also grateful to Vincent Enouf from Unité de Génétique Moléculaire des Virus à ARN—UMR3569 CNRS, Université de Paris, Centre National de Référence Virus des Infections Respiratoires (dont la grippe); to Plateforme de Microbiologie Mutualisée (P2M) and the Pasteur International Bioresources network (PIBnet); to Institut Pasteur Paris for providing the resources for Illumina sequencing; and finally, to F. Sgarlatta for proofreading the manuscript.
Published: 2022
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13. Prevalence and characterization of piperaquine, mefloquine and artemisinin derivatives triple-resistant Plasmodium falciparum in Cambodia

Author: Mélissa Mairet-Khedim, Camille Roesch, Nimol Khim, Sreynet Srun, Anthony Bouillon, Saorin Kim, Sopheakvatey Ke, Chhayleang Kauy, Nimol Kloeung, Rotha Eam, Chanra Khean, Chanvong Kul, Sophy Chy, Rithea Leang, Pascal Ringwald, Jean-Christophe Barale, Benoit Witkowski, Microbiologie structurale - Structural Microbiology (Microb. Struc. (UMR_3528 / U-Pasteur_5)), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Malaria Translational Research Unit (MTRU), Institut Pasteur du Cambodge, Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP)-Institut Pasteur [Paris] (IP)-Université Paris Cité (UPCité), Malaria Molecular Epidemiology (MMEU), Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP), National Center for Malariology, Ministry of Health [Phnom Penh], Organisation Mondiale de la Santé / World Health Organization Office (OMS / WHO), and This study was funded by the Bill & Melinda Gates Foundation and Global Fund/UNOPS through WHO and by the Institut Pasteur Department of International Affairs. M.M.K. was supported by the 5% initiative (MIVS-ACT, grant #15SANIN211).
Subjects: Pharmacology, Microbiology (medical), Infectious Diseases, [SDV]Life Sciences [q-bio], Pharmacology (medical)
Abstract: Background In early 2016, in Preah Vihear, Northern Cambodia, artesunate/mefloquine was used to cope with dihydroartemisinin/piperaquine-resistant Plasmodium falciparum parasites. Following this policy, P. falciparum strains harbouring molecular markers associated with artemisinin, piperaquine and mefloquine resistance have emerged. However, the lack of a viable alternative led Cambodia to adopt artesunate/mefloquine countrywide, raising concerns about a surge of triple-resistant P. falciparum strains. Objectives To assess the prevalence of triple-resistant parasites after artesunate/mefloquine implementation countrywide in Cambodia and to characterize their phenotype. Methods For this multicentric study, 846 samples were collected from 2016 to 2019. Genotyping of molecular markers associated with artemisinin, piperaquine and mefloquine resistance was coupled with phenotypic analyses. Results Only four triple-resistant P. falciparum isolates (0.47%) were identified during the study period. These parasites combined the pfk13 polymorphism with pfmdr1 amplification, pfpm2 amplification and/or pfcrt mutations. They showed significantly higher tolerance to artemisinin, piperaquine and mefloquine and also to the mefloquine and piperaquine combination. Conclusions The use of artesunate/mefloquine countrywide in Cambodia has not led to a massive increase of triple-resistant P. falciparum parasites. However, these parasites circulate in the population, and exhibit clear resistance to piperaquine, mefloquine and their combination in vitro. This study demonstrates that P. falciparum can adapt to more complex drug associations, which should be considered in future therapeutic designs.
Published: 2022
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14. Shedding X-ray Light on the Role of Magnesium in the Activity of Mycobacterium tuberculosis Salicylate Synthase (MbtI) for Drug Design

Author: Arianna Gelain, Fiorella Meneghetti, Josè Camilla Sammartino, Giulio Poli, Elena Pini, Stefania Villa, Laurent R. Chiarelli, Marco Bellinzoni, Giovanni Stelitano, Alessio Porta, Giangiacomo Beretta, Tiziano Tuccinardi, M. Mori, Università degli Studi di Milano [Milano] (UNIMI), Università degli Studi di Pavia, University of Pisa - Università di Pisa, Temple University [Philadelphia], Pennsylvania Commonwealth System of Higher Education (PCSHE), Microbiologie structurale - Structural Microbiology (Microb. Struc. (UMR_3528 / U-Pasteur_5)), Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS)-Université de Paris (UP), This work was funded by University of Milan (Linea B) and the Italian Ministry of Education, University and Research (MIUR): Dipartimenti di Eccellenza Program (2018–2022) - Dept. of Biology and Biotechnology 'L. Spallanzani', University of Pavia. Partial support was also provided by institutional grants from Institut Pasteur and CNRS., Università degli Studi di Milano = University of Milan (UNIMI), Università degli Studi di Pavia = University of Pavia (UNIPV), and Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité)
Subjects: Models, Molecular, Siderophore, MESH: Mycobacterium tuberculosis, Protein Conformation, MESH: Drug Design, Crystallography, X-Ray, 01 natural sciences, MESH: Protein Conformation, MESH: Structure-Activity Relationship, Models, MESH: Magnesium, Drug Discovery, Magnesium, Ternary complex, chemistry.chemical_classification, 0303 health sciences, Crystallography, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], biology, ATP synthase, 3. Good health, Biochemistry, Molecular Medicine, MESH: Models, Molecular, [PHYS.PHYS.PHYS-BIO-PH]Physics [physics]/Physics [physics]/Biological Physics [physics.bio-ph], Lyases, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, Cofactor, Article, Mycobacterium tuberculosis, 03 medical and health sciences, Structure-Activity Relationship, [CHIM.CRIS]Chemical Sciences/Cristallography, Structure–activity relationship, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, 030304 developmental biology, Rational design, Molecular, MESH: Crystallography, X-Ray, biology.organism_classification, MESH: Lyases, 0104 chemical sciences, 010404 medicinal & biomolecular chemistry, Enzyme, chemistry, Drug Design, X-Ray, biology.protein, [INFO.INFO-BI]Computer Science [cs]/Bioinformatics [q-bio.QM]
Abstract: International audience; The Mg2+-dependent Mycobacterium tuberculosis salicylate synthase (MbtI) is a key enzyme involved in the biosynthesis of siderophores. Because iron is essential for the survival and pathogenicity of the microorganism, this protein constitutes an attractive target for antitubercular therapy, also considering the absence of homologous enzymes in mammals. An extension of the structure-activity relationships of our furan-based candidates allowed us to disclose the most potent competitive inhibitor known to date (10, Ki = 4 μM), which also proved effective on mycobacterial cultures. By structural studies, we characterized its unexpected Mg2+-independent binding mode. We also investigated the role of the Mg2+ cofactor in catalysis, analyzing the first crystal structure of the MbtI-Mg2+-salicylate ternary complex. Overall, these results pave the way for the development of novel antituberculars through the rational design of improved MbtI inhibitors.
Published: 2020
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15. Essential dynamic interdependence of FtsZ and SepF for Z-ring and septum formation in Corynebacterium glutamicum

Author: Quentin Gaday, Anne Marie Wehenkel, Adrià Sogues, Sylvain Trépout, Rosario Durán, Alexandre Chenal, Mathilde Ben Assaya, Martín Graña, Pedro M. Alzari, Mariano A. Martinez, Alexis Voegele, Patrick England, Ahmed Haouz, Michael S. VanNieuwenhze, Microbiologie structurale - Structural Microbiology (Microb. Struc. (UMR_3528 / U-Pasteur_5)), Institut Pasteur [Paris] (IP)-Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), Bioinformatics / Bioinformática [Montevideo], Institut Pasteur de Montevideo, Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP), Biochimie des Interactions Macromoléculaires / Biochemistry of Macromolecular Interactions, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Department of Chemistry, Indiana University, Indiana University [Bloomington], Indiana University System-Indiana University System, Biophysique Moléculaire (Plate-forme), Cristallographie (Plateforme) - Crystallography (Platform), Institut Curie [Paris], Chimie et modélisation pour la biologie du cancer (CMBC), Institut Curie [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Chimie du CNRS (INC)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Analytical Biochemistry and Proteomics / Bioquímica y Proteómica Analíticas [Montevideo], This work was partially supported by grants from the Institut Pasteur (Paris), the CNRS (France) and the Agence Nationale de la Recherche (PhoCellDiv, ANR-18-CE11-0017-01). A.S. is part of the Pasteur-Paris University (PPU) International Ph.D Program, funded by the European Union’s Horizon 2020 research and innovation program under the Marie Sklodowska-Curie grant agreement No 665807. Q.G. was funded by MTCI Ph.D school (ED 563), A.V. was supported by a DIM MalInf (infectious diseases) grant. M.G. acknowledges support from Programa de Desarrollo de las Ciencias Básicas and Sistema Nacional de Investigación e Innovación, Uruguay., We thank A. Ducret for help with MicrobeJ, F. Gubellini for help with electron microscopy, M. Bott and M. Baumgart for the pk19-P3323-lcpA plasmid and help with corynebacterial genetics, and H. Gramajo for the pTGR5 plasmid. We gratefully acknowledge the core facilities at the Institut Pasteur C2RT, in particular G. Pehau-Arnaudet (UBI), B. Raynal, S. Brule (PFBMI), P. Weber, C. Pissis (PFC), and J. Fernandes (UtechS PBI/Imagopole, supported by France BioImaging, ANR-10–INSB–04, Investments for the Future). We thank the staff of ESRF and of EMBL-Grenoble for assistance and support in using beamlines ID30B and ID23-1, and the staff of SOLEIL Synchrotron for assistance in using the beamline Disco. We acknowledge the PICT-IBISA for providing access to the cryo-EM facility at Orsay. Finally, we would like to thank the reviewers for their coments and suggestions, which have helped us to improve the quality of the manuscript., ANR-18-CE11-0017,PhoCellDiv,Mécanismes moléculaires phospho-dépendants de l'assemblage et de la régulation du divisome bactérien(2018), European Project: 665807,H2020,H2020-MSCA-COFUND-2014,PASTEURDOC(2015), BENEDIC, Bénédicte, APPEL À PROJETS GÉNÉRIQUE 2018 - Mécanismes moléculaires phospho-dépendants de l'assemblage et de la régulation du divisome bactérien - - PhoCellDiv2018 - ANR-18-CE11-0017 - AAPG2018 - VALID, Institut Pasteur International Docotal Program - PASTEURDOC - - H20202015-10-01 - 2020-10-01 - 665807 - VALID, Institut Pasteur [Paris]-Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), and Université Paris Diderot - Paris 7 (UPD7)-Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS)
Subjects: 0301 basic medicine, Cell division, [SDV]Life Sciences [q-bio], ved/biology.organism_classification_rank.species, Cell, General Physics and Astronomy, Plasma protein binding, physiological processes, Corynebacterium glutamicum, lcsh:Science, Peptide sequence, 0303 health sciences, Multidisciplinary, biology, Chemistry, Cell biology, [SDV] Life Sciences [q-bio], medicine.anatomical_structure, biological phenomena, cell phenomena, and immunity, Dimerization, Cell division site, Protein Binding, Science, 030106 microbiology, Sequence alignment, macromolecular substances, Bacterial physiology, General Biochemistry, Genetics and Molecular Biology, Article, 03 medical and health sciences, Bacterial Proteins, medicine, Amino Acid Sequence, Model organism, Mode of action, FtsZ, X-ray crystallography, 030304 developmental biology, 030306 microbiology, ved/biology, General Chemistry, Gene Expression Regulation, Bacterial, biology.organism_classification, Cytoskeletal proteins, 030104 developmental biology, biology.protein, bacteria, lcsh:Q, Sequence Alignment, Function (biology), Cytokinesis, Bacteria
Abstract: The mechanisms of Z-ring assembly and regulation in bacteria are poorly understood, particularly in non-model organisms. Actinobacteria, a large bacterial phylum that includes the pathogen Mycobacterium tuberculosis, lack the canonical FtsZ-membrane anchors and Z-ring regulators described for E. coli. Here we investigate the physiological function of Corynebacterium glutamicum SepF, the only cell division-associated protein from Actinobacteria known to interact with the conserved C-terminal tail of FtsZ. We show an essential interdependence of FtsZ and SepF for formation of a functional Z-ring in C. glutamicum. The crystal structure of the SepF–FtsZ complex reveals a hydrophobic FtsZ-binding pocket, which defines the SepF homodimer as the functional unit, and suggests a reversible oligomerization interface. FtsZ filaments and lipid membranes have opposing effects on SepF polymerization, indicating that SepF has multiple roles at the cell division site, involving FtsZ bundling, Z-ring tethering and membrane reshaping activities that are needed for proper Z-ring assembly and function., The mechanisms of Z-ring assembly and regulation in bacteria are poorly understood, particularly in non-model organisms. Here, Sogues et al. study the interaction between FtsZ and SepF in Corynebacterium glutamicum, showing an essential interdependence of these proteins for formation of a functional Z-ring.
Published: 2020
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16. Enabling reactive microscopy with MicroMator

Author: Zachary R. Fox, Steven Fletcher, Achille Fraisse, Chetan Aditya, Sebastián Sosa-Carrillo, Julienne Petit, Sébastien Gilles, François Bertaux, Jakob Ruess, Gregory Batt, InBio - Méthodes Expérimentales et Computationnelles pour la Modélisation des Processus Cellulaires / Experimental and Computational Methods for Modeling Cellular Processes (INBIO), Institut Pasteur [Paris] (IP)-Inria de Paris, Institut National de Recherche en Informatique et en Automatique (Inria)-Institut National de Recherche en Informatique et en Automatique (Inria)-Université Paris Cité (UPCité), Los Alamos National Laboratory (LANL), Microbiologie structurale - Structural Microbiology (Microb. Struc. (UMR_3528 / U-Pasteur_5)), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Service Expérimentation et Développement (SED [Saclay]), Inria Saclay - Ile de France, Institut National de Recherche en Informatique et en Automatique (Inria)-Institut National de Recherche en Informatique et en Automatique (Inria), This work was supported by ANR grants CyberCircuits (ANR‐18‐CE91‐0002), MEMIP (ANR‐16‐CE33‐0018), and Cogex (ANR‐16‐CE12‐0025), by the H2020 Fet‐Open COSY‐BIO grant (grant agreement no. 766840) and by the Inria IPL grant COSY. We acknowledge the support of the U.S. Department of Energy through the LANL/LDRD Program and the Center for Non Linear Studies for this work. We thank Anne-Marie Wehenkel for her detailed comments., ANR-18-CE91-0002,CyberCircuits,Circuits cybergénétiques pour tester la composabilité des réseaux génétiques(2018), ANR-16-CE33-0018,MEMIP,Modèles à effets mixtes de processus intracellulaires: méthodes, outils et applications(2016), ANR-16-CE12-0025,COGEX,Contrôle automatisé de l'expression des gènes(2016), and European Project: 766840,H2020,COSY-BIO(2017)
Subjects: Machine Learning, Microscopy, Multidisciplinary, Image Processing, Computer-Assisted, General Physics and Astronomy, Saccharomyces cerevisiae, General Chemistry, [SDV.BIBS]Life Sciences [q-bio]/Quantitative Methods [q-bio.QM], Software, General Biochemistry, Genetics and Molecular Biology
Abstract: Posté sur BioRxiv le 10 juin 2021; International audience; Microscopy image analysis has recently made enormous progress both in terms of accuracy and speed thanks to machine learning methods and improved computational resources. This greatly facilitates the online adaptation of microscopy experimental plans using real-time information of the observed systems and their environments. Applications in which reactiveness is needed are multifarious. Here we report MicroMator, an open and flexible software for defining and driving reactive microscopy experiments. It provides a Python software environment and an extensible set of modules that greatly facilitate the definition of events with triggers and effects interacting with the experiment. We provide a pedagogic example performing dynamic adaptation of fluorescence illumination on bacteria, and demonstrate MicroMator’s potential via two challenging case studies in yeast to single-cell control and single-cell recombination, both requiring real-time tracking and light targeting at the single-cell level.
Published: 2022
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17. Human peroxiredoxin 6 is essential for malaria parasites and provides a host-based drug target

Author: Matthias Paulus Wagner, Pauline Formaglio, Olivier Gorgette, Jerzy Michal Dziekan, Christèle Huon, Isabell Berneburg, Stefan Rahlfs, Jean-Christophe Barale, Sheldon I. Feinstein, Aron B. Fisher, Didier Ménard, Zbynek Bozdech, Rogerio Amino, Lhousseine Touqui, Chetan E. Chitnis, Biologie de Plasmodium et Vaccins - Malaria Parasite Biology and Vaccines, Institut Pasteur [Paris] (IP)-Université Paris Cité (UPCité), Infection et Immunité paludéennes - Malaria Infection and Immunity, Plateforme BioImagerie Ultrastructurale – Ultrastructural BioImaging Platform (UTechS UBI), Institut Pasteur [Paris] (IP), Nanyang Technological University [Singapour], Justus-Liebig-Universität Gießen = Justus Liebig University (JLU), Microbiologie structurale - Structural Microbiology (Microb. Struc. (UMR_3528 / U-Pasteur_5)), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Malaria Translational Research Unit (MTRU), Institut Pasteur du Cambodge, Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP)-Institut Pasteur [Paris] (IP), Perelman School of Medicine, University of Pennsylvania, Génétique du paludisme et résistance - Malaria Genetics and Resistance, Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris Cité (UPCité), Dynamique des interactions Hôte pathogène, Université de Strasbourg (UNISTRA), Institut de Parasitologie et de Pathologie Tropicale (IPPTS), Mucoviscidose et bronchopathies chroniques : biopathologie et phénotype cliniques - Cystic Fibrosis and Bronchial Diseases, Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-CHU Saint-Antoine [AP-HP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU)-Université Paris Cité (UPCité), This work was funded by the following grants: Fondation pour la Recherche Médicale grant (FDT202001010791) to M.P.W., Agence Nationale de la Recherche grant (ANR-17-CE15-0010) to C.E.C., Pasteur Innov grant from Institut Pasteur to C.E.C., Laboratoire d’Excellence (LabEx) 'French Parasitology Alliance For Health Care' (ANR-11-LABX-0024-PARAFRAP) to C.E.C. and D.M., Laboratoire d’Excellence 'Integrative Biology of Emerging Infectious Diseases' (grant no. ANR-10-LABX-62-IBEID) to P.F., NTU Presidential Postdoctoral Fellowship grant (NTU/PPF/2019) to J.M.D., Singaporean Ministry of Education grant (MOE-T2EP30120-0015) to J.M.D. and Z.B., and LOEWE Center DRUID (Projects B3) within the Hessian Excellence Program to I.B., We thank Gordon Langsley for fruitful discussions and critical inputs. We also thank the electron microscopy Ultrastructural BioImaging (UBI) platform, Institut Pasteur, Paris, for providing ready access to the electron microscopy facility. The authors are grateful to the Central Animal Facility of Institut Pasteur for providing support with animal importation, care, housing, and breeding. We thank the Image Analysis Hub (IAH), Institut Pasteur, Paris, for their help with image analysis. M.P.W. was part of the Pasteur - Paris University (PPU) International PhD Program and European Union’s Horizon 2020 research and innovation program under the Marie Sklodowska-Curie grant agreement no. 665807., 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), ANR-10-LABX-0062,IBEID,Integrative Biology of Emerging Infectious Diseases(2010), European Project: 665807,H2020,H2020-MSCA-COFUND-2014,PASTEURDOC(2015), and School of Biological Sciences
Subjects: Plasmodium falciparum, Drug Resistance, malaria, PRDX6, General Biochemistry, Genetics and Molecular Biology, molecular parasitology, Antimalarials, Hemoglobins, Mice, Oximes, Animals, Humans, endocytosis, host cell cytosol uptake, oxidative stress, [SDV.MP.PAR]Life Sciences [q-bio]/Microbiology and Parasitology/Parasitology, Malaria, Falciparum, CP: Microbiology, Biological sciences [Science], lipid peroxidation, hemoglobin, Lipids, Artemisinins, Endocytosis, CP: Metabolism, artemisinin, Benzaldehydes, Artemisinin, Peroxiredoxin VI
Abstract: The uptake and digestion of host hemoglobin by malaria parasites during blood-stage growth leads to significant oxidative damage of membrane lipids. Repair of lipid peroxidation damage is crucial for parasite survival. Here, we demonstrate that Plasmodium falciparum imports a host antioxidant enzyme, peroxiredoxin 6 (PRDX6), during hemoglobin uptake from the red blood cell cytosol. PRDX6 is a lipid-peroxidation repair enzyme with phospholipase A2 (PLA2) activity. Inhibition of PRDX6 with a PLA2 inhibitor, Darapladib, increases lipid-peroxidation damage in the parasite and disrupts transport of hemoglobin-containing vesicles to the food vacuole, causing parasite death. Furthermore, inhibition of PRDX6 synergistically reduces the survival of artemisinin-resistant parasites following co-treatment of parasite cultures with artemisinin and Darapladib. Thus, PRDX6 is a host-derived drug target for development of antimalarial drugs that could help overcome artemisinin resistance. Ministry of Education (MOE) Nanyang Technological University Published version M.P.W. was part of the Pasteur - Paris University (PPU) International PhD Program and European Union’s Horizon 2020 research and innovation program under the Marie Sklodowska-Curie grant agreement no. 665807. This work was funded by the following grants: Fondation pour la Recherche Me ́ dicale grant (FDT202001010791) to M.P.W.; Agence Nationale de la Recherche grant (ANR-17-CE15-0010) to C.E.C.; Pasteur Innov grant from Institut Pasteur to C.E.C.; Laboratoire d’Excellence (LabEx) ‘‘French Parasitology Alliance For Health Care’’ (ANR-11-LABX-0024-PARAFRAP) to C.E.C. and D.M.; Laboratoire d’Excellence ‘‘Integrative Biology of Emerging Infectious Diseases’’ (grant no. ANR-10-LABX-62-IBEID) to P.F.; NTU Presidential Postdoctoral Fellowship grant (NTU/PPF/2019) to J.M.D.; Singaporean Ministry of Education grant (MOE-T2EP30120-0015) to J.M.D. and Z.B.; and LOEWE Center DRUID (Projects B3) within the Hessian Excellence Program to I.B.
Published: 2022
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18. The complexin C-terminal amphipathic helix stabilizes the fusion pore open state by sculpting membranes

