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94 results on '"Alain Roussel"'

1. Structure–function analysis of PorXFj, the PorX homolog from Flavobacterium johnsioniae, suggests a role of the CheY-like domain in type IX secretion motor activity

Author: Mariotte Zammit, Julia Bartoli, Christine Kellenberger, Pauline Melani, Alain Roussel, Eric Cascales, and Philippe Leone
Subjects: Type IX secretion system, Flavobacterium johnsoniae, Two-component system, Response regulator, Crystal structure, CheY, Medicine, Science
Abstract: Abstract The type IX secretion system (T9SS) is a large multi-protein transenvelope complex distributed into the Bacteroidetes phylum and responsible for the secretion of proteins involved in pathogenesis, carbohydrate utilization or gliding motility. In Porphyromonas gingivalis, the two-component system PorY sensor and response regulator PorX participate to T9SS gene regulation. Here, we present the crystal structure of PorXFj, the Flavobacterium johnsoniae PorX homolog. As for PorX, the PorXFj structure is comprised of a CheY-like N-terminal domain and an alkaline phosphatase-like C-terminal domain separated by a three-helix bundle central domain. While not activated and monomeric in solution, PorXFj crystallized as a dimer identical to active PorX. The CheY-like domain of PorXFj is in an active-like conformation, and PorXFj possesses phosphodiesterase activity, in agreement with the observation that the active site of its phosphatase-like domain is highly conserved with PorX.
Published: 2024
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2. Antiviral activity of intracellular nanobodies targeting the influenza virus RNA-polymerase core.

Author: Mélissa Bessonne, Jessica Morel, Quentin Nevers, Bruno Da Costa, Allison Ballandras-Colas, Florian Chenavier, Magali Grange, Alain Roussel, Thibaut Crépin, and Bernard Delmas
Subjects: Immunologic diseases. Allergy, RC581-607, Biology (General), QH301-705.5
Abstract: Influenza viruses transcribe and replicate their genome in the nucleus of the infected cells, two functions that are supported by the viral RNA-dependent RNA-polymerase (FluPol). FluPol displays structural flexibility related to distinct functional states, from an inactive form to conformations competent for replication and transcription. FluPol machinery is constituted by a structurally-invariant core comprising the PB1 subunit stabilized with PA and PB2 domains, whereas the PA endonuclease and PB2 C-domains can pack in different configurations around the core. To get insights into the functioning of FluPol, we selected single-domain nanobodies (VHHs) specific of the influenza A FluPol core. When expressed intracellularly, some of them exhibited inhibitory activity on type A FluPol, but not on the type B one. The most potent VHH (VHH16) binds PA and the PA-PB1 dimer with an affinity below the nanomolar range. Ectopic intracellular expression of VHH16 in virus permissive cells blocks multiplication of different influenza A subtypes, even when induced at late times post-infection. VHH16 was found to interfere with the transport of the PA-PB1 dimer to the nucleus, without affecting its handling by the importin β RanBP5 and subsequent steps in FluPol assembly. Using FluPol mutants selected after passaging in VHH16-expressing cells, we identified the VHH16 binding site at the interface formed by PA residues with the N-terminus of PB1, overlapping or close to binding sites of two host proteins, ANP32A and RNA-polymerase II RPB1 subunit which are critical for virus replication and transcription, respectively. These data suggest that the VHH16 neutralization is likely due to several activities, altering the import of the PA-PB1 dimer into the nucleus as well as inhibiting specifically virus transcription and replication. Thus, the VHH16 binding site represents a new Achilles' heel for FluPol and as such, a potential target for antiviral development.
Published: 2024
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3. SARS-CoV-2 detection using a nanobody-functionalized voltammetric device

Author: Quentin Pagneux, Alain Roussel, Hiba Saada, Christian Cambillau, Béatrice Amigues, Vincent Delauzun, Ilka Engelmann, Enagnon Kazali Alidjinou, Judith Ogiez, Anne Sophie Rolland, Emmanuel Faure, Julien Poissy, Alain Duhamel, Rabah Boukherroub, David Devos, and Sabine Szunerits
Subjects: Medicine
Abstract: Pagneux et al. developed a nanobody-functionalized electrochemical platform with smartphone readout to detect SARS-CoV-2 in clinical samples. They demonstrated that their device can rapidly and accurately detect SARS-CoV-2 in saliva and nasopharyngeal swab samples and discriminate between this virus and other respiratory viruses.
Published: 2022
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4. Biosynthetic proteins targeting the SARS-CoV-2 spike as anti-virals.

Author: Stéphanie Thébault, Nathalie Lejal, Alexis Dogliani, Amélie Donchet, Agathe Urvoas, Marie Valerio-Lepiniec, Muriel Lavie, Cécile Baronti, Franck Touret, Bruno Da Costa, Clara Bourgon, Audrey Fraysse, Audrey Saint-Albin-Deliot, Jessica Morel, Bernard Klonjkowski, Xavier de Lamballerie, Jean Dubuisson, Alain Roussel, Philippe Minard, Sophie Le Poder, Nicolas Meunier, and Bernard Delmas
Subjects: Immunologic diseases. Allergy, RC581-607, Biology (General), QH301-705.5
Abstract: The binding of the SARS-CoV-2 spike to angiotensin-converting enzyme 2 (ACE2) promotes virus entry into the cell. Targeting this interaction represents a promising strategy to generate antivirals. By screening a phage-display library of biosynthetic protein sequences build on a rigid alpha-helicoidal HEAT-like scaffold (named αReps), we selected candidates recognizing the spike receptor binding domain (RBD). Two of them (F9 and C2) bind the RBD with affinities in the nM range, displaying neutralisation activity in vitro and recognizing distinct sites, F9 overlapping the ACE2 binding motif. The F9-C2 fusion protein and a trivalent αRep form (C2-foldon) display 0.1 nM affinities and EC50 of 8-18 nM for neutralization of SARS-CoV-2. In hamsters, F9-C2 instillation in the nasal cavity before or during infections effectively reduced the replication of a SARS-CoV-2 strain harbouring the D614G mutation in the nasal epithelium. Furthermore, F9-C2 and/or C2-foldon effectively neutralized SARS-CoV-2 variants (including delta and omicron variants) with EC50 values ranging from 13 to 32 nM. With their high stability and their high potency against SARS-CoV-2 variants, αReps provide a promising tool for SARS-CoV-2 therapeutics to target the nasal cavity and mitigate virus dissemination in the proximal environment.
Published: 2022
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5. A new non-classical fold of varroa odorant-binding proteins reveals a wide open internal cavity

Author: Beatrice Amigues, Jiao Zhu, Anais Gaubert, Simona Arena, Giovanni Renzone, Philippe Leone, Isabella Maria Fischer, Harald Paulsen, Wolfgang Knoll, Andrea Scaloni, Alain Roussel, Christian Cambillau, and Paolo Pelosi
Subjects: Medicine, Science
Abstract: Abstract Odorant-binding proteins (OBPs), as they occur in insects, form a distinct class of proteins that apparently has no closely related representatives in other animals. However, ticks, mites, spiders and millipedes contain genes encoding proteins with sequence similarity to insect OBPs. In this work, we have explored the structure and function of such non-insect OBPs in the mite Varroa destructor, a major pest of honey bee. Varroa OBPs present six cysteines paired into three disulphide bridges, but with positions in the sequence and connections different from those of their insect counterparts. VdesOBP1 structure was determined in two closely related crystal forms and appears to be a monomer. Its structure assembles five α-helices linked by three disulphide bridges, one of them exhibiting a different connection as compared to their insect counterparts. Comparison with classical OBPs reveals that the second of the six α-helices is lacking in VdesOBP1. Ligand-binding experiments revealed molecules able to bind only specific OBPs with a moderate affinity, suggesting that either optimal ligands have still to be identified, or post-translational modifications present in the native proteins may be essential for modulating binding activity, or else these OBPs might represent a failed attempt in evolution and are not used by the mites.
Published: 2021
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6. Preanalytical Issues and Cycle Threshold Values in SARS-CoV‑2 Real-Time RT-PCR Testing: Should Test Results Include These?

Author: Ilka Engelmann, Enagnon Kazali Alidjinou, Judith Ogiez, Quentin Pagneux, Sana Miloudi, Ilyes Benhalima, Mahdi Ouafi, Famara Sane, Didier Hober, Alain Roussel, Christian Cambillau, David Devos, Rabah Boukherroub, and Sabine Szunerits
Subjects: Chemistry, QD1-999
Published: 2021
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7. Activity and Crystal Structure of the Adherent-Invasive Escherichia coli Tle3/Tli3 T6SS Effector/Immunity Complex Determined Using an AlphaFold2 Predicted Model

Author: Thi Thu Hang Le, Christine Kellenberger, Marie Boyer, Pierre Santucci, Nicolas Flaugnatti, Eric Cascales, Alain Roussel, Stéphane Canaan, Laure Journet, and Christian Cambillau
Subjects: type VI secretion system, phospholipase, immunity, adherent-invasive Escherichia coli (AIEC), protein secretion, X-ray structure, Biology (General), QH301-705.5, Chemistry, QD1-999
Abstract: The type VI secretion system (T6SS) delivers enzymatic effectors into target cells to destroy them. Cells of the same strain protect themselves against effectors with immunity proteins that specifically inhibit effectors. Here, we report the identification and characterization of a Tle3 phospholipase effector and its cognate immunity protein Tli3—an outer membrane lipoprotein from adherent-invasive Escherichia coli (AIEC). Enzymatic assays demonstrate that purified Tle3AIEC has a phospholipase A1, and not A2, activity and that its toxicity is neutralized by the cognate immunity protein Tli3AIEC. Tli3AIEC binds Tle3 in a 1:1 stoichiometric ratio. Tle3AIEC, Tli3AIEC and the Tle3AIEC-Tli3AIEC complex were purified and subjected to crystallization. The Tle3AIEC-Tli3AIEC complex structure could not be solved by SeMet phasing, but only by molecular replacement when using an AlphaFold2 prediction model. Tle3AIEC exhibits an α/β-hydrolase fold decorated by two protruding segments, including a N-terminus loop. Tli3AIEC displays a new fold of three stacked β-sheets and a protruding loop that inserts in Tle3AIECcatalytic crevice. We showed, experimentally, that Tle3AIEC interacts with the VgrG AIEC cargo protein and AlphaFold2 prediction of the VgrGAIEC-Tle3AIEC complex reveals a strong interaction between the VgrGAIEC C-terminus adaptor and Tle3AIEC N-terminal loop.
Published: 2023
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8. Structure Prediction and Analysis of Hepatitis E Virus Non-Structural Proteins from the Replication and Transcription Machinery by AlphaFold2

Author: Adeline Goulet, Christian Cambillau, Alain Roussel, and Isabelle Imbert
Subjects: Hepatitis E virus, nonstructural proteins, viral replication/transcription enzymes, AlphaFold2, macro domain, helicase, Microbiology, QR1-502
Abstract: Hepatitis E virus (HEV) is a major cause of acute viral hepatitis in humans globally. Considered for a long while a public health issue only in developing countries, the HEV infection is now a global public health concern. Most human infections are caused by the HEV genotypes 1, 2, 3 and 4 (HEV-1 to HEV-4). Although HEV-3 and HEV-4 can evolve to chronicity in immunocompromised patients, HEV-1 and HEV-2 lead to self-limited infections. HEV has a positive-sense single-stranded RNA genome of ~7.2 kb that is translated into a large pORF1 replicative polyprotein, essential for the viral RNA genome replication and transcription. Unfortunately, the composition and structure of these replicases are still unknown. The recent release of the powerful machine-learning protein structure prediction software AlphaFold2 (AF2) allows us to accurately predict the structure of proteins and their complexes. Here, we used AF2 with the replicase encoded by the polyprotein pORF1 of the human-infecting HEV-3. The boundaries and structures reveal five domains or nonstructural proteins (nsPs): the methyltransferase, Zn-binding domain, macro, helicase, and RNA-dependent RNA polymerase, reliably predicted. Their substrate-binding sites are similar to those observed experimentally for other related viral proteins. Precisely knowing enzyme boundaries and structures is highly valuable to recombinantly produce stable and active proteins and perform structural, functional and inhibition studies.
Published: 2022
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9. A simple and versatile microfluidic device for efficient biomacromolecule crystallization and structural analysis by serial crystallography

Author: Raphaël de Wijn, Oliver Hennig, Jennifer Roche, Sylvain Engilberge, Kevin Rollet, Pablo Fernandez-Millan, Karl Brillet, Heike Betat, Mario Mörl, Alain Roussel, Eric Girard, Christoph Mueller-Dieckmann, Gavin C. Fox, Vincent Olieric, José A. Gavira, Bernard Lorber, and Claude Sauter
Subjects: macromolecule, crystallization, counter-diffusion, microfluidics, seeding, ligand soaking, trace fluorescent labeling, serial crystallography, room temperature, protein structure, ChipX3, Crystallography, QD901-999
Abstract: Determining optimal conditions for the production of well diffracting crystals is a key step in every biocrystallography project. Here, a microfluidic device is described that enables the production of crystals by counter-diffusion and their direct on-chip analysis by serial crystallography at room temperature. Nine `non-model' and diverse biomacromolecules, including seven soluble proteins, a membrane protein and an RNA duplex, were crystallized and treated on-chip with a variety of standard techniques including micro-seeding, crystal soaking with ligands and crystal detection by fluorescence. Furthermore, the crystal structures of four proteins and an RNA were determined based on serial data collected on four synchrotron beamlines, demonstrating the general applicability of this multipurpose chip concept.
Published: 2019
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10. Anchoring the T6SS to the cell wall: Crystal structure of the peptidoglycan binding domain of the TagL accessory protein.

Author: Van Son Nguyen, Silvia Spinelli, Éric Cascales, Alain Roussel, Christian Cambillau, and Philippe Leone
Subjects: Medicine, Science
Abstract: The type VI secretion system (T6SS) is a widespread mechanism of protein delivery into target cells, present in more than a quarter of all sequenced Gram-negative bacteria. The T6SS constitutes an important virulence factor, as it is responsible for targeting effectors in both prokaryotic and eukaryotic cells. The T6SS comprises a tail structure tethered to the cell envelope via a trans-envelope complex. In most T6SS, the membrane complex is anchored to the cell wall by the TagL accessory protein. In this study, we report the first crystal structure of a peptidoglycan-binding domain of TagL. The fold is conserved with members of the OmpA/Pal/MotB family, and more importantly, the peptidoglycan binding site is conserved. This structure further exemplifies how proteins involved in anchoring to the cell wall for different cellular functions rely on an interaction network with peptidoglycan strictly conserved.
Published: 2021
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11. Type IX secretion system PorM and gliding machinery GldM form arches spanning the periplasmic space

Author: Philippe Leone, Jennifer Roche, Maxence S. Vincent, Quang Hieu Tran, Aline Desmyter, Eric Cascales, Christine Kellenberger, Christian Cambillau, and Alain Roussel
Subjects: Science
Abstract: No structural data for the bacterial type IX secretion system (T9SS) are available so far. Here, the authors present the crystal structures of the periplasmic domains from two major T9SS components PorM and GldM, which span most of the periplasmic space, and propose a putative model of the T9SS core membrane complex.
Published: 2018
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12. Blocking Antibodies Targeting the CD39/CD73 Immunosuppressive Pathway Unleash Immune Responses in Combination Cancer Therapies

