Eric Durand

Aix-Marseille Université, Marsiglia, Provence-Alpes-Côte d'Azur, France

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Publications (30)136.64 Total impact

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    ABSTRACT: 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)[mdash]TssJ, TssM and TssL[mdash]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.
    Full-text · Article · Jul 2015 · Nature
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    ABSTRACT: The type VI secretion system (T6SS) is a secretion pathway widespread in Gram-negative bacteria that targets toxins in both prokaryotic and eukaryotic cells. Although most T6SSs identified so far are involved in inter-bacterial competition, a few are directly required for full virulence of pathogens. The T6SS comprises 13 core proteins that assemble a large complex structurally and functionally similar to a phage contractile tail structure anchored to the cell envelope by a trans-membrane spanning stator. The central part of this stator, TssM, is a 1129-amino-acid protein anchored in the inner membrane that binds to the TssJ outer membrane lipoprotein. In this study, we have raised camelid antibodies against the purified TssM periplasmic domain. We report the crystal structure of two specific nanobodies that bind to TssM in the nanomolar range. Interestingly, the most potent nanobody, nb25, competes with the TssJ lipoprotein for TssM binding in vitro suggesting that TssJ and the nb25 CDR3 loop share the same TssM binding site or causes a steric hindrance preventing TssM-TssJ complex formation. Indeed, periplasmic production of the nanobodies displacing the TssM-TssJ interaction inhibits the T6SS function in vivo. This study illustrates the power of nanobodies to specifically target and inhibit bacterial secretion systems.
    Full-text · Article · Mar 2015 · PLoS ONE
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    ABSTRACT: The type VI secretion system (T6SS) is a macromolecular machine that delivers protein effectors into both prokaryotic and eukaryotic cells, therefore participating in interbacterial competition and virulence. The T6SS is functionally and structurally similar to the contractile bacteriophage cell puncturing device: the contraction of a sheath-like structure is believed to propel an inner tube terminated by a spike towards target cells, allowing the delivery of effectors. In this review, we summarize recent advances in the identification and characterization of T6SS effector proteins, highlighting the broad repertoire of enzymatic activities, and discuss recent findings relating to the secretion mechanisms.
    Full-text · Article · Sep 2014 · Trends in Microbiology
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    ABSTRACT: The structural characterization of various constructs of the Measles virus (MeV) Phosphoprotein (P) multimerization domain (PMD) has brought to light significant discrepancies in the quaternary structure due to both crystal constraints and the flexible nature of this coiled-coil. Indeed, despite a conserved tetrameric parallel coiled-coil core, structural comparison unveiled significant deformations in the C-terminal extremities that even led to the partial unfolding of the coiled-coil. These deformations were induced by intermolecular interactions within the crystal, as well as by the crystallization condition. These deformations also suggest that PMD has the ability to adapt to external mechanical constrains. Using a combination of biophysical methods (size-exclusion chromatography, circular dichroism and small angle X-ray scattering), we assessed the differential flexibility of the C-terminal region of the MeV PMD in solution. Taken together, these results show that crystal packing can be used to "freeze" in a certain state, parts of proteins known to be in a dynamic folding-unfolding equilibrium. They also bring awareness that conclusions about function and mechanism based on analysis of a single crystal structure of a known dynamic protein can be easily biased, and they challenge to some extent the assumption that coiled-coil structures can be reliably predicted from the amino acid sequence.
    Full-text · Article · Aug 2014 · Acta Crystallographica Section A: Foundations and Advances
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    ABSTRACT: The structures of two constructs of the measles virus (MeV) phosphoprotein (P) multimerization domain (PMD) are reported and are compared with a third structure published recently by another group [Communie et al. (2013), J. Virol. 87 , 7166–7169]. Although the three structures all have a tetrameric and parallel coiled-coil arrangement, structural comparison unveiled considerable differences in the quaternary structure and unveiled that the three structures suffer from significant structural deformation induced by intermolecular interactions within the crystal. These results show that crystal packing can bias conclusions about function and mechanism based on analysis of a single crystal structure, and they challenge to some extent the assumption according to which coiled-coil structures can be reliably predicted from the amino-acid sequence. Structural comparison also highlighted significant differences in the extent of disorder in the C-terminal region of each monomer. The differential flexibility of the C-terminal region is also supported by size-exclusion chromatography and small-angle X-ray scattering studies, which showed that MeV PMD exists in solution as a dynamic equilibrium between two tetramers of different compaction. Finally, the possible functional implications of the flexibility of the C-terminal region of PMD are discussed.
    Full-text · Article · Jun 2014 · Acta Crystallographica Section D Biological Crystallography
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    ABSTRACT: The Type VI secretion system (T6SS) delivers protein effectors to diverse cell types including prokaryotic and eukaryotic cells, therefore participating in inter-bacterial competition and pathogenesis. The T6SS is constituted of an envelope-spanning complex anchoring a cytoplasmic tubular edifice. This tubular structure is evolutionarily, functionally and structurally related to the tail of contractile phages. It is composed of an inner tube tipped by a spike complex, and engulfed within a sheath-like structure. This structure assembles onto a platform called "baseplate" that is connected to the membrane sub-complex. The T6SS functions as a nano-crossbow: upon contraction of the sheath, the inner tube is propelled towards the target cell, allowing effector delivery. This review focuses on the architecture and biogenesis of this fascinating secretion machine, highlighting recent advances regarding the assembly of the membrane or tail complexes. This article is part of a Special Issue entitled: Protein Trafficking & Secretion.
    Full-text · Article · Mar 2014 · Biochimica et Biophysica Acta
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    Dataset: Figure S1