Author: Kevin C. Courtney, Lanxi Wu, Taraknath Mandal, Mark Swift, Zhao Zhang, Mohammad Alaghemandi, Zhenyong Wu, Mazdak M. Bradberry, Claire Deo, Luke D. Lavis, Niels Volkmann, Dorit Hanein, Qiang Cui, Huan Bao, Edwin R. Chapman, University of Wisconsin-Madison, Boston University [Boston] (BU), Indian Institute of Technology Kanpur (IIT Kanpur), The Scintillon Institute, Howard Hughes Medical Institute (HHMI), European Molecular Biology Laboratory [Heidelberg] (EMBL), Imagerie structurale - Structural Image Analysis, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Microbiologie structurale - Structural Microbiology (Microb. Struc. (UMR_3528 / U-Pasteur_5)), Études structurales de machines moléculaires in cellulo - Structural studies of macromolecular machines in cellula, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Université Paris Cité (UPCité), The Scripps Research Institute [La Jolla, San Diego], This work was supported by Pew Charitable Trust grant no. 864K625 (E.R.C. and D.H.), National Institutes of Health grant nos. MH061876 and NS097362 (E.R.C.), P01-GM121203 (N.V.) and DP2GM140920 (H.B.). Equipment for the cryo-CLEM and in situ cellular tomography workflow used in this work was funded by National Institutes of Health grant nos. S10-OD012372 (D.H.), S10-OD026926 (D.H.), P01-GM121203 (N.V.) and R01-AI132378 (N.V., D.H.), and Pew Charitable Trust grant no. 864K625. The computational component is supported by the grant no. NSF-DMS1661900 (Q.C.) Computational resources from the Extreme Science and Engineering Discovery Environment (XSEDE), which is supported by NSF grant no. OCI-1053575 (Q.C.), are greatly appreciated, and computations are also supported in part by the Shared Computing Cluster, which is administered by Boston University’s Research Computing Services. E.R.C. is an Investigator of the Howard Hughes Medical Institute.
Subjects: Protein Conformation, alpha-Helical, Protein Stability, [SDV]Life Sciences [q-bio], Lipid Bilayers, Nerve Tissue Proteins, Molecular Dynamics Simulation, Membrane Fusion, Article, Peptide Fragments, Adaptor Proteins, Vesicular Transport, HEK293 Cells, Structural Biology, Nuclear Pore, Animals, Drosophila Proteins, Humans, Synaptic Vesicles, Caenorhabditis elegans Proteins, Molecular Biology
Abstract: International audience; Neurotransmitter release is mediated by proteins that drive synaptic vesicle fusion with the presynaptic plasma membrane. While soluble N-ethylmaleimide sensitive factor attachment protein receptors (SNAREs) form the core of the fusion apparatus, additional proteins play key roles in the fusion pathway. Here, we report that the C-terminal amphipathic helix of the mammalian accessory protein, complexin (Cpx), exerts profound effects on membranes, including the formation of pores and the efficient budding and fission of vesicles. Using nanodisc-black lipid membrane electrophysiology, we demonstrate that the membrane remodeling activity of Cpx modulates the structure and stability of recombinant exocytic fusion pores. Cpx had particularly strong effects on pores formed by small numbers of SNAREs. Under these conditions, Cpx increased the current through individual pores 3.5-fold, and increased the open time fraction from roughly 0.1 to 1.0. We propose that the membrane sculpting activity of Cpx contributes to the phospholipid rearrangements that underlie fusion by stabilizing highly curved membrane fusion intermediates.
Published: 2022
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19. Gp29 LysA of mycobacteriophage TM4 can hydrolyze peptidoglycan through an N-acetyl-muramoyl-L-alanine amidase activity

Author: Estefanía Urdániz, Mariano Martín, Florencia Payaslián, Lucas Alfredo Defelipe, Martín Dodes, Mariano Martinez, Pedro M. Alzari, Gabriela Cabrera, Marcelo Adrián Martí, Mariana Piuri, Facultad de Ciencias Exactas y Naturales [Buenos Aires] (FCEyN), Universidad de Buenos Aires [Buenos Aires] (UBA), Universidad Nacional de Córdoba [Argentina], European Molecular Biology Laboratory [Hamburg] (EMBL), Microbiologie structurale - Structural Microbiology (Microb. Struc. (UMR_3528 / U-Pasteur_5)), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), This research was supported by Agencia Nacional de Promoción Científica y Tecnológica (PIDC-0015/2015) to MP. MM and FP are CONICET fellows. LAD was funded by EMBL Interdisciplinary Postdoc Programme under Marie Curie Actions COFUND 664726., and European Project: 664726,H2020,H2020-MSCA-COFUND-2014,EI3POD(2015)
Subjects: Endolysin, [SDV]Life Sciences [q-bio], Mycobacterium smegmatis, MESH: Micrococcus, Biophysics, N-acetyl-muramoyl-L-alanine amidase, Peptidoglycan, Galactans, Biochemistry, Mass Spectrometry, MESH: Muramic Acids, Micrococcus, Analytical Chemistry, Viral Proteins, LysA, MESH: Mycobacteriophages, Endopeptidases, Escherichia coli, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, MESH: Endopeptidases, Molecular Biology, MESH: Mycobacterium smegmatis, MESH: Mass Spectrometry, Mycobacteriophage, MESH: Peptidoglycan, MESH: Escherichia coli, Hydrolysis, Computational Biology, MESH: Galactans, Mycobacteriophages, N-Acetylmuramoyl-L-alanine Amidase, MESH: Viral Proteins, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, Muramic Acids, MESH: N-Acetylmuramoyl-L-alanine Amidase, [INFO.INFO-BI]Computer Science [cs]/Bioinformatics [q-bio.QM], MESH: Hydrolysis, MESH: Computational Biology
Abstract: International audience; Bacteriophage endolysins are crucial for progeny release at the end of the lytic cycle. Mycobacteriophage's genomes carry a lysin A essential gene, whose product cleaves the peptidoglycan (PG) layer and a lysin B, coding for an esterase, that cleaves the linkage between the mycolic acids and the arabinogalactan-PG complex. Lysin A mycobacteriophage proteins are highly modular and in gp29 (LysA) of phage TM4 three distinctive domains were identified. By bioinformatics analysis the central module was previously found to be similar to an amidase-2 domain family with an N-acetylmuramoyl-L-alanine amidase activity. We demonstrated experimentally that purified LysA is able to lyse a suspension of Micrococcus lysodeikticus and can promote cell lysis when expressed in E. coli and Mycobacterium smegmatis. After incubation of LysA with MDP (Muramyl dipeptide, N-acetyl-muramyl-L-alanyl-D-isoglutamine) we detected the presence of N-acetylmuramic acid (NAcMur) and L-Ala-D-isoGlutamine (L-Ala-D-isoGln) corroborating the proposed muramidase activity of this enzyme. This protein was stabilized at acidic pH in the presence of Zn consistent with the increase of the enzymatic activity under these conditions. By homology modeling, we predicted that the Zn ion is coordinated by His 226, His 335, and Asp 347 and we also identified the amino acid Glu 290 as the catalytic residue. LysA activity was completely abolished in derived mutants on these key residues, suggesting that the PG hydrolysis solely relies on the central domain of the protein.
Published: 2022
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20. Topoisomerase I (TOP1) dynamics: conformational transition from open to closed states

Author: Diane T. Takahashi, Danièle Gadelle, Keli Agama, Evgeny Kiselev, Hongliang Zhang, Emilie Yab, Stephanie Petrella, Patrick Forterre, Yves Pommier, Claudine Mayer, Biotechnologie et signalisation cellulaire (BSC), Université de Strasbourg (UNISTRA)-Institut de recherche de l'Ecole de biotechnologie de Strasbourg (IREBS)-Centre National de la Recherche Scientifique (CNRS), Microbiologie structurale - Structural Microbiology (Microb. Struc. (UMR_3528 / U-Pasteur_5)), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Institut de Biologie Intégrative de la Cellule (I2BC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Center for Cancer Research, National Cancer Institute [Bethesda] (NCI-NIH), National Institutes of Health [Bethesda] (NIH)-National Institutes of Health [Bethesda] (NIH), Université Paris Diderot - Paris 7 (UPD7), These studies have been supported by a European Research Council (ERC) Grant from the European Union’s Seventh Framework Program (FP/2007-2013) (Project EVOMOBIL-ERC) [340440 to P.F.], l′Agence Nationale de la Recherche [Project ESSPOIR ANR-17-CE12-0032 to T.T., D.G., S.P., and C.M.]. Grants from the Institut Pasteur (Paris), the CNRS (France), and the Université de Paris (France). The studies have also been supported by the Center for Cancer Research, the Intramural Program of the National Cancer Institute, National Institutes of Health [Z01-BC-006161 to K.A., E.K., H.Z., and Y.P.], We are grateful to the core facilities of the Institut Pasteur C2RT, and in particular the scientist from the Crystallography Platform: Ahmed Haouz, Patrick Weber, and Cedric Pissis for performing robot-driven crystallization trials and helpful discussions. We also wish to thank Ariel Mechaly for helping with phasing the SeMet TOP1 structure, and also acknowledge the staff of ESRF and SOLEIL Synchrotrons for assistance and support using beamlines ID29 (Grenoble, France) and SWING and PX2 (Saclay, France)., ANR-17-CE12-0032,ESSPOIR,Comprendre la structure de SPO11, ses fonctions et ses interactions(2017), and European Project: 340440,EC:FP7:ERC,ERC-2013-ADG,EVOMOBIL(2014)
Subjects: Models, Molecular, Multidisciplinary, DNA Repair, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Protein Conformation, Science, DNA damage and repair, General Physics and Astronomy, DNA, General Chemistry, Sciences du Vivant [q-bio]/Biochimie, Biologie Moléculaire, Crystallography, X-Ray, Article, General Biochemistry, Genetics and Molecular Biology, DNA-Binding Proteins, DNA Topoisomerases, Type I, Catalytic Domain, Humans, Camptothecin, Structural biology, Sequence Alignment, DNA Damage, X-ray crystallography
Abstract: Eukaryotic topoisomerases I (TOP1) are ubiquitous enzymes removing DNA torsional stress. However, there is little data concerning the three-dimensional structure of TOP1 in the absence of DNA, nor how the DNA molecule can enter/exit its closed conformation. Here, we solved the structure of thermostable archaeal Caldiarchaeum subterraneum CsTOP1 in an apo-form. The enzyme displays an open conformation resulting from one substantial rotation between the capping (CAP) and the catalytic (CAT) modules. The junction between these two modules is a five-residue loop, the hinge, whose flexibility permits the opening/closing of the enzyme and the entry of DNA. We identified a highly conserved tyrosine near the hinge as mediating the transition from the open to closed conformation upon DNA binding. Directed mutagenesis confirmed the importance of the hinge flexibility, and linked the enzyme dynamics with sensitivity to camptothecin, a TOP1 inhibitor targeting the TOP1 enzyme catalytic site in the closed conformation., Topoisomerase I (TOP1) relaxes both positive and negative supercoils by nicking DNA and after rotation of the broken DNA strand closes the nick. Here, the authors present the DNA free crystal structure of TOP1 from the hyperthermophilic archaeon Caldiarchaeum subterraneum in the open form and discuss the mechanism of how DNA enters the catalytic site of TOP1.
Published: 2022
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21. Lipophilic quinolone derivatives: Synthesis and in vitro antibacterial evaluation

Author: Sadowski, Elodie, Bercot, Beatrice, Chauffour, Aurélie, Gomez, Catherine, Varon, Emmanuelle, Mainardis, Mary, Sougakoff, Wladimir, Mayer, Claudine, Sachon, Emmanuelle, Anquetin, Guillaume, Aubry, Alexandra, Centre d'Immunologie et des Maladies Infectieuses (CIMI), Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), CHU Pitié-Salpêtrière [AP-HP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU), Laboratoire des biomolécules (LBM UMR 7203), Chimie Moléculaire de Paris Centre (FR 2769), École normale supérieure - Paris (ENS-PSL), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Ecole Nationale Supérieure de Chimie de Paris - Chimie ParisTech-PSL (ENSCP), Université Paris sciences et lettres (PSL)-Ecole Superieure de Physique et de Chimie Industrielles de la Ville de Paris (ESPCI Paris), Université Paris sciences et lettres (PSL)-Institut de Chimie du CNRS (INC)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-École normale supérieure - Paris (ENS-PSL), Université Paris sciences et lettres (PSL)-Institut de Chimie du CNRS (INC)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Département de Chimie - ENS Paris, Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Infection, Anti-microbiens, Modélisation, Evolution (IAME (UMR_S_1137 / U1137)), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris Cité (UPCité)-Université Sorbonne Paris Nord, Hopital Saint-Louis [AP-HP] (AP-HP), Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP), Laboratoire Génomique, bioinformatique et chimie moléculaire (GBCM), Conservatoire National des Arts et Métiers [CNAM] (CNAM), HESAM Université - Communauté d'universités et d'établissements Hautes écoles Sorbonne Arts et métiers université (HESAM)-HESAM Université - Communauté d'universités et d'établissements Hautes écoles Sorbonne Arts et métiers université (HESAM), Centre Hospitalier Intercommunal de Créteil (CHIC), Laboratoire des sciences de l'ingénieur, de l'informatique et de l'imagerie (ICube), École Nationale du Génie de l'Eau et de l'Environnement de Strasbourg (ENGEES)-Université de Strasbourg (UNISTRA)-Institut National des Sciences Appliquées - Strasbourg (INSA Strasbourg), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut National de Recherche en Informatique et en Automatique (Inria)-Les Hôpitaux Universitaires de Strasbourg (HUS)-Centre National de la Recherche Scientifique (CNRS)-Matériaux et Nanosciences Grand-Est (MNGE), Université de Strasbourg (UNISTRA)-Université de Haute-Alsace (UHA) Mulhouse - Colmar (Université de Haute-Alsace (UHA))-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Université de Haute-Alsace (UHA) Mulhouse - Colmar (Université de Haute-Alsace (UHA))-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Réseau nanophotonique et optique, Université de Strasbourg (UNISTRA)-Université de Haute-Alsace (UHA) Mulhouse - Colmar (Université de Haute-Alsace (UHA))-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS), Microbiologie structurale - Structural Microbiology (Microb. Struc. (UMR_3528 / U-Pasteur_5)), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Spectrométrie de masse de Sorbonne Université [IBPS] (MS3U), Institut de Biologie Paris Seine (IBPS), Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), Interfaces, Traitements, Organisation et Dynamique des Systèmes (ITODYS (UMR_7086)), Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Université Paris Cité (UPCité), The work was supported by grants from the order of Malta Grants for Leprosy Research, QUIET Lutech MA077/SL00091 – ref UPMCX13042 from the SATT LUTECH and ANR DETONATOR., ANR-20-CE18-0030,DETONATOR,Décryptage du mode d'action de nouveaux antituberculeux prometteurs(2020), Centre d'Immunologie et de Maladies Infectieuses (CIMI), Centre National de Référence des Mycobactéries et de la Résistance aux Antituberculeux [CHU Pitié-Salpêtrière], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU), Centre National de la Recherche Scientifique (CNRS)-Sorbonne Université (SU)-Département de Chimie - ENS Paris, École normale supérieure - Paris (ENS Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-École normale supérieure - Paris (ENS Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Chimie Moléculaire de Paris Centre (FR 2769), Institut de Chimie du CNRS (INC)-École normale supérieure - Paris (ENS Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Centre National de la Recherche Scientifique (CNRS)-Ecole Nationale Supérieure de Chimie de Paris - Chimie ParisTech-PSL (ENSCP), Université Paris sciences et lettres (PSL)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Ecole Nationale Supérieure de Chimie de Paris - Chimie ParisTech-PSL (ENSCP), Université Paris sciences et lettres (PSL)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Paris (UP)-Université Sorbonne Paris Nord, Service de Bactériologie et d'Hygiène Hospitalière [CHU Pitié-Salpêtrière], Institut National des Sciences Appliquées - Strasbourg (INSA Strasbourg), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS)-École Nationale du Génie de l'Eau et de l'Environnement de Strasbourg (ENGEES)-Réseau nanophotonique et optique, Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Université de Haute-Alsace (UHA) Mulhouse - Colmar (Université de Haute-Alsace (UHA))-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Université de Haute-Alsace (UHA) Mulhouse - Colmar (Université de Haute-Alsace (UHA))-Matériaux et nanosciences d'Alsace (FMNGE), Institut de Chimie du CNRS (INC)-Université de Strasbourg (UNISTRA)-Université de Haute-Alsace (UHA) Mulhouse - Colmar (Université de Haute-Alsace (UHA))-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC)-Université de Strasbourg (UNISTRA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS)-Université de Paris (UP), Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université de Paris (UP), Université de Paris (UP), Noel, Anne-Laure, Décryptage du mode d'action de nouveaux antituberculeux prometteurs - - DETONATOR2020 - ANR-20-CE18-0030 - AAPG2020 - VALID, and Centre National de Référence des Mycobactéries et de la Résistance aux Antituberculeux [CHU Pitié-Salpêtrière] (CNR-MyRMA)
Subjects: Dose-Response Relationship, Drug, Molecular Structure, [CHIM.THER] Chemical Sciences/Medicinal Chemistry, ESKAPE, Microbial Sensitivity Tests, [CHIM.THER]Chemical Sciences/Medicinal Chemistry, Quinolones, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, Neisseria gonorrhoeae, Anti-Bacterial Agents, Antibacterial, Structure-Activity Relationship, Synthesis, [SDV.MP.BAC] Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, Hydrophobic and Hydrophilic Interactions, Fluoroquinolones
Abstract: International audience; This paper reports on the design of a series of 10 novel lipophilic piperazinyl derivatives of the 1-cyclopropyl-6-fluoro-8-methoxy-4-oxo-1,4-dihydroquinoline-3-carboxylic acid, their synthesis, their characterisation by 1H, 13C and 19F NMR, IR spectroscopy and HRMS, as well as their biological activity against bacteria of medical interest. Among these derivatives, 2 were as potent as the parent quinolone against Neisseriagonorrhoeae whereas all the compounds displayed lower activity than the parent quinolone against other bacteria of medical interest. Our results showing that the increased lipophilicity was deleterious for antibacterial activity may help to design new quinolone derivatives in the future, especially lipophilic quinolones which have been poorly investigated previously.
Published: 2022
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22. Elimination of PknL and MSMEG_4242 in Mycobacterium smegmatis alters the character of the outer cell envelope and selects for mutations in Lsr2