Author: Ivan Perrot, Henri-Alexandre Michaud, Marc Giraudon-Paoli, Séverine Augier, Aurélie Docquier, Laurent Gros, Rachel Courtois, Cécile Déjou, Diana Jecko, Ondine Becquart, Hélène Rispaud-Blanc, Laurent Gauthier, Benjamin Rossi, Stéphanie Chanteux, Nicolas Gourdin, Beatrice Amigues, Alain Roussel, Armand Bensussan, Jean-François Eliaou, Jérémy Bastid, François Romagné, Yannis Morel, Emilie Narni-Mancinelli, Eric Vivier, Carine Paturel, and Nathalie Bonnefoy
Subjects: Biology (General), QH301-705.5
Abstract: Summary: Immune checkpoint inhibitors have revolutionized cancer treatment. However, many cancers are resistant to ICIs, and the targeting of additional inhibitory signals is crucial for limiting tumor evasion. The production of adenosine via the sequential activity of CD39 and CD73 ectoenzymes participates to the generation of an immunosuppressive tumor microenvironment. In order to disrupt the adenosine pathway, we generated two antibodies, IPH5201 and IPH5301, targeting human membrane-associated and soluble forms of CD39 and CD73, respectively, and efficiently blocking the hydrolysis of immunogenic ATP into immunosuppressive adenosine. These antibodies promoted antitumor immunity by stimulating dendritic cells and macrophages and by restoring the activation of T cells isolated from cancer patients. In a human CD39 knockin mouse preclinical model, IPH5201 increased the anti-tumor activity of the ATP-inducing chemotherapeutic drug oxaliplatin. These results support the use of anti-CD39 and anti-CD73 monoclonal antibodies and their combination with immune checkpoint inhibitors and chemotherapies in cancer. : The production of adenosine via CD39 and CD73 ectoenzymes participates in an immunosuppressive tumor microenvironment. Perrot et al. generated two antibodies, IPH5201 and IPH5301, targeting human CD39 and CD73, respectively. In vitro and in vivo data support the use of anti-CD39 and anti-CD73 mAbs in combination cancer therapies. Keywords: CD39, CD73, cancer immunotherapies, therapeutic antibodies, adenosine pathway, tumor micro-environment, immunosuppression
Published: 2019
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13. Author Correction: Crystal structure of Type IX secretion system PorE C-terminal domain from Porphyromonas gingivalis in complex with a peptidoglycan fragment

Author: Nhung Thi Trang Trinh, Hieu Quang Tran, Quyen Van Dong, Christian Cambillau, Alain Roussel, and Philippe Leone
Subjects: Medicine, Science
Abstract: An amendment to this paper has been published and can be accessed via a link at the top of the paper.
Published: 2021
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14. Unraveling the Self-Assembly of the Pseudomonas aeruginosa XcpQ Secretin Periplasmic Domain Provides New Molecular Insights into Type II Secretion System Secreton Architecture and Dynamics

Author: Badreddine Douzi, Nhung T. T. Trinh, Sandra Michel-Souzy, Aline Desmyter, Geneviève Ball, Pascale Barbier, Artemis Kosta, Eric Durand, Katrina T. Forest, Christian Cambillau, Alain Roussel, and Romé Voulhoux
Subjects: Pseudomonas aeruginosa, secretin, dynamics, protein structure-function, stoichiometry, type II secretion system, Microbiology, QR1-502
Abstract: ABSTRACT The type II secretion system (T2SS) releases large folded exoproteins across the envelope of many Gram-negative pathogens. This secretion process therefore requires specific gating, interacting, and dynamics properties mainly operated by a bipartite outer membrane channel called secretin. We have a good understanding of the structure-function relationship of the pore-forming C-terminal domain of secretins. In contrast, the high flexibility of their periplasmic N-terminal domain has been an obstacle in obtaining the detailed structural information required to uncover its molecular function. In Pseudomonas aeruginosa, the Xcp T2SS plays an important role in bacterial virulence by its capacity to deliver a large panel of toxins and degradative enzymes into the surrounding environment. Here, we revealed that the N-terminal domain of XcpQ secretin spontaneously self-assembled into a hexamer of dimers independently of its C-terminal domain. Furthermore, and by using multidisciplinary approaches, we elucidate the structural organization of the XcpQ N domain and demonstrate that secretin flexibility at interdimer interfaces is mandatory for its function. IMPORTANCE Bacterial secretins are large homooligomeric proteins constituting the outer membrane pore-forming element of several envelope-embedded nanomachines essential in bacterial survival and pathogenicity. They comprise a well-defined membrane-embedded C-terminal domain and a modular periplasmic N-terminal domain involved in substrate recruitment and connection with inner membrane components. We are studying the XcpQ secretin of the T2SS present in the pathogenic bacterium Pseudomonas aeruginosa. Our data highlight the ability of the XcpQ N-terminal domain to spontaneously oligomerize into a hexamer of dimers. Further in vivo experiments revealed that this domain adopts different conformations essential for the T2SS secretion process. These findings provide new insights into the functional understanding of bacterial T2SS secretins.
Published: 2017
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15. Crystal structure and self-interaction of the type VI secretion tail-tube protein from enteroaggregative Escherichia coli.

Author: Badreddine Douzi, Silvia Spinelli, Stéphanie Blangy, Alain Roussel, Eric Durand, Yannick R Brunet, Eric Cascales, and Christian Cambillau
Subjects: Medicine, Science
Abstract: The type VI secretion system (T6SS) is a widespread machine used by bacteria to control their environment and kill or disable bacterial species or eukaryotes through toxin injection. The T6SS comprises a central tube formed of stacked hexamers of hemolysin co-regulated proteins (Hcp) and terminated by a trimeric valine-glycine repeat protein G (VgrG) component, the cell puncturing device. A contractile tail sheath, formed by the TssB and TssC proteins, surrounds this tube. This syringe-like machine has been compared to an inverted phage, as both Hcp and VgrG share structural homology with tail components of Caudovirales. Here we solved the crystal structure of a tryptophan-substituted double mutant of Hcp1 from enteroaggregative Escherichia coli and compared it to the structures of other Hcps. Interestingly, we observed that the purified Hcp native protein is unable to form tubes in vitro. To better understand the rationale for observation, we measured the affinity of Hcp1 hexamers with themselves by surface plasmon resonance. The intra-hexamer interaction is weak, with a KD value of 7.2 µM. However, by engineering double cysteine mutants at defined positions, tubes of Hcp1 gathering up to 15 stacked hexamers formed in oxidative conditions. These results, together with those available in the literature regarding TssB and TssC, suggest that assembly of the T6SS tube differs significantly from that of Sipho- or Myoviridae.
Published: 2014
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16. Molecular engineering of fungal GH5 and GH26 beta-(1,4)-mannanases toward improvement of enzyme activity.

Author: Marie Couturier, Julia Féliu, Sophie Bozonnet, Alain Roussel, and Jean-Guy Berrin
Subjects: Medicine, Science
Abstract: Microbial mannanases are biotechnologically important enzymes since they target the hydrolysis of hemicellulosic polysaccharides of softwood biomass into simple molecules like manno-oligosaccharides and mannose. In this study, we have implemented a strategy of molecular engineering in the yeast Yarrowia lipolytica to improve the specific activity of two fungal endo-mannanases, PaMan5A and PaMan26A, which belong to the glycoside hydrolase (GH) families GH5 and GH26, respectively. Following random mutagenesis and two steps of high-throughput enzymatic screening, we identified several PaMan5A and PaMan26A mutants that displayed improved kinetic constants for the hydrolysis of galactomannan. Examination of the three-dimensional structures of PaMan5A and PaMan26A revealed which of the mutated residues are potentially important for enzyme function. Among them, the PaMan5A-G311S single mutant, which displayed an impressive 8.2-fold increase in kcat /KM due to a significant decrease of KM, is located within the core of the enzyme. The PaMan5A-K139R/Y223H double mutant revealed modification of hydrolysis products probably in relation to an amino-acid substitution located nearby one of the positive subsites. The PaMan26A-P140L/D416G double mutant yielded a 30% increase in kcat /KM compared to the parental enzyme. It displayed a mutation in the linker region (P140L) that may confer more flexibility to the linker and another mutation (D416G) located at the entrance of the catalytic cleft that may promote the entrance of the substrate into the active site. Taken together, these results show that the directed evolution strategy implemented in this study was very pertinent since a straightforward round of random mutagenesis yielded significantly improved variants, in terms of catalytic efiiciency (kcat/KM).
Published: 2013
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17. Functional analysis of PGRP-LA in Drosophila immunity.

Author: Mathilde Gendrin, Anna Zaidman-Rémy, Nichole A Broderick, Juan Paredes, Mickaël Poidevin, Alain Roussel, and Bruno Lemaitre
Subjects: Medicine, Science
Abstract: PeptidoGlycan Recognition Proteins (PGRPs) are key regulators of the insect innate antibacterial response. Even if they have been intensively studied, some of them have yet unknown functions. Here, we present a functional analysis of PGRP-LA, an as yet uncharacterized Drosophila PGRP. The PGRP-LA gene is located in cluster with PGRP-LC and PGRP-LF, which encode a receptor and a negative regulator of the Imd pathway, respectively. Structure predictions indicate that PGRP-LA would not bind to peptidoglycan, pointing to a regulatory role of this PGRP. PGRP-LA expression was enriched in barrier epithelia, but low in the fat body. Use of a newly generated PGRP-LA deficient mutant indicates that PGRP-LA is not required for the production of antimicrobial peptides by the fat body in response to a systemic infection. Focusing on the respiratory tract, where PGRP-LA is strongly expressed, we conducted a genome-wide microarray analysis of the tracheal immune response of wild-type, Relish, and PGRP-LA mutant larvae. Comparing our data to previous microarray studies, we report that a majority of genes regulated in the trachea upon infection differ from those induced in the gut or the fat body. Importantly, antimicrobial peptide gene expression was reduced in the tracheae of larvae and in the adult gut of PGRP-LA-deficient Drosophila upon oral bacterial infection. Together, our results suggest that PGRP-LA positively regulates the Imd pathway in barrier epithelia.
Published: 2013
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18. Crystal structure of Diedel, a marker of the immune response of Drosophila melanogaster.

Author: Franck Coste, Cordula Kemp, Vanessa Bobezeau, Charles Hetru, Christine Kellenberger, Jean-Luc Imler, and Alain Roussel
Subjects: Medicine, Science
Abstract: BACKGROUND:The Drosophila melanogaster gene CG11501 is up regulated after a septic injury and was proposed to act as a negative regulator of the JAK/STAT signaling pathway. Diedel, the CG11501 gene product, is a small protein of 115 residues with 10 cysteines. METHODOLOGY/PRINCIPAL FINDINGS:We have produced Diedel in Drosophila S2 cells as an extra cellular protein thanks to its own signal peptide and solved its crystal structure at 1.15 Å resolution by SIRAS using an iodo derivative. Diedel is composed of two sub domains SD1 and SD2. SD1 is made of an antiparallel β-sheet covered by an α-helix and displays a ferredoxin-like fold. SD2 reveals a new protein fold made of loops connected by four disulfide bridges. Further structural analysis identified conserved hydrophobic residues on the surface of Diedel that may constitute a potential binding site. The existence of two conformations, cis and trans, for the proline 52 may be of interest as prolyl peptidyl isomerisation has been shown to play a role in several physiological mechanisms. The genome of D. melanogaster contains two other genes coding for proteins homologous to Diedel, namely CG43228 and CG34329. Strikingly, apart from Drosophila and the pea aphid Acyrthosiphon pisum, Diedel-related sequences were exclusively identified in a few insect DNA viruses of the Baculoviridae and Ascoviridae families. CONCLUSION/SIGNIFICANCE:Diedel, a marker of the Drosophila antimicrobial/antiviral response, is a member of a small family of proteins present in drosophilids, aphids and DNA viruses infecting lepidopterans. Diedel is an extracellular protein composed of two sub-domains. Two special structural features (hydrophobic surface patch and cis/trans conformation for proline 52) may indicate a putative interaction site, and support an extra cellular signaling function for Diedel, which is in accordance with its proposed role as negative regulator of the JAK/STAT signaling pathway.
Published: 2012
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19. Monalysin, a novel ß-pore-forming toxin from the Drosophila pathogen Pseudomonas entomophila, contributes to host intestinal damage and lethality.

Author: Onya Opota, Isabelle Vallet-Gély, Renaud Vincentelli, Christine Kellenberger, Ioan Iacovache, Manuel Rodrigo Gonzalez, Alain Roussel, Françoise-Gisou van der Goot, and Bruno Lemaitre
Subjects: Immunologic diseases. Allergy, RC581-607, Biology (General), QH301-705.5
Abstract: Pseudomonas entomophila is an entomopathogenic bacterium that infects and kills Drosophila. P. entomophila pathogenicity is linked to its ability to cause irreversible damages to the Drosophila gut, preventing epithelium renewal and repair. Here we report the identification of a novel pore-forming toxin (PFT), Monalysin, which contributes to the virulence of P. entomophila against Drosophila. Our data show that Monalysin requires N-terminal cleavage to become fully active, forms oligomers in vitro, and induces pore-formation in artificial lipid membranes. The prediction of the secondary structure of the membrane-spanning domain indicates that Monalysin is a PFT of the ß-type. The expression of Monalysin is regulated by both the GacS/GacA two-component system and the Pvf regulator, two signaling systems that control P. entomophila pathogenicity. In addition, AprA, a metallo-protease secreted by P. entomophila, can induce the rapid cleavage of pro-Monalysin into its active form. Reduced cell death is observed upon infection with a mutant deficient in Monalysin production showing that Monalysin plays a role in P. entomophila ability to induce intestinal cell damages, which is consistent with its activity as a PFT. Our study together with the well-established action of Bacillus thuringiensis Cry toxins suggests that production of PFTs is a common strategy of entomopathogens to disrupt insect gut homeostasis.
Published: 2011
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20. Sensing of COVID-19 spike protein in nasopharyngeal samples using a portable surface plasmon resonance diagnostic system

Author: Hiba Saada, Quentin Pagneux, James Wei, Ludovic Live, Alain Roussel, Alexis Dogliani, Lycia Die Morini, Ilka Engelmann, Enagnon Kazali Alidjinou, Anne Sophie Rolland, Emmanuel Faure, Julien Poissy, Julien Labreuche, Gil Lee, Peng Li, Gerard Curran, Anass Jawhari, Jhonny A. Yunda, Sorin Melinte, Axel Legay, Jean-Luc Gala, David Devos, Rabah Boukherroub, Sabine Szunerits, Institut d’Électronique, de Microélectronique et de Nanotechnologie - UMR 8520 (IEMN), Centrale Lille-Université de Lille-Centre National de la Recherche Scientifique (CNRS)-Université Polytechnique Hauts-de-France (UPHF)-JUNIA (JUNIA), Université catholique de Lille (UCL)-Université catholique de Lille (UCL), Affinité Instruments, 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), Pathogenèse virale du diabète de type 1 - ULR 3610 (Laboratoire de Virologie), Université de Lille-Centre Hospitalier Régional Universitaire [Lille] (CHRU Lille), Lille Neurosciences & Cognition - U 1172 (LilNCog), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Lille-Centre Hospitalier Régional Universitaire [Lille] (CHRU Lille), 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), Evaluation des technologies de santé et des pratiques médicales - ULR 2694 (METRICS), Magnostics, Coherent Optical Microscopy and X-rays (COMiX), Institut FRESNEL (FRESNEL), Aix Marseille Université (AMU)-École Centrale de Marseille (ECM)-Centre National de la Recherche Scientifique (CNRS)-Aix Marseille Université (AMU)-École Centrale de Marseille (ECM)-Centre National de la Recherche Scientifique (CNRS), Biosensing Diagnostics, Université Catholique de Louvain = Catholic University of Louvain (UCL), NanoBioInterfaces - IEMN (NBI - IEMN), Université catholique de Lille (UCL)-Université catholique de Lille (UCL)-Centrale Lille-Université de Lille-Centre National de la Recherche Scientifique (CNRS)-Université Polytechnique Hauts-de-France (UPHF)-JUNIA (JUNIA), The project is funded by the Horizon 2020 framework programme of the European Union under grant agreement No. 101016038 (CorDial-S). This work was supported by theBelgian F.R.S. – FNRS under Grant No. R.8008.21. Financial support by ANR via CorDial-Flu is in addition acknowledged., European Project: 101016038,H2020,H2020-SC1-PHE-CORONAVIRUS-2020-2-CNECT, CorDial-S(2020), IEMN, Collection, Portable and fast surface plasmon resonance point-of-care test for COVID-19 - CorDial-S - - H20202020-12-01 - 2021-11-30 - 101016038 - VALID, UCL - SST/ICTM/ELEN - Pôle en ingénierie électrique, and UCL - SSS/IREC/CTMA - Centre de technologies moléculaires appliquées (plate-forme technologique)
Subjects: Machine Learning, VHH-72-Fc, [SPI]Engineering Sciences [physics], Diagnostic system, Plasmon resonance, COVID-19 spike protein, [SPI] Engineering Sciences [physics], SARS-CoV-2, COVID-19, Surface Plasmon Resonance
Abstract: International audience; Rapid, yet sensitive and accurate testing concepts are critical in the control of spreading diseases. With the COVID-19 pandemic still ongoing, the need for efficient, fast and accurate testing of the infection state of children and the elderly traveling as well as people with symptoms has not declined. Most current methods, which are highly sensitive, are rather slow and cannot be applied at the point of care. While rapid antigenic tests ascertain only high viral burden, here, we demonstrate an alternative, rapid point-of-care diagnostics with the ability to sense low viral loads. The goal of a portable and fast quantitative diagnostic device has been achieved via the use of VHH-72-Fc, a nanobody featuring high binding strength to the spike 1 glycoprotein of the SARS-CoV-2 viral envelope, a surface plasmon resonance sensing approach, and machine learning for predicting the cut-off value between positive and negative nasopharyngeal swab samples. The concept was validated on 119 nasopharyngeal samples, 50 positive and 69 negative, as identified by reverse transcription quantitative polymerase chain reaction (RT-qPCR) tests, showing a 88% positive percentage agreement and a 92% negative percentage agreement, as compared to RT-qPCR. Simple artificial neural network data processing revealed the influence in the sampling time to achieve unique performance in terms of speed, specificity and sensitivity. These sensing features combined with no sample preparation and portability of the diagnostic device suggest that this approach is well-adapted to be operated in hospital or laboratory located diagnostic centres.
Published: 2022

