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Search Results: 1 - 10 of 14770 matches for " Christian Cambillau "
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Structures and host-adhesion mechanisms of lactococcal siphophages
Silvia Spinelli,David Veesler,Cecilia Bebeacua,Christian Cambillau
Frontiers in Microbiology , 2014, DOI: 10.3389/fmicb.2014.00003
Abstract: The Siphoviridae family of bacteriophages is the largest viral family on earth and comprises members infecting both bacteria and archaea. Lactococcal siphophages infect the Gram-positive bacterium Lactococcus lactis, which is widely used for industrial milk fermentation processes (e.g. cheese production). As a result, lactococcal phages have become one of the most thoroughly characterized class of phages from a genomic standpoint. They exhibit amazing and intriguing characteristics. First, each phage has a strict specificity towards a unique or a handful of L. lactis host strains. Second, most lactococcal phages possess a large organelle at their tail tip (termed the baseplate), bearing the receptor binding proteins and mediating host adsorption. The recent accumulation of structural and functional data revealed the modular structure of their building blocks, their different mechanisms of activation and the fine specificity of their receptor binding proteins. These results also illustrated similarities and differences between lactococcal Siphoviridae and Gram-negative infecting Myoviridae.
Revised Mimivirus major capsid protein sequence reveals intron-containing gene structure and extra domain
Sa?d Azza, Christian Cambillau, Didier Raoult, Marie Suzan-Monti
BMC Molecular Biology , 2009, DOI: 10.1186/1471-2199-10-39
Abstract: This study describes the full length 3430 bp Capsid coding gene and characterises the 593 amino acids long corresponding Capsid protein 1. The recombinant full length protein allowed the production of a specific monoclonal antibody able to detect the Capsid protein 1 within the viral particle. This protein appeared to be post-translationnally modified by glycosylation and phosphorylation. We proposed a secondary structure prediction of APM Capsid protein 1 compared to the Capsid protein structure of Paramecium Bursaria Chlorella Virus 1, another member of the Nucleo-Cytoplasmic Large DNA virus family.The characterisation of the full length L425 Capsid coding gene of Acanthamoebae polyphaga Mimivirus provides new insights into the structure of the main Capsid protein. The production of a full length recombinant protein will be useful for further structural studies.Acanthamoebae polyphaga Mimivirus was described for the first time in 2003 [1]. It was isolated from amoebae growing in water sample from a cooling tower during an outbreak of pneumonia in an English hospital. Compared to other members of the Nucleo-Cytoplasmic Large DNA Viruses (NCLDV) family [2], APM has very particular characteristics due to its size, structure, genome sequence [3], and replication cycle through a specific virus factory [4]. The virus particle was shown to be icosahedral, with a capsid shell diameter of 5000 ? covered by long fibers and appears to have at least two lipid membranes beneath its Capsid protein [5]. The APM 1.2-Mb genome encodes at least 911 proteins [Genbank: NC_006450] [6]. Proteomic analysis of proteins extracted from purified viral particles allowed the identification of 114 proteins including the Capsid protein D13L, coded by the L425 gene. The D13L Capsid protein was shown to be the most abundant and major glycoprotein of APM [7] and is thought to be the main component of the outermost protein shell layer. The protein sequence was deduced from the first available Methi
Expanding the molecular toolbox for Lactococcus lactis: construction of an inducible thioredoxin gene fusion expression system
Fran?ois P Douillard, Mary O'Connell-Motherway, Christian Cambillau, Douwe van Sinderen
Microbial Cell Factories , 2011, DOI: 10.1186/1475-2859-10-66
Abstract: Mimicking thioredoxin gene fusion systems available for E. coli, two nisin-inducible expression vectors were constructed to over-produce various proteins in L. lactis as thioredoxin fusion proteins. In this study, we demonstrate that our novel L. lactis fusion partner expression vectors allow high-level expression of soluble heterologous proteins Tuc2009 ORF40, Bbr_0140 and Tuc2009 BppU/BppL that were previously insoluble or not expressed using existing L. lactis expression vectors. Over-expressed proteins were subsequently purified by Ni-TED affinity chromatography. Intact heterologous proteins were detected by immunoblotting analyses. We also show that the thioredoxin moiety of the purified fusion protein was specifically and efficiently cleaved off by enterokinase treatment.This study is the first