Author: William R. Jacobs, Laurent Kremer, Estalina Báez-Ramírez, Albertus Viljoen, Catherine Vilchèze, Howard E. Takiff, Pedro M. Alzari, Mamadou Daffé, Andrej Benjak, Gustavo Lopez, Gwenaëlle André-Leroux, Elba Guerrero, Séverine Carrère-Kremer, Françoise Laval, Carlos Andrés Aranaga, Luis J. Querales, Stewart T. Cole, Instituto Venezolano de Investigaciones Cientificas (IVIC), Institut de Recherche en Infectiologie de Montpellier (IRIM), Université de Montpellier (UM)-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), Ecole Polytechnique Fédérale de Lausanne (EPFL), Microbiologie structurale - Structural Microbiology (Microb. Struc. (UMR_3528 / U-Pasteur_5)), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Albert Einstein College of Medicine [New York], Shenzhen Nanshan Center for Chronic Disease Control [Shenzhen, China] (ShenZhenCDC), The work for this project was supported by Ecos Nord project PI-200000300 (to P.A. & H.T.), Misión Ciencia Project, 'Identificación y caracterización de blancos especificos para nuevos fármacos contra enfermedades transmisibles' (to H.T.) , Fonacit Project S1-2001000853 (to H.T.), the Sanming Project of Medicine in Shenzhen (grant number SZSM201603029), the Swiss National Science Foundation, grant number 31003A_162641 (to STC), NIH, NIAID Grant # R01AI026170 (to W.R.J. Jr.), Fondation pour la Recherche Médicale (FRM) (DEQ20150331719) (to L.K.), Kremer, Laurent, Université de Toulouse (UT)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées, and Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS)-Université de Paris (UP)
Subjects: Kinase, [SDV]Life Sciences [q-bio], Mutant, Lsr2, Motility, Applied Microbiology and Biotechnology, Microbiology, Serine, 03 medical and health sciences, Tuberculosis, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], Molecular Biology, Gene, Transposase, ComputingMilieux_MISCELLANEOUS, 030304 developmental biology, Mycobacterial envelope, 0303 health sciences, biology, QH573-671, 030306 microbiology, Chemistry, Mycobacterium smegmatis, Cell Biology, biology.organism_classification, Phenotype, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, Cell biology, [SDV] Life Sciences [q-bio], Biofilms, PknL, Cell envelope, [SDV.MP.BAC] Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, Cytology
Abstract: International audience; Four serine/threonine kinases are present in all mycobacteria: PknA, PknB, PknG and PknL. PknA and PknB are essential for growth and replication, PknG regulates metabolism, but little is known about PknL. Inactivation of pknL and adjacent regulator MSMEG_4242 in rough colony M. smegmatis mc2155 produced both smooth and rough colonies. Upon restreaking rough colonies, smooth colonies appeared at a frequency of ~ 1/250. Smooth mutants did not form biofilms, showed increased sliding motility and anomalous lipids on thin-layer chromatography, identified by mass spectrometry as lipooligosaccharides and perhaps also glycopeptidolipids. RNA-seq and Sanger sequencing revealed that all smooth mutants had inactivated lsr2 genes due to mutations and different IS1096 insertions. When complemented with lsr2, the colonies became rough, anomalous lipids disappeared and sliding motility decreased. Smooth mutants showed increased expression of IS1096 transposase TnpA and MSMEG_4727, which encodes a protein similar to PKS5. When MSMEG_4727 was deleted, smooth pknL/MSMEG_4242/lsr2 mutants reverted to rough, formed good biofilms, their motility decreased slightly and their anomalous lipids disappeared. Rough delpknL/del4242 mutants formed poor biofilms and showed decreased, aberrant sliding motility and both phenotypes were complemented with the two deleted genes. Inactivation of lsr2 changes colony morphology from rough to smooth, augments sliding motility and increases expression of MSMEG_4727 and other enzymes synthesizing lipooligosaccharides, apparently preventing biofilm formation. Similar morphological phase changes occur in other mycobacteria, likely reflecting environmental adaptations. PknL and MSMEG_4242 regulate lipid components of the outer cell envelope and their absence selects for lsr2 inactivation. A regulatory, phosphorylation cascade model is proposed.
Published: 2021
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23. Artemisinin-independent inhibitory activity of Artemisia sp. infusions against different Plasmodium stages including relapse-causing hypnozoites

Author: Valérie Soulard, Nathalie Dereuddre-Bosquet, Cyrille Y. Botté, Roger Le Grand, Nadia Amanzougaghene, Jean-François Franetich, Amélie Vantaux, Kutub Ashraf, Christophe-Sébastien Arnold, Benoit Witkowski, Mallaury Bordessoulles, Jean-Christophe Barale, Teun Bousema, Romain Duval, Georges Snounou, Guillaume Cazals, Shahin Tajeri, Geert-Jan van Gemert, Dominique Mazier, Centre d'Immunologie et des Maladies Infectieuses (CIMI), Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), Institut Pasteur du Cambodge, Réseau International des Instituts Pasteur (RIIP), Institute for Advanced Biosciences / Institut pour l'Avancée des Biosciences (Grenoble) (IAB), Centre Hospitalier Universitaire [Grenoble] (CHU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Etablissement français du sang - Auvergne-Rhône-Alpes (EFS)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes (UGA), Institut des Biomolécules Max Mousseron [Pôle Chimie Balard] (IBMM), 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), Radboud University Medical Center [Nijmegen], Immunologie des Maladies Virales et Autoimmunes (IMVA - U1184), Université Paris-Sud - Paris 11 (UP11)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Microbiologie structurale - Structural Microbiology (Microb. Struc. (UMR_3528 / U-Pasteur_5)), Institut Pasteur [Paris]-Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPC), Mère et enfant en milieu tropical : pathogènes, système de santé et transition épidémiologique (MERIT - UMR_D 216), Institut de Recherche pour le Développement (IRD)-Université Paris Cité (UPC), Malaria Translational Research Pasteur International Unit, Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP)-Institut Pasteur [Paris] (IP), 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), Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM), Infectious Diseases Models for Innovative Therapies (IDMIT), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Institut de Recherche pour le Développement (IRD)-Université Paris Cité (UPCité), K Ashraf was supported by Labex ParaFrap-IRD PhD South program (ANR-11-LABX-0024). S Tajeri acknowledges postdoctoral funding support from Fondation pour la Recherche Médicale (FRM) for the PALUKILL project. N Amanzougaghene was supported by Agence nationale de la recherche, project Plasmodrug, and Fondation Sorbonne Université. The work performed by CY Botté and C-S Arnold are supported by Fondation pour la Recherche Médicale (FRM, EQU202103012700), Agence Nationale de la Recherche, France (grant ANR-21-CE44-0010-01, Project ApicoLipidAdapt), Région Auvergne Rhône Alpes (Grant AuRA IRICE GEMELI), Finovi program (Apicolipid project), Laboratoire d’Excellence Parafrap, France (grant ANR-11-LABX-0024), LIA-IRP CNRS Program (Apicolipid project), CEFIPRA-MESRI (Project 6003-1), IDEX Université Grenoble-Alpes and MESRI PhD program fellowship. The investigations on P. cynomolgi were funded through a grant from the Agence Nationale de la Recherche, France (ANR-17-CE13-0025-01), IDMIT infrastructure is supported by the French government 'Programme d’Investissements d’Avenir' (PIA), under grants ANR-11-INBS-0008 (INBS IDMIT). This work benefited from the support of Maison de l’Artémisia, ITMO I3M, as well as equipment and services from the CELIS cell culture core facility (Paris Brain Institute), a platform supported through the ANR grants, ANR-10-IAIHU-06 and ANR-11-INBS-0011-NeurATRIS, We would like to acknowledge the help of QUANT microscopy platform of the Paris Brain Institute specially David Akbar, Claire Lovo, and Aymeric Millécamps for their help in analysis of microscopic images and sporozoite motility videos. We thank Laurent Rénia, Noah Sather, Philippe Grellier, and Scott Lindner for the kind gift of anti-Plasmodium antibodies listed in the Materials and Methods section. The kind help of Sivchheng Phal and Chansophea Chhin (Institut Pasteur du Cambodge, Phnom Penh, Cambodia) with P. vivax experiments is sincerely thanked. We are grateful to Lucile Cornet-Vernet and Pierre Lutgen for providing us Artemisia plants., ANR-11-LABX-0024,ParaFrap,Alliance française contre les maladies parasitaires(2011), ANR-18-CE18-0009,plasmodrug,Développement d'un nouvel antipaludique ciblant plusieurs étapes du cycle de Plasmodium(2018), ANR-21-CE44-0010,ApicoLipidAdapt,Elucider les mécanismes d'adaptation métaboliques et lipidiques du parasite Toxoplasma gondii en fonction de l'environnement nutritionnel de son hôte(2021), ANR-17-CE13-0025,IMHyp,Recherches sur l'Hypnozoïte du Paludisme(2017), ANR-11-INBS-0008,IDMIT,Infrastructure nationale pour la modélisation des maladies infectieuses humaines(2011), ANR-11-INBS-0011,NeurATRIS,Infrastructure de Recherche Translationnelle pour les Biothérapies en Neurosciences(2011), Witkowski, Benoit, Laboratoires d'excellence - Alliance française contre les maladies parasitaires - - ParaFrap2011 - ANR-11-LABX-0024 - LABX - VALID, APPEL À PROJETS GÉNÉRIQUE 2018 - Développement d'un nouvel antipaludique ciblant plusieurs étapes du cycle de Plasmodium - - plasmodrug2018 - ANR-18-CE18-0009 - AAPG2018 - VALID, Elucider les mécanismes d'adaptation métaboliques et lipidiques du parasite Toxoplasma gondii en fonction de l'environnement nutritionnel de son hôte - - ApicoLipidAdapt2021 - ANR-21-CE44-0010 - AAPG2021 - VALID, Recherches sur l'Hypnozoïte du Paludisme - - IMHyp2017 - ANR-17-CE13-0025 - AAPG2017 - VALID, Infrastructures - Infrastructure nationale pour la modélisation des maladies infectieuses humaines - - IDMIT2011 - ANR-11-INBS-0008 - INBS - VALID, Infrastructures - Infrastructure de Recherche Translationnelle pour les Biothérapies en Neurosciences - - NeurATRIS2011 - ANR-11-INBS-0011 - INBS - VALID, Centre d'Immunologie et de Maladies Infectieuses (CIMI), Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS)-Université de Paris (UP), Institut de Recherche pour le Développement (IRD)-Université de Paris (UP), Mère et enfant en milieu tropical : pathogènes, système de santé et transition épidémiologique (MERIT - UMR_D 261), Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM)-Ecole Nationale Supérieure de Chimie de Montpellier (ENSCM), Université de Montpellier (UM), Immunologie des maladies virales, auto-immunes, hématologiques et bactériennes (IMVA-HB), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris-Saclay, Institut de Biologie François JACOB (JACOB), 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), and Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité)
Subjects: artemisia afra, Plasmodium berghei, Health, Toxicology and Mutagenesis, [SDV]Life Sciences [q-bio], Plasmodium vivax, lnfectious Diseases and Global Health Radboud Institute for Molecular Life Sciences [Radboudumc 4], Drug resistance, Plant Science, Pharmacology, Artemisia annua, Plasmodium, 0302 clinical medicine, [SDV.MHEP.MI]Life Sciences [q-bio]/Human health and pathology/Infectious diseases, Parasite hosting, Artemisia annua, artemisia afra, Plasmodium cynomolgi, Plasmodium falciparum, Plasmodium berghei, hypnozoite, hepatic stage, Apicoplast, mitochondria, Artemisia afra, Artemisinin, 0303 health sciences, Ecology, biology, hepatic stage, [SDV.BIBS]Life Sciences [q-bio]/Quantitative Methods [q-bio.QM], 3. Good health, mitochondria, [SDV] Life Sciences [q-bio], medicine.drug, Plasmodium cynomolgi, 030231 tropical medicine, Plasmodium falciparum, [SDV.CAN]Life Sciences [q-bio]/Cancer, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, Biochemistry, Genetics and Molecular Biology (miscellaneous), 03 medical and health sciences, All institutes and research themes of the Radboud University Medical Center, Apicoplast, parasitic diseases, medicine, [SDV.BV]Life Sciences [q-bio]/Vegetal Biology, [SDV.MP.PAR]Life Sciences [q-bio]/Microbiology and Parasitology/Parasitology, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, 030304 developmental biology, biology.organism_classification, medicine.disease, lnfectious Diseases and Global Health Radboud Institute for Health Sciences [Radboudumc 4], Artemisia, Malaria, hypnozoite
Abstract: Artemisinin-based combination therapies (ACT) are the frontline treatments against malaria worldwide. Recently the use of traditional infusions fromArtemisia annua(from which artemisinin is obtained) orA. afra(lacking artemisinin) has been controversially advocated. Such unregulated plant-based remedies are strongly discouraged as they might constitute sub-optimal therapies and promote drug resistance. Here, we conducted the first comparative study of the anti-malarial effects of both plant infusionsin vitroagainst the asexual erythrocytic stages ofP. falciparumand the pre-erythrocytic (i. e., liver) stages of variousPlasmodiumspecies. Low concentrations of either infusion accounted for significant inhibitory activities across every parasite species and stage studied. We show that these antiplasmodial effects were essentially artemisinin-independent and were additionally monitored by observations of the parasite apicoplast and mitochondrion. In particular, the infusions significantly incapacitated sporozoites, and forP. vivaxandP. cynomolgi,disrupted the hypnozoites. This provides the first indication that compounds other than 8-aminoquinolines could be effective antimalarials against relapsing parasites. These observations advocate for further screening to uncover urgently needed novel antimalarial lead compounds.
Published: 2021
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24. Actinobacteria challenge the paradigm: A unique protein architecture for a well-known, central metabolic complex

Author: Marco Bellinzoni, Pedro M. Alzari, Lu Yang, Eduardo M. Bruch, Bertrand Raynal, Alexandra Boyko, Norik Lexa-Sapart, Pierre Vilela, Microbiologie structurale - Structural Microbiology (Microb. Struc. (UMR_3528 / U-Pasteur_5)), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Wuhan Institute of Biological Products, Biophysique Moléculaire (plateforme) - Molecular Biophysics (platform), This work was funded by the Agence Nationale de la Recherche (ANR) through the projects SUPERCPLX (ANR-13-JSV8-0003) and METACTINO (ANR-18-CE92-0003), both granted to M.B., and by institutional grants from the Institut Pasteur and the CNRS., We are grateful to the core facilities at Institut Pasteur C2RT (Centre for Technological Resources and Research), in particular to A. Haouz, P. Weber, and C. Pissis (Crystallography), S. Brûlé and B. Baron (Molecular Biophysics), M. Matondo, T. Chaze, and T. Douché (Proteomics), and J.-M. Winter, S. Tachon, and M. Vos (NanoImaging). We also gratefully acknowledge F. Gubellini (Structural Microbiology Unit) for her help in EM sample preparation and J.J. Pierella Karlusich (Ecole Normale Superieure, Paris), A. Thureau (Synchrotron Soleil), and V. Bunik (Lomonosov University, Moscow) for many helpful discussions. The NanoImaging Core at Institut Pasteur was created with the help of a grant from the French Government’s 'Investissements d’Avenir' program (EQUIPEX CACSICE—'Centre d’analyse de systèmes complexes dans les environnements complexes,' ANR-11-EQPX-0008) and is acknowledged for support with cryo-EM sample preparation, image acquisition, and analysis. We also acknowledge the synchrotron sources Soleil (Saint-Aubin, France) and ESRF (Grenoble, France) for granting access to their facilities and their staff for helpful assistance on the respective beamlines., ANR-13-JSV8-0003,SUPERCPLX,Structure, fonction et régulation d'un supercomplexe métabolique clé chez les Actinobacteries(2013), ANR-18-CE92-0003,METACTINO,Déchiffrer la plasticité métabolique des Actinobactéries(2018), Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS)-Université de Paris (UP), and Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS)
Subjects: Biochemical Phenomena, Molecular Conformation, Dehydrogenase, Computational biology, Crystallography, X-Ray, Actinobacteria, Metabolic engineering, 03 medical and health sciences, Pyruvate dehydrogenase complex, pyruvate dehydrogenase, Integrative structural biology, Pyruvic Acid, Protein oligomerization, Ketoglutarate Dehydrogenase Complex, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], Gene, 030304 developmental biology, chemistry.chemical_classification, 0303 health sciences, Multidisciplinary, Bacteria, biology, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], 030302 biochemistry & molecular biology, Computational Biology, Mycobacterium tuberculosis, Biological Sciences, biology.organism_classification, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, Kinetics, Enzyme, chemistry, Acyltransferase, Macromolecular complexes, central metabolism, Plasmids
Abstract: International audience; α-oxoacid dehydrogenase complexes are large, tripartite enzymatic machineries carrying out key reactions in central metabolism. Extremely conserved across the tree of life, they have been, so far, all considered to be structured around a high–molecular weight hollow core, consisting of up to 60 subunits of the acyltransferase component. We provide here evidence that Actinobacteria break the rule by possessing an acetyltranferase component reduced to its minimally active, trimeric unit, characterized by a unique C-terminal helix bearing an actinobacterial specific insertion that precludes larger protein oligomerization. This particular feature, together with the presence of an odhA gene coding for both the decarboxylase and the acyltransferase domains on the same polypetide, is spread over Actinobacteria and reflects the association of PDH and ODH into a single physical complex. Considering the central role of the pyruvate and 2-oxoglutarate nodes in central metabolism, our findings pave the way to both therapeutic and metabolic engineering applications.
Published: 2021
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25. A Tetratricopeptide Repeat Scaffold Couples Signal Detection to OdhI Phosphorylation in Metabolic Control by the Protein Kinase PknG