21. Structural and functional analyses of the Porphyromonas gingivalis type IX secretion system PorN protein

Author: Olivier, Fuchsbauer, Ignacio, Lunar Silva, Eric, Cascales, Alain, Roussel, and Philippe, Leone
Subjects: Structure-Activity Relationship, Bacterial Proteins, Virulence Factors, Periplasm, Humans, Bacterial Secretion Systems, Porphyromonas gingivalis
Abstract: Porphyromonas gingivalis, the major human pathogen bacterium associated with periodontal diseases, secretes virulence factors through the Bacteroidetes-specific type IX secretion system (T9SS). Effector proteins of the T9SS are recognized by the complex via their conserved C-terminal domains (CTDs). Among the 18 proteins essential for T9SS function in P. gingivalis, PorN is a periplasmic protein that forms large ring-shaped structures in association with the PorK outer membrane lipoprotein. PorN also mediates contacts with the PorM subunit of the PorLM energetic module, and with the effector's CTD. However, no information is available on the PorN structure and on the implication of PorN domains for T9SS assembly and effector recognition. Here we present the crystal structure of PorN at 2.0-Å resolution, which represents a novel fold with no significant similarity to any known structure. In agreement with in silico analyses, we also found that the N- and C-terminal regions of PorN are intrinsically disordered. Our functional studies showed that the N-terminal disordered region is involved in PorN dimerization while the C-terminal disordered region is involved in the interaction with PorK. Finally, we determined that the folded PorN central domain is involved in the interaction with PorM, as well as with the effector's CTD. Altogether, these results lay the foundations for a more comprehensive model of T9SS architecture and effector transport.
Published: 2021

22. SARS-CoV-2 detection using a nanobody-functionalized voltammetric device

Author: Quentin Pagneux, Alain Roussel, Hiba Saada, Christian Cambillau, Béatrice Amigues, Vincent Delauzun, Ilka Engelmann, Enagnon Kazali Alidjinou, Judith Ogiez, Anne Sophie Rolland, Emmanuel Faure, Julien Poissy, Alain Duhamel, Rabah Boukherroub, David Devos, Sabine Szunerits, Institut d’Électronique, de Microélectronique et de Nanotechnologie - UMR 8520 (IEMN), Centrale Lille-Université de Lille-Centre National de la Recherche Scientifique (CNRS)-Université Polytechnique Hauts-de-France (UPHF)-JUNIA (JUNIA), Université catholique de Lille (UCL)-Université catholique de Lille (UCL), Architecture et fonction des macromolécules biologiques (AFMB), Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Institut des Sciences Chimiques de Rennes (ISCR), Université de Rennes (UR)-Institut National des Sciences Appliquées - Rennes (INSA Rennes), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Ecole Nationale Supérieure de Chimie de Rennes (ENSCR)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Pathogenèse virale du diabète de type 1 - ULR 3610 (Laboratoire de Virologie), Université de Lille-Centre Hospitalier Régional Universitaire [Lille] (CHRU Lille), Lille Neurosciences & Cognition - U 1172 (LilNCog), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Lille-Centre Hospitalier Régional Universitaire [Lille] (CHRU Lille), 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), Evaluation des technologies de santé et des pratiques médicales - ULR 2694 (METRICS), NanoBioInterfaces - IEMN (NBI - IEMN), Université catholique de Lille (UCL)-Université catholique de Lille (UCL)-Centrale Lille-Université de Lille-Centre National de la Recherche Scientifique (CNRS)-Université Polytechnique Hauts-de-France (UPHF)-JUNIA (JUNIA), Financial support from the Centre National de la Recherche Scientifique (CNRS), the University of Lille, I-SITE via the COVID task force and the Hauts-de-France region via ANR Resilience (CorDial-FLU) is acknowledged. The unrestricted help of Jean-Pierre Voisin from Htds and his team is acknowledged. Support of the SATT Nord as well as Eurasanté in this project is acknowledged. This work was supported by the French Infrastructure for Integrated Structural Biology (FRISBI) ANR-10-INBS-0005. We want to thank the technicians of virology laboratory of CHU Lille, and all nurses, medical doctors involved in the project. The interest from Palmsense in the work is also acknowledged., ANR-10-INBS-0005,FRISBI,Infrastructure Française pour la Biologie Structurale Intégrée(2010), ANR-21-HDF1-0003,CorDial-FLU,Dispositive portable de diagnostic pour différencier le virus la grippe de celui de Covid-19(2021), IEMN, Collection, Infrastructure Française pour la Biologie Structurale Intégrée - - FRISBI2010 - ANR-10-INBS-0005 - INBS - VALID, and Dispositive portable de diagnostic pour différencier le virus la grippe de celui de Covid-19 - - CorDial-FLU2021 - ANR-21-HDF1-0003 - PRHDF - VALID
Subjects: [SDV] Life Sciences [q-bio], [SPI]Engineering Sciences [physics], [SPI] Engineering Sciences [physics], [SDV]Life Sciences [q-bio], Diagnostic markers, Nanobiotechnology
Abstract: Background An ongoing need during the COVID-19 pandemic has been the requirement for accurate and efficient point-of-care testing platforms to distinguish infected from non-infected people, and to differentiate SARS-CoV-2 infections from other viruses. Electrochemical platforms can detect the virus via its envelope spike protein by recording changes in voltammetric signals between samples. However, this remains challenging due to the limited sensitivity of these sensing platforms. Methods Here, we report on a nanobody-functionalized electrochemical platform for the rapid detection of whole SARS-CoV-2 viral particles in complex media such as saliva and nasopharyngeal swab samples. The sensor relies on the functionalization of gold electrode surface with highly-oriented Llama nanobodies specific to the spike protein receptor binding domain (RBD). The device provides results in 10 min of exposure to 200 µL of unprocessed samples with high specificity to SARS-CoV-2 viral particles in human saliva and nasopharyngeal swab samples. Results The developed sensor could discriminate between different human coronavirus strains and other respiratory viruses, with 90% positive and 90% negative percentage agreement on 80 clinical samples, as compared to RT-qPCR. Conclusions We believe this diagnostic concept, also validated for RBD mutants and successfully tested on Delta variant samples, to be a powerful tool to detect patients’ infection status, easily extendable to other viruses and capable of overcoming sensing-related mutation effects.
Published: 2021

23. A new non-classical fold of varroa odorant-binding proteins reveals a wide open internal cavity

Author: Paolo Pelosi, Anais Gaubert, Philippe Leone, Jiao Zhu, Wolfgang Knoll, Béatrice Amigues, Giovanni Renzone, Simona Arena, Harald Paulsen, Andrea Scaloni, Christian Cambillau, Alain Roussel, Isabella Maria Fischer, Architecture et fonction des macromolécules biologiques (AFMB), and Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)
Subjects: Models, Molecular, Protein Folding, Protein Conformation, Odorant binding, Disulphide bridges, Insect, Crystallography, X-Ray, Ligands, Receptors, Odorant, Biochemistry, Ligand-binding assays, Conserved Sequence, Phylogeny, media_common, 0303 health sciences, Multidisciplinary, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], biology, varroa, odorant-binding, proteins, 030302 biochemistry & molecular biology, Varroa destructor, Medicine, Insect Proteins, Varroa, Structural biology, Protein Binding, Evolution, Science, Varroidae, media_common.quotation_subject, Article, 03 medical and health sciences, [CHIM.CRIS]Chemical Sciences/Cristallography, Mite, Animals, Amino Acid Sequence, Cysteine, Gene, Fluorescent Dyes, 030304 developmental biology, Binding Sites, Sequence Homology, Amino Acid, biology.organism_classification, Three-dimensional structure, Kinetics, Odorant-binding protein, biology.protein, Sequence Alignment
Abstract: Odorant-binding proteins (OBPs), as they occur in insects, form a distinct class of proteins that apparently has no closely related representatives in other animals. However, ticks, mites, spiders and millipedes contain genes encoding proteins with sequence similarity to insect OBPs. In this work, we have explored the structure and function of such non-insect OBPs in the mite Varroa destructor, a major pest of honey bee. Varroa OBPs present six cysteines paired into three disulphide bridges, but with positions in the sequence and connections different from those of their insect counterparts. VdesOBP1 structure was determined in two closely related crystal forms and appears to be a monomer. Its structure assembles five α-helices linked by three disulphide bridges, one of them exhibiting a different connection as compared to their insect counterparts. Comparison with classical OBPs reveals that the second of the six α-helices is lacking in VdesOBP1. Ligand-binding experiments revealed molecules able to bind only specific OBPs with a moderate affinity, suggesting that either optimal ligands have still to be identified, or post-translational modifications present in the native proteins may be essential for modulating binding activity, or else these OBPs might represent a failed attempt in evolution and are not used by the mites.
Published: 2021

24. Macromolecular interactions in vitro, comparing classical and novel approaches

Author: Jean-Baptiste Charbonnier, Christophe Quétard, Pierre Soule, Paloma F. Varela, Guillaume Bec, Adrián Velázquez-Campoy, Christine Ebel, Stephan Uebel, Patrick England, Magali Aumont-Nicaise, David Stroebel, Alain Roussel, Christophe Velours, Agence Nationale de la Recherche (France), 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), Microbiologie cellulaire et moléculaire et pathogénicité (MCMP), Université Bordeaux Segalen - Bordeaux 2-Centre National de la Recherche Scientifique (CNRS), Max Planck Institute of Biochemistry (MPIB), Max-Planck-Gesellschaft, Biophysique Moléculaire (plateforme) - Molecular Biophysics (platform), Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), University of Zaragoza - Universidad de Zaragoza [Zaragoza], Instituto de Investigación Sanitaria de Aragón [Zaragoza] (IIS Aragón), Centro de Investigación Biomédica en Red en el Área temática de Enfermedades Hepáticas y Digestivas (CIBERehd), Liver Unit, Clínica Universitaria, CIBER-EHD, Fundación Agencia Aragonesa para la Investigación y el Desarrollo (ARAID), Institut de biologie de l'ENS Paris (IBENS), Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Département de Biologie - ENS Paris, École normale supérieure - Paris (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)-École normale supérieure - Paris (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), Architecture et Réactivité de l'ARN (ARN), Institut de biologie moléculaire et cellulaire (IBMC), Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), NanoTemper Technologies GmbH [München], ForteBio-Sartorius [Dourdan], Institut de biologie structurale (IBS - UMR 5075), Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA), Architecture et fonction des macromolécules biologiques (AFMB), Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), JBC is supported by ARC program (SLS220120605310), French Infrastructure for Integrated Structural Biology (FRISBI) ANR-10-INSB-05, ANR-18-CE44-0008, ANR-20-CE11-0026, INCA 2016-1-PL BIO-11 and INCA SLX4 INCA 2016-159., ANR-10-INBS-0005,FRISBI,Infrastructure Française pour la Biologie Structurale Intégrée(2010), ANR-18-CE44-0008,DNAPAR18,Etude de l'ADP-ribosylation d'ADN et de son rôle dans la réponse aux dommages à l'ADN(2018), ANR-20-CE11-0026,BreakDance,Caractérisation de la chorégraphie orchestrée par l'hétérodimère Ku sur les cassures double-brin de l'ADN(2020), Microbiologie Fondamentale et Pathogénicité (MFP), Max-Planck-Institut für Biochemie = Max Planck Institute of Biochemistry (MPIB), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Département de Biologie - ENS Paris, École normale supérieure - Paris (ENS-PSL), 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)-École normale supérieure - Paris (ENS-PSL), 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)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), and Institut de biologie de l'ENS Paris (UMR 8197/1024) (IBENS)
Subjects: 0301 basic medicine, 030103 biophysics, Macromolecular Substances, Computer science, Biophysics, Computational biology, Calorimetry, Ligands, Artificial binders, Protein–protein interaction, Analytical Ultracentrifugation, 03 medical and health sciences, Humans, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Molecular scale biophysics, Macromolecular interactions, Molecular interactions, Microscale thermophoresis, Proteins, Isothermal titration calorimetry, DNA, General Medicine, 3. Good health, 030104 developmental biology, Artifcial binders, Target protein, Double-stranded DNA breaks repair factors, Macromolecule
Abstract: 18 pags., 4 figs. -- This article has been updated, a correction to this article was published on 27 April 2021, Biophysical quantification of protein interactions is central to unveil the molecular mechanisms of cellular processes. Researchers can choose from a wide panel of biophysical methods that quantify molecular interactions in different ways, including both classical and more novel techniques. We report the outcome of an ARBRE-MOBIEU training school held in June 2019 in Gif-sur-Yvette, France (https://mosbio.sciencesconf.org/). Twenty European students benefited from a week’s training with theoretical and practical sessions in six complementary approaches: (1) analytical ultracentrifugation with or without a fluorescence detector system (AUC-FDS), (2) isothermal titration calorimetry (ITC), (3) size exclusion chromatography coupled to multi-angle light scattering (SEC-MALS), (4) bio-layer interferometry (BLI), (5) microscale thermophoresis (MST) and, (6) switchSENSE. They implemented all these methods on two examples of macromolecular interactions with nanomolar affinity: first, a protein–protein interaction between an artificial alphaRep binder, and its target protein, also an alphaRep; second, a protein-DNA interaction between a DNA repair complex, Ku70/Ku80 (hereafter called Ku), and its cognate DNA ligand. We report the approaches used to analyze the two systems under study and thereby showcase application of each of the six techniques. The workshop provided students with improved understanding of the advantages and limitations of different methods, enabling future choices concerning approaches that are most relevant or informative for specific kinds of sample and interaction., JBC is supported by ARC program (SLS220120605310), French Infrastructure for Integrated Structural Biology (FRISBI) ANR10-INSB-05, ANR-18-CE44-0008, ANR-20-CE11-0026, INCA 2016- 1-PL BIO-11 and INCA SLX4 INCA 2016-159.
Published: 2021

25. Crystal structures of two camelid nanobodies raised against GldL, a component of the type IX secretion system from Flavobacterium johnsoniae

Author: Philippe Leone, Anaïs Gaubert, Alain Roussel, Pauline Melani, Christian Cambillau, and Thi Trang Nhung Trinh
Subjects: Models, Molecular, Camelus, Gliding motility, Biophysics, Context (language use), Crystal structure, Crystallography, X-Ray, Biochemistry, Flavobacterium, Research Communications, 03 medical and health sciences, Bacterial Proteins, Protein Domains, Structural Biology, Genetics, Animals, Scattering, Radiation, Molecular replacement, Secretion, Flavobacterium johnsoniae, Bacterial Secretion Systems, 030304 developmental biology, 0303 health sciences, 030306 microbiology, Chemistry, Single-Domain Antibodies, Condensed Matter Physics, Kinetics, Cytoplasm, Thermodynamics, Protein Multimerization, Function (biology)
Abstract: GldL is an inner-membrane protein that is essential for the function of the type IX secretion system (T9SS) in Flavobacterium johnsoniae. The complex that it forms with GldM is supposed to act as a new rotary motor involved in the gliding motility of the bacterium. In the context of structural studies of GldL to gain information on the assembly and function of the T9SS, two camelid nanobodies were selected, produced and purified. Their interaction with the cytoplasmic domain of GldL was characterized and their crystal structures were solved. These nanobodies will be used as crystallization chaperones to help in the crystallization of the cytoplasmic domain of GldL and could also help to solve the structure of the complex using molecular replacement.
Published: 2021

26. Preanalytical issues and cycle threshold values in SARS-CoV-2 real-time RT-PCR testing:should test results include these?