description of a thioredoxin gene fusion expression system, purposely developed to circumvent problems associated with protein over-expression in L. lactis. It was shown to prevent protein insolubility and degradation, allowing sufficient production of soluble proteins for further structural and functional characterization.The food-grade bacterium L. lactis subsp. cremoris in conjunction with the Nisin Inducible Controlled Expression (NICE) system [1-3] has been extensively used over the last few decades as a valuable bacterial expression system for large-scale production of homologous or heterologous proteins [4], metabolic studies [5], or membrane proteins [6]. The NICE system is based on the well characterized nisin-dependent, quorum-sensing mechanism of L. lactis [2,3,7]. It was initially exploited in L. lactis for heterologous protein overexpression and subsequently implemented in several other Gram-positive bacteria [2,3,7-10]. Typically, the genetically-engineered strain L. lactis subsp. cremoris NZ9000 is employed as expression host, as its chromosome contains the signal transduction genes nisR and nisK involved in the nisin-induced transcriptional control of th
Dissection of the TssB-TssC Interface during Type VI Secretion Sheath Complex Formation
Xiang Y. Zhang, Yannick R. Brunet, Laureen Logger, Badreddine Douzi, Christian Cambillau, Laure Journet, Eric Cascales
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0081074
Abstract: The Type VI secretion system (T6SS) is a versatile machine that delivers toxins into either eukaryotic or bacterial cells. At a molecular level, the T6SS is composed of a membrane complex that anchors a long cytoplasmic tubular structure to the cell envelope. This structure is thought to resemble the tail of contractile bacteriophages. It is composed of the Hcp protein that assembles into hexameric rings stacked onto each other to form a tube similar to the phage tail tube. This tube is proposed to be wrapped by a structure called the sheath, composed of two proteins, TssB and TssC. It has been shown using fluorescence microscopy that the TssB and TssC proteins assemble into a tubular structure that cycles between long and short conformations suggesting that, similarly to the bacteriophage sheath, the T6SS sheath undergoes elongation and contraction events. The TssB and TssC proteins have been shown to interact and a specific α-helix of TssB is required for this interaction. Here, we confirm that the TssB and TssC proteins interact in enteroaggregative E. coli. We further show that this interaction requires the N-terminal region of TssC and the conserved α-helix of TssB. Using site-directed mutagenesis coupled to phenotypic analyses, we demonstrate that an hydrophobic motif located in the N-terminal region of this helix is required for interaction with TssC, sheath assembly and T6SS function.
Ligands for Pheromone-Sensing Neurons Are Not Conformationally Activated Odorant Binding Proteins
Carolina Gomez-Diaz,Jaime H. Reina,Christian Cambillau,Richard Benton
PLOS Biology , 2013, DOI: 10.1371/journal.pbio.1001546
Abstract: Pheromones form an essential chemical language of intraspecific communication in many animals. How olfactory systems recognize pheromonal signals with both sensitivity and specificity is not well understood. An important in vivo paradigm for this process is the detection mechanism of the sex pheromone (Z)-11-octadecenyl acetate (cis-vaccenyl acetate [cVA]) in Drosophila melanogaster. cVA-evoked neuronal activation requires a secreted odorant binding protein, LUSH, the CD36-related transmembrane protein SNMP, and the odorant receptor OR67d. Crystallographic analysis has revealed that cVA-bound LUSH is conformationally distinct from apo (unliganded) LUSH. Recombinantly expressed mutant versions of LUSH predicted to enhance or diminish these structural changes produce corresponding alterations in spontaneous and/or cVA-evoked activity when infused into olfactory sensilla, leading to a model in which the ligand for pheromone receptors is not free cVA, but LUSH that is “conformationally activated” upon cVA binding. Here we present evidence that contradicts this model. First, we demonstrate that the same LUSH mutants expressed transgenically affect neither basal nor pheromone-evoked activity. Second, we compare the structures of apo LUSH, cVA/LUSH, and complexes of LUSH with non-pheromonal ligands and find no conformational property of cVA/LUSH that can explain its proposed unique activated state. Finally, we show that high concentrations of cVA can induce neuronal activity in the absence of LUSH, but not SNMP or OR67d. Our findings are not consistent with the model that the cVA/LUSH complex acts as the pheromone ligand, and suggest that pheromone molecules alone directly activate neuronal receptors.