Author: María-Natalia Lisa, Adrià Sogues, Nathalie Barilone, Meike Baumgart, Magdalena Gil, Martín Graña, Rosario Durán, Ricardo M. Biondi, Marco Bellinzoni, Michael Bott, Pedro M. Alzari, Instituto de Biología Molecular y Celular de Rosario [Rosario] (IBR), Consejo Nacional de Investigaciones Científicas y Técnicas [Buenos Aires] (CONICET)-Universidad Nacional de Rosario [Santa Fe], Microbiologie structurale - Structural Microbiology (Microb. Struc. (UMR_3528 / U-Pasteur_5)), Institut Pasteur [Paris] (IP)-Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), Institute of Bio- and Geosciences [Jülich] (IBG), Forschungszentrum Jülich GmbH | Centre de recherche de Juliers, Helmholtz-Gemeinschaft = Helmholtz Association-Helmholtz-Gemeinschaft = Helmholtz Association, Institut Pasteur de Montevideo, Réseau International des Instituts Pasteur (RIIP), Universitätsklinikum Frankfurt, M.-N.L. received fellowships from the EMBO (European Molecular Biology Organization) and the Fondation pour la Recherche Médicale (FRM, France). This study was funded by grants from the Institut Pasteur, the CNRS (France), the Agence Nationale de la Recherche (ANR, France, contract ANR-09-BLAN-0400) and the European Commission Seventh Framework Program (contract HEALTH-F3-2011-260872). M.-N.L. acknowledges support from the Agencia I+D+I (grant PICT 2017-1932). R.M.B. acknowledges support from DFG (grant BI 1044/12-1)., ANR-09-BLAN-0400,MuKit(2009), and European Project: LSHP-CT
Subjects: protein kinases, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], bacterial signaling, Glutamic Acid, protein structure-function, Gene Expression Regulation, Bacterial, Mycobacterium tuberculosis, Protein Serine-Threonine Kinases, equipment and supplies, complex mixtures, Microbiology, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, QR1-502, Corynebacterium glutamicum, Bacterial Proteins, ddc:570, bacteria, Tetratricopeptide Repeat, Amino Acids, Phosphorylation, Protein Processing, Post-Translational, Research Article, Signal Transduction
Abstract: Signal transduction is essential for bacteria to adapt to changing environmental conditions. Among many forms of posttranslational modifications, reversible protein phosphorylation has evolved as a ubiquitous molecular mechanism of protein regulation in response to specific stimuli. The Ser/Thr protein kinase PknG modulates the fate of intracellular glutamate by controlling the phosphorylation status of the 2-oxoglutarate dehydrogenase regulator OdhI, a function that is conserved among diverse actinobacteria. PknG has a modular organization characterized by the presence of regulatory domains surrounding the catalytic domain. Here, we present an investigation using in vivo experiments, as well as biochemical and structural methods, of the molecular basis of the regulation of PknG from Corynebacterium glutamicum (CgPknG), in the light of previous knowledge available for the kinase from Mycobacterium tuberculosis (MtbPknG). We found that OdhI phosphorylation by CgPknG is regulated by a conserved mechanism that depends on a C-terminal domain composed of tetratricopeptide repeats (TPRs) essential for metabolic homeostasis. Furthermore, we identified a conserved structural motif that physically connects the TPR domain to a β-hairpin within the flexible N-terminal region that is involved in docking interactions with OdhI. Based on our results and previous reports, we propose a model in which the TPR domain of PknG couples signal detection to the specific phosphorylation of OdhI. Overall, the available data indicate that conserved PknG domains in distant actinobacteria retain their roles in kinase regulation in response to nutrient availability.
Published: 2021
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26. Prevalence of ExoY Activity in Pseudomonas aeruginosa Reference Panel Strains and Impact on Cytotoxicity in Epithelial Cells

Author: Silistre, Hazel, Raoux-Barbot, Dorothée, Mancinelli, Federica, Sangouard, Flora, Dupin, Alice, Belyy, Alexander, Deruelle, Vincent, Renault, Louis, Ladant, Daniel, Touqui, Lhousseine, Mechold, Undine, Microbiologie structurale - Structural Microbiology (Microb. Struc. (UMR_3528 / U-Pasteur_5)), Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS)-Université de Paris (UP), Département de Biologie structurale et Chimie - Department of Structural Biology and Chemistry, Institut Pasteur [Paris], 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)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Université de Paris (UP), Biochimie des Interactions Macromoléculaires / Biochemistry of Macromolecular Interactions, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Institut de Biologie Intégrative de la Cellule (I2BC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Centre de Recherche Saint-Antoine (CR Saint-Antoine), Sorbonne Université (SU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-CHU Saint-Antoine [AP-HP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU), ANR-18-CE44-0004,activExoY,Role et spécificités fonctionnelles des facteurs de virulence de type ExoY à activité Nucléotidyl Cyclase dépendante de l'actine dans les infections de bactéries à Gram-négatif(2018), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Institut Pasteur [Paris] (IP)-Université Paris Cité (UPCité), École Pratique des Hautes Études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Université Paris Cité (UPCité), Centre de Recherche Saint-Antoine (CRSA), Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU), Gestionnaire, HAL Sorbonne Université 5, and APPEL À PROJETS GÉNÉRIQUE 2018 - Role et spécificités fonctionnelles des facteurs de virulence de type ExoY à activité Nucléotidyl Cyclase dépendante de l'actine dans les infections de bactéries à Gram-négatif - - activExoY2018 - ANR-18-CE44-0004 - AAPG2018 - VALID
Subjects: cystic fibrosis, cGMP, [SDV.MP]Life Sciences [q-bio]/Microbiology and Parasitology, ExoY, Pseudomonas aeruginosa, [SDV.MP] Life Sciences [q-bio]/Microbiology and Parasitology, Microbiology, nucleotidyl cyclase, Original Research
Abstract: International audience; ExoY is among the effectors that are injected by the type III secretion system (T3SS) of Pseudomonas aeruginosa into host cells. Inside eukaryotic cells, ExoY interacts with F-actin, which stimulates its potent nucleotidyl cyclase activity to produce cyclic nucleotide monophosphates (cNMPs). ExoY has broad substrate specificity with GTP as a preferential substrate in vitro. How ExoY contributes to the virulence of P. aeruginosa remains largely unknown. Here, we examined the prevalence of active ExoY among strains from the international P. aeruginosa reference panel, a collection of strains that includes environmental and clinical isolates, commonly used laboratory strains, and sequential clonal isolates from cystic fibrosis (CF) patients and thus represents the large diversity of this bacterial species. The ability to secrete active ExoY was determined by measuring the F-actin stimulated guanylate cyclase (GC) activity in bacterial culture supernatants. We found an overall ExoY activity prevalence of about 60% among the 40 examined strains with no significant difference between CF and non-CF isolates. In parallel, we used cellular infection models of human lung epithelial cells to compare the cytotoxic effects of isogenic reference strains expressing active ExoY or lacking the exoY gene. We found that P. aeruginosa strains lacking ExoY were in fact more cytotoxic to the epithelial cells than those secreting active ExoY. This suggests that under certain conditions, ExoY might partly alleviate the cytotoxic effects of other virulence factors of P. aeruginosa.
Published: 2021
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27. Conformational transitions in the active site of mycobacterial 2-oxoglutarate dehydrogenase upon binding phosphonate analogues of 2-oxoglutarate: From a Michaelis-like complex to ThDP adducts

Author: Alexandra Boyko, Tristan Wagner, Victoria I. Bunik, Marco Bellinzoni, Pedro M. Alzari, Microbiologie structurale - Structural Microbiology (Microb. Struc. (UMR_3528 / U-Pasteur_5)), Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS)-Université de Paris (UP), Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University (MSU), We acknowledge the synchrotron sources Soleil (Saint-Aubin, France) and ESRF (Grenoble, France) for granting access to their facilities, and their staff for helpful assistance on the respective beamlines. This work was partially supported by institutional grants from the Institut Pasteur and the CNRS. V. I. Bunik greatly acknowledges current financial support of the work of her group on the phosphonates by RSF grant 18-14-00116., We are grateful to Bruno Baron (Molecular Biophysics Platform, Institut Pasteur) for his helpful assistance with circular dichroism experiments, and to Ahmed Haouz and Patrick Weber (Crystallography Platform, Institut Pasteur) for carrying out robot-driven crystallization screenings., and Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité)
Subjects: Stereochemistry, [PHYS.PHYS.PHYS-BIO-PH]Physics [physics]/Physics [physics]/Biological Physics [physics.bio-ph], Kinetics, Organophosphonates, MESH: Catalytic Domain, 2-Oxoglutarate dehydrogenase, MESH: Succinates, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, MESH: Ketoglutarate Dehydrogenase Complex, Mycobacterium, Adduct, 03 medical and health sciences, chemistry.chemical_compound, Structural Biology, Oxidoreductase, Catalytic Domain, MESH: Mycobacterium, [CHIM.CRIS]Chemical Sciences/Cristallography, MESH: Protein Binding, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Ketoglutarate Dehydrogenase Complex, MESH: Oxidoreductases, 030304 developmental biology, chemistry.chemical_classification, 0303 health sciences, MESH: Kinetics, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], biology, MESH: Ketoglutaric Acids, 030302 biochemistry & molecular biology, Active site, Succinates, Phosphonate, 2-Oxoglutarate phosphonate analogues, Enzyme, chemistry, Covalent bond, MESH: Thiamine Pyrophosphate, biology.protein, Thiamine diphosphate, Ketoglutaric Acids, Thiamine, Induced conformational transition, [INFO.INFO-BI]Computer Science [cs]/Bioinformatics [q-bio.QM], Thiamine Pyrophosphate, Oxidoreductases, MESH: Organophosphonates, Protein Binding
Abstract: International audience; Mycobacterial KGD, the thiamine diphosphate (ThDP)-dependent E1o component of the 2-oxoglutarate dehydrogenase complex (OGDHC), is known to undergo significant conformational changes during catalysis with two distinct conformational states, previously named as the early and late state. In this work, we employ two phosphonate analogues of 2-oxoglutarate (OG), i.e. succinyl phosphonate (SP) and phosphono ethyl succinyl phosphonate (PESP), as tools to isolate the first catalytic steps and understand the significance of conformational transitions for the enzyme regulation. The kinetics showed a more efficient inhibition of mycobacterial E1o by SP (Ki 0.043 ± 0.013 mM) than PESP (Ki 0.88 ± 0.28 mM), consistent with the different circular dichroism spectra of the corresponding complexes. PESP allowed us to get crystallographic snapshots of the Michaelis-like complex, the first one for 2-oxo acid dehydrogenases, followed by the covalent adduction of the inhibitor to ThDP, mimicking the pre-decarboxylation complex. In addition, covalent ThDP-phosphonate complexes obtained with both compounds by co-crystallization were in the late conformational state, probably corresponding to slowly dissociating enzyme-inhibitor complexes. We discuss the relevance of these findings in terms of regulatory features of the mycobacterial E1o enzymes, and in the perspective of developing tools for species-specific metabolic regulation.
Published: 2019
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28. Cn29, a novel orphan peptide found in the venom of the scorpion Centruroides noxius: Structure and function

Author: R.L.L. Mendoza, J.I. Guijarro, Lourival D. Possani, Jimena I. Cid-Uribe, Muriel Delepierre, Georgina B. Gurrola, Fredy V. Coronas, Universidad Nacional Autónoma de México (UNAM), Plateforme technologique de RMN biologique - Biological NMR Technological Platform, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Microbiologie structurale - Structural Microbiology (Microb. Struc. (UMR_3528 / U-Pasteur_5)), Institut Pasteur [Paris]-Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), The 800 MHz NMR spectrometer of the Institut Pasteur was partially funded by the Région Ile de France (SESAME 2014 NMRCHR grant no 4014526)., The authors acknowledge the experiments run by Dr. Froylan Gómez Laguna, Dr. Estuardo López Vera and Dr. Rita Restano-Cassulini aimed at identifying a pharmacological function for Cn29 and Catherine Simenel (Biological NMR Technological Platform) for her help with the use of the 800 MHz spectrometer., Universidad Nacional Autónoma de México = National Autonomous University of Mexico (UNAM), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), and Institut Pasteur [Paris] (IP)-Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS)
Subjects: Models, Molecular, Scorpion Venoms, Venom, Peptide, Toxicology, complex mixtures, Gryllidae, Scorpions, Mice, 03 medical and health sciences, chemistry.chemical_compound, Solid-phase synthesis, Sequence Analysis, Protein, Centruroides noxius, Toxicity Tests, Peptide synthesis, Animals, Amino Acid Sequence, 3D-structure, Protein secondary structure, 030304 developmental biology, chemistry.chemical_classification, 0303 health sciences, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], 030302 biochemistry & molecular biology, RNA, NMR, Amino acid, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], Biochemistry, chemistry, Scorpion peptide, Peptides
Abstract: International audience; A peptide (Cn29) from the venom of the scorpion Centruroides noxius (about 2% of the soluble venom) was purified and its primary and three-dimensional structures were determined. The peptide contains 27 amino acids with primary sequence: LCLSCRGGDYDCRVKGTCENGKCVCGS. The peptide is tightly packed by three disulfide linkages formed between C2-C23, C5-C18 and C12-C25. Since the native peptide was obtained in limited amounts, the full synthetic peptide was prepared using the standard F-moc-based solid phase synthesis method of Merrifield. The native and synthetic peptides were shown to be identical by sequencing, HPLC separation and mass spectrometry. The solution structure of the peptide solved from NMR data shows that it consists of a well-defined N-terminal region without regular secondary structure extending from Leu 1 to Asp 9, followed by a short helical fragment from Tyr10 to Val14 and two short β strands (Thr17-Glu19 and Lys22-Val24). The primary and tertiary structures of Cn29 are different from all other scorpion peptides described in the literature. Transcriptome analysis of RNA obtained from C. noxius confirmed the expression of a gene coding for Cn29 in its venom gland. Initial experiments were conducted to identify its possible function: lethality tests in mice and insects as well as ion-channel binding using in vitro electrophysiological assays. None of the physiological or biological tests displayed any activity for this peptide, which at present is considered to be another orphan peptide found in scorpion venoms. The peptide is thus the first example of a novel structural component present in scorpion venoms.
Published: 2019
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29. A systematic review of Mycobacterium leprae DNA gyrase mutations and their impact on fluoroquinolone resistance

Author: Alexandra Aubry, Emmanuelle Cambau, Aurélie Chauffour, Florence Morel, Claudine Mayer, Stéphanie Petrella, Florence Reibel, Centre d'Immunologie et des Maladies Infectieuses (CIMI), Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), CHU Pitié-Salpêtrière [AP-HP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU), Groupe Hospitalier Nord Essonne [Longjumeau], Microbiologie structurale - Structural Microbiology (Microb. Struc. (UMR_3528 / U-Pasteur_5)), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Infection, Anti-microbiens, Modélisation, Evolution (IAME (UMR_S_1137 / U1137)), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris Cité (UPCité)-Université Sorbonne Paris Nord, Centre d'Immunologie et de Maladies Infectieuses (CIMI), Centre National de Référence des Mycobactéries et de la Résistance aux Antituberculeux [CHU Pitié-Salpêtrière], Sorbonne Université (SU)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP), Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS)-Université de Paris (UP), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Paris (UP)-Université Sorbonne Paris Nord, Centre National de Référence des Mycobactéries et de la Résistance aux Antituberculeux [CHU Pitié-Salpêtrière] (CNR-MyRMA), Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU), and Gestionnaire, HAL Sorbonne Université 5
Subjects: Microbiology (medical), [SDV.IMM] Life Sciences [q-bio]/Immunology, Microbial Sensitivity Tests, Biology, Gene mutation, DNA gyrase, 03 medical and health sciences, Antibiotic resistance, Drug Resistance, Bacterial, Genotype, Humans, fluoroquinolones, Mycobacterium leprae, Resistance substitutions, GyrA, 030304 developmental biology, GyrB, Genetics, 0303 health sciences, Numbering system, 030306 microbiology, General Medicine, biology.organism_classification, Fluoroquinolone resistance, 3. Good health, Multiple drug resistance, Infectious Diseases, DNA Gyrase, Mutation, [SDV.IMM]Life Sciences [q-bio]/Immunology, Mutations
Abstract: Background The fact that Mycobacterium leprae does not grow in vitro remains a challenge in the survey of its antimicrobial resistance (AMR). Mainly molecular methods are used to diagnose AMR in M. leprae to provide reliable data concerning mutations and their impact. Fluoroquinolones (FQs) are efficient for the treatment of leprosy and the main second-line drugs in case of multidrug resistance. Objectives This study aimed at performing a systematic review (a) to characterize all DNA gyrase gene mutations described in clinical isolates of M. leprae, (b) to distinguish between those associated with FQ resistance or susceptibility and (c) to delineate a consensus numbering system for M. leprae GyrA and GyrB. Data sources Data source was PubMed. Study eligibility criteria Publications reporting genotypic susceptibility-testing methods and gyrase gene mutations in M. leprae clinical strains. Results In 25 studies meeting our inclusion criteria, 2884 M. leprae isolates were analysed (2236 for gyrA only (77%) and 755 for both gyrA and gyrB (26%)): 3.8% of isolates had gyrA mutations (n = 110), mostly at position 91 (n = 75, 68%) and 0.8% gyrB mutations (n = 6). Since we found discrepancies regarding the location of substitutions associated with FQ resistance, we established a consensus numbering system to properly number the mutations. We also designed a 3D model of the M. leprae DNA gyrase to predict the impact of mutations whose role in FQ-susceptibility has not been demonstrated previously. Conclusions Mutations in DNA gyrase are observed in 4% of the M. leprae clinical isolates. To solve discrepancies among publications and to distinguish between mutations associated with FQ resistance or susceptibility, the consensus numbering system we proposed as well as the 3D model of the M. leprae gyrase for the evaluation of the impact of unknown mutations in FQ resistance, will provide help for resistance surveillance.
Published: 2021
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30. Proteome remodeling in the Mycobacterium tuberculosis PknG knockout: Molecular evidence for the role of this kinase in cell envelope biogenesis and hypoxia response