Author: Christian Cambillau, Mahdi Ouafi, David Devos, Enagnon Kazali Alidjinou, Sana Miloudi, Judith Ogiez, Alain Roussel, Ilka Engelmann, Famara Sane, Quentin Pagneux, Rabah Boukherroub, Ilyes Benhalima, Sabine Szunerits, Didier Hober, Centre d'Immunologie de Marseille - Luminy (CIML), Aix Marseille Université (AMU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Pathogenèse virale du diabète de type 1 - ULR 3610 (Laboratoire de Virologie), Université de Lille-Centre Hospitalier Régional Universitaire [Lille] (CHRU Lille), Institut d’Électronique, de Microélectronique et de Nanotechnologie - UMR 8520 (IEMN), Centrale Lille-Université de Lille-Centre National de la Recherche Scientifique (CNRS)-Université Polytechnique Hauts-de-France (UPHF)-JUNIA (JUNIA), Université catholique de Lille (UCL)-Université catholique de Lille (UCL), Architecture et fonction des macromolécules biologiques (AFMB), Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Lille Neurosciences & Cognition - U 1172 (LilNCog), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Lille-Centre Hospitalier Régional Universitaire [Lille] (CHRU Lille), NanoBioInterfaces - IEMN (NBI - IEMN), Université catholique de Lille (UCL)-Université catholique de Lille (UCL)-Centrale Lille-Université de Lille-Centre National de la Recherche Scientifique (CNRS)-Université Polytechnique Hauts-de-France (UPHF)-JUNIA (JUNIA), + H2020 Grant agreement ID: 101016038 CorDial-S PORTABLE AND FAST SURFACE PLASMON RESONANCE POINT-OF-CARE TEST FOR COVID-19, ANR-21-HDF1-0003,CorDial-FLU,Dispositive portable de diagnostic pour différencier le virus la grippe de celui de Covid-19(2021), European Project: 101016038,H2020,H2020-SC1-PHE-CORONAVIRUS-2020-2-CNECT, CorDial-S(2020), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Aix Marseille Université (AMU), Lille Neurosciences & Cognition - U 1172 (LilNCog (ex-JPARC)), Centrale Lille-Université de Lille-Centre National de la Recherche Scientifique (CNRS)-Université Polytechnique Hauts-de-France (UPHF)-JUNIA (JUNIA)-Centrale Lille-Université de Lille-Centre National de la Recherche Scientifique (CNRS)-Université Polytechnique Hauts-de-France (UPHF)-JUNIA (JUNIA), IEMN, Collection, Dispositive portable de diagnostic pour différencier le virus la grippe de celui de Covid-19 - - CorDial-FLU2021 - ANR-21-HDF1-0003 - PRHDF - VALID, Portable and fast surface plasmon resonance point-of-care test for COVID-19 - CorDial-S - - H20202020-12-01 - 2021-11-30 - 101016038 - VALID, Centre d'Immunologie de Marseille - Luminy [CIML], Pathogenèse virale du diabète de type 1- ULR 3610 [Laboratoire de Virologie], Institut d’Électronique, de Microélectronique et de Nanotechnologie (IEMN) - UMR 8520 [IEMN], Architecture et fonction des macromolécules biologiques [AFMB], Lille Neurosciences & Cognition - U 1172 [LilNCog (ex-JPARC)], NanoBioInterfaces - IEMN [NBI - IEMN], Pathogenèse virale du diabète de type 1- ULR 3610 (Laboratoire de Virologie), Institut d’Électronique, de Microélectronique et de Nanotechnologie (IEMN) - UMR 8520 (IEMN), Ecole Centrale de Lille-Université de Lille-Centre National de la Recherche Scientifique (CNRS)-Université Polytechnique Hauts-de-France (UPHF)-JUNIA (JUNIA), and Ecole Centrale de Lille-Université de Lille-Centre National de la Recherche Scientifique (CNRS)-Université Polytechnique Hauts-de-France (UPHF)-JUNIA (JUNIA)-Ecole Centrale de Lille-Université de Lille-Centre National de la Recherche Scientifique (CNRS)-Université Polytechnique Hauts-de-France (UPHF)-JUNIA (JUNIA)
Subjects: Virus inactivation, Coronavirus disease 2019 (COVID-19), General Chemical Engineering, Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), viruses, Extraction, Computational biology, Biology, Assays, 03 medical and health sciences, [SDV.MHEP.MI]Life Sciences [q-bio]/Human health and pathology/Infectious diseases, Genetics, QD1-999, 030304 developmental biology, Infectivity, [SDV.MP.VIR] Life Sciences [q-bio]/Microbiology and Parasitology/Virology, [SDV.MHEP.ME] Life Sciences [q-bio]/Human health and pathology/Emerging diseases, 0303 health sciences, Cycle threshold, [SDV.MHEP.ME]Life Sciences [q-bio]/Human health and pathology/Emerging diseases, 030306 microbiology, General Chemistry, Gold standard (test), 3. Good health, Chemistry, Real-time polymerase chain reaction, Perspective, [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology, [SDV.MHEP.MI] Life Sciences [q-bio]/Human health and pathology/Infectious diseases, Infectious diseases, Diagnostic imaging, RNA extraction
Abstract: International audience; Since the emergence of SARS-CoV-2 pandemic, clinical laboratories worldwide are overwhelmed with SARS-CoV-2 testing using the current gold standard: real-time reverse-transcription polymerase chain reaction (RT-PCR) assays. The large numbers of suspected cases led to shortages in numerous reagents such as specimen transport and RNA extraction buffers. We try to provide some answers on how strongly preanalytical issues affect RT-PCR results by reviewing the utility of different transport buffer media and virus inactivation procedures and comparing the literature data with our own recent findings. We show that various viral inactivation procedures and transport buffers are available and are less of a bottleneck for PCR-based methods. However, efficient alternative lysis buffers remain more difficult to find, and several fast RT-PCR assays are not compatible with guanidine-containing media, making this aspect more of a challenge in the current crisis. Furthermore, the availability of different SARS-CoV-2-specific RT-PCR kits with different sensitivities makes the definition of a general cutoff level for the cycle threshold (Ct) value challenging. Only a few studies have considered how Ct values relate to viral infectivity and how preanalytical issues might affect viral infectivity and RNA detection. We review the current data on the correlation between Ct values and viral infectivity. The presence of the SARS-CoV-2 viral genome in its own is not sufficient proof of infectivity and caution is needed in evaluation of the infectivity of samples. The correlation between Ct values and viral infectivity revealed an RT-PCR cutoff value of 34 cycles for SARS-CoV-2 infectivity using a laboratory-developed RT-PCR assay targeting the RdRp gene. While ideally each clinical laboratory should perform its own correlation, we believe this perspective article could be a reference point for others, in particular medical doctors and researchers interested in COVID-19 diagnostics, and a first step toward harmonization.
Published: 2021

27. The role of the surface ligand on the performance of electrochemical SARS-CoV-2 antigen biosensors

Author: David Devos, Christian Cambillau, Henri Happy, Quentin Pagneux, Vladyslav Mishyn, Enagnon Kazali Alidjinou, Ilka Engelmann, Rabah Boukherroub, Abir Swaidan, Sabine Szunerits, Alain Roussel, Institut d’Électronique, de Microélectronique et de Nanotechnologie - UMR 8520 (IEMN), Centrale Lille-Université de Lille-Centre National de la Recherche Scientifique (CNRS)-Université Polytechnique Hauts-de-France (UPHF)-JUNIA (JUNIA), NanoBioInterfaces - IEMN (NBI - IEMN), Centrale Lille-Université de Lille-Centre National de la Recherche Scientifique (CNRS)-Université Polytechnique Hauts-de-France (UPHF)-JUNIA (JUNIA)-Centrale Lille-Université de Lille-Centre National de la Recherche Scientifique (CNRS)-Université Polytechnique Hauts-de-France (UPHF)-JUNIA (JUNIA), Architecture et fonction des macromolécules biologiques (AFMB), Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Lille Neurosciences & Cognition - U 1172 (LilNCog (ex-JPARC)), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Lille-Centre Hospitalier Régional Universitaire [Lille] (CHRU Lille), Centre d'Immunologie de Marseille - Luminy (CIML), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Aix Marseille Université (AMU), Pathogenèse virale du diabète de type 1 - ULR 3610 (Laboratoire de Virologie), Université de Lille-Centre Hospitalier Régional Universitaire [Lille] (CHRU Lille), Carbon - IEMN (CARBON-IEMN), Centre National de la Recherche Scientifique (CNRS)Centre National de la Recherche Scientifique (CNRS), University of Lille, I-SITE, via the COVID task force, Hauts-de-France region via ANR Resilience (CorDial-FLU), French Infrastructure for Integrated Structural Biology (FRISBI) [ANR-10-INBS-0005], ANR-10-INBS-0005,FRISBI,Infrastructure Française pour la Biologie Structurale Intégrée(2010), ANR-21-HDF1-0003,CorDial-FLU,Dispositive portable de diagnostic pour différencier le virus la grippe de celui de Covid-19(2021), Université catholique de Lille (UCL)-Université catholique de Lille (UCL), Université catholique de Lille (UCL)-Université catholique de Lille (UCL)-Centrale Lille-Université de Lille-Centre National de la Recherche Scientifique (CNRS)-Université Polytechnique Hauts-de-France (UPHF)-JUNIA (JUNIA), Lille Neurosciences & Cognition - U 1172 (LilNCog), Aix Marseille Université (AMU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Carbon - IEMN (CARBON - IEMN), IEMN, Collection, Infrastructure Française pour la Biologie Structurale Intégrée - - FRISBI2010 - ANR-10-INBS-0005 - INBS - VALID, and Dispositive portable de diagnostic pour différencier le virus la grippe de celui de Covid-19 - - CorDial-FLU2021 - ANR-21-HDF1-0003 - PRHDF - VALID
Subjects: Coronavirus disease 2019 (COVID-19), Computer science, [SPI] Engineering Sciences [physics], Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), Point-of-care testing, Point-of-Care Systems, Medical laboratory, Economic shortage, 02 engineering and technology, Review, Biosensing Techniques, point-of-care (poc) technologies, Ligands, 01 natural sciences, Biochemistry, Analytical Chemistry, [SPI]Engineering Sciences [physics], COVID-19 Testing, Health care, Humans, field effect transistors, Antigens, Viral, Disease surveillance, business.industry, 010401 analytical chemistry, COVID-19, Electrochemical Techniques, 021001 nanoscience & nanotechnology, biosensors, 0104 chemical sciences, 3. Good health, sars-cov-2, Risk analysis (engineering), electrochemistry, surface ligands, Analytics, Point-of-Care Testing, 0210 nano-technology, business
Abstract: International audience; Point-of-care (POC) technologies and testing programs hold great potential to significantly improve diagnosis and disease surveillance. POC tests have the intrinsic advantage of being able to be performed near the patient or treatment facility, owing to their portable character. With rapid results often in minutes, these diagnostic platforms have a high positive impact on disease management. POC tests are, in addition, advantageous in situations of a shortage of skilled personnel and restricted availability of laboratory-based analytics. While POC testing programs are widely considered in addressing health care challenges in low-income health systems, the ongoing pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections could largely benefit from fast, efficient, accurate, and cost-effective point-of-care testing (POCT) devices for limiting COVID-19 spreading. The unrestrained availability of SARS-CoV-2 POC tests is indeed one of the adequate means of better managing the COVID-19 outbreak. A large number of novel and innovative solutions to address this medical need have emerged over the last months. Here, we critically elaborate the role of the surface ligands in the design of biosensors to cope with the current viral outbreak situation. Their notable effect on electrical and electrochemical sensors' design will be discussed in some given examples.
Published: 2021

28. Correction to: Macromolecular interactions in vitro, comparing classical and novel approaches

Author: Christine Ebel, Christophe Velours, Paloma F. Varela, Guillaume Bec, Magali Aumont-Nicaise, Patrick England, David Stroebel, Christophe Quétard, Adrián Velázquez-Campoy, Alain Roussel, Stephan Uebel, Pierre Soule, and Jean-Baptiste Charbonnier
Subjects: Chemistry, Biophysics, Membrane biology, General Medicine, Computational biology, In vitro
Abstract: Correction to: European Biophysics Journal https://doi.org/10.1007/s00249-021-01517-5
Published: 2020

29. Extracellular vesicles as a platform to study cell-surface membrane proteins

Author: Magali Grange, Vincent Delauzun, Laurent Gauthier, Alain Roussel, Philippe Leone, Anaïs Gaubert, Béatrice Amigues, Architecture et fonction des macromolécules biologiques (AFMB), Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Innate Pharma Research Labs [Marseille], Innate Pharma, ANR-10-INBS-0005,FRISBI,Infrastructure Française pour la Biologie Structurale Intégrée(2010), and ANR-14-EBOL-0001,STOP-EBOLA,Neutralization du virus Ebola par des nanobodies de camélidés(2014)
Subjects: Detergents, Protein-ligand interaction, GPI-Linked Proteins, Ligands, Extracellular vesicles, General Biochemistry, Genetics and Molecular Biology, Mass Spectrometry, law.invention, 03 medical and health sciences, Extracellular Vesicles, Protein structure, law, Antibody generation, [CHIM.CRIS]Chemical Sciences/Cristallography, Humans, Cloning, Molecular, Molecular Biology, 5'-Nucleotidase, 030304 developmental biology, 0303 health sciences, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Chemistry, 030302 biochemistry & molecular biology, Cryoelectron Microscopy, Membrane Proteins, Extracellular vesicle, Dynamic Light Scattering, Recombinant Proteins, Microscopy, Electron, Membrane, HEK293 Cells, Membrane protein, Solubilization, Biophysics, Recombinant DNA, Immunization, Cell surface membrane, Plasmids
Abstract: International audience; Keywords Extracellular vesicle production Membrane protein expression Membrane protein-ligand interaction Cell-surface receptor Electron microscopy Immunization Highlights Membrane protein interaction studies using extracellular vesicles Extracellular vesicles as a tool for structure determination of membrane proteins Extracellular vesicles use for immunization Abstract Producing intact recombinant membrane proteins for structural studies is an inherently challenging task due to their requirement for a cell-lipid environment. Most procedures developed involve isolating the protein by detergent solubilization and further reconstitutions into artificial membranes. These procedures are highly time consuming and suffer from further drawbacks, including low yields and high cost. We describe here an alternative method for rapidly obtaining recombinant cell-surface membrane proteins displayed on extracellular vesicles (EVs) derived from cells in culture. Interaction between these membrane proteins and ligands can be analyzed directly on EVs. Moreover, EVs can also be used for protein structure determination or immunization purposes.
Published: 2019