    Full-text · Dataset · Mar 2014
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    Dataset: Figure S2

    Full-text · Dataset · Mar 2014
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    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.
    Full-text · Article · Feb 2014 · PLoS ONE
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    ABSTRACT: The Type VI secretion system (T6SS) is a macromolecular machine that mediates bacteria-host or bacteria-bacteria interactions. The T6SS core apparatus assembles from 13 proteins that form two sub-assemblies: a phage-like complex and a trans-envelope complex. The Hcp, VgrG, TssE, and TssB/C subunits are structurally and functionally related to components of the tail of contractile bacteriophages. This phage-like structure is thought to be anchored to the membrane by a trans-envelope complex composed of the TssJ, TssL, and TssM proteins. However, how the two sub-complexes are connected remains unknown. Here we identify TssK, a protein that establishes contacts with the two T6SS sub-complexes through direct interactions with TssL, Hcp, and TssC. TssK is a cytoplasmic protein assembling trimers that display a three-armed shape, as revealed by TEM and SAXS analyses. Fluorescence microscopy experiments further demonstrate the requirement of TssK for sheath assembly. Our results suggest a central role for TssK by linking both complexes during T6SS assembly.
    No preview · Article · Aug 2013 · Journal of Biological Chemistry
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    ABSTRACT: The Type VI secretion system (T6SS) is a macromolecular complex widespread in Gram-negative bacteria. Although several T6SS are required for virulence towards host models, most are necessary to eliminate competitor bacteria. Other functions, such as resistance to amoeba predation, biofilm formation or adaptation to environmental conditions have also been reported. This multitude of functions is reflected by the large repertoire of regulatory mechanisms shown to control T6SS expression, production or activation. Here, we demonstrate that one T6SS gene cluster encoded within the Yersinia pseudotuberculosis genome, T6SS-4, is regulated by OmpR, the response regulator of the two-component system EnvZ-OmpR. We first identified OmpR in a transposon mutagenesis screen. OmpR does not control the expression of the four other Y. pseudotuberculosis T6SS gene clusters and of an isolated vgrG gene, and responds to osmotic stresses to bind to and activate the T6SS-4 promoter. Finally, we show that T6SS-4 promotes Y. pseudotuberculosis survival in high osmolarity conditions and resistance to deoxycholate.
    Full-text · Article · Jun 2013 · PLoS ONE
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    ABSTRACT: Vibrio cholerae is the cause of the diarrheal disease cholera. V. cholerae produces RtxA, a large toxin of the MARTX family, which is targeted to the host cell cytosol, where its actin cross-linking domain (ACD) cross-links G-actin, leading to F-actin depolymerization, cytoskeleton rearrangements, and cell rounding. These effects on the cytoskeleton prevent phagocytosis and bacterial engulfment by macrophages, thus preventing V. cholerae clearance from the gut. The V. cholerae Type VI secretion-associated VgrG1 protein also contains a C-terminal ACD, which shares 61% identity with MARTX ACD and has been shown to covalently cross-link G-actin. Here, we purified the VgrG1 C-terminal domain and determined its crystal structure. The VgrG1 ACD exhibits a V-shaped three-dimensional structure, formed of 12 β-strands and nine α-helices. Its active site comprises five residues that are conserved in MARTX ACD toxin, within a conserved area of ∼10 Å radius. We showed that less than 100 ACD molecules are sufficient to depolymerize the actin filaments of a fibroblast cell in vivo. Mutagenesis studies confirmed that Glu-16 is critical for the F-actin depolymerization function. Co-crystals with divalent cations and ATP reveal the molecular mechanism of the MARTX/VgrG toxins and offer perspectives for their possible inhibition.
    Preview · Article · Aug 2012 · Journal of Biological Chemistry