Author: Wehenkel, Anne Marie, Lima, Analía, Leyva, Alejandro, Rivera, Bernardina, Portela, María Magdalena, Gil, Magdalena, Cascioferro, Alessandro, Lisa, María-Natalia, Wehenkel, Annemarie, Bellinzoni, Marco, Carvalho, Paulo, Batthyány, Carlos, Alvarez, María, Brosch, Roland, Alzari, Pedro, Durán, Rosario, Microbiologie structurale - Structural Microbiology (Microb. Struc. (UMR_3528 / U-Pasteur_5)), Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS)-Université de Paris (UP), Institut Pasteur de Montevideo, Réseau International des Instituts Pasteur (RIIP), Instituto de Investigaciones Biológicas Clemente Estable [Montevideo] (IIBCE), Universidad de la República Uruguay, Pathogénomique mycobactérienne intégrée - Integrated Mycobacterial Pathogenomics, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Instituto de Biología Molecular y Celular de Rosario [Rosario] (IBR), Consejo Nacional de Investigaciones Científicas y Técnicas [Buenos Aires] (CONICET)-Universidad Nacional de Rosario [Santa Fe], Fiocruz Paraná - Instituto Carlos Chagas / Carlos Chagas Institute [Curitiba, Brésil] (ICC), Fundação Oswaldo Cruz (FIOCRUZ), Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP), Universidad de la República [Montevideo] (UCUR), This work was supported by grants from Agencia Nacional de Investigación e Innovación, Uruguay (ANII, FCE_3_2013_1_100358 and FCE_1_2014_1_104045) and FOCEM - Fondo para la Convergencia Estructural del Mercosur (COF 03/11). MG and BR were supported by fellowships from ANII [POS_NAC_2012_1_8824, POS_NAC_2015_1_109755, POS_FCE_2015_1_1005186]. AC and RB were supported by the Agence Nationale pour la Recherche (France)., Universidad de la República [Montevideo] (UDELAR), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), and Fundação Oswaldo Cruz / Oswaldo Cruz Foundation (FIOCRUZ)
Subjects: 0301 basic medicine, Proteomics, Tuberculosis, Proteome, [PHYS.PHYS.PHYS-BIO-PH]Physics [physics]/Physics [physics]/Biological Physics [physics.bio-ph], Quantitative proteomics, Mutant, Biophysics, Serine threonine protein kinase, PknG, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, Protein Serine-Threonine Kinases, Biochemistry, Mycobacterium tuberculosis, 03 medical and health sciences, Bacterial Proteins, medicine, [CHIM.CRIS]Chemical Sciences/Cristallography, Humans, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Hrp-1, Serine/Threonine protein kinase, Hypoxia, Protein kinase A, ComputingMilieux_MISCELLANEOUS, Biological Phenomena, 030304 developmental biology, Msl3, 0303 health sciences, 030102 biochemistry & molecular biology, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], biology, 030306 microbiology, Kinase, medicine.disease, biology.organism_classification, 3. Good health, Cell biology, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], 030104 developmental biology, Mas, [INFO.INFO-BI]Computer Science [cs]/Bioinformatics [q-bio.QM], Biogenesis, Intracellular
Abstract: International audience; Mycobacterium tuberculosis, the etiological agent of tuberculosis, is among the deadliest human pathogens. One of M. tuberculosis's pathogenic hallmarks is its ability to persist in a dormant state in the host. Thus, this pathogen has developed mechanisms to withstand stressful conditions found in the human host. Particularly, the Ser/Thr-protein kinase PknG has gained relevance since it regulates nitrogen metabolism and facilitates bacterial survival inside macrophages. Nevertheless, the molecular mechanisms underlying these effects are far from being elucidated. To further investigate these issues, we performed quantitative proteomic analyses of protein extracts from M. tuberculosis H37Rv and a mutant lacking pknG. We found that in the absence of PknG the mycobacterial proteome was remodeled since 5.7% of the proteins encoded by M. tuberculosis presented significant changes in its relative abundance compared with the wild-type. The main biological processes affected by pknG deletion were cell envelope components biosynthesis and response to hypoxia. Thirteen DosR-regulated proteins were underrepresented in the pknG deletion mutant, including Hrp-1, which was 12.5-fold decreased according to Parallel Reaction Monitoring experiments. Altogether, our results allow us to postulate that PknG regulation of bacterial adaptation to stress conditions might be an important mechanism underlying its reported effect on intracellular bacterial survival. SIGNIFICANCE: PknG is a Ser/Thr kinase from Mycobacterium tuberculosis with key roles in bacterial metabolism and bacterial survival within the host. However, at present the molecular mechanisms underlying these functions remain largely unknown. In this work, we evaluate the effect of pknG deletion on M. tuberculosis proteome using different approaches. Our results clearly show that the global proteome was remodeled in the absence of PknG and shed light on new molecular mechanism underlying PknG role. Altogether, this work contributes to a better understanding of the molecular bases of the adaptation of M. tuberculosis, one of the most deadly human pathogens, to its host.
Published: 2021
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31. Reverse-Complement Equivariant Networks for DNA Sequences

Author: Jean-Philippe Vert, Vincent Mallet, Microbiologie structurale - Structural Microbiology (Microb. Struc. (UMR_3528 / U-Pasteur_5)), Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS)-Université de Paris (UP), Centre de Bioinformatique (CBIO), MINES ParisTech - École nationale supérieure des mines de Paris, Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL), Google Brain, Paris, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), and Mines Paris - PSL (École nationale supérieure des mines de Paris)
Subjects: Pointwise, 0303 health sciences, Theoretical computer science, Computer science, [SDV.BIBS]Life Sciences [q-bio]/Quantitative Methods [q-bio.QM], Convolutional neural network, DNA sequencing, Set (abstract data type), 03 medical and health sciences, 0302 clinical medicine, Fragment (logic), Encoding (memory), DECIPHER, A-DNA, 030217 neurology & neurosurgery, 030304 developmental biology
Abstract: As DNA sequencing technologies keep improving in scale and cost, there is a growing need to develop machine learning models to analyze DNA sequences, e.g., to decipher regulatory signals from DNA fragments bound by a particular protein of interest. As a double helix made of two complementary strands, a DNA fragment can be sequenced as two equivalent, so-called Reverse Complement (RC) sequences of nucleotides. To take into account this inherent symmetry of the data in machine learning models can facilitate learning. In this sense, several authors have recently proposed particular RC-equivariant convolutional neural networks (CNNs). However, it remains unknown whether other RC-equivariant architectures exist, which could potentially increase the set of basic models adapted to DNA sequences for practitioners. Here, we close this gap by characterizing the set of all linear RC-equivariant layers, and show in particular that new architectures exist beyond the ones already explored. We further discuss RC-equivariant pointwise nonlinearities adapted to different architectures, as well as RC-equivariant embeddings of k-mers as an alternative to one-hot encoding of nucleotides. We show experimentally that the new architectures can outperform existing ones.
Published: 2021
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32. 3D architecture and structural flexibility revealed in the subfamily of large glutamate dehydrogenases by a mycobacterial enzyme

Author: Jorge P. López-Alonso, Roberto Melero, María-Natalia Lisa, Melisa Lázaro, Mikel Valle, Eduardo M. Bruch, Charlotte Huet, Alexandra Dodu, Sandra Delgado, Pedro M. Alzari, Luciano A. Abriata, Basque Research and Technology Alliance (BRTA), Centro Nacional de Biotecnología [Madrid] (CNB-CSIC), Consejo Superior de Investigaciones Científicas [Madrid] (CSIC), Microbiologie structurale - Structural Microbiology (Microb. Struc. (UMR_3528 / U-Pasteur_5)), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Université de Strasbourg (UNISTRA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Ecole Polytechnique Fédérale de Lausanne (EPFL), Instituto de Biología Molecular y Celular de Rosario [Rosario] (IBR), Consejo Nacional de Investigaciones Científicas y Técnicas [Buenos Aires] (CONICET)-Universidad Nacional de Rosario [Santa Fe], Plataforma de Biología Estructural y Metabolómica [Rosario] (PLABEM), This work was supported by the grant PICT 2017-1932, from the Agencia Nacional de Promoción de la Investigación, el Desarrollo Tecnológico y la Innovación (Agencia I + D + i, Argentina), received by M.N.L., and the grant PGC2018-098996-B-100 from the Spanish Ministerio de Ciencia e Innovación, received by M.V., M.V. thanks the AEI (Agencia Estatal de Investigación) for the Severo Ochoa Excellence Accreditation (SEV-2016-0644)., and univOAK, Archive ouverte
Subjects: Models, Molecular, Subfamily, domain, Medicine (miscellaneous), Plasma protein binding, Crystallography, X-Ray, small-angle scattering, 0302 clinical medicine, Glutamate Dehydrogenase, Catalytic Domain, refinement, Biology (General), chemistry.chemical_classification, 0303 health sciences, biology, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Chemistry, Mycobacterium smegmatis, Glutamate receptor, Recombinant Proteins, Biochemistry, Metabolic enzymes, Pathogens, General Agricultural and Biological Sciences, Protein Binding, QH301-705.5, [SDV.BBM.BS] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Protein domain, Allosteric regulation, system, Article, General Biochemistry, Genetics and Molecular Biology, 03 medical and health sciences, Bacterial Proteins, Protein Domains, Functional studies, gene, X-ray crystallography, 030304 developmental biology, Glutamate dehydrogenase, Cryoelectron Microscopy, biology.organism_classification, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, Kinetics, Enzyme, suite, cryo-em, Protein quaternary structure, Protein Multimerization, [SDV.MP.BAC] Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, metabolism, 030217 neurology & neurosurgery
Abstract: Glutamate dehydrogenases (GDHs) are widespread metabolic enzymes that play key roles in nitrogen homeostasis. Large glutamate dehydrogenases composed of 180 kDa subunits (L-GDHs180) contain long N- and C-terminal segments flanking the catalytic core. Despite the relevance of L-GDHs180 in bacterial physiology, the lack of structural data for these enzymes has limited the progress of functional studies. Here we show that the mycobacterial L-GDH180 (mL-GDH180) adopts a quaternary structure that is radically different from that of related low molecular weight enzymes. Intersubunit contacts in mL-GDH180 involve a C-terminal domain that we propose as a new fold and a flexible N-terminal segment comprising ACT-like and PAS-type domains that could act as metabolic sensors for allosteric regulation. These findings uncover unique aspects of the structure-function relationship in the subfamily of L-GDHs., Lázaro et. al. report the first 3D structure of a large glutamate dehydrogenase (L-GDH), the one corresponding to the Mycobacterium smegmatis enzyme composed of 180 kDa subunits (mL-GDH180), obtained by X-ray crystallography and cryo-electron microscopy. This structure reveals that mL-GDH180 assembles as tetramers with the N- and C-terminal domains being involved in inter-subunit contacts and unveils unique features of the subfamily of L-GDHs.
Published: 2021
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33. Potential role of the X circular code in the regulation of gene expression

Author: Christian J. Michel, Julie Dawn Thompson, Olivier Poch, Claudine Mayer, Raymond Ripp, Laboratoire des sciences de l'ingénieur, de l'informatique et de l'imagerie (ICube), École Nationale du Génie de l'Eau et de l'Environnement de Strasbourg (ENGEES)-Université de Strasbourg (UNISTRA)-Institut National des Sciences Appliquées - Strasbourg (INSA Strasbourg), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS)-Matériaux et nanosciences d'Alsace (FMNGE), Institut de Chimie du CNRS (INC)-Université de Strasbourg (UNISTRA)-Université de Haute-Alsace (UHA) Mulhouse - Colmar (Université de Haute-Alsace (UHA))-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC)-Université de Strasbourg (UNISTRA)-Université de Haute-Alsace (UHA) Mulhouse - Colmar (Université de Haute-Alsace (UHA))-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Réseau nanophotonique et optique, Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Université de Haute-Alsace (UHA) Mulhouse - Colmar (Université de Haute-Alsace (UHA))-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA), Microbiologie structurale - Structural Microbiology (Microb. Struc. (UMR_3528 / U-Pasteur_5)), Université Paris Diderot - Paris 7 (UPD7)-Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), This work was supported by Institute funds from the French Centre National de la Recherche Scientifique and the University of Strasbourg. This work was supported by French Infrastructure Institut Français de Bioinformatique (IFB) ANR-11-INBS-0013, and the ANR Grants Elixir-Excelerate: GA-676559 and RAinRARE: ANR-18-RAR3-0006-02., The authors would like to thank the BigEst and BICS Bioinformatics Platforms for their assistance., ANR-11-INBS-0013,IFB (ex Renabi-IFB),Institut français de bioinformatique(2011), ANR-18-RAR3-0006,RAinRARE(2018), European Project: 676559,H2020,H2020-INFRADEV-1-2015-1,ELIXIR-EXCELERATE(2015), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut National de Recherche en Informatique et en Automatique (Inria)-Les Hôpitaux Universitaires de Strasbourg (HUS)-Centre National de la Recherche Scientifique (CNRS)-Matériaux et Nanosciences Grand-Est (MNGE), Université de Strasbourg (UNISTRA)-Université de Haute-Alsace (UHA) Mulhouse - Colmar (Université de Haute-Alsace (UHA))-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Université de Haute-Alsace (UHA) Mulhouse - Colmar (Université de Haute-Alsace (UHA))-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Réseau nanophotonique et optique, Université de Strasbourg (UNISTRA)-Université de Haute-Alsace (UHA) Mulhouse - Colmar (Université de Haute-Alsace (UHA))-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Paris Diderot, Equipe HAL, Infrastructures - Institut français de bioinformatique - - IFB (ex Renabi-IFB)2011 - ANR-11-INBS-0013 - INBS - VALID, ERA-NET E-RARE-3 - - RAinRARE2018 - ANR-18-RAR3-0006 - E-Rare-3 - VALID, ELIXIR-EXCELERATE: Fast-track ELIXIR implementation and drive early user exploitation across the life-sciences. - ELIXIR-EXCELERATE - - H20202015-09-01 - 2019-08-31 - 676559 - VALID, and Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité)
Subjects: Statistics and Probability, Reading Frames, Genetic code, [SDV]Life Sciences [q-bio], Computational biology, Biology, Genome, General Biochemistry, Genetics and Molecular Biology, 03 medical and health sciences, 0302 clinical medicine, Plasmid, [SDV.BBM.GTP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN], Gene expression, [INFO]Computer Science [cs], Nucleotide Motifs, Codon, Gene, 030304 developmental biology, Regulation of gene expression, 0303 health sciences, Applied Mathematics, Circular code, Translation (biology), General Medicine, Ribosomal RNA, Codon optimization, Gene Expression Regulation, Modeling and Simulation, Codon usage bias, Transfer RNA, [SDV.BBM.GTP] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN], Codon usage, Ribosomes, 030217 neurology & neurosurgery
Abstract: The X circular code is a set of 20 trinucleotides (codons) that has been identified in the protein-coding genes of most organisms (bacteria, archaea, eukaryotes, plasmids, viruses). It has been shown previously that the X circular code has the important mathematical property of being an error-correcting code. Thus, motifs of the X circular code, i.e. a series of codons belonging to X, which are significantly enriched in the genes, allow identification and maintenance of the reading frame in genes. X motifs have also been identified in many transfer RNA (tRNA) genes and in important functional regions of the ribosomal RNA (rRNA), notably in the peptidyl transferase center and the decoding center. Here, we investigate the potential role of X motifs as functional elements in the regulation of gene expression. Surprisingly, the definition of a simple parameter identifies several relations between the X circular code and gene expression. First, we identify a correlation between the 20 codons of the X circular code and the optimal codons/dicodons that have been shown to influence translation efficiency. Using previously published experimental data, we then demonstrate that the presence of X motifs in genes can be used to predict the level of gene expression. Based on these observations, we propose the hypothesis that the X motifs represent a new genetic signal, contributing to the maintenance of the correct reading frame and the optimization and regulation of gene expression.Author SummaryThe standard genetic code is used by (quasi-) all organisms to translate information in genes into proteins. Recently, other codes have been identified in genomes that increase the versatility of gene decoding. Here, we focus on the circular codes, an important class of genome codes, that have the ability to detect and maintain the reading frame during translation. Motifs of the X circular code are enriched in protein-coding genes from most organisms from bacteria to eukaryotes, as well as in important molecules in the gene translation machinery, including transfer RNA (tRNA) and ribosomal RNA (rRNA). Based on these observations, it has been proposed that the X circular code represents an ancestor of the standard genetic code, that was used in primordial systems to simultaneously decode a smaller set of amino acids and synchronize the reading frame. Using previously published experimental data, we highlight several links between the presence of X motifs in genes and more efficient gene expression, supporting the hypothesis that the X circular code still contributes to the complex dynamics of gene regulation in extant genomes.
Published: 2021
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34. Energy metabolism | 2-Oxoglutarate Dehydrogenase Complex

Author: Victoria I. Bunik, Tristan Wagner, Marco Bellinzoni, Department of Chemistry, Lomonosov Moscow State University, Lomonosov Moscow State University (MSU), Sechenov First Moscow State Medical University, Max Planck Institute for Marine Microbiology, Max-Planck-Gesellschaft, Microbiologie structurale - Structural Microbiology (Microb. Struc. (UMR_3528 / U-Pasteur_5)), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), and Joseph Jez
Subjects: 0303 health sciences, 2-oxoglutarate dehydrogenase, 030302 biochemistry & molecular biology, DHTKD1 or E1a, Carboligase, OGDHL or E1o, Multienzyme complex, Side reaction with molecular oxygen, Vitamin, DLST or E2o or ODO2, 03 medical and health sciences, Thiamine diphosphate, DLDH or DLD or E3, OGDH or E1o or ODO1 or KGD, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], Dihydrolipoamide dehydrogenase, Dihydrolipoamide succinyl transferase, ComputingMilieux_MISCELLANEOUS, 030304 developmental biology
Abstract: International audience; 2-Oxoglutarate dehydrogenase (ogdh gene) is an ubiquitous enzyme with additional isoenzymes (ogdhl and dhtkd1 genes) characterized in animals. Using thiamine (vitamin B1) diphosphate, the isoenzymes catalyze oxidative decarboxylation of 2-oxoglutarate or 2-oxoadipate. The coupled generation of succinyl- or glutaryl-CoA and NADH requires self-assembled multienzyme complexes. The complex core is formed by dihydrolipoyl succinyltransferase (dlst gene), binding multiple copies of 2-oxoglutarate dehydrogenase and dihydrolipoamide dehydrogenase (dldh gene). The components stoichiometry depends on the complex sources and/or isolation conditions. Components of the 2-oxoglutarate dehydrogenase complex also catalyze carboligase reaction or reaction with molecular oxygen, which are of potential physiological significance.
Published: 2021
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35. Hidden kinetic traps in multidomain folding highlight the presence of a misfolded but functionally competent intermediate