30. Cas9 Allosteric Inhibition by the Anti-CRISPR Protein AcrIIA6

Author: Christian Cambillau, Béatrice Amigues, Geneviève M. Rousseau, Sylvain Moineau, Claire Zimberger, Alain Roussel, Olivier Fuchsbauer, Antonio Chaves-Sanjuan, Yannick Doyon, Alexis Duringer, Adeline Goulet, Sébastien Lévesque, Minja Velimirovic, Paolo Swuec, Silvia Spinelli, Daniel Agudelo, Martino Bolognesi, Architecture et fonction des macromolécules biologiques (AFMB), Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Università degli Studi di Milano [Milano] (UNIMI), Centre de recherche du CHU de Québec-Université Laval (CRCHUQ), CHU de Québec–Université Laval, Université Laval [Québec] (ULaval)-Université Laval [Québec] (ULaval), Faculté de médecine dentaire [Université Laval, Québec], Université Laval [Québec] (ULaval), Dpt de Microbiologie-Infectiologie et d’Immunologie [Laval], Department of Biosciences [Milano], Félix d'Hérelle Reference Center for Bacterial Viruses, Groupe de Recherche en Écologie Buccale (GREB), Centre National de la Recherche Scientifique (CNRS), Aix Marseille Université (AMU), NSERC of Canada, Canada RGPIN-2014-05132/RGPIN-2014-05698Fonds de la Recherche en Sante du Quebec FRQS 254294United States Department of Health & Human ServicesNational Institutes of Health (NIH) - USA R01-GM129325/P41-GM103311, ANR-18-CE11-0016,PHARE,Vengeance de phages: analyses structurales et fonctionnelles de protéines anti CRISPR-Cas9(2018), ANR-10-INBS-0005,FRISBI,Infrastructure Française pour la Biologie Structurale Intégrée(2010), Hôpital de l'Enfant-Jésus [CHU Québec] (HEJ), Faculté de médecine de l'Université Laval [Québec] (ULaval), ANR-10-INSB-05-01 INSB ANR-10-INSB-0501, Fonds de la recherche du Québec-Santé (FRQS 254294)NIH R01-GM129325 and P41-GM103311, Discovery program, RGPIN-2014-05132, RGPIN-2014-05698, Università degli Studi di Milano = University of Milan (UNIMI), Laboratoire d'ingénierie des systèmes macromoléculaires (LISM), and Aix Marseille Université (AMU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)
Subjects: bacteriophages, Protein Conformation, [SDV]Life Sciences [q-bio], Allosteric regulation, anti-CRISPR protein, Virulence, cryo-electron microscopy, Biology, Genome, Structure-Activity Relationship, Viral Proteins, 03 medical and health sciences, chemistry.chemical_compound, 0302 clinical medicine, Allosteric Regulation, CRISPR-Associated Protein 9, Hydrolase, Escherichia coli, Humans, Streptococcus thermophilus, CRISPR, Clustered Regularly Interspaced Short Palindromic Repeats, Streptococcus thermophilus Cas9, Molecular Biology, 030304 developmental biology, 0303 health sciences, Binding Sites, Cas9, DNA, Cell Biology, biology.organism_classification, 3. Good health, Cell biology, St1Cas9, Kinetics, chemistry, Mutation, CRISPR-Cas Systems, CRISPR-Cas9, K562 Cells, 030217 neurology & neurosurgery, Bacteria, Protein Binding
Abstract: International audience; Molecular CellArticleCas9 Allosteric Inhibitionby the Anti-CRISPR Protein AcrIIA6Olivier Fuchsbauer,1,2,9Paolo Swuec,3,4,9Claire Zimberger,1,2Be ́atrice Amigues,1,2Se ́bastien Levesque,5Daniel Agudelo,5Alexis Duringer,5Antonio Chaves-Sanjuan,4Silvia Spinelli,1,2Genevie`ve M. Rousseau,6,7Minja Velimirovic,5Martino Bolognesi,3,4Alain Roussel,1,2Christian Cambillau,1,2Sylvain Moineau,6,7,8Yannick Doyon,5and Adeline Goulet1,2,10,*1Architecture et Fonction des Macromole ́cules Biologiques, Centre National de la Recherche Scientifique (CNRS), Campus de Luminy, Case932, 13288 Marseille Cedex 09, France2Architecture et Fonction des Macromole ́cules Biologiques, Aix-Marseille Universite ́, Campus de Luminy, Case 932, 13288 Marseille Cedex09, France3Dipartimento di Bioscienze, Universita`degli Studi di Milano, Via Celoria 26, 20133 Milano, Italy4Centro di Ricerca Pediatrica Romeo ed Enrica Invernizzi, Universita`degli Studi di Milano, Via Celoria 26, 20133 Milano, Italy5Centre Hospitalier Universitaire de Que ́bec–Universite ́Laval Research Center, Que ́bec City, QC G1V 4G2, Canada6De ́partement de biochimie, de microbiologie, et de bio-informatique, Faculte ́des sciences et de ge ́nie, Universite ́Laval, Que ́bec City, QC,G1V 0A6, Canada7Groupe de recherche en e ́cologie buccale, Faculte ́de me ́decine dentaire, Universite ́Laval, Que ́bec City, QC, G1V 0A6, Canada8Fe ́lix d’He ́relle Reference Center for Bacterial Viruses, Faculte ́de me ́decine dentaire, Universite ́Laval, Que ́bec City, QC, G1V 0A6, Canada9These authors contributed equally10Lead Contact*Correspondence:adeline.goulet@afmb.univ-mrs.frhttps://doi.org/10.1016/j.molcel.2019.09.012SUMMARYIn the arms race against bacteria, bacteriophageshave evolved diverse anti-CRISPR proteins (Acrs)that block CRISPR-Cas immunity. Acrs play key rolesin the molecular coevolution of bacteria with theirpredators, use a variety of mechanisms of action,and provide tools to regulate Cas-based genomemanipulation. Here, we present structural and func-tional analyses of AcrIIA6, an Acr from virulentphages, exploring its unique anti-CRISPR action.Our cryo-EM structures and functional data ofAcrIIA6 binding toStreptococcus thermophilusCas9 (St1Cas9) show that AcrIIA6 acts as an allo-steric inhibitor and induces St1Cas9 dimerization.AcrIIA6 reduces St1Cas9 binding affinity for DNAand prevents DNA binding within cells. The PAMand AcrIIA6 recognition sites are structurally closeand allosterically linked. Mechanistically, AcrIIA6 af-fects the St1Cas9 conformational dynamics associ-ated with PAM binding. Finally, we identify a naturalSt1Cas9 variant resistant to AcrIIA6 illustratingAcr-driven mutational escape and molecular diversi-fication of Cas9 proteins
Published: 2019

31. Blocking Antibodies Targeting the CD39/CD73 Immunosuppressive Pathway Unleash Immune Responses in Combination Cancer Therapies

Author: Hélène Rispaud-Blanc, Jean-François Eliaou, Cécile Dejou, Rachel Courtois, Béatrice Amigues, Emilie Narni-Mancinelli, Nathalie Bonnefoy, Jérémy Bastid, Armand Bensussan, Marc Giraudon-Paoli, Aurélie Docquier, François Romagné, O. Becquart, Carine Paturel, Ivan Perrot, Benjamin Rossi, Yannis Morel, Laurent Gauthier, Henri-Alexandre Michaud, Laurent Gros, Eric Vivier, Nicolas Gourdin, Alain Roussel, Diana Jecko, Stéphanie Chanteux, Severine Augier, Innate Pharma, Institut de Recherche en Cancérologie de Montpellier (IRCM - U1194 Inserm - UM), CRLCC Val d'Aurelle - Paul Lamarque-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Montpellier (UM), Centre Hospitalier Régional Universitaire [Montpellier] (CHRU Montpellier), Centre de Recherche en Cancérologie de Lyon (UNICANCER/CRCL), Centre Léon Bérard [Lyon]-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Architecture et fonction des macromolécules biologiques (AFMB), Centre National de la Recherche Scientifique (CNRS)-Aix Marseille Université (AMU)-Institut National de la Recherche Agronomique (INRA), Immunologie, dermatologie, oncologie, Oncodermatologie, immunologie et cellules souches cutanées (IDO (U976 / UMR_S 976)), Université Paris Diderot - Paris 7 (UPD7)-Institut National de la Santé et de la Recherche Médicale (INSERM), OREGA Biotech, Centre d'Immunologie de Marseille - Luminy (CIML), Aix Marseille Université (AMU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris Diderot - Paris 7 (UPD7), CRLCC Val d'Aurelle - Paul Lamarque-Université de Montpellier (UM)-Institut National de la Santé et de la Recherche Médicale (INSERM), Biologie et physiopathologie cutanées : expression génique, signalisation et thérapie, Université Nice Sophia Antipolis (... - 2019) (UNS), Université Côte d'Azur (UCA)-Université Côte d'Azur (UCA)-IFR50-Institut National de la Santé et de la Recherche Médicale (INSERM), IFR50, Université Côte d'Azur (UCA)-Université Côte d'Azur (UCA)-Faculté de Médecine Nice, Dynamique Musculaire et Métabolisme (DMEM), Institut National de la Recherche Agronomique (INRA)-Université de Montpellier (UM), AB Science SA, Cellules Souches et Radiations (SCSR (U967 / UMR-E_008)), Institut National de la Santé et de la Recherche Médicale (INSERM)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris Diderot - Paris 7 (UPD7)-Université Paris-Sud - Paris 11 (UP11), Centre de Recherche en Cancérologie de Lyon (CRCL), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre Léon Bérard [Lyon]-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Institut Curie, Institut National de la Recherche Agronomique (INRA)-Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), Recherche & Développement, Université Paris-Sud - Paris 11 (UP11)-Université Paris Diderot - Paris 7 (UPD7)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM), and Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Aix Marseille Université (AMU)
Subjects: 0301 basic medicine, Adenosine, T-Lymphocytes, medicine.medical_treatment, Mice, Adenosine Triphosphate, 0302 clinical medicine, Tumor Microenvironment, Medicine, Gene Knock-In Techniques, 5'-Nucleotidase, Melanoma, lcsh:QH301-705.5, Mice, Knockout, biology, Apyrase, Immunosuppression, 3. Good health, Oxaliplatin, Survival Rate, [SDV.IMM]Life Sciences [q-bio]/Immunology, Antibody, medicine.drug, medicine.drug_class, Antineoplastic Agents, [SDV.CAN]Life Sciences [q-bio]/Cancer, Monoclonal antibody, General Biochemistry, Genetics and Molecular Biology, 03 medical and health sciences, Immune system, Antigens, CD, Cell Line, Tumor, Blocking antibody, Animals, Humans, Antibodies, Blocking, business.industry, Cancer, [SDV.IMM.IMM]Life Sciences [q-bio]/Immunology/Immunotherapy, medicine.disease, Immune checkpoint, Mice, Inbred C57BL, Disease Models, Animal, 030104 developmental biology, lcsh:Biology (General), Leukocytes, Mononuclear, biology.protein, Cancer research, business, 030217 neurology & neurosurgery
Abstract: Summary: Immune checkpoint inhibitors have revolutionized cancer treatment. However, many cancers are resistant to ICIs, and the targeting of additional inhibitory signals is crucial for limiting tumor evasion. The production of adenosine via the sequential activity of CD39 and CD73 ectoenzymes participates to the generation of an immunosuppressive tumor microenvironment. In order to disrupt the adenosine pathway, we generated two antibodies, IPH5201 and IPH5301, targeting human membrane-associated and soluble forms of CD39 and CD73, respectively, and efficiently blocking the hydrolysis of immunogenic ATP into immunosuppressive adenosine. These antibodies promoted antitumor immunity by stimulating dendritic cells and macrophages and by restoring the activation of T cells isolated from cancer patients. In a human CD39 knockin mouse preclinical model, IPH5201 increased the anti-tumor activity of the ATP-inducing chemotherapeutic drug oxaliplatin. These results support the use of anti-CD39 and anti-CD73 monoclonal antibodies and their combination with immune checkpoint inhibitors and chemotherapies in cancer. : The production of adenosine via CD39 and CD73 ectoenzymes participates in an immunosuppressive tumor microenvironment. Perrot et al. generated two antibodies, IPH5201 and IPH5301, targeting human CD39 and CD73, respectively. In vitro and in vivo data support the use of anti-CD39 and anti-CD73 mAbs in combination cancer therapies. Keywords: CD39, CD73, cancer immunotherapies, therapeutic antibodies, adenosine pathway, tumor micro-environment, immunosuppression
Published: 2019
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32. Feedforward interference cancellation system applied to the 800 MHz CDMA cellular band

Author: Alain Roussel
Published: 2018

33. A phospholipase A1 antibacterial Type VI secretion effector interacts directly with the C-terminal domain of the VgrG spike protein for delivery

Author: Christine Kellenberger, Eric Cascales, Alain Roussel, Laure Journet, Marie-Stéphanie Aschtgen, Stéphane Canaan, Thi Thu Hang Le, Nicolas Flaugnatti, Van Son Nguyen, Christian Cambillau, and Stéphanie Blangy
Subjects: 0301 basic medicine, Effector, 030106 microbiology, Protein domain, Periplasmic space, Biology, Phospholipase, Microbiology, Cell biology, 03 medical and health sciences, 030104 developmental biology, Biochemistry, Phospholipase A1, Secretion, Bacterial outer membrane, Molecular Biology, Type VI secretion system
Abstract: The Type VI secretion system (T6SS) is a multiprotein machine that delivers protein effectors in both prokaryotic and eukaryotic cells, allowing interbacterial competition and virulence. The mechanism of action of the T6SS requires the contraction of a sheath-like structure that propels a needle towards target cells, allowing the delivery of protein effectors. Here, we provide evidence that the entero-aggregative Escherichia coli Sci-1 T6SS is required to eliminate competitor bacteria. We further identify Tle1, a toxin effector encoded by this cluster and showed that Tle1 possesses phospholipase A1 and A2 activities required for the interbacterial competition. Self-protection of the attacker cell is secured by an outer membrane lipoprotein, Tli1, which binds Tle1 in a 1:1 stoichiometric ratio with nanomolar affinity, and inhibits its phospholipase activity. Tle1 is delivered into the periplasm of the prey cells using the VgrG1 needle spike protein as carrier. Further analyses demonstrate that the C-terminal extension domain of VgrG1, including a transthyretin-like domain, is responsible for the interaction with Tle1 and its subsequent delivery into target cells. Based on these results, we propose an additional mechanism of transport of T6SS effectors in which cognate effectors are selected by specific motifs located at the C-terminus of VgrG proteins.
Published: 2016

34. Tissue-Specific Regulation of Drosophila NF-κB Pathway Activation by Peptidoglycan Recognition Protein SC

Author: Julien Royet, Christine Kellenberger, Florence Capo, Bernard Charroux, Delphine Chaduli, Denis Costechareyre, Alexandre Fabre, and Alain Roussel
Subjects: 0301 basic medicine, Regulation of gene expression, Genetics, Innate immune system, Mutant, Pattern recognition receptor, Biology, 3. Good health, Cell biology, 03 medical and health sciences, chemistry.chemical_compound, 030104 developmental biology, chemistry, Transcriptional regulation, Immunology and Allergy, Peptidoglycan, Signal transduction, Receptor
Abstract: In Drosophila, peptidoglycan (PGN) is detected by PGN recognition proteins (PGRPs) that act as pattern recognition receptors. Some PGRPs such as PGRP-LB or PGRP-SCs are able to cleave PGN, therefore reducing the amount of immune elicitors and dampening immune deficiency (IMD) pathway activation. The precise role of PGRP-SC is less well defined because the PGRP-SC genes (PGRP-SC1a, PGRP-SC1b and PGRP-SC2) lie very close on the chromosome and have been studied using a deletion encompassing the three genes. By generating PGRP-SC-specific mutants, we reevaluated the roles of PGRP-LB, PGRP-SC1 and PGRP-SC2, respectively, during immune responses. We showed that these genes are expressed in different gut domains and that they follow distinct transcriptional regulation. Loss-of-function mutant analysis indicates that PGRP-LB is playing a major role in IMD pathway activation and bacterial load regulation in the gut, although PGRP-SCs are expressed at high levels in this organ. We also demonstrated that PGRP-SC2 is the main negative regulator of IMD pathway activation in the fat body. Accordingly, we showed that mutants for either PGRP-LB or PGRP-SC2 displayed a distinct susceptibility to bacteria depending on the infection route. Lastly, we demonstrated that PGRP-SC1 and PGRP-SC2 are required in vivo for full Toll pathway activation by Gram-positive bacteria.
Published: 2015