Crystal Structure and Self-Interaction of the Type VI Secretion Tail-Tube Protein from Enteroaggregative Escherichia coli
Badreddine Douzi, Silvia Spinelli, Stéphanie Blangy, Alain Roussel, Eric Durand, Yannick R. Brunet, Eric Cascales, Christian Cambillau
PLOS ONE , 2014, DOI: 10.1371/journal.pone.0086918
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.
Crystal Structure of ATVORF273, a New Fold for a Thermo- and Acido-Stable Protein from the Acidianus Two-Tailed Virus
Catarina Felisberto-Rodrigues, Stéphanie Blangy, Adeline Goulet, Gisle Vestergaard, Christian Cambillau, Roger A. Garrett, Miguel Ortiz-Lombardía
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0045847
Abstract: Acidianus two-tailed virus (ATV) infects crenarchaea of the genus Acidianus living in terrestrial thermal springs at extremely high temperatures and low pH. ATV is a member of the Bicaudaviridae virus family and undergoes extra-cellular development of two tails, a process that is unique in the viral world. To understand this intriguing phenomenon, we have undertaken structural studies of ATV virion proteins and here we present the crystal structure of one of these proteins, ATV. ATV forms tetramers in solution and a molecular envelope is provided for the tetramer, computed from small-angle X-ray scattering (SAXS) data. The crystal structure has properties typical of hyperthermostable proteins, including a relatively high number of salt bridges. However, the protein also exhibits flexible loops and surface pockets. Remarkably, ATV displays a new protein fold, consistent with the absence of homologues of this protein in public sequence databases.
Effect of Rickettsial Toxin VapC on Its Eukaryotic Host
Gilles Audoly, Renaud Vincentelli, Sophie Edouard, Kalliopi Georgiades, Oleg Mediannikov, Grégory Gimenez, Cristina Socolovschi, Jean-Louis Mège, Christian Cambillau, Didier Raoult
PLOS ONE , 2011, DOI: 10.1371/journal.pone.0026528
Abstract: Rickettsia are intracellular bacteria typically associated with arthropods that can be transmitted to humans by infected vectors. Rickettsia spp. can cause mild to severe human disease with a possible protection effect of corticosteroids when antibiotic treatments are initiated. We identified laterally transferred toxin-antitoxin (TA) genetic elements, including vapB/C, in several Rickettsia genomes and showed that they are functional in bacteria and eukaryotic cells. We also generated a plaque assay to monitor the formation of lytic plaques over time and demonstrated that chloramphenicol accelerates host cell lysis of vapB/C-containing Rickettsia. Whole-genome expression, TUNEL and FISH assays on the infected cells following exposure to the antibiotic revealed early apoptosis of host cells, which was linked to over-transcription of bacterial vapB/C operons and subsequent cytoplasmic VapC toxin release. VapC that is expressed in Escherichia coli and Saccharomyces cerevisiae or microinjected into mammalian cells is toxic through RNase activity and is prevented by dexamethasone. This study provides the first biological evidence that toxin–antitoxin elements act as pathogenic factors in bacterial host cells, confirming comparative genomic evidence of their role in bacterial pathogenicity. Our results suggest that early mortality following antibiotic treatment of some bacterial infections can be prevented by administration of dexamethasone.