Author: Maurizio Brunori, Angelo Toto, Candice Gautier, Lorenzo Visconti, Stefano Gianni, Yanlei Zhu, Florence Cordier, Francesca Troilo, Francesca Malagrinò, Nicolas Wolff, Institut Pasteur, Fondation Cenci Bolognetti - Istituto Pasteur Italia, Fondazione Cenci Bolognetti, Réseau International des Instituts Pasteur (RIIP), Università degli Studi di Roma 'La Sapienza' = Sapienza University [Rome] (UNIROMA), Microbiologie structurale - Structural Microbiology (Microb. Struc. (UMR_3528 / U-Pasteur_5)), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Récepteurs Canaux - Channel Receptors, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), The project has received funding from the European Union’s Horizon 2020 research and innovation program under the Marie Sklodowska-Curie grant agreement PDZnet No 675341. Work partly supported by grants from the Italian Ministero dell’Istruzione dell’Università e della Ricerca (Progetto di Interesse ‘Invecchiamento’ to S.G.), Sapienza University of Rome (B52F16003410005, RP11715C34AEAC9B and RM1181641C2C24B9 to S.G), the Associazione Italiana per la Ricerca sul Cancro (Individual Grant -MFAG 2016, 18701 to S.G.) the Istituto Pasteur Italia (Teresa Ariaudo Research Project 2018, to A.T.). F.M. was supported by a fellowship from the Associazione Italiana per la Ricerca sul Cancro.The authors also acknowledge the Conseil Régional d’Ile de France for financial support through the SESAME 2014 NMRCHR program No. 14014526 (800 MHz spectrometer)., European Project: 675341,H2020-MSCA-ITN-2015,PDZNet(2016), Università degli Studi di Roma 'La Sapienza' = Sapienza University [Rome], Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS)-Université de Paris (UP), Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur [Paris], Gautier, C., Troilo, F., Cordier, F., Malagrino, Francesca, Toto, A., Visconti, L., Zhu, Y., Brunori, M., Wolff, N., and Gianni, S.
Subjects: Scaffold protein, Protein Folding, PDZ domain, PDZ Domains, Sequence (biology), 03 medical and health sciences, 030304 developmental biology, Kinetic, 0303 health sciences, Multidisciplinary, Misfolding, Tandem, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Chemistry, 030302 biochemistry & molecular biology, Proteins, Folding, Biological Sciences, Ligand (biochemistry), Folding (chemistry), [SDV.BBM.BP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biophysics, Biophysics and Computational Biology, Kinetics, Proteome, folding, kinetics, misfolding, Biophysics, Protein folding
Abstract: Significance Much of our current knowledge on protein folding is based on work focused on isolated domains. In this study, using a combination of NMR and kinetic experiments, we depict the folding pathway of a multidomain construct comprising two PDZ domains in tandem, belonging to the protein Whirlin. We demonstrate the presence of a misfolded intermediate that competes with productive folding. Interestingly, we show that, unexpectedly, this misfolded state retains the native-like functional ability to bind its physiological ligand, representing a clear example of a functionally competent misfolded state. On the basis of these results and a comparative analysis of the amino acidic sequences of Whirlin from different species, we propose a possible physiological role of the misfolded intermediate., Although more than 75% of the proteome is composed of multidomain proteins, current knowledge of protein folding is based primarily on studies of isolated domains. In this work, we describe the folding mechanism of a multidomain tandem construct comprising two distinct covalently bound PDZ domains belonging to a protein called Whirlin, a scaffolding protein of the hearing apparatus. In particular, via a synergy between NMR and kinetic experiments, we demonstrate the presence of a misfolded intermediate that competes with productive folding. In agreement with the view that tandem domain swapping is a potential source of transient misfolding, we demonstrate that such a kinetic trap retains native-like functional activity, as shown by the preserved ability to bind its physiological ligand. Thus, despite the general knowledge that protein misfolding is intimately associated with dysfunction and diseases, we provide a direct example of a functionally competent misfolded state. Remarkably, a bioinformatics analysis of the amino acidic sequence of Whirlin from different species suggests that the tendency to perform tandem domain swapping between PDZ1 and PDZ2 is highly conserved, as demonstrated by their unexpectedly high sequence identity. On the basis of these observations, we discuss on a possible physiological role of such misfolded intermediate.
Published: 2020
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36. Solid-State Phase Transformation and Self-Assembly of Amorphous Nanoparticles into Higher-Order Mineral Structures

Author: Daniel J. Kelly, Nagarajan Murali, Paul G. Falkowski, Thierry Azaïs, Guillaume Laurent, Gérard Pehau-Arnaudet, Viacheslav Manichev, Margarita Rivers, Stanislas Von Euw, Department of Marine and Coastal Sciences [New Brunswick], School of Environmental and Biological Sciences [New Brunswick], Rutgers, The State University of New Jersey [New Brunswick] (RU), Rutgers University System (Rutgers)-Rutgers University System (Rutgers)-Rutgers, The State University of New Jersey [New Brunswick] (RU), Rutgers University System (Rutgers)-Rutgers University System (Rutgers), Trinity Centre for Bioengineering [Dublin] (TCBE), Trinity Biomedical Sciences Institute [Dublin], Trinity College Dublin-Trinity College Dublin, Spectroscopie, Modélisation, Interfaces pour L'Environnement et la Santé (LCMCP-SMiLES), Laboratoire de Chimie de la Matière Condensée de Paris (LCMCP), Institut de Chimie du CNRS (INC)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), Department of Chemistry and Chemical Biology [Piscataway], Rutgers University [Camden], Institute of Advanced Materials, Devices, and Nanotechnology [Piscataway], Microbiologie structurale - Structural Microbiology (Microb. Struc. (UMR_3528 / U-Pasteur_5)), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Plateforme BioImagerie Ultrastructurale – Ultrastructural BioImaging Platform (UTechS UBI), Institut Pasteur [Paris] (IP), Department of Physics [Wellesley], Wellesley College, National Science Foundation under Grant No. EF 1416785, European Union’s Horizon 2020 research and innovation program under the Marie Sklodowska-Curie grant agreement No. 793861, Ulysses scheme of the Irish Research Council, ANR-11-EQPX-0008,CACSICE,Centre d'analyse de systèmes complexes dans les environnements complexes(2011), Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS)-Université de Paris (UP), and Institut Pasteur [Paris]
Subjects: Nanoparticle, Crystal growth, General Chemistry, [CHIM.MATE]Chemical Sciences/Material chemistry, 010402 general chemistry, 01 natural sciences, Biochemistry, Catalysis, Amorphous calcium carbonate, 0104 chemical sciences, Amorphous solid, law.invention, chemistry.chemical_compound, Colloid and Surface Chemistry, Chemical engineering, chemistry, law, [CHIM.ANAL]Chemical Sciences/Analytical chemistry, Molecule, Particle, Self-assembly, Crystallization
Abstract: International audience; Materials science has been informed by nonclassical pathways to crystallization, based on biological processes, about the fabrication of damage-tolerant composite materials. Various biomineralizing taxa, such as stony corals, deposit metastable, magnesium-rich, amorphous calcium carbonate nanoparticles that further assemble and transform into higher-order mineral structures. Here, we examine a similar process in abiogenic conditions using synthetic, amorphous calcium magnesium carbonate nanoparticles. Applying a combination of high-resolution imaging and in situ solid-state nuclear magnetic resonance spectroscopy, we reveal the underlying mechanism of the solid-state phase transformation of these amorphous nanoparticles into crystals under aqueous conditions. These amorphous nanoparticles are covered by a hydration shell of bound water molecules. Fast chemical exchanges occur: the hydrogens present within the nanoparticles exchange with the hydrogens from the surface-bound H2O molecules which, in turn, exchange with the hydrogens of the free H2O molecule of the surrounding aqueous medium. This cascade of chemical exchanges is associated with an enhanced mobility of the ions/molecules that compose the nanoparticles which, in turn, allow for their rearrangement into crystalline domains via solid-state transformation. Concurrently, the starting amorphous nanoparticles aggregate and form ordered mineral structures through crystal growth by particle attachment. Sphere-like aggregates and spindle-shaped structures were, respectively, formed from relatively high or low weights per volume of the same starting amorphous nanoparticles. These results offer promising prospects for exerting control over such a nonclassical pathway to crystallization to design mineral structures that could not be achieved through classical ion-by-ion growth.
Published: 2020
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37. Characterization of accessory genes in coronavirus genomes

Author: Christian Jean Michel, Claudine Mayer, Olivier Poch, Julie Dawn Thompson, Laboratoire des sciences de l'ingénieur, de l'informatique et de l'imagerie (ICube), École Nationale du Génie de l'Eau et de l'Environnement de Strasbourg (ENGEES)-Université de Strasbourg (UNISTRA)-Institut National des Sciences Appliquées - Strasbourg (INSA Strasbourg), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut National de Recherche en Informatique et en Automatique (Inria)-Les Hôpitaux Universitaires de Strasbourg (HUS)-Centre National de la Recherche Scientifique (CNRS)-Matériaux et Nanosciences Grand-Est (MNGE), Université de Strasbourg (UNISTRA)-Université de Haute-Alsace (UHA) Mulhouse - Colmar (Université de Haute-Alsace (UHA))-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Université de Haute-Alsace (UHA) Mulhouse - Colmar (Université de Haute-Alsace (UHA))-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Réseau nanophotonique et optique, Université de Strasbourg (UNISTRA)-Université de Haute-Alsace (UHA) Mulhouse - Colmar (Université de Haute-Alsace (UHA))-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS), Microbiologie structurale - Structural Microbiology (Microb. Struc. (UMR_3528 / U-Pasteur_5)), Institut Pasteur [Paris] (IP)-Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), This work was supported by the Institut Français de Bioinformatique (IFB), ANR-11-INBS-0013, the ANR projects Elixir-Excelerate: GA-676559 and RAinRARE: ANR-18-RAR3–0006-02, and institute funds from the French Centre National de la Recherche Scientifique and the University of Strasbourg., ANR-11-INBS-0013,IFB (ex Renabi-IFB),Institut français de bioinformatique(2011), ANR-18-RAR3-0006,RAinRARE(2018), European Project: 676559,H2020,H2020-INFRADEV-1-2015-1,ELIXIR-EXCELERATE(2015), Institut National des Sciences Appliquées - Strasbourg (INSA Strasbourg), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS)-École Nationale du Génie de l'Eau et de l'Environnement de Strasbourg (ENGEES)-Réseau nanophotonique et optique, Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Université de Haute-Alsace (UHA) Mulhouse - Colmar (Université de Haute-Alsace (UHA))-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Université de Haute-Alsace (UHA) Mulhouse - Colmar (Université de Haute-Alsace (UHA))-Matériaux et nanosciences d'Alsace (FMNGE), Institut de Chimie du CNRS (INC)-Université de Strasbourg (UNISTRA)-Université de Haute-Alsace (UHA) Mulhouse - Colmar (Université de Haute-Alsace (UHA))-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC)-Université de Strasbourg (UNISTRA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Université Paris Diderot - Paris 7 (UPD7)-Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Thompson, Julie, Infrastructures - Institut français de bioinformatique - - IFB (ex Renabi-IFB)2011 - ANR-11-INBS-0013 - INBS - VALID, ERA-NET E-RARE-3 - - RAinRARE2018 - ANR-18-RAR3-0006 - E-Rare-3 - VALID, and ELIXIR-EXCELERATE: Fast-track ELIXIR implementation and drive early user exploitation across the life-sciences. - ELIXIR-EXCELERATE - - H20202015-09-01 - 2019-08-31 - 676559 - VALID
Subjects: Genes, Viral, viruses, Accessory genes, Genome, Viral, ORF prediction, lcsh:Infectious and parasitic diseases, Evolution, Molecular, Viral Matrix Proteins, 03 medical and health sciences, Betacoronavirus, Open Reading Frames, Viral Proteins, 0302 clinical medicine, [SDV.BBM.GTP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN], Circular code motifs, Animals, Humans, lcsh:RC109-216, Viral Regulatory and Accessory Proteins, 030212 general & internal medicine, Codon, 030304 developmental biology, 0303 health sciences, SARS-CoV-2, Research, Computational Biology, COVID-19, SARS-CoV, 3. Good health, Coronavirus, Severe acute respiratory syndrome-related coronavirus, Spike Glycoprotein, Coronavirus, [SDV.BBM.GTP] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN]
Abstract: Background: The Covid19 infection is caused by the SARS-CoV-2 virus, a novel member of the coronavirus (CoV) family. CoV genomes code for a ORF1a / ORF1ab polyprotein and four structural proteins widely studied as major drug targets. The genomes also contain a variable number of open reading frames (ORFs) coding for accessory proteins that are not essential for virus replication, but appear to have a role in pathogenesis. The accessory proteins have been less well characterized and are difficult to predict by classical bioinformatics methods.Methods: We propose a computational tool GOFIX to characterize potential ORFs in virus genomes. In particular, ORF coding potential is estimated by searching for enrichment in motifs of the X circular code, that is known to be over-represented in the reading frames of viral genes.Results: We applied GOFIX to study the SARS-CoV-2 and related genomes including SARS-CoV and SARS-like viruses from bat, civet and pangolin hosts, focusing on the accessory proteins. Our analysis provides evidence supporting the presence of overlapping ORFs 7b, 9b and 9c in all the genomes and thus helps to resolve some differences in current genome annotations. In contrast, we predict that ORF3b is not functional in all genomes. Novel putative ORFs were also predicted, including a truncated form of the ORF10 previously identified in SARS-CoV-2 and a little known ORF overlapping the Spike protein in Civet-CoV and SARS-CoV.Conclusions: Our findings contribute to characterizing sequence properties of accessory genes of SARS coronaviruses, and especially the newly acquired genes making use of overlapping reading frames.
Published: 2020
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38. Central role and structure of the membrane pseudokinase YukC in the antibacterial Bacillus subtilis Type VIIb Secretion System

Author: Thierry Doan, Maïalène Chabalier, Matteo Tassinari, Pedro M. Alzari, Eric Cascales, Quentin Gaday, Rémi Fronzes, Marco Bellinzoni, Mathilde Ben-Assaya, Mariano Martinez, Francesca Gubellini, Microbiologie structurale - Structural Microbiology (Microb. Struc. (UMR_3528 / U-Pasteur_5)), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Sorbonne Université (SU), Institut Européen de Chimie et Biologie (IECB), Université de Bordeaux (UB)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Laboratoire d'ingénierie des systèmes macromoléculaires (LISM), Aix Marseille Université (AMU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), and M.T. was supported by a scholarship from the Pasteur-Paris University (PPU) International PhD Program.
Subjects: protein complexes, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], biology, interaction network, Chemistry, Protein subunit, pseudokinase, Bacillus subtilis, bacterial two-hybrid assay, biology.organism_classification, Molecular machine, Transmembrane protein, Cell biology, nervous system, Mechanism of action, medicine, Type VIIb Secretion System, Secretion, bacterial competition, medicine.symptom, crystallographic structure, Structural motif, Function (biology)
Abstract: Posté dans BioRxiv le 9 mai 2020; We previously linked TSHZ3 haploinsufficiency to autism spectrum disorder (ASD) and showed that embryonic or postnatal Tshz3 deletion in mice results in behavioral traits relevant to the two core domains of ASD, namely social interaction deficits and repetitive behaviors. Here, we provide evidence that cortical projection neurons (CPNs) and striatal cholinergic interneurons (SCINs) are two main and complementary players in the TSHZ3-linked ASD syndrome. We show that in the cerebral cortex, TSHZ3 is expressed in CPNs and in a proportion of GABA interneurons, while not in cholinergic interneurons or glial cells. TSHZ3-expressing cells, which are predominantly SCINs in the striatum, represent a low proportion of neurons in the ascending cholinergic projection system. We then characterized two new conditional knockout (cKO) models generated by crossing Tshz3 flox/flox with Emx1-Cre ( Emx1-cKO ) or Chat-Cre ( Chat-cKO ) mice to decipher the respective role of CPNs and SCINs. Emx1-cKO mice show altered excitatory synaptic transmission onto CPNs and plasticity at corticostriatal synapses, with neither cortical neuron loss nor impaired layer distribution. These animals present social interaction deficits but no repetitive patterns of behavior. Chat-cKO mice exhibit no loss of SCINs but changes in the electrophysiological properties of these interneurons, associated with repetitive patterns of behavior without social interaction deficits. Therefore, dysfunction in either CPNs or SCINs segregates with a distinct ASD behavioral trait. These findings provide novel insights onto the implication of the corticostriatal circuitry in ASD by revealing an unexpected neuronal dichotomy in the biological background of the two core behavioral domains of this disorder.
Published: 2020
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39. Conformational plasticity of the response regulator CpxR, a key player in Gammaproteobacteria virulence and drug-resistance

Author: Pedro M. Alzari, Nathalie Sassoon, Ariel E. Mechaly, Ahmed Haouz, Alejandro Buschiazzo, Jean-Michel Betton, Microbiologie structurale - Structural Microbiology (Microb. Struc. (UMR_3528 / U-Pasteur_5)), Institut Pasteur [Paris]-Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), Université Paris Diderot - Paris 7 (UPD7), Cristallographie (Plateforme) - Crystallography (Platform), Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Département de Microbiologie - Department of Microbiology, Institut Pasteur [Paris], Institut Pasteur de Montevideo, Réseau International des Instituts Pasteur (RIIP), Institut Pasteur [Paris] (IP)-Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Institut Pasteur [Paris] (IP), and Cristallographie (Plate-forme)
Subjects: 0301 basic medicine, Virulence, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], biology, Chemistry, Gene Expression Regulation, Bacterial, Drug resistance, Plasticity, biology.organism_classification, medicine.disease_cause, Cell biology, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], 03 medical and health sciences, Response regulator, 030104 developmental biology, Bacterial Proteins, X-Ray Diffraction, Structural Biology, Transcription (biology), Gammaproteobacteria, Transcriptional regulation, medicine, Escherichia coli
Abstract: International audience; The transcriptional regulator CpxR mediates an adaptive response to envelope stress, tightly linked to virulence and antibiotics resistance in several Gammaproteobacteria pathogens. In this work, we integrated crystallographic and small-angle X-ray scattering data to gain insights into the structure and conformational plasticity of CpxR from Escherichia coli. CpxR dimerizes through two alternative interaction surfaces. Moreover, widely different CpxR conformations coexist in solution, from compact to fully extended ones. The possible functional implications of these structural features are discussed.
Published: 2018
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40. The synthesis and kinetic evaluation of aryl α-aminophosphonates as novel inhibitors of T. cruzi trans-sialidase

Author: Andrew G. Watts, A. Carlos Frasch, Ricardo Resende, Jean M. H. van den Elsen, Susan J. Crennell, Patricia Marcé, Pedro M. Alzari, Zexin Chen, University of Bath [Bath], Microbiologie structurale - Structural Microbiology (Microb. Struc. (UMR_3528 / U-Pasteur_5)), Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS)-Université de Paris (UP), Instituto de Investigaciones Biotecnológicas [San Martín] (IIB-INTECH), Consejo Nacional de Investigaciones Científicas y Técnicas [Buenos Aires] (CONICET)-Universidad Nacional de San Martin (UNSAM), RR was supported by a University Research Scholarship from the University of Bath., and Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité)
Subjects: 0301 basic medicine, Trans-sialidase, chemistry.chemical_compound, Drug Discovery, MESH: Trypanocidal Agents, Enzyme Inhibitors, Amination, Binding affinities, chemistry.chemical_classification, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], biology, MESH: Neuraminidase, General Medicine, Trypanocidal Agents, 3. Good health, Molecular Docking Simulation, T cruzi trans-sialidase, Biochemistry, MESH: Enzyme Inhibitors, Non competitive, MESH: Organophosphonates, MESH: Trypanosoma cruzi, [PHYS.PHYS.PHYS-BIO-PH]Physics [physics]/Physics [physics]/Biological Physics [physics.bio-ph], Trypanosoma cruzi, Kinetic analysis, MESH: Glycoproteins, Organophosphonates, Neuraminidase, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, 03 medical and health sciences, SDG 3 - Good Health and Well-being, MESH: Molecular Docking Simulation, [CHIM.CRIS]Chemical Sciences/Cristallography, Humans, MESH: Chagas Disease, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Chagas Disease, MESH: Amination, Glycoproteins, Pharmacology, MESH: Humans, 030102 biochemistry & molecular biology, Inhibitors, Aryl, Organic Chemistry, biology.organism_classification, 030104 developmental biology, Enzyme, chemistry, Non-competitive, [INFO.INFO-BI]Computer Science [cs]/Bioinformatics [q-bio.QM], α-aminophosphonates
Abstract: The trans-sialidase protein expressed by Trypanosoma cruzi is an important enzyme in the life cycle of this human pathogenic parasite and is considered a promising target for the development of new drug treatments against Chagas' disease. Here we describe α-amino phosphonates as a novel class of inhibitor of T. cruzi trans-sialidase. Molecular modelling studies were initially used to predict the active-site binding affinities for a series of amino phosphonates, which were subsequently synthesised and their IC 50s determined in vitro. The measured inhibitory activities show some correlation with the predictions from molecular modelling, with 1-napthyl derivatives found to be the most potent inhibitors having IC 50s in the low micromolar range. Interestingly, kinetic analysis of the mode of inhibition demonstrated that the α-aminophosphonates tested here operate in a non-competitive manner.
Published: 2018
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41. The stress sigma factor σS/RpoS counteracts Fur repression of genes involved in iron and manganese metabolism and modulates the ionome of Salmonella enterica serovar Typhimurium