35. Towards a complete structural deciphering of Type VI secretion system

Author: Badreddine Douzi, Van Son Nguyen, Eric Cascales, Eric Durand, Alain Roussel, Christian Cambillau, Laboratoire de l'intégration, du matériau au système (IMS), Université Sciences et Technologies - Bordeaux 1-Institut Polytechnique de Bordeaux-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), Architecture et fonction des macromolécules biologiques (AFMB), Institut National de la Recherche Agronomique (INRA)-Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Aix Marseille Université (AMU), and Centre National de la Recherche Scientifique (CNRS)-Aix Marseille Université (AMU)-Institut National de la Recherche Agronomique (INRA)
Subjects: 0301 basic medicine, Models, Molecular, Future studies, Cryo-electron microscopy, Bacterial Proteins/*chemistry/ultrastructure, Protein Conformation, [SDV]Life Sciences [q-bio], 030106 microbiology, Crystallography, X-Ray, 03 medical and health sciences, Protein structure, Bacterial Proteins, Structural Biology, Gram-Negative Bacteria, Prokaryotic cells, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], Molecular Biology, ComputingMilieux_MISCELLANEOUS, Type VI secretion system, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Effector, Chemistry, Cryoelectron Microscopy, Gram-Negative Bacteria/*chemistry/ultrastructure, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, [SDV.BBM.MN]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular Networks [q-bio.MN], Type VI Secretion Systems, Type VI Secretion Systems/*chemistry/ultrastructure, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], Medium resolution, [SDV.BBM.BP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biophysics, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], Biophysics, Tail structure
Abstract: International audience; The Type VI secretion system (T6SS) is a dynamic nanomachine present in many Gram-negative bacteria. Using a contraction mechanism similar to that of myophages, bacteriocins or anti-feeding prophages, it injects toxic effectors into both eukaryotic and prokaryotic cells. T6SS assembles three large ensembles: the trans-membrane complex (TMC), the baseplate and the tail. Recently, the tail structure has been elucidated by cryo electron microscopy (cryoEM) in extended and contracted forms. The structure of the trans-membrane complex has been deciphered using a combination of X-ray crystallography and EM. However, the structural characterisation of the baseplate lags behind and should be the target of future studies. Finally, cryo-tomography should provide low/medium resolution maps allowing to assemble the different parts ultimately leading to a complete structural description of T6SS.
Published: 2018

36. Type IX secretion system PorM and gliding machinery GldM form extended arches spanning the periplasmic space

Author: Alain Roussel, Christian Cambillau, Eric Cascales, Jennifer Roche, Christine Kellenberger, Aline Desmyter, Maxence S. Vincent, Philippe Leone, Quang Hieu Tran, Architecture et fonction des macromolécules biologiques (AFMB), Institut National de la Recherche Agronomique (INRA)-Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), Physiologie de la reproduction et des comportements [Nouzilly] (PRC), Centre National de la Recherche Scientifique (CNRS)-Université de Tours-Institut Français du Cheval et de l'Equitation [Saumur]-Institut National de la Recherche Agronomique (INRA), 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), Centre National de la Recherche Scientifique (CNRS)-Aix Marseille Université (AMU)-Institut National de la Recherche Agronomique (INRA), and Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Aix Marseille Université (AMU)
Subjects: 0301 basic medicine, crystal structure, bacterial pathogenesis, Operon, Protein Conformation, Science, [SDV]Life Sciences [q-bio], 030106 microbiology, General Physics and Astronomy, Flavobacterium, General Biochemistry, Genetics and Molecular Biology, Article, gliding machinery, 03 medical and health sciences, Protein structure, Bacterial Proteins, Escherichia coli, Inner membrane, Animals, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], lcsh:Science, Porphyromonas gingivalis, Bacterial Secretion Systems, ComputingMilieux_MISCELLANEOUS, Multidisciplinary, biology, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Chemistry, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, [SDV.BBM.MN]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular Networks [q-bio.MN], General Chemistry, Periplasmic space, biology.organism_classification, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, Cell biology, Transport protein, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], 030104 developmental biology, Helix, Periplasm, lcsh:Q, Bacterial outer membrane, Camelids, New World, dental diseases, type IX secretion system
Abstract: Type IX secretion system (T9SS), exclusively present in the Bacteroidetes phylum, has been studied mainly in Flavobacterium johnsoniae and Porphyromonas gingivalis. Among the 18 genes, essential for T9SS function, a group of four, porK-N (P. gingivalis) or gldK-N (F. johnsoniae) belongs to a co-transcribed operon that expresses the T9SS core membrane complex. The central component of this complex, PorM (or GldM), is anchored in the inner membrane by a trans-membrane helix and interacts through the outer membrane PorK-N complex. There is a complete lack of available atomic structures for any component of T9SS, including the PorKLMN complex. Here we report the crystal structure of the GldM and PorM periplasmic domains. Dimeric GldM and PorM, each contain four domains of ~180-Å length that span most of the periplasmic space. These and previously reported results allow us to propose a model of the T9SS core membrane complex as well as its functional behavior., No structural data for the bacterial type IX secretion system (T9SS) are available so far. Here, the authors present the crystal structures of the periplasmic domains from two major T9SS components PorM and GldM, which span most of the periplasmic space, and propose a putative model of the T9SS core membrane complex.
Published: 2018

37. TssA: The cap protein of the Type VI secretion system tail

Author: Laure Journet, Yoann G. Santin, Eric Durand, Abdelrahim Zoued, Alain Roussel, Christian Cambillau, Eric Cascales, Laboratoire d'ingénierie des systèmes macromoléculaires (LISM), Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Aix Marseille Université (AMU), Architecture et fonction des macromolécules biologiques (AFMB), Centre National de la Recherche Scientifique (CNRS)-Aix Marseille Université (AMU)-Institut National de la Recherche Agronomique (INRA), Aix Marseille Université (AMU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), and Institut National de la Recherche Agronomique (INRA)-Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS)
Subjects: 0301 basic medicine, [SDV]Life Sciences [q-bio], 030106 microbiology, [SDV.BC.BC]Life Sciences [q-bio]/Cellular Biology/Subcellular Processes [q-bio.SC], General Biochemistry, Genetics and Molecular Biology, 03 medical and health sciences, Escherichia coli, Bacteriophages, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], ComputingMilieux_MISCELLANEOUS, Type VI secretion system, biology, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Effector, Chemistry, Escherichia coli Proteins, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, [SDV.BBM.MN]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular Networks [q-bio.MN], Type VI Secretion Systems, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, Cell biology, Transport protein, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], 030104 developmental biology, Biochemistry, Cytoplasm, Enteroaggregative Escherichia coli, Chaperone (protein), biology.protein, Biogenesis, Catabolite activator protein
Abstract: The Type VI secretion system (T6SS) is a multiprotein and mosaic apparatus that delivers protein effectors into prokaryotic or eukaryotic cells. Recent data on the enteroaggregative Escherichia coli (EAEC) T6SS have provided evidence that the TssA protein is a key component during T6SS biogenesis. The T6SS comprises a trans-envelope complex that docks the baseplate, a cytoplasmic complex that represents the assembly platform for the tail. The T6SS tail is structurally, evolutionarily and functionally similar to the contractile tails of bacteriophages. We have shown that TssA docks to the membrane complex, recruits the baseplate complex and initiates and coordinates the polymerization of the inner tube with that of the sheath. Here, we review these recent findings, discuss the variations within TssA-like proteins, speculate on the role of EAEC TssA in T6SS biogenesis and propose future research perspectives.
Published: 2017

38. Camelid nanobodies used as crystallization chaperones for different constructs of PorM, a component of the type IX secretion system from Porphyromonas gingivalis

Author: Christian Cambillau, Christine Kellenberger, Alain Roussel, Aline Desmyter, Anaïs Gaubert, Jennifer Roche, Philippe Leone, Thi Trang Nhung Trinh, Yoan Duhoo, Architecture et fonction des macromolécules biologiques (AFMB), Institut National de la Recherche Agronomique (INRA)-Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS)-Aix Marseille Université (AMU)-Institut National de la Recherche Agronomique (INRA), Architecture et fonction des macromolécules biologiques ( AFMB ), and Centre National de la Recherche Scientifique ( CNRS ) -Aix Marseille Université ( AMU ) -Institut National de la Recherche Agronomique ( INRA )
Subjects: MESH: Sequence Homology, Amino Acid, [SDV.NEU.NB]Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC]/Neurobiology, Plasma protein binding, MESH: Amino Acid Sequence, Biochemistry, MESH: Recombinant Proteins, [SDV.MHEP.MI]Life Sciences [q-bio]/Human health and pathology/Infectious diseases, MESH: Animals, [ SDV.BIBS ] Life Sciences [q-bio]/Quantitative Methods [q-bio.QM], Peptide sequence, MESH: Bacterial Proteins, MESH : Porphyromonas gingivalis, MESH : Protein Conformation, alpha-Helical, [SDV.MHEP.ME]Life Sciences [q-bio]/Human health and pathology/Emerging diseases, [ SDV.MHEP.ME ] Life Sciences [q-bio]/Human health and pathology/Emerging diseases, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], MESH: Escherichia coli, MESH : Amino Acid Sequence, MESH : Protein Binding, MESH: Camelus, [SDV.BIBS]Life Sciences [q-bio]/Quantitative Methods [q-bio.QM], MESH : Sequence Homology, Amino Acid, MESH : Genetic Vectors, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], [ SDV.MHEP.MI ] Life Sciences [q-bio]/Human health and pathology/Infectious diseases, MESH : Crystallization, [ SDV.NEU.NB ] Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC]/Neurobiology, [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology, MESH: Camelids, New World, MESH: Porphyromonas gingivalis, MESH: Models, Molecular, Camelus, MESH: Gene Expression, MESH : Cloning, Molecular, Biophysics, MESH: Sequence Alignment, [ SDV.MP.VIR ] Life Sciences [q-bio]/Microbiology and Parasitology/Virology, MESH: Single-Domain Antibodies, Microbiology, 03 medical and health sciences, Bacterial Proteins, Genetics, MESH: Protein Binding, Secretion, Molecular replacement, Protein Interaction Domains and Motifs, 5fwo, MESH: Cloning, Molecular, Amino Acid Sequence, Porphyromonas gingivalis, MESH: Protein Conformation, alpha-Helical, [ SDV.IMM.II ] Life Sciences [q-bio]/Immunology/Innate immunity, MESH: Protein Interaction Domains and Motifs, MESH : Molecular Chaperones, Periplasmic space, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, MESH : Camelids, New World, MESH : Gene Expression, 030104 developmental biology, MESH: Binding Sites, Protein Conformation, beta-Strand, PorM, Molecular Chaperones, type IX secretion system, 0301 basic medicine, Models, Molecular, Protein Conformation, alpha-Helical, MESH : Escherichia coli, [SDV.BIO]Life Sciences [q-bio]/Biotechnology, Gene Expression, [ SDV.MP.BAC ] Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, Crystallography, X-Ray, [SDV.IMM.II]Life Sciences [q-bio]/Immunology/Innate immunity, MESH : Bacterial Secretion Systems, Research Communications, [ SDV.BBM.BC ] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], Structural Biology, MESH: Genetic Vectors, 5lmj, MESH : Bacterial Proteins, Cloning, Molecular, MESH: Bacterial Secretion Systems, Bacterial Secretion Systems, MESH : Protein Conformation, beta-Strand, MESH: Crystallization, 5lmw, MESH: Kinetics, MESH : Sequence Alignment, MESH : Camelus, Condensed Matter Physics, Recombinant Proteins, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], MESH : Single-Domain Antibodies, Thermodynamics, MESH: Protein Conformation, beta-Strand, nb02, MESH : Kinetics, nb01, MESH: Thermodynamics, MESH: Molecular Chaperones, Crystallization, Camelids, New World, Protein Binding, crystallization chaperones, MESH : Recombinant Proteins, MESH : Models, Molecular, Genetic Vectors, Context (language use), Biology, MESH : Peptide Library, Peptide Library, Escherichia coli, Animals, nb130, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], Peptide library, MESH : Thermodynamics, nb19, Binding Sites, Sequence Homology, Amino Acid, [ SDV.SP.PHARMA ] Life Sciences [q-bio]/Pharmaceutical sciences/Pharmacology, [ SDV.BIO ] Life Sciences [q-bio]/Biotechnology, Single-Domain Antibodies, biology.organism_classification, MESH: Crystallography, X-Ray, Kinetics, [SDV.SP.PHARMA]Life Sciences [q-bio]/Pharmaceutical sciences/Pharmacology, MESH : Animals, MESH: Peptide Library, MESH : Crystallography, X-Ray, camelid nanobodies, Sequence Alignment, 5lz0, MESH : Binding Sites, MESH : Protein Interaction Domains and Motifs, [ SDV.BBM.BS ] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM]
Abstract: PorM is a membrane protein that is involved in the assembly of the type IX secretion system (T9SS) inPorphyromonas gingivalis, a major bacterial pathogen that is responsible for periodontal disease in humans. In the context of structural studies of PorM to better understand T9SS assembly, four camelid nanobodies were selected, produced and purified, and their specific interaction with the N-terminal or C-terminal part of the periplasmic domain of PorM was investigated. Diffracting crystals were also obtained, and the structures of the four nanobodies were solved by molecular replacement. Furthermore, two nanobodies were used as crystallization chaperones and turned out to be valuable tools in the structure-determination process of the periplasmic domain of PorM.
Published: 2017

39. Characterization of the Porphyromonas gingivalis Type IX Secretion Trans-envelope PorKLMNP Core Complex

Author: Julien Stathopulos, Abdelrahim Zoued, Christine Kellenberger, Maxence S. Vincent, Alain Roussel, Philippe Leone, Mickaël J. Canestrari, Christian Cambillau, Eric Cascales, Bérengère Ize, Laboratoire d'ingénierie des systèmes macromoléculaires (LISM), Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Aix Marseille Université (AMU), Architecture et fonction des macromolécules biologiques (AFMB), Centre National de la Recherche Scientifique (CNRS)-Aix Marseille Université (AMU)-Institut National de la Recherche Agronomique (INRA), Aix Marseille Université (AMU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), and Institut National de la Recherche Agronomique (INRA)-Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS)
Subjects: 0301 basic medicine, Gliding motility, [SDV]Life Sciences [q-bio], 030106 microbiology, channel, membrane proteins, Biochemistry, Flavobacterium, 03 medical and health sciences, membrane complex, protein secretion, Tannerella forsythia, Secretion, Type IX secretion, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Porphyromonas, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], Molecular Biology, Porphyromonas gingivalis, periodontitis, biology, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Chemistry, toxins, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, [SDV.BBM.MN]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular Networks [q-bio.MN], Cell Biology, biology.organism_classification, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, Cell biology, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], Bacterial adhesin, [SDV.BBM.BP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biophysics, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], stomatognathic diseases, Secretory protein, Membrane protein, T9SS, protein transport, gingipains, gliding motility, Cell envelope, gingivitis
Abstract: International audience; The transport of proteins at the cell surface of Bacteriodetes depends on a secretory apparatus known as Type IX secretion system (T9SS). This machine is responsible for the cell surface exposition of various proteins such as adhesins required for gliding motility in Flavobacteria, S-layer components in Tannerella forsythia and tooth tissue-degrading enzymes in the oral pathogen Porphyromonas gingivalis. While a number of subunits of the T9SS have been identified, we lack details on the architecture of this secretion apparatus. Here we provide evidence that five of the genes encoding the core complex of the T9SS are co-transcribed, and that the gene products are distributed in the cell envelope. Protein-protein interaction studies then revealed that these proteins oligomerize and interact through a dense network of contacts.
Published: 2017

40. Characterization of the

Author: Maxence S, Vincent, Mickaël J, Canestrari, Philippe, Leone, Julien, Stathopulos, Bérengère, Ize, Abdelrahim, Zoued, Christian, Cambillau, Christine, Kellenberger, Alain, Roussel, and Eric, Cascales
Subjects: stomatognathic diseases, Protein Subunits, Bacterial Proteins, Genes, Bacterial, Bacteroidaceae Infections, Humans, Protein Interaction Maps, Crystallography, X-Ray, Bacterial Secretion Systems, Porphyromonas gingivalis, Microbiology
Abstract: The transport of proteins at the cell surface of Bacteroidetes depends on a secretory apparatus known as type IX secretion system (T9SS). This machine is responsible for the cell surface exposition of various proteins, such as adhesins, required for gliding motility in Flavobacterium, S-layer components in Tannerella forsythia, and tooth tissue-degrading enzymes in the oral pathogen Porphyromonas gingivalis. Although a number of subunits of the T9SS have been identified, we lack details on the architecture of this secretion apparatus. Here we provide evidence that five of the genes encoding the core complex of the T9SS are co-transcribed and that the gene products are distributed in the cell envelope. Protein-protein interaction studies then revealed that these proteins oligomerize and interact through a dense network of contacts.
Published: 2016