  • No preview · Article · Apr 2012 · Journal of Biological Chemistry
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    ABSTRACT: The Type VI secretion system (T6SS) is a macromolecular system distributed in Gram-negative bacteria, responsible for the secretion of effector proteins into target cells. The T6SS has a broad versatility as it can target both eukaryotic and prokaryotic cells. It is therefore involved in host pathogenesis or killing neighboring bacterial cells to colonize a new niche. At the architecture level, the T6SS core apparatus is composed of 13 proteins, which assemble in two subcomplexes. One of these subcomplexes, composed of subunits that share structural similarities with bacteriophage tail and baseplate components, is anchored to the cell envelope by the membrane subcomplex. This latter is constituted of at least three proteins, TssL, TssM, and TssJ. The crystal structure of the TssJ outer membrane lipoprotein and its interaction with the inner membrane TssM protein have been recently reported. TssL and TssM share sequence homology and characteristics with two components of the Type IVb secretion system (T4bSS), IcmH/DotU and IcmF, respectively. In this study, we report the crystal structure of the cytoplasmic domain of the TssL inner membrane protein from the enteroaggregative Escherichia coli Sci-1 T6SS. It folds as a hook-like structure composed of two three-helix bundles. Two TssL molecules associate to form a functional complex. Although the TssL trans-membrane segment is the main determinant of self-interaction, contacts between the cytoplasmic domains are required for TssL function. Based on sequence homology and secondary structure prediction, we propose that the TssL structure is the prototype for the members of the TssL and IcmH/DotU families.
    Full-text · Article · Feb 2012 · Journal of Biological Chemistry
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    ABSTRACT: Author Summary Type VI secretion systems (T6SS) are specialized secretion machines responsible for the transport of virulence factors. T6SS are versatile as they are able to target both eukaryotic and prokaryotic cells. They therefore play an important role in pathogenesis by acting directly on the host, as well as eliminating competing bacteria from the niche. At a molecular level, T6SS are composed of a minimum of 13 proteins called core-components, all required for the activity of the secretion system. These core-components can be divided in two groups: soluble proteins having a common evolution history with bacteriophage T4 subunits, and membrane or membrane-associated proteins required for anchoring the bacteriophage-like structure to the envelope. Here, we report the crystal structure of one of the membrane-associated core component, the TssJ lipoprotein. The structure exhibits a transthyretin fold supplemented with additional structural elements. One of these, a loop connecting two beta-strands, is responsible for the interaction of the TssJ lipoprotein with the C-terminal domain of the inner membrane protein TssM. We propose that these two proteins link the two membranes and form a channel accommodating the bacteriophage-like structure. These results provide important new insights to understand the biogenesis of these secretion apparati.
    Full-text · Article · Nov 2011 · PLoS Pathogens
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    ABSTRACT: In Gram-negative bacteria, type II secretion systems assemble a piston-like structure, called pseudopilus, which expels exoproteins out of the cell. The pseudopilus is constituted by a major pseudopilin that when overproduced multimerizes into a long cell surface structure named hyper-pseudopilus. Pseudomonas aeruginosa possesses two type II secretion systems, Xcp and Hxc. Although major pseudopilins are exchangeable among type II secretion systems, we show that XcpT and HxcT are not. We demonstrate that HxcT does not form a hyper-pseudopilus and is different in amino acid sequence and multimerization properties. Using structure-based mutagenesis, we observe that five mutations are sufficient to revert HxcT into a functional XcpT-like protein, which also becomes capable of forming a hyper-pseudopilus. Phylogenetic and experimental analysis showed that the whole Hxc system was acquired by P. aeruginosa PAO1 and other Pseudomonas species through horizontal gene transfer. We thus identified a new type II secretion subfamily, of which the P. aeruginosa Hxc system is the archetype. This finding demonstrates how similar bacterial machineries evolve toward distinct mechanisms that may contribute specific functions.
    Full-text · Article · Jul 2011 · Journal of Biological Chemistry