Author: Selma Metaane, Véronique Monteil, Sophie Ayrault, Louise Bordier, Corinne Levi-Meyreuis, Françoise Norel, Biochimie des Interactions Macromoléculaires / Biochemistry of Macromolecular Interactions, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Microbiologie structurale - Structural Microbiology (Microb. Struc. (UMR_3528 / U-Pasteur_5)), Laboratoire des Sciences du Climat et de l'Environnement [Gif-sur-Yvette] (LSCE), Université de Versailles Saint-Quentin-en-Yvelines (UVSQ)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut national des sciences de l'Univers (INSU - CNRS)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Géochimie Des Impacts (GEDI), Université de Versailles Saint-Quentin-en-Yvelines (UVSQ)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut national des sciences de l'Univers (INSU - CNRS)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Université de Versailles Saint-Quentin-en-Yvelines (UVSQ)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut national des sciences de l'Univers (INSU - CNRS)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Université Paris-Saclay, This work was supported by the French National Research Agency (ANR-19-CE44-0005-01 to FN, https://anr.fr) and the Foundation for Medical Research (FRM FDM201806005943 to SM, https://www.frm.org)., and ANR-19-CE44-0005,PERIOMET,Ionome et remodelage métabolique dans la persistance et le réveil de Salmonella quiescentes(2019)
Subjects: Ions, Salmonella typhimurium, Manganese, Multidisciplinary, [SDV]Life Sciences [q-bio], Iron, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, Sigma Factor, Gene Expression Regulation, Bacterial, Serogroup, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, Bacterial Proteins, bacteria, [CHIM]Chemical Sciences, Magnesium
Abstract: In many Gram-negative bacteria, the stress sigma factor of RNA polymerase, σS/RpoS, remodels global gene expression to reshape the physiology of quiescent cells and ensure their survival under non-optimal growth conditions. In the foodborne pathogen Salmonella enterica serovar Typhimurium, σS is also required for biofilm formation and virulence. We have previously identified sRNAs genes positively controlled by σS in Salmonella, including the two paralogous sRNA genes, ryhB1 and ryhB2/isrE. Expression of ryhB1 and ryhB2 is repressed by the ferric uptake regulator Fur when iron is available. In this study, we show that σS alleviates Fur-mediated repression of the ryhB genes and of additional Fur target genes. Moreover, σS induces transcription of the manganese transporter genes mntH and sitABCD and prevents their repression, not only by Fur, but also by the manganese-responsive regulator MntR. These findings prompted us to evaluate the impact of a ΔrpoS mutation on the Salmonella ionome. Inductively coupled plasma mass spectrometry analyses revealed a significant effect of the ΔrpoS mutation on the cellular concentration of manganese, magnesium, cobalt and potassium. In addition, transcriptional fusions in several genes involved in the transport of these ions were regulated by σS. This study suggests that σS controls fluxes of ions that might be important for the fitness of quiescent cells. Consistent with this hypothesis, the ΔrpoS mutation extended the lag phase of Salmonella grown in rich medium supplemented with the metal ion chelator EDTA, and this effect was abolished when magnesium, but not manganese or iron, was added back. These findings unravel the importance of σS and magnesium in the regrowth potential of quiescent cells.
Published: 2022
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42. Arabidopsis thaliana Hcc1 is a Sco‐like metallochaperone for Cu A assembly in Cytochrome c Oxidase

Author: María-Natalia Lisa, Marcos N. Morgada, Pedro M. Alzari, Alejandro J. Vila, Estefanía Giannini, María-Eugenia Llases, Instituto de Biología Molecular y Celular de Rosario [Rosario] (IBR), Consejo Nacional de Investigaciones Científicas y Técnicas [Buenos Aires] (CONICET)-Universidad Nacional de Rosario [Santa Fe], Plataforma de Biología Estructural y Metabolómica [Rosario] (PLABEM), Facultad de Ciencias Bioquımicas y Farmaceuticas [Rosario] (FBIOyF), Universidad Nacional de Rosario [Santa Fe], Microbiologie structurale - Structural Microbiology (Microb. Struc. (UMR_3528 / U-Pasteur_5)), Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS)-Université de Paris (UP), We acknowledge the synchrotron source in ESRF (Grenoble, France) for granting access to their facilities, and their staff members for the helpful assistance. We acknowledge funding from the grant PICT 2012‐1285, from the Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT, Argentina). MNL and AJV are staff members of the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET, Argentina). MEL, MM and EG are recipient of fellowships from CONICET. The NMR spectrometer was supported with grants from ANPCyT. We also thank support from the Argentina‐France ECOS exchange program (ECOS A15B01) to AJV and PMA., We thank Ahmed Haouz and Patrick Weber (Institut Pasteur) for their help with robot‐driven crystallization screenings. We thank M. Avecilla for the excellent technical assistance in this work., and Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité)
Subjects: 0301 basic medicine, Cellular respiration, [PHYS.PHYS.PHYS-BIO-PH]Physics [physics]/Physics [physics]/Biological Physics [physics.bio-ph], Protein Data Bank (RCSB PDB), [SDV.BC]Life Sciences [q-bio]/Cellular Biology, Biochemistry, 03 medical and health sciences, 0302 clinical medicine, Cytochrome-c-Oxidase, [CHIM.CRIS]Chemical Sciences/Cristallography, Cytochrome c oxidase, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Molecular Biology, Gene, CuA, biology, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Chemistry, Cell Biology, Plant development, 030104 developmental biology, Sco, 030220 oncology & carcinogenesis, copper, biology.protein, metallochaperone, [INFO.INFO-BI]Computer Science [cs]/Bioinformatics [q-bio.QM]
Abstract: International audience; The assembly of the CuA site in Cytochrome c Oxidase (COX) is a critical step for aerobic respiration in COX-dependent organisms. Several gene products have been associated with the assembly of this copper site, the most conserved of them belonging to the Sco family of proteins, which have been shown to perform different roles in different organisms. Plants express two orthologs of Sco proteins: Hcc1 and Hcc2. Hcc1 is known to be essential for plant development and for COX maturation, but its precise function has not been addressed until now. Here, we report the biochemical, structural and functional characterization of Arabidopsis thaliana Hcc1 protein (here renamed Sco1). We solved the crystal structure of the Cu+1 -bound soluble domain of this protein, revealing a tri coordinated environment involving a CxxxCxn H motif. We show that AtSco1 is able to work as a copper metallochaperone, inserting two Cu+1 ions into the CuA site in a model of CoxII. We also show that AtSco1 does not act as a thiol-disulfide oxido-reductase. Overall, this information sheds new light on the biochemistry of Sco proteins, highlighting the diversity of functions among them despite their high structural similarities. DATABASE: PDB entry 6N5U (Crystal structure of Arabidopsis thaliana ScoI with copper bound).
Published: 2020
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43. Unexpected electron spin density on the axial methionine ligand in Cu A suggests its involvement in electron pathways

Author: Alejandro J. Vila, Daniel H. Murgida, María-Eugenia Llases, Estefanía Giannini, María-Natalia Lisa, Marcos N. Morgada, María A. Castro, Pedro M. Alzari, Instituto de Biología Molecular y Celular de Rosario [Rosario] (IBR), Consejo Nacional de Investigaciones Científicas y Técnicas [Buenos Aires] (CONICET)-Universidad Nacional de Rosario [Santa Fe], Facultad de Ciencias Bioquımicas y Farmaceuticas [Rosario] (FBIOyF), Universidad Nacional de Rosario [Santa Fe], Instituto de Química Física de los Materiales, Medio Ambiente y Energía [Buenos Aires] (INQUIMAE), Consejo Nacional de Investigaciones Científicas y Técnicas [Buenos Aires] (CONICET)-Facultad de Ciencias Exactas y Naturales [Buenos Aires] (FCEyN), Universidad de Buenos Aires [Buenos Aires] (UBA)-Universidad de Buenos Aires [Buenos Aires] (UBA), Microbiologie structurale - Structural Microbiology (Microb. Struc. (UMR_3528 / U-Pasteur_5)), Institut Pasteur [Paris]-Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), Plataforma de Biología Estructural y Metabolómica [Rosario] (PLABEM), and Institut Pasteur [Paris] (IP)-Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS)
Subjects: Stereochemistry, [PHYS.PHYS.PHYS-BIO-PH]Physics [physics]/Physics [physics]/Biological Physics [physics.bio-ph], [SDV.BC]Life Sciences [q-bio]/Cellular Biology, 010402 general chemistry, 01 natural sciences, Catalysis, Cofactor, purl.org/becyt/ford/1 [https], Metal, 03 medical and health sciences, chemistry.chemical_compound, Delocalized electron, Electron transfer, Materials Chemistry, [CHIM.CRIS]Chemical Sciences/Cristallography, Cytochrome c oxidase, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, purl.org/becyt/ford/1.6 [https], 030304 developmental biology, 0303 health sciences, Methionine, biology, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Chemistry, Cytochrome c, Metals and Alloys, General Chemistry, Nuclear magnetic resonance spectroscopy, electron transfer, NMR, 0104 chemical sciences, Surfaces, Coatings and Films, Electronic, Optical and Magnetic Materials, visual_art, Ceramics and Composites, biology.protein, visual_art.visual_art_medium, [INFO.INFO-BI]Computer Science [cs]/Bioinformatics [q-bio.QM]
Abstract: The CuA center is a paradigm for the study of long-range biological electron transfer. This metal center is an essential cofactor for terminal oxidases like cytochrome c oxidase, the enzymatic complex responsible for cellular respiration in eukaryotes and in most bacteria. CuA acts as an electron hub by transferring electrons from reduced cytochrome c to the catalytic site of the enzyme where dioxygen reduction takes place. Different electron transfer pathways have been proposed involving a weak axial methionine ligand residue, conserved in all CuA sites. This hypothesis has been challenged by theoretical calculations indicating the lack of electron spin density in this ligand. Here we report an NMR study with selectively labeled methionine in a native CuA. NMR spectroscopy discloses the presence of net electron spin density in the methionine axial ligand in the two alternative ground states of this metal center. Similar spin delocalization observed on two second sphere mutants further supports this evidence. These data provide a novel view of the electronic structure of CuA centers and support previously neglected electron transfer pathways. Fil: Morgada, Marcos Nicolás. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina Fil: Llases, María Eugenia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina Fil: Giannini, Estefanía. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina Fil: Castro, Maria Ana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; Argentina Fil: Alzari, Pedro M.. Instituto Pasteur; Francia Fil: Murgida, Daniel Horacio. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; Argentina Fil: Lisa, María Natalia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina Fil: Vila, Alejandro Jose. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina
Published: 2020
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44. Population genomics and antimicrobial resistance in Corynebacterium diphtheriae

Author: Sarah L. Baines, Julie Toubiana, Annick Carmi-Leroy, Marina Barros-Pinkelnig, Carla Rodrigues, Anne Marie Wehenkel, Xavier Didelot, Sylvain Brisse, Leonardo G. Panunzi, Edgar Badell, Melanie Hennart, Quentin Gaday, Melody Dazas, Brisse, Sylvain, Integrative Biology of Emerging Infectious Diseases - - IBEID2010 - ANR-10-LABX-0062 - LABX - VALID, Klebsiella pneumoniae: from ecology to source attribution and transmission control - MedVetKlebs (a component of European Joint Programme One Health) - - H2020-SFS-2017-12018-01-01 - 2019-12-31 - 773830 - VALID, Biodiversité et Epidémiologie des Bactéries pathogènes - Biodiversity and Epidemiology of Bacterial Pathogens, Institut Pasteur [Paris] (IP), Collège Doctoral, Sorbonne Université (SU), Institut Français de Bioinformatique (IFB-CORE), Institut National de Recherche en Informatique et en Automatique (Inria)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Microbiologie structurale - Structural Microbiology (Microb. Struc. (UMR_3528 / U-Pasteur_5)), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), The Peter Doherty Institute for Infection and Immunity [Melbourne], University of Melbourne-The Royal Melbourne Hospital, Centre national de Référence des Corynebactéries du Complexe Diphtheriae - National Reference Center Corynebacteria of the diphtheriae complex (CNR), University of Warwick [Coventry], 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), Université Paris Cité (UPCité), MH was supported financially by a PhD grant from the European Joint Programme One Health, which has received funding from the European Union’s Horizon 2020 Research and Innovation Programme under Grant Agreement No. 773830. LGP was supported financially by a grant from the Institut Français de Bioinformatique, the national infrastructure for services in bioinformatics created in the frame of the French Government’s Investissement d’Avenir program. QG was funded by MTCI PhD school (ED 563). CR was supported by a Pasteur-Roux fellowship from Institut Pasteur. SLB received support from a Victorian Fellowship, provided by VESKI and funded by the State Government of Victoria, Australia. The National Reference Center for Corynebacteria of the Diphtheriae complex receives support from Institut Pasteur and Public Health France (Santé Publique France, Saint Maurice, France). This work was supported financially by the French government’s Investissement d’Avenir program Laboratoire d’Excellence 'Integrative Biology of Emerging Infectious Diseases' (ANR-10-LABX-62-IBEID). AW and QG receive support from Institut Pasteur and the CNRS (France)., We thank Vincent Enouf and the P2M core facility of Institut Pasteur for genomic sequencing. We are indebted to Collection de l’Institut Pasteur for providing reference strains of ribotypes, which were deposited following their described by Grimont and colleagues in 2004. We thank Valerie Bouchez for help with the Oxford Nanopore Technologies sequencing., ANR-10-LABX-0062,IBEID,Integrative Biology of Emerging Infectious Diseases(2010), European Project: 773830,H2020-SFS-2017-1,MedVetKlebs (a component of European Joint Programme One Health)(2018), Institut Pasteur [Paris], Institut Pasteur [Paris]-Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), The Royal Melbourne Hospital-University of Melbourne, Collège doctoral [Sorbonne universités], Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS)-Université de Paris (UP), Université de Paris (UP), and European Project: 773830,H2020-SFS-2017-1,MedVetKlebs (a component of European Joint Programme One Health EJP)(2018)
Subjects: 0301 basic medicine, Antibiotic resistance, Biovar, [SDV]Life Sciences [q-bio], lcsh:Medicine, Diphtheria Toxin, Prospective Studies, Genetics (clinical), Phylogeny, Genetics, education.field_of_study, biology, Diphtheria, Genomics, Antimicrobial, C. diphtheriae, 3. Good health, Anti-Bacterial Agents, Horizontal gene transfer, Molecular Medicine, Macrolides, DNA, Bacterial, Genome-wide association study, lcsh:QH426-470, 030106 microbiology, Population, Microbial Sensitivity Tests, [SDV.BID]Life Sciences [q-bio]/Biodiversity, Mobile genetic element, 03 medical and health sciences, Drug Resistance, Bacterial, medicine, [SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology, Humans, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, education, Molecular Biology, Corynebacterium diphtheriae, Diphtheria toxin, Research, lcsh:R, biology.organism_classification, medicine.disease, QR, lcsh:Genetics, 030104 developmental biology, Genes, Bacterial, [SDV.SPEE] Life Sciences [q-bio]/Santé publique et épidémiologie, [SDV.SPEE]Life Sciences [q-bio]/Santé publique et épidémiologie, Metagenomics, Multilocus Sequence Typing, [SDV.BID] Life Sciences [q-bio]/Biodiversity
Abstract: BackgroundCorynebacterium diphtheriae, the agent of diphtheria, is a genetically diverse bacterial species. Although antimicrobial resistance has emerged against several drugs including first-line penicillin, the genomic determinants and population dynamics of resistance are largely unknown for this neglected human pathogen.MethodsHere, we analyzed the associations of antimicrobial susceptibility phenotypes, diphtheria toxin production, and genomic features inC. diphtheriae. We used 247 strains collected over several decades in multiple world regions, including the 163 clinical isolates collected prospectively from 2008 to 2017 in France mainland and overseas territories.ResultsPhylogenetic analysis revealed multiple deep-branching sublineages, grouped into a Mitis lineage strongly associated with diphtheria toxin production and a largely toxin gene-negative Gravis lineage with few toxin-producing isolates including the 1990s ex-Soviet Union outbreak strain. The distribution of susceptibility phenotypes allowed proposing ecological cutoffs for most of the 19 agents tested, thereby defining acquired antimicrobial resistance. Penicillin resistance was found in 17.2% of prospective isolates. Seventeen (10.4%) prospective isolates were multidrug-resistant (≥ 3 antimicrobial categories), including four isolates resistant to penicillin and macrolides. Homologous recombination was frequent (r/m = 5), and horizontal gene transfer contributed to the emergence of antimicrobial resistance in multiple sublineages. Genome-wide association mapping uncovered genetic factors of resistance, including an accessory penicillin-binding protein (PBP2m) located in diverse genomic contexts. Genepbp2mis widespread in otherCorynebacteriumspecies, and its expression inC. glutamicumdemonstrated its effect against several beta-lactams. A novel 73-kbC. diphtheriaemultiresistance plasmid was discovered.ConclusionsThis work uncovers the dynamics of antimicrobial resistance inC. diphtheriaein the context of phylogenetic structure, biovar, and diphtheria toxin production and provides a blueprint to analyze re-emerging diphtheria.
Published: 2020
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45. High-Throughput Crystallization Pipeline at the Crystallography Core Facility of the Institut Pasteur

Author: Frederick Saul, Patrick Weber, Rafael Navaza, Ahmed Haouz, Cédric Pissis, Ariel E. Mechaly, Pedro M. Alzari, Cristallographie (Plateforme) - Crystallography (Platform), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Microbiologie structurale - Structural Microbiology (Microb. Struc. (UMR_3528 / U-Pasteur_5)), Institut Pasteur [Paris] (IP)-Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), We are grateful for funding from Réseau National des Génopoles (contract RG-2002-08) and ANR GIS-IBISA (France), European Commission support for projects SPINE (contract QLG2-CT-2002-00988) and X-TB (contract QLK2-CT-2001-02018), and continuous financial support from Institut Pasteur., Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), and Institut Pasteur [Paris]-Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS)
Subjects: MESH: Databases, Protein, Computer science, macromolecular crystallography pipeline, Pipeline (computing), [SDV]Life Sciences [q-bio], 030303 biophysics, Pharmaceutical Science, MESH: Academies and Institutes, Crystallography, X-Ray, Article, Analytical Chemistry, Structural genomics, law.invention, 03 medical and health sciences, Data sequences, law, Drug Discovery, Humans, crystal optimization, Physical and Theoretical Chemistry, Crystallization, Databases, Protein, Throughput (business), 030304 developmental biology, 0303 health sciences, MESH: Humans, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Organic Chemistry, Academies and Institutes, Computational Biology, MESH: Crystallography, X-Ray, Core Facility, Crystallography, Chemistry (miscellaneous), Molecular Medicine, high-throughput crystallization screening, MESH: Computational Biology
Abstract: International audience; The availability of whole-genome sequence data, made possible by significant advances in DNA sequencing technology, led to the emergence of structural genomics projects in the late 1990s. These projects not only significantly increased the number of 3D structures deposited in the Protein Data Bank in the last two decades, but also influenced present crystallographic strategies by introducing automation and high-throughput approaches in the structure-determination pipeline. Today, dedicated crystallization facilities, many of which are open to the general user community, routinely set up and track thousands of crystallization screening trials per day. Here, we review the current methods for high-throughput crystallization and procedures to obtain crystals suitable for X-ray diffraction studies, and we describe the crystallization pipeline implemented in the medium-scale crystallography platform at the Institut Pasteur (Paris) as an example.
Published: 2019
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46. Expanding the type IIB DNA topoisomerase family: identification of new topoisomerase and topoisomerase-like proteins in mobile genetic elements