41. Multifunctional Natural Killer Cell Engagers Targeting NKp46 Trigger Protective Tumor Immunity

Author: Béatrice Amigues, Frédéric Bosco, Hélène Rispaud-Blanc, Gwendoline Grondin, Romain Remark, Guillaume Habif, Ariane Morel, Stéphanie Cornen, Flavien Caraguel, Nadia Anceriz, Laurent Gauthier, Emilie Narni-Mancinelli, Alain Roussel, Cédric Cesari, Franceline Guillot, Audrey Blanchard-Alvarez, Melody Sapet, Sylvia Trichard, Yannis Morel, Benjamin Rossi, Eric Vivier, François Romagné, Sandrine Arrufat, Cellules Souches et Radiations (SCSR (U967 / UMR-E_008)), Institut National de la Santé et de la Recherche Médicale (INSERM)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris Diderot - Paris 7 (UPD7)-Université Paris-Sud - Paris 11 (UP11), Innate Pharma, Architecture et fonction des macromolécules biologiques (AFMB), Institut National de la Recherche Agronomique (INRA)-Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), Institut Curie, Centre d'Immunologie de Marseille - Luminy (CIML), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Aix Marseille Université (AMU), Stabilité génétique, Cellules Souches et Radiations (SCSR (U_967)), 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)-Université Paris Cité (UPCité), Institut Curie [Paris], Aix Marseille Université (AMU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), ANR-17-RHUS-0007,PIONEER,Precision Immuno-Oncology for advanced Non small cell lung cancer patients with PD-1 ICI Resistance(2017), Université Paris-Sud - Paris 11 (UP11)-Université Paris Diderot - Paris 7 (UPD7)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM), 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)-Université de Paris (UP), and Centre National de la Recherche Scientifique (CNRS)-Aix Marseille Université (AMU)-Institut National de la Recherche Agronomique (INRA)
Subjects: Cytotoxicity, Immunologic, medicine.drug_class, medicine.medical_treatment, Biology, Monoclonal antibody, General Biochemistry, Genetics and Molecular Biology, Natural killer cell, Mice, 03 medical and health sciences, Antineoplastic Agents, Immunological, 0302 clinical medicine, Immune system, Cancer immunotherapy, Immunity, Antibodies, Bispecific, medicine, Animals, Antigens, Ly, Humans, 030304 developmental biology, 0303 health sciences, Natural Cytotoxicity Triggering Receptor 1, Neoplasms, Experimental, Tumor antigen, 3. Good health, Killer Cells, Natural, medicine.anatomical_structure, Immunoglobulin G, Cancer cell, Cancer research, biology.protein, [SDV.IMM]Life Sciences [q-bio]/Immunology, Antibody, 030217 neurology & neurosurgery
Abstract: Summary Over the last decade, various new therapies have been developed to promote anti-tumor immunity. Despite interesting clinical results in hematological malignancies, the development of bispecific killer-cell-engager antibody formats directed against tumor cells and stimulating anti-tumor T cell immunity has proved challenging, mostly due to toxicity problems. We report here the generation of trifunctional natural killer (NK) cell engagers (NKCEs), targeting two activating receptors, NKp46 and CD16, on NK cells and a tumor antigen on cancer cells. Trifunctional NKCEs were more potent in vitro than clinical therapeutic antibodies targeting the same tumor antigen. They had similar in vivo pharmacokinetics to full IgG antibodies and no off-target effects and efficiently controlled tumor growth in mouse models of solid and invasive tumors. Trifunctional NKCEs thus constitute a new generation of molecules for fighting cancer. Video Abstract
Published: 2019

42. Cytokine Diedel and a viral homologue suppress the IMD pathway in Drosophila

Author: Cordula Kemp, Olivier Lamiable, Alain Roussel, Laurent Daeffler, Christine Kellenberger, Nadège Pelte, Laurent Troxler, João Trindade Marques, Jules A. Hoffmann, Michael Boutros, Jean-Luc Imler, Architecture et fonction des macromolécules biologiques (AFMB), Centre National de la Recherche Scientifique (CNRS)-Aix Marseille Université (AMU)-Institut National de la Recherche Agronomique (INRA), and Institut National de la Recherche Agronomique (INRA)-Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS)
Subjects: 0301 basic medicine, Sindbis virus, viruses, Molecular Sequence Data, Mutant, Cell, 03 medical and health sciences, chemistry.chemical_compound, Immune system, RNA interference, medicine, Animals, Drosophila Proteins, Amino Acid Sequence, Gene, ComputingMilieux_MISCELLANEOUS, Multidisciplinary, Sequence Homology, Amino Acid, biology, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Alphavirus Infections, Intracellular parasite, fungi, Immunity, Biological Sciences, biology.organism_classification, Survival Analysis, Virology, [SDV.BIBS]Life Sciences [q-bio]/Quantitative Methods [q-bio.QM], Up-Regulation, 3. Good health, Cell biology, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], Drosophila melanogaster, 030104 developmental biology, medicine.anatomical_structure, chemistry, Mutation, Cytokines, Sindbis Virus, DNA, Signal Transduction
Abstract: Viruses are obligatory intracellular parasites that suffer strong evolutionary pressure from the host immune system. Rapidly evolving viral genomes can adapt to this pressure by acquiring genes that counteract host defense mechanisms. For example, many vertebrate DNA viruses have hijacked cellular genes encoding cytokines or cytokine receptors to disrupt host cell communication. Insect viruses express suppressors of RNA interference or apoptosis, highlighting the importance of these cell intrinsic antiviral mechanisms in invertebrates. Here, we report the identification and characterization of a family of proteins encoded by insect DNA viruses that are homologous to a 12-kDa circulating protein encoded by the virus-induced Drosophila gene diedel (die). We show that die mutant flies have shortened lifespan and succumb more rapidly than controls when infected with Sindbis virus. This reduced viability is associated with deregulated activation of the immune deficiency (IMD) pathway of host defense and can be rescued by mutations in the genes encoding the homolog of IKKγ or IMD itself. Our results reveal an endogenous pathway that is exploited by insect viruses to modulate NF-κB signaling and promote fly survival during the antiviral response.
Published: 2016

43. Binding of the RamR Repressor to Wild-Type and Mutated Promoters of the ramA Gene Involved in Efflux-Mediated Multidrug Resistance in Salmonella enterica Serovar Typhimurium

Author: Axel Cloeckaert, Etienne Giraud, Bertrand Castaing, Franck Coste, Sylvie Canepa, Marie-Christine Maurel, Françoise Culard, Alain Roussel, Sylvie Baucheron, Centre de biophysique moléculaire (CBM), Université d'Orléans (UO)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC), Infectiologie et Santé Publique (UMR ISP), Institut National de la Recherche Agronomique (INRA)-Université de Tours, and Institut National de la Recherche Agronomique (INRA)-Université de Tours (UT)
Subjects: Salmonella typhimurium, serovar Thyphimurium, chemistry.chemical_compound, résonnance plasmonique de surface, Transcription (biology), Drug Resistance, Multiple, Bacterial, MESH: Animals, Pharmacology (medical), Promoter Regions, Genetic, MESH: Bacterial Proteins, Genetics, MESH: Gene Expression Regulation, Bacterial, 0303 health sciences, MESH: Transcription Factors, DNA-Binding Proteins, MESH: Cattle, Infectious Diseases, Salmonella enterica, Multidrug Resistance-Associated Proteins, MESH: Multidrug Resistance-Associated Proteins, Protein Binding, MESH: Mutation, MESH: Trans-Activators, Repressor, gène ramA, Biology, 03 medical and health sciences, Bacterial Proteins, Mechanisms of Resistance, MESH: Promoter Regions, Genetic, Animals, Humans, MESH: Protein Binding, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, TetR, Binding site, 030304 developmental biology, Pharmacology, Binding Sites, MESH: Humans, BIOCHIMIE, 030306 microbiology, Wild type, MESH: Salmonella typhimurium, Promoter, Gene Expression Regulation, Bacterial, MESH: Drug Resistance, Multiple, Bacterial, biology.organism_classification, Molecular biology, Multiple drug resistance, MESH: Binding Sites, chemistry, Mutation, Trans-Activators, Cattle, MESH: DNA-Binding Proteins, Transcription Factors
Abstract: The transcriptional activator RamA is involved in multidrug resistance (MDR) by increasing expression of the AcrAB-TolC RND-type efflux system in several pathogenic Enterobacteriaceae . In Salmonella enterica serovar Typhimurium ( S. Typhimurium), ramA expression is negatively regulated at the local level by RamR, a transcriptional repressor of the TetR family. We here studied the DNA-binding activity of the RamR repressor with the ramA promoter (P ramA ). As determined by high-resolution footprinting, the 28-bp-long RamR binding site covers essential features of P ramA , including the −10 conserved region, the transcriptional start site of ramA , and two 7-bp inverted repeats. Based on the RamR footprint and on electrophoretic mobility shift assays (EMSAs), we propose that RamR interacts with P ramA as a dimer of dimers, in a fashion that is structurally similar to the QacR-DNA binding model. Surface plasmon resonance (SPR) measurements indicated that RamR has a 3-fold-lower affinity ( K D [equilibrium dissociation constant] = 191 nM) for the 2-bp-deleted P ramA of an MDR S. Typhimurium clinical isolate than for the wild-type P ramA ( K D = 66 nM). These results confirm the direct regulatory role of RamR in the repression of ramA transcription and precisely define how an alteration of its binding site can give rise to an MDR phenotype.
Published: 2012

44. Tissue- and ligand-specific sensing of gram-negative infection in drosophila by PGRP-LC isoforms and PGRP-LE

Author: Alain Roussel, Claudine Neyen, Bruno Lemaitre, and Mickael Poidevin
Subjects: Gene isoform, Immunology, Molecular Sequence Data, Biology, Real-Time Polymerase Chain Reaction, 03 medical and health sciences, chemistry.chemical_compound, 0302 clinical medicine, Immune system, Tracheal cytotoxin, Immunology and Allergy, Animals, Drosophila Proteins, Protein Isoforms, Gram-Positive Bacterial Infections, 030304 developmental biology, Genetics, 0303 health sciences, Microscopy, Confocal, Base Sequence, Reverse Transcriptase Polymerase Chain Reaction, Pattern recognition receptor, Antibacterial Response, biology.organism_classification, Immunohistochemistry, Immunity, Innate, Cell biology, chemistry, Drosophila, Peptidoglycan, Carrier Proteins, Peptidoglycan binding, 030217 neurology & neurosurgery, Bacteria
Abstract: The Drosophila antimicrobial response is one of the best characterized systems of pattern recognition receptor-mediated defense in metazoans. Drosophila senses Gram-negative bacteria via two peptidoglycan recognition proteins (PGRPs), membrane-bound PGRP-LC and secreted/cytosolic PGRP-LE, which relay diaminopimelic acid (DAP)-type peptidoglycan sensing to the Imd signaling pathway. In the case of PGRP-LC, differential splicing of PGRP domain-encoding exons to a common intracellular domain-encoding exon generates three receptor isoforms, which differ in their peptidoglycan binding specificities. In this study, we used Phi31-mediated recombineering to generate fly lines expressing specific isoforms of PGRP-LC and assessed the tissue-specific roles of PGRP-LC isoforms and PGRP-LE in the antibacterial response. Our in vivo studies demonstrate the key role of PGRP-LCx in sensing DAP-type peptidoglycan-containing Gram-negative bacteria or Gram-positive bacilli during systemic infection. We also highlight the contribution of PGRP-LCa/x heterodimers to the systemic immune response to Gram-negative bacteria through sensing of tracheal cytotoxin (TCT), whereas PGRP-LCy may have a minor role in antagonizing the immune response. Our results reveal that both PGRP-LC and PGRP-LE contribute to the intestinal immune response, with a predominant role of cytosolic PGRP-LE in the midgut, the central section of endodermal origin where PGRP-LE is enriched. Our in vivo model also definitively establishes TCT as the long-distance elicitor of systemic immune responses to intestinal bacteria observed in a loss-of-tolerance model. In conclusion, our study delineates how a combination of extracellular sensing by PGRP-LC isoforms and intracellular sensing through PGRP-LE provides sophisticated mechanisms to detect and differentiate between infections by different DAP-type bacteria in Drosophila.
Published: 2012
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45. A phospholipase A1 antibacterial Type VI secretion effector interacts directly with the C-terminal domain of the VgrG spike protein for delivery

Author: Nicolas, Flaugnatti, Thi Thu Hang, Le, Stéphane, Canaan, Marie-Stéphanie, Aschtgen, Van Son, Nguyen, Stéphanie, Blangy, Christine, Kellenberger, Alain, Roussel, Christian, Cambillau, Eric, Cascales, and Laure, Journet
Subjects: Models, Molecular, Bacterial Proteins, Protein Domains, Virulence, Multigene Family, Bacterial Toxins, Escherichia coli, Animals, Type VI Secretion Systems, Caenorhabditis elegans, Phospholipases A1
Abstract: The Type VI secretion system (T6SS) is a multiprotein machine that delivers protein effectors in both prokaryotic and eukaryotic cells, allowing interbacterial competition and virulence. The mechanism of action of the T6SS requires the contraction of a sheath-like structure that propels a needle towards target cells, allowing the delivery of protein effectors. Here, we provide evidence that the entero-aggregative Escherichia coli Sci-1 T6SS is required to eliminate competitor bacteria. We further identify Tle1, a toxin effector encoded by this cluster and showed that Tle1 possesses phospholipase A1 and A2 activities required for the interbacterial competition. Self-protection of the attacker cell is secured by an outer membrane lipoprotein, Tli1, which binds Tle1 in a 1:1 stoichiometric ratio with nanomolar affinity, and inhibits its phospholipase activity. Tle1 is delivered into the periplasm of the prey cells using the VgrG1 needle spike protein as carrier. Further analyses demonstrate that the C-terminal extension domain of VgrG1, including a transthyretin-like domain, is responsible for the interaction with Tle1 and its subsequent delivery into target cells. Based on these results, we propose an additional mechanism of transport of T6SS effectors in which cognate effectors are selected by specific motifs located at the C-terminus of VgrG proteins.
Published: 2015