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    ABSTRACT: In Gram-negative bacteria, type II secretion systems assemble a piston-like structure, called pseudopilus, which expels exoproteins out of the cell. The pseudopilus is constituted by a major pseudopilin that when overproduced multimerizes into a long cell surface structure named hyper-pseudopilus. Pseudomonas aeruginosa possesses two type II secretion systems, Xcp and Hxc. Although major pseudopilins are exchangeable among type II secretion systems, we show that XcpT and HxcT are not. We demonstrate that HxcT does not form a hyper-pseudopilus and is different in amino acid sequence and multimerization properties. Using structure-based mutagenesis, we observe that five mutations are sufficient to revert HxcT into a functional XcpT-like protein, which also becomes capable of forming a hyper-pseudopilus. Phylogenetic and experimental analysis showed that the whole Hxc system was acquired by P. aeruginosa PAO1 and other Pseudomonas species through horizontal gene transfer. We thus identified a new type II secretion subfamily, of which the P. aeruginosa Hxc system is the archetype. This finding demonstrates how similar bacterial machineries evolve toward distinct mechanisms that may contribute specific functions.
    Full-text · Article · May 2011 · Journal of Biological Chemistry
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    ABSTRACT: Pseudomonas aeruginosa utilizes the type II secretion machinery to transport virulence factors through the outer membrane into the extracellular space. Five proteins in the type II secretion system share sequence homology with pilin subunits of type IV pili and are called the pseudopilins. The major pseudopilin XcpT(G) assembles into an intraperiplasmic pilus and is thought to act in a piston-like manner to push substrates through an outer membrane secretin. The other four minor pseudopilins, XcpU(H), XcpV(I), XcpW(J) and XcpX(K), play less well defined roles in pseudopilus formation. It was recently discovered that these four minor pseudopilins form a quaternary complex that is presumed to initiate the formation of the pseudopilus and to localize to its tip. Here, the structure of XcpW(J) was refined to 1.85 Å resolution. The structure revealed the type IVa pilin fold with an embellished variable antiparallel β-sheet as also found in the XcpW(J) homologue enterotoxigenic Escherichia coli GspJ(W) and the XcpU(H) homologue Vibrio cholerae EpsU(H). It is proposed that the exposed surface of this sheet may cradle the long N-terminal α1 helix of another pseudopilin. The final 31 amino acids of the XcpW(J) structure are instrinsically disordered. Deletion of this unstructured region of XcpW(J) did not prevent type II secretion in vivo.
    Full-text · Article · Feb 2011 · Acta Crystallographica Section D Biological Crystallography
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    ABSTRACT: Type IV secretion (T4S) systems are involved in secretion of virulence factors such as toxins or transforming molecules, or bacterial conjugation. T4S systems are composed of 12 proteins named VirB1-B11 and VirD4. Among them, three ATPases are involved in the assembly of the T4S system and/or provide energy for substrate transfer, VirB4, VirB11 and VirD4. The X-ray crystal structures of VirB11 and VirD4 have already been solved but VirB4 has proven to be reluctant to any structural investigation so far. Here, we have used small-angle X-ray scattering to obtain the first structural models for the membrane-extracted, dimeric form of the TraB protein, the VirB4 homolog encoded by the E. coli pKM101 plasmid, and for the monomeric soluble form of the LvhB4 protein, the VirB4 homolog of the T4S system encoded by the Legionella pneumophila lvh operon. We have obtained the low resolution structures of the full-length TraB and of its N- and C-terminal halves. From these SAXS models, we derive the internal organisation of TraB. We also show that the two TraB N- and C-terminal domains are independently involved in the dimerisation of the full-length protein. These models provide the first structural insights into the architecture of VirB4 proteins. In particular, our results highlight the modular arrangement and functional relevance of the dimeric-membrane-bound form of TraB.
    Full-text · Article · Jan 2011 · BMC Structural Biology
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    Full-text · Article · Jan 2011

Publication Stats

646 Citations
136.64 Total Impact Points

Institutions

  • 2009-2015
    • Aix-Marseille Université
      • Unité de Recherche d'Architecture et Fonction des Macromolécules Biologiques (UMR 7257 AFMB)
      Marsiglia, Provence-Alpes-Côte d'Azur, France
  • 2007-2014
    • French National Centre for Scientific Research
      • Institut de Microbiologie de la Méditerranée
      Lutetia Parisorum, Île-de-France, France
    • Utrecht University
      Utrecht, Utrecht, Netherlands
  • 2009-2011
    • Birkbeck, University of London
      • Institute of Structural and Molecular Biology
      Londinium, England, United Kingdom