Author: Claudine Mayer, Danièle Gadelle, Violette Da Cunha, Mart Krupovic, Patrick Forterre, Tomio S. Takahashi, Institut de Biologie Intégrative de la Cellule (I2BC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Biologie Cellulaire des Archées (ARCHEE), Département Microbiologie (Dpt Microbio), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Institut de Biologie Intégrative de la Cellule (I2BC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Virologie des archées - Archaeal Virology, Institut Pasteur [Paris], Microbiologie structurale - Structural Microbiology (Microb. Struc. (UMR_3528 / U-Pasteur_5)), Université Paris Diderot - Paris 7 (UPD7)-Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), European Research Council (ERC) Grant from the European Union's Seventh Framework Program (FP/2007-2013) (Project EVOMOBIL-ERC) [340440 to V.D.C., P.F.], l’Agence Nationale de la Recherche [Project ESSPOIR ANR-17-CE12-0032 to T.T., D.G., C.M., Project ENVIRA ANR-17-CE15-0005-01 to M.K.], The authors would like to thank Dr Jacques Oberto, Dr Ryan Catchpole and Dr Stephanie Petrella for valuable discussions, ANR-17-CE15-0005,ENVIRA,Remodelage de la membrane cytoplasmique par les virus enveloppés d'archées(2017), ANR-17-CE12-0032,ESSPOIR,Comprendre la structure de SPO11, ses fonctions et ses interactions(2017), European Project: 340440,EC:FP7:ERC,ERC-2013-ADG,EVOMOBIL(2014), Institut Pasteur [Paris] (IP), and Institut Pasteur [Paris] (IP)-Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS)
Subjects: Genetics, 0303 health sciences, Subfamily, Applied Mathematics, Topoisomerase, [SDV]Life Sciences [q-bio], Archaeal Viruses, Standard Article, Biology, Computer Science Applications, 03 medical and health sciences, chemistry.chemical_compound, 0302 clinical medicine, Plasmid, chemistry, Structural Biology, Extrachromosomal DNA, biology.protein, Mobile genetic elements, Molecular Biology, Gene, 030217 neurology & neurosurgery, DNA, 030304 developmental biology
Abstract: The control of DNA topology by DNA topoisomerases is essential for virtually all DNA transactions in the cell. These enzymes, present in every organism, exist as several non-homologous families. We previously identified a small group of atypical type IIB topoisomerases, called Topo VIII, mainly encoded by plasmids. Here, taking advantage of the rapid expansion of sequence databases, we identified new putative Topo VIII homologs. Our analyses confirm the exclusivity of the corresponding genes to mobile genetic elements (MGE) and extend their distribution to nine different bacterial phyla and one archaeal superphylum. Notably, we discovered another subfamily of topoisomerases, dubbed ‘Mini-A’, including distant homologs of type IIB topoisomerases and encoded by extrachromosomal and integrated bacterial and archaeal viruses. Interestingly, a short, functionally uncharacterized motif at the C-terminal extremity of type IIB topoisomerases appears sufficient to discriminate between Mini-A, Topo VI and Topo VIII subfamilies. This motif could be a key element for understanding the differences between the three subfamilies. Collectively, this work leads to an updated model for the origin and evolution of the type IIB topoisomerase family and raises questions regarding the role of topoisomerases during replication of MGE in bacteria and archaea.
Published: 2019
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47. In vitro activity of ferroquine against artemisinin-based combination therapy (ACT)-resistant Plasmodium falciparum isolates from Cambodia

Author: Jean Christophe Barale, Chanra Khean, Nimol Khim, Sopheakvatey Ke, Saorin Kim, Benoit Witkowski, Nimol Kloeung, Didier Leboulleux, Pascal Ringwald, Didier Leroy, Didier Menard, Chhayleang Kauy, Rithea Leang, Rotha Eam, Flore Nardella, Alain Pellet, Melissa Mairet-Khedim, Malaria Molecular Epidemiology, Institut Pasteur du Cambodge, Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP), Biologie des Interactions Hôte-Parasite - Biology of Host-Parasite Interactions, Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Evotec, National Center of Parasitology (CNM), National Malaria Center [Phnom Penh], National Center for Malariology, Ministry of Health [Phnom Penh], World Health Organisation (WHO), Organisation Mondiale de la Santé / World Health Organization Office (OMS / WHO), Microbiologie structurale - Structural Microbiology (Microb. Struc. (UMR_3528 / U-Pasteur_5)), Université Paris Diderot - Paris 7 (UPD7)-Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Malaria : parasites et hôtes - Malaria : parasites and hosts, Institut Pasteur [Paris], Medicines for Malaria Venture (MMV), Université de Genève (UNIGE), This study was supported by Medicines for Malaria Venture (project RD/13/0002). M. M.-K. and B. W. were supported by 5% initiative [molecular and in vitro surveillance of ACT partner drug efficacy in the Greater Mekong Subregion (MIVS-ACT), grant #15SANIN211]. F. N. was supported by Foundation Pierre Ledoux., We would like to thank the patients who participated in TESs and the Cambodian Health Centre medical staff for their contribution, 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)-Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), Institut Pasteur [Paris] (IP), Université de Genève = University of Geneva (UNIGE), Barale, Jean-Christophe, and Malaria Molecular Epidemiology (MMEU)
Subjects: 0301 basic medicine, Microbiology (medical), Combination therapy, Metallocenes, Plasmodium falciparum, 030231 tropical medicine, Drug Resistance, Antimalarials, Inhibitory Concentration 50, 03 medical and health sciences, 0302 clinical medicine, Parasitic Sensitivity Tests, Piperaquine, medicine, Humans, Parasite hosting, Pharmacology (medical), [SDV.MP.PAR]Life Sciences [q-bio]/Microbiology and Parasitology/Parasitology, Ferrous Compounds, Malaria, Falciparum, Artemisinin, Pharmacology, biology, Mefloquine, Haplotype, medicine.disease, biology.organism_classification, Virology, Artemisinins, 3. Good health, 030104 developmental biology, Infectious Diseases, Aminoquinolines, Drug Therapy, Combination, Cambodia, [SDV.MP.PAR] Life Sciences [q-bio]/Microbiology and Parasitology/Parasitology, Malaria, medicine.drug
Abstract: Background Cambodia is the epicentre of resistance emergence for virtually all antimalarial drugs. Selection and spread of parasites resistant to artemisinin-based combination therapy (ACT) is a major threat for malaria elimination, hence the need to renew the pool of effective treatments. Objectives To determine whether ACT resistance haplotypes could have an effect on ferroquine in vitro antimalarial activity. Methods In vitro susceptibility to ferroquine was measured for 80 isolates from Cambodia characterized for their molecular resistance profile to artemisinin, piperaquine and mefloquine. Results Among the 80 isolates tested, the overall median (IQR) IC50 of ferroquine was 10.9 nM (8.7–18.3). The ferroquine median (IQR) IC50 was 8.9 nM (8.1–11.8) for Pfk13 WT parasites and was 12.9 nM (9.5–20.0) for Pfk13 C580Y parasites with no amplification of Pfpm2 and Pfmdr1 genes. The median (IQR) IC50 of ferroquine for Pfk13 C580Y parasites with amplification of the Pfpm2 gene was 17.2 nM (14.5–20.5) versus 9.1 nM (7.9–10.7) for Pfk13 C580Y parasites with amplification of the Pfmdr1 gene. Conclusions Ferroquine exerts promising efficacy against ACT-resistant isolates. Whereas Pfpm2 amplification was associated with the highest parasite tolerance to ferroquine, the susceptibility range observed was in accordance with those measured in ACT resistance-free areas. This enables consideration of ferroquine as a relevant therapeutic option against ACT-resistant malaria.
Published: 2019
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48. Could Mycolactone Inspire New Potent Analgesics? Perspectives and Pitfalls

Author: Denis Dupoiron, Laurent Marsollier, Priscille Brodin, Marie-Line Reynaert, Edouard Yeramian, 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 Cancérologie de l'Ouest [Angers/Nantes] (UNICANCER/ICO), UNICANCER, Microbiologie structurale - Structural Microbiology (Microb. Struc. (UMR_3528 / U-Pasteur_5)), Institut Pasteur [Paris] (IP)-Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), ATOMycA (CRCINA-ÉQUIPE 6), Centre de Recherche en Cancérologie et Immunologie Nantes-Angers (CRCINA), 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)-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), Financial support for this work was provided by the region Pays de la Loire (Kalmos project), the Agence Nationale de Recherche (ANR-17-CE18-0001), the Institut Pasteur of Lille (granted to P.B. and M.L.R.), the Inserm Program ATIP avenir (granted to L.M), the region Pays de la Loire (Kalmos project) and Angers University., ANR-17-CE18-0001,AT2R-TRAAK-BIOANALGESICS,Caractérisations moléculaires et cellulaires de voies de signalisation de la douleur: contrôle de la douleur dans l'ulcère de buruli comme source d'inspiration pour la conception rationnelle de nouveaux analgésiques puissants(2017), Institut Pasteur [Paris]-Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), 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)-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), Bernardo, Elizabeth, and Caractérisations moléculaires et cellulaires de voies de signalisation de la douleur: contrôle de la douleur dans l'ulcère de buruli comme source d'inspiration pour la conception rationnelle de nouveaux analgésiques puissants - - AT2R-TRAAK-BIOANALGESICS2017 - ANR-17-CE18-0001 - AAPG2017 - VALID
Subjects: Buruli ulcer, Health, Toxicology and Mutagenesis, Analgesic, lcsh:Medicine, Pain, neurons, mycolactone, [SDV.CAN]Life Sciences [q-bio]/Cancer, Review, Disease, Toxicology, Bioinformatics, 03 medical and health sciences, chemistry.chemical_compound, 0302 clinical medicine, Immune system, [SDV.CAN] Life Sciences [q-bio]/Cancer, Animals, Humans, Medicine, Mycolactone, 030304 developmental biology, Analgesics, 0303 health sciences, biology, business.industry, lcsh:R, analgesia, biology.organism_classification, medicine.disease, AT2R, drug development, 3. Good health, chemistry, Drug development, Infectious disease (medical specialty), Mycobacterium ulcerans, Macrolides, business, 030217 neurology & neurosurgery
Abstract: International audience; Pain currently represents the most common symptom for which medical attention is sought by patients. The available treatments have limited effectiveness and significant side-effects. In addition, most often, the duration of analgesia is short. Today, the handling of pain remains a major challenge. One promising alternative for the discovery of novel potent analgesics is to take inspiration from Mother Nature; in this context, the detailed investigation of the intriguing analgesia implemented in Buruli ulcer, an infectious disease caused by the bacterium Mycobacterium ulcerans and characterized by painless ulcerative lesions, seems particularly promising. More precisely, in this disease, the painless skin ulcers are caused by mycolactone, a polyketide lactone exotoxin. In fact, mycolactone exerts a wide range of effects on the host, besides being responsible for analgesia, as it has been shown notably to modulate the immune response or to provoke apoptosis. Several cellular mechanisms and different targets have been proposed to account for the analgesic effect of the toxin, such as nerve degeneration, the inhibition of inflammatory mediators and the activation of angiotensin II receptor 2. In this review, we discuss the current knowledge in the field, highlighting possible controversies. We first discuss the different pain-mimicking experimental models that were used to study the effect of mycolactone. We then detail the different variants of mycolactone that were used in such models. Overall, based on the results and the discussions, we conclude that the development of mycolactone-derived molecules can represent very promising perspectives for new analgesic drugs, which could be effective for specific pain indications. Key Contribution: This review develops arguments and issues around the development of new potent analgesic drugs derived from mycolactone, a toxin secreted by Mycobacterium ulcerans. To this end, the various modes of action of the toxin potentially underlying its analgesic properties are detailed and discussed in the context of various pain models-also concerning different variants of mycolactone.
Published: 2019
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49. Effect of well drilling on Buruli ulcer incidence in Benin: a case-control, quantitative survey

Author: Degnonvi, Horace, Fleuret, Sebastien, Coudereau, Clément, Gnimavo, Ronald, Giffon, Sigrid, Yeramian, Edouard, Johnson, Roch, Marion, Estelle, ATOMycA (CRCINA-ÉQUIPE 6), 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)-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 Interfacultaire de Formation et de Recherche en Environnement pour le Développement Durable [Cotonou, Benin], University of Abomey Calavi (UAC), Espaces et Sociétés (ESO), Institut de Géographie et d'Aménagement Régional de l'Université de Nantes (IGARUN), Université de Nantes (UN)-Université de Nantes (UN)-Centre National de la Recherche Scientifique (CNRS)-Université de Rennes 2 (UR2), Université de Rennes (UNIV-RENNES)-Université de Rennes (UNIV-RENNES)-AGROCAMPUS OUEST, Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro)-Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro)-Université d'Angers (UA)-Université de Caen Normandie (UNICAEN), Normandie Université (NU)-Normandie Université (NU)-Le Mans Université (UM), Centre de Diagnostic et de Traitement de la lèpre et de l’ulcère de Buruli [Pobè, Benin], Fondation Raoul Follereau [Pobe, Benin], Microbiologie structurale - Structural Microbiology (Microb. Struc. (UMR_3528 / U-Pasteur_5)), Université Paris Diderot - Paris 7 (UPD7)-Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Fondation Francaise Raoul Follereau, French National Institute of Health and Medical Research, and Région Pays de Loire., 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)-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), Université d’Abomey-Calavi = University of Abomey Calavi (UAC), Université de Caen Normandie (UNICAEN), Normandie Université (NU)-Normandie Université (NU)-Le Mans Université (UM)-Université d'Angers (UA)-AGROCAMPUS OUEST-Université de Rennes 2 (UR2)-Centre National de la Recherche Scientifique (CNRS)-Institut de Géographie et d'Aménagement Régional de l'Université de Nantes (IGARUN), Université de Nantes (UN)-Université de Nantes (UN), Institut Pasteur [Paris] (IP)-Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), Bernardo, Elizabeth, Normandie Université (NU)-Normandie Université (NU)-Le Mans Université (UM)-Université d'Angers (UA)-AGROCAMPUS OUEST-Université de Rennes 2 (UR2), and Université de Rennes (UNIV-RENNES)-Université de Rennes (UNIV-RENNES)-Centre National de la Recherche Scientifique (CNRS)-Institut de Géographie et d'Aménagement Régional de l'Université de Nantes (IGARUN)
Subjects: [SDV.CAN] Life Sciences [q-bio]/Cancer, Case-Control Studies, Drinking Water, Incidence, Water Wells, Benin, Humans, Hygiene, [SDV.CAN]Life Sciences [q-bio]/Cancer, Sanitation, Buruli Ulcer, Hand Disinfection
Abstract: International audience; BACKGROUND:Buruli ulcer is the third most common mycobacterial disease worldwide. The public health burden of this neglected tropical disease is large, particularly in poor areas of west and central Africa. The development of appropriate preventive strategies is hampered by an incomplete understanding of the epidemiology and transmission of the disease. We investigated the effect of the drilling of wells on Buruli ulcer incidence.METHODS:In this case-control, quantitative survey, we obtained field data for Buruli ulcer incidence over a 10-year period from a specialised centre that collected data for the Ouémé and Plateau departments in Benin, and data for well drilling from the Ministry of Energy, Water and Mines in Benin. The coordinates of the wells drilled were obtained during site visits. A case-control study was then done to investigate the role of well water use in protecting against Buruli ulcer.FINDINGS:We found a strong inverse correlation between the incidence of Buruli ulcer and the number of new wells drilled in the Bonou municipality (r2=0·8818). A case-control study (106 cases and 212 controls) showed that regular use of the water from the wells for washing, bathing, drinking, or cooking was protective against Buruli ulcer (adjusted odds ratio 0·1, 95% CI 0·04-0·44; p=0·0012).INTERPRETATION:This study opens up new possibilities for developing an effective yet affordable policy to fight the disease on a substantial geographical scale. Our study shows that providing access to protected water is an efficient and feasable way to reduce the incidence of Buruli ulcer.FUNDING:Fondation Francaise Raoul Follereau, French National Institute of Health and Medical Research, and Région Pays de Loire.
Published: 2019
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50. Novel mechanistic insights into physiological signaling pathways mediated by mycobacterial Ser/Thr protein kinases

Author: Marco Bellinzoni, Pedro M. Alzari, Anne Marie Wehenkel, Rosario Durán, Microbiologie structurale - Structural Microbiology (Microb. Struc. (UMR_3528 / U-Pasteur_5)), Université Paris Diderot - Paris 7 (UPD7)-Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Institut Pasteur de Montevideo, Réseau International des Instituts Pasteur (RIIP), This work was partially supported by grants from the Institut Pasteur, Paris, France, the Institut Pasteur de Montevideo, Uruguay and the ANR (Agence Nationale de la Recherche), France [research contracts ANR-13-JSV8-0003-01, ANR-18-CE11-0017-01 and ANR-18-CE92-0003-01]., ANR-18-CE11-0017,PhoCellDiv,Mécanismes moléculaires phospho-dépendants de l'assemblage et de la régulation du divisome bactérien(2018), ANR-18-CE92-0003,METACTINO,Déchiffrer la plasticité métabolique des Actinobactéries(2018), ANR-13-JSV8-0003,SUPERCPLX,Structure, fonction et régulation d'un supercomplexe métabolique clé chez les Actinobacteries(2013), and Institut Pasteur [Paris] (IP)-Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS)
Subjects: 0301 basic medicine, 030106 microbiology, Immunology, Phosphatase, Glutamic Acid, Peptidoglycan, Protein Serine-Threonine Kinases, Biology, Microbiology, Mycobacterium tuberculosis, Serine, 03 medical and health sciences, Transduction (genetics), 0302 clinical medicine, Bacterial Proteins, Genetics, Protein phosphorylation, Tyrosine, Genetics (clinical), Kinase, biology.organism_classification, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, 3. Good health, Cell biology, 030104 developmental biology, Infectious Diseases, Signal transduction, Intracellular, 030215 immunology, Signal Transduction
Abstract: International audience; Protein phosphorylation is known to be one of the keystones of signal sensing and transduction in all living organisms. Once thought to be essentially confined to the eukaryotic kingdoms, reversible phosphorylation on serine, threonine and tyrosine residues, has now been shown to play a major role in many prokaryotes, where the number of Ser/Thr protein kinases (STPKs) equals or even exceeds that of two component systems. Mycobacterium tuberculosis, the etiological agent of tuberculosis, is one of the most studied organisms for the role of STPK-mediated signaling in bacteria. Driven by the interest and tractability of these enzymes as potential therapeutic targets, extensive studies revealed the remarkable conservation of protein kinases and their cognate phosphatases across evolution, and their involvement in bacterial physiology and virulence. Here, we present an overview of the current knowledge of mycobacterial STPKs structures and kinase activation mechanisms, and we then focus on PknB and PknG, two well-characterized STPKs that are essential for the intracellular survival of the bacillus. We summarize the mechanistic evidence that links PknB to the regulation of peptidoglycan synthesis in cell division and morphogenesis, and the major findings that establishes PknG as a master regulator of central carbon and nitrogen metabolism. Two decades after the discovery of STPKs in M. tuberculosis, the emerging landscape of O-phosphosignaling is starting to unveil how eukaryotic-like kinases can be engaged in unique, non-eukaryotic-like, signaling mechanisms in mycobacteria.
Published: 2019
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