46. Biogenesis and structure of a Type VI secretion membrane core complex

Author: Abdelrahim Zoued, Annick Dujeancourt, Laureen Logger, Rémi Fronzes, Benjamin Bardiaux, Eric Cascales, Van Son Nguyen, Silvia Spinelli, Aline Desmyter, Marie-Stéphanie Aschtgen, Eric Durand, Christian Cambillau, Gérard Pehau-Arnaudet, Alain Roussel, Laboratoire d'ingénierie des systèmes macromoléculaires (LISM), Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Aix Marseille Université (AMU), Architecture et fonction des macromolécules biologiques (AFMB), Centre National de la Recherche Scientifique (CNRS)-Aix Marseille Université (AMU)-Institut National de la Recherche Agronomique (INRA), Biologie Structurale de la Sécrétion Bactérienne, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Bioinformatique structurale - Structural Bioinformatics, Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur [Paris], This work was funded by Agence Nationale de la Recherche (ANR) grants ANR-10-JCJC-1303-03 to E.C., Bip:Bip to R.F., ANR-14-CE14-0006-02 to C.C. and E.C., and supported by the French Infrastructure for Integrated Structural Biology (FRISBI) ANR-10-INSB-05-01. E.D. was supported by a post-doctoral fellowship from the Fondation pour la Recherche Médicale (SPF20101221116) and ANR grants Bip:Bip and ANR-10-JCJC-1303-03. V.S.N. was supported by a PhD grant from the French Embassy in Vietnam (792803C). A.Z., L.L. and M.S.A. were recipients of doctoral fellowships from the French Ministère de la Recherche. A.Z. received a Fondation pour la Recherche Médicale fellowship (FDT20140931060)., ANR-10-JCJC-1303,A Fun T6SS,Assemblage et Fonction des Systèmes de Sécrétion de Type VI bactériens(2010), ANR-10-INBS-0005,FRISBI,Infrastructure Française pour la Biologie Structurale Intégrée(2010), ANR-14-CE14-0006,B-War,Guerre bactérienne: Architecture et Fonction du Système de Sécrétion de Type VI(2014), Institut Pasteur [Paris] - Centre National de la Recherche Scientifique (CNRS), Aix Marseille Université (AMU) - Institut National de la Santé et de la Recherche Médicale (INSERM) - Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS) - Aix Marseille Université (AMU) - Institut National de la Recherche Agronomique (INRA), Microscopie ultrastructurale - Ultrapole (CITECH), Institut Pasteur [Paris], Bioinformatique structurale, This work was funded by Agence Nationale de la Recherche (ANR) grants ANR-10-JCJC-1303-03 to E.C., Bip:Bip to R.F., ANR-14-CE14-0006-02 to C.C. and E.C., and supported by the French Infrastructure for Integrated Structural Biology (FRISBI) ANR-10-INSB-05-01. E.D. was supported by a post-doctoral fellowship from the Fondation pour la Recherche Médicale (SPF20101221116) and ANR grants Bip:Bip and ANR-10-JCJC-1303-03. V.S.N. was supported by a PhD grant from the French Embassy in Vietnam (792803C). A.Z., L.L. and M.S.A. were recipients of doctoral fellowships from the French Ministère de la Recherche. A.Z. received a Fondation pour la Recherche Médicale fellowship (FDT20140931060), We thank O. Francetic for providing anti-DglA and anti-OmpF antibodies. We thank E. Marza, P. Violinova Krasteva and H. Remaut for comments on the manuscript, and T. Mignot, M. Guzzo and L. Espinosa for advice about the fluorescence microscopy experiments and the statistical analyses. We also thank the members of the R.F. and E.C. research groups for discussions and suggestions, and R. Lloubès, J. Sturgis and A. Galinier for encouragement. We thank the ERSF and Soleil Synchrotron radiation facilities for beamline allocation, ANR-10-JCJC-1303, A Fun T6SS, Assemblage et Fonction des Systèmes de Sécrétion de Type VI bactériens(2010), ANR-14-CE14-0006, B-War, Guerre bactérienne: Architecture et Fonction du Système de Sécrétion de Type VI(2014), ANR-10-INBS-05-01/10-INBS-0005, FRISBI, Infrastructure Française pour la Biologie Structurale Intégrée(2010), CASCALES, ERIC, Jeunes Chercheuses et Jeunes Chercheurs - Assemblage et Fonction des Systèmes de Sécrétion de Type VI bactériens - - A Fun T6SS2010 - ANR-10-JCJC-1303 - JCJC - VALID, Infrastructure Française pour la Biologie Structurale Intégrée - - FRISBI2010 - ANR-10-INBS-0005 - INBS - VALID, Appel à projets générique - Guerre bactérienne: Architecture et Fonction du Système de Sécrétion de Type VI - - B-War2014 - ANR-14-CE14-0006 - Appel à projets générique - VALID, Aix Marseille Université (AMU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Institut National de la Recherche Agronomique (INRA)-Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), and ANR-10-INBS-05-01/10-INBS-0005,FRISBI,Infrastructure Française pour la Biologie Structurale Intégrée(2010)
Subjects: Models, Molecular, Cytoplasm, MESH : Bacterial Secretion Systems, MESH : Escherichia coli/chemistry, MESH: Escherichia coli Proteins, MESH : Cytoplasm/chemistry, [SDV.BC.BC]Life Sciences [q-bio]/Cellular Biology/Subcellular Processes [q-bio.SC], Crystallography, X-Ray, [SDV.BBM.BM] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, MESH : Multiprotein Complexes/biosynthesis, MESH: Protein Structure, Tertiary, Bacterial secretion, MESH : Membrane Proteins/biosynthesis, MESH: Lipopeptides, MESH : Escherichia coli Proteins/chemistry, MESH: Bacterial Secretion Systems, Bacterial Secretion Systems, [SDV.BBM.BC] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], MESH : Cell Membrane/chemistry, "structure of a type VI", MESH : Membrane Proteins/chemistry, MESH : Escherichia coli Proteins/biosynthesis, 0303 health sciences, [SDV.BIBS] Life Sciences [q-bio]/Quantitative Methods [q-bio.QM], Multidisciplinary, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], MESH: Escherichia coli, Escherichia coli Proteins, MESH: Periplasm, MESH : Escherichia coli/metabolism, MESH: Protein Subunits, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], Biochemistry, Periplasm, MESH : Cytoplasm/metabolism, MESH: Membrane Proteins, Cell envelope, Bacterial outer membrane, Porosity, MESH : Lipopeptides/biosynthesis, MESH : Protein Structure, Tertiary, MESH: Models, Molecular, [SDV.BBM.BS] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], MESH : Models, Molecular, MESH : Lipopeptides/chemistry, MESH: Microscopy, Electron, Biology, MESH : Protein Subunits/biosynthesis, Lipopeptides, 03 medical and health sciences, MESH: Porosity, Escherichia coli, [SDV.BC.BC] Life Sciences [q-bio]/Cellular Biology/Subcellular Processes [q-bio.SC], Electron microscopy, Secretion, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], "Biogenesis", MESH : Cell Membrane/metabolism, "secretion membrane", MESH : Protein Subunits/chemistry, 030304 developmental biology, Type VI secretion system, X-ray crystallography, MESH : Periplasm/metabolism, MESH : Periplasm/chemistry, 030306 microbiology, MESH: Cytoplasm, Cell Membrane, Membrane Proteins, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, Periplasmic space, MESH: Multiprotein Complexes, MESH: Crystallography, X-Ray, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, MESH : Microscopy, Electron, MESH : Multiprotein Complexes/chemistry, Protein Structure, Tertiary, Microscopy, Electron, Protein Subunits, Membrane protein, Multiprotein Complexes, Biophysics, [SDV.MP.BAC] Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, MESH : Porosity, MESH : Crystallography, X-Ray, Biogenesis, MESH: Cell Membrane
Abstract: International audience; Bacteria share their ecological niches with other microbes. The bacterial type VI secretion system is one of the key players in microbial competition, as well as being an important virulence determinant during bacterial infections. It assembles a nano-crossbow-like structure in the cytoplasm of the attacker cell that propels an arrow made of a haemolysin co-regulated protein (Hcp) tube and a valine–glycine repeat protein G (VgrG) spike and punctures the prey’s cell wall. The nano-crossbow is stably anchored to the cell envelope of the attacker by a membrane core complex. Here we show that this complex is assembled by the sequential addition of three type VI subunits (Tss)— TssJ, TssM and TssL—and present a structure of the fully assembled complex at 11.6 A ̊ resolution, determined by negative-stain electron microscopy. With overall C5 symmetry, this 1.7-megadalton complex comprises a large base in the cytoplasm. It extends in the periplasm via ten arches to form a double-ring structure containing the carboxy-terminal domain of TssM (TssMct) and TssJ that is anchored in the outer membrane. The crystal structure of the TssMct–TssJ complex coupled to whole-cell accessibility studies suggest that large conformational changes induce transient pore formation in the outer membrane, allowing passage of the attacking Hcp tube/VgrG spike.
Published: 2015

47. X-ray and Cryo-electron Microscopy Structures of Monalysin Pore-forming Toxin Reveal Multimerization of the Pro-form

Author: Philippe Leone, Cecilia Bebeacua, Bruno Lemaitre, Christine Kellenberger, Alain Roussel, Onya Opota, Igor Orlov, Bruno P. Klaholz, Christian Cambillau, Architecture et fonction des macromolécules biologiques (AFMB), Institut National de la Recherche Agronomique (INRA)-Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), and Centre National de la Recherche Scientifique (CNRS)-Aix Marseille Université (AMU)-Institut National de la Recherche Agronomique (INRA)
Subjects: Models, Molecular, Pore Forming Cytotoxic Proteins, animal structures, Cryo-electron microscopy, Stereochemistry, Bacterial Toxins, Molecular Sequence Data, Aerolysin, Biology, Crystallography, X-Ray, Cleavage (embryo), Biochemistry, Cell membrane, Protein structure, Pseudomonas, medicine, Amino Acid Sequence, Molecular Biology, ComputingMilieux_MISCELLANEOUS, Pore-forming toxin, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Sequence Homology, Amino Acid, Cell Membrane, Cryoelectron Microscopy, Cell Biology, [SDV.BIBS]Life Sciences [q-bio]/Quantitative Methods [q-bio.QM], Transmembrane protein, Protein Structure, Tertiary, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], medicine.anatomical_structure, Membrane, Protein Structure and Folding, Mutation, Protein Multimerization, Crystallization, Protein Binding
Abstract: beta-Barrel pore-forming toxins (beta-PFT), a large family of bacterial toxins, are generally secreted as water-soluble monomers and can form oligomeric pores in membranes following proteolytic cleavage and interaction with cell surface receptors. Monalysin has been recently identified as a beta-PFT that contributes to the virulence of Pseudomonas entomophila against Drosophila. It is secreted as a pro-protein that becomes active upon cleavage. Here we report the crystal and cryo-electron microscopy structure of the pro-form of Monalysin as well as the crystal structures of the cleaved form and of an inactive mutant lacking the membrane-spanning region. The overall structure of Monalysin displays an elongated shape, which resembles those of beta-poreforming toxins, such as Aerolysin, but is devoid of a receptorbinding domain. X-ray crystallography, cryo-electron microscopy, and light-scattering studies show that pro-Monalysin forms a stable doughnut-like 18-mer complex composed of two disk-shaped nonamers held together by N-terminal swapping of the pro-peptides. This observation is in contrast with the monomeric pro-form of the other beta-PFTs that are receptor-dependent for membrane interaction. The membrane-spanning region of pro-Monalysin is fully buried in the center of the doughnut, suggesting that upon cleavage of pro-peptides, the two disk-shaped nonamers can, and have to, dissociate to leave the transmembrane segments free to deploy and lead to pore formation. In contrast with other toxins, the delivery of 18 subunits at once, nearby the cell surface, may be used to bypass the requirement of receptor-dependent concentration to reach the threshold for oligomerization into the pore-forming complex.
Published: 2015

48. Production, crystallization and X-ray diffraction analysis of a complex between a fragment of the TssM T6SS protein and a camelid nanobody

Author: Christian Cambillau, Aline Desmyter, Thi Thu Hang Le, Alain Roussel, Van Son Nguyen, Silvia Spinelli, Eric Cascales, Christine Kellenberger, Architecture et fonction des macromolécules biologiques (AFMB), Institut National de la Recherche Agronomique (INRA)-Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), Laboratoire d'ingénierie des systèmes macromoléculaires (LISM), Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Aix Marseille Université (AMU), Aix Marseille Université (AMU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), and Centre National de la Recherche Scientifique (CNRS)-Aix Marseille Université (AMU)-Institut National de la Recherche Agronomique (INRA)
Subjects: [SDV]Life Sciences [q-bio], Molecular Sequence Data, Biophysics, Myoviridae, Biology, Crystallography, X-Ray, medicine.disease_cause, Biochemistry, Research Communications, Structural Biology, Escherichia coli, Genetics, medicine, Animals, Inner membrane, Molecular replacement, Amino Acid Sequence, Bacterial Secretion Systems, ComputingMilieux_MISCELLANEOUS, Type VI secretion system, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Escherichia coli Proteins, Membrane Proteins, Single-Domain Antibodies, Condensed Matter Physics, biology.organism_classification, [SDV.BIBS]Life Sciences [q-bio]/Quantitative Methods [q-bio.QM], Peptide Fragments, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], Membrane, Membrane protein, biological sciences, health occupations, bacteria, Crystallization, Bacterial outer membrane, Camelids, New World
Abstract: The type VI secretion system (T6SS) is a machine evolved by Gram-negative bacteria to deliver toxin effectors into target bacterial or eukaryotic cells. The T6SS is functionally and structurally similar to the contractile tail of theMyoviridaefamily of bacteriophages and can be viewed as a syringe anchored to the bacterial membrane by a transenvelope complex. The membrane complex is composed of three proteins: the TssM and TssL inner membrane components and the TssJ outer membrane lipoprotein. The TssM protein is central as it interacts with both TssL and TssJ, therefore linking the membranes. Using controlled trypsinolysis, a 32.4 kDa C-terminal fragment of enteroaggregativeEscherichia coliTssM (TssM32Ct) was purified. A nanobody obtained from llama immunization, nb25, exhibited subnanomolar affinity for TssM32Ct. Crystals of the TssM32Ct–nb25 complex were obtained and diffracted to 1.9 Å resolution. The crystals belonged to space groupP64, with unit-cell parametersa = b = 95.23,c= 172.95 Å. Molecular replacement with a model nanobody indicated the presence of a dimer of TssM32Ct–nb25 in the asymmetric unit.
Published: 2015

49. Crystallization and preliminary X-ray analysis of the C-terminal fragment of PorM, a subunit of the Porphyromonas gingivalis type IX secretion system

Author: Philippe Leone, Julien Stathopulos, Christian Cambillau, Alain Roussel, Eric Cascales, 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), Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Aix Marseille Université (AMU), Architecture et fonction des macromolécules biologiques (AFMB), Institut National de la Recherche Agronomique (INRA)-Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), and Centre National de la Recherche Scientifique (CNRS)-Aix Marseille Université (AMU)-Institut National de la Recherche Agronomique (INRA)
Subjects: Proteolysis, Protein subunit, [SDV]Life Sciences [q-bio], Biophysics, macromolecular substances, Crystallography, X-Ray, medicine.disease_cause, Biochemistry, Research Communications, 03 medical and health sciences, Bacterial Proteins, Structural Biology, Genetics, medicine, Secretion, Bacterial Secretion Systems, Escherichia coli, Porphyromonas gingivalis, Pathogen, ComputingMilieux_MISCELLANEOUS, 030304 developmental biology, 0303 health sciences, medicine.diagnostic_test, biology, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], 030306 microbiology, Periplasmic space, Condensed Matter Physics, biology.organism_classification, [SDV.BIBS]Life Sciences [q-bio]/Quantitative Methods [q-bio.QM], Protein Structure, Tertiary, Protein Subunits, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], Membrane protein, Chromatography, Gel, Crystallization, human activities
Abstract: PorM is a membrane protein involved in the assembly of the type IX secretion system (T9SS) fromPorphyromonas gingivalis, a major bacterial pathogen responsible for periodontal disease in humans. The periplasmic domain of PorM was overexpressed inEscherichia coliand purified. A fragment of the purified protein was obtained by limited proteolysis. Crystals of this fragment belonged to the tetragonal space groupP43212. Native and MAD data sets were recorded to 2.85 and 3.1 Å resolution, respectively, using synchrotron radiation.
Published: 2015

50. An Archaeal Peptidase Assembles into Two Different Quaternary Structures

Author: M. Asunción Durá, Bruno Franzetti, Christine Ebel, Frédéric M. D. Vellieux, Celine Fabry, Rob W.H. Ruigrok, Alain Roussel, Guy Schoehn, and Véronique Receveur-Bréchot
Subjects: 0303 health sciences, biology, 030302 biochemistry & molecular biology, Peptidase complex, Cell Biology, biology.organism_classification, Biochemistry, Aminopeptidase, 03 medical and health sciences, Pyrococcus horikoshii, Crystallography, Octahedron, Hydrolase, Tetrahedron, Molecular Biology, Internal organization, 030304 developmental biology, Archaea
Abstract: Cellular proteolysis involves large oligomeric peptidases that play key roles in the regulation of many cellular processes. The cobalt-activated peptidase TET1 from the hyperthermophilic Archaea Pyrococcus horikoshii (PhTET1) was found to assemble as a 12-subunit tetrahedron and as a 24-subunit octahedral particle. Both quaternary structures were solved by combining x-ray crystallography and cryoelectron microscopy data. The internal organization of the PhTET1 particles reveals highly self-compartmentalized systems made of networks of access channels extended by vast catalytic chambers. The two edifices display aminopeptidase activity, and their organizations indicate substrate navigation mechanisms different from those described in other large peptidase complexes. Compared with the tetrahedron, the octahedron forms a more expanded hollow structure, representing a new type of giant peptidase complex. PhTET1 assembles into two different quaternary structures because of quasi-equivalent contacts that previously have only been identified in viral capsids.
Published: 2006

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