Towards a Structural Comprehension of Bacterial Type VI Secretion Systems: Characterization of the TssJ-TssM Complex of an Escherichia coli Pathovar
Catarina Felisberto-Rodrigues,Eric Durand,Marie-Stéphanie Aschtgen,Stéphanie Blangy,Miguel Ortiz-Lombardia,Badreddine Douzi,Christian Cambillau ,Eric Cascales
PLOS Pathogens , 2011, DOI: 10.1371/journal.ppat.1002386
Abstract: Type VI secretion systems (T6SS) are trans-envelope machines dedicated to the secretion of virulence factors into eukaryotic or prokaryotic cells, therefore required for pathogenesis and/or for competition towards neighboring bacteria. The T6SS apparatus resembles the injection device of bacteriophage T4, and is anchored to the cell envelope through a membrane complex. This membrane complex is composed of the TssL, TssM and TagL inner membrane anchored proteins and of the TssJ outer membrane lipoprotein. Here, we report the crystal structure of the enteroaggregative Escherichia coli Sci1 TssJ lipoprotein, a two four-stranded β-sheets protein that exhibits a transthyretin fold with an additional α-helical domain and a protruding loop. We showed that TssJ contacts TssM through this loop since a loop depleted mutant failed to interact with TssM in vitro or in vivo. Biophysical analysis of TssM and TssJ-TssM interaction suggest a structural model of the membrane-anchored outer shell of T6SS. Collectively, our results provide an improved understanding of T6SS assembly and encourage structure-aided drug design of novel antimicrobials targeting T6SS.
Crystal structure of AFV3-109, a highly conserved protein from crenarchaeal viruses
Jenny Keller, Nicolas Leulliot, Christian Cambillau, Valérie Campanacci, Stéphanie Porciero, David Prangishvili, Patrick Forterre, Diego Cortez, Sophie Quevillon-Cheruel, Herman van Tilbeurgh
Virology Journal , 2007, DOI: 10.1186/1743-422x-4-12
Abstract: Studies on viral diversity in geothermally heated aquatic environments, at temperatures above 80°C, resulted in isolation of about two dozens of double-stranded DNA viruses infecting members of the third domain of life, the Archaea [1]. The viruses have diverse unusual morphotypes, not encountered among dsDNA viruses of the Bacteria or Eukarya. Moreover, the detailed analysis of their genomes led to the conclusion that hyperthermophilic archaeal viruses are evolutionarily unrelated to other known viruses, and form a singular group in the viral world with a unique origin, or more likely, multiple origins [2]. Based on morphological and genomic characteristics, these viruses have been assigned to seven novel viral families: stiff, rod-shaped Rudiviridae [3], filamentous Lipothrixviridae [4-7], spindle-shaped Fuselloviridae [8-10], droplet-shaped SNDV [11], spherical "Globuloviridae" [12,13], bottle-shaped "Ampullaviridae" [14], and two-tailed "Bicaudaviridae" [15]. Three more hyperthermophilic archaeal viruses, the icosahedral STIV [16], spindle-shaped STSV1 [17] and PSV [18] have still not been classified.A most prominent feature of the genomes of hyperthermophilic archaeal viruses is an extremely low number of genes coding for proteins homologous to any sequences in the existing sequence databases, be it proteins of other viruses or those of cellular life forms [2]. A few encoded proteins, functions of which have been recognised and confirmed biochemically, include the dUTPase [3] and the Holliday junction resolvase [19] of the rudiviruses SIRV1 and SIRV2 and the integrase/recombinase of the fusellovirus SSV1 [20]. The viruses from different families share a very small pool of genes with putative functions, including predicted transcription regulators, glycosylases, ATPase, as well as small proteins of unknown function from an uncharacterized YddF family [2]. The later protein family has three bacterial representatives in Bacillus subtilis, Clostridium beijerincki,
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