Eric Durand

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

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Publications (26)107.45 Total impact

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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.
    Biochimica et Biophysica Acta 03/2014; · 4.66 Impact Factor
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    ABSTRACT: We herein report the structure of two constructs of the measles virus (MeV) phosphoprotein (P) multimerization domain (PMD) and compare them with a third one published recently by another group (Communie et al., 2013). Although the three structures have all a tetrameric and parallel coiled-coil arrangement, structural comparison unveiled considerable differences in the quaternary structure. Indeed, the three structures suffering 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 that 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.
    Acta Crystallographica Section D Biological Crystallography 01/2014; · 14.10 Impact Factor
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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.
    PLoS ONE 01/2014; 9(2):e86918. · 3.53 Impact Factor
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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.
    Trends in microbiology. 01/2014;
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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 analysis. 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.
    Journal of Biological Chemistry 08/2013; · 4.65 Impact Factor
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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.
    PLoS ONE 01/2013; 8(6):e66615. · 3.53 Impact Factor
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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 depolymerisation, 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 contains also 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 9 α-helices. Its active site comprises five residues that are conserved in MARTX ACD toxin, within a conserved area of ~10 A 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.
    Journal of Biological Chemistry 08/2012; · 4.65 Impact Factor
  • Journal of Biological Chemistry 04/2012; 17(287):14157-68. · 4.65 Impact Factor
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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.
    Journal of Biological Chemistry 02/2012; 287(17):14157-68. · 4.65 Impact Factor
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    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.
    PLoS Pathogens 11/2011; 7(11):e1002386. · 8.14 Impact Factor
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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.
    Journal of Biological Chemistry 07/2011; 286(27):24407-24416. · 4.65 Impact Factor
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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.
    Journal of Biological Chemistry 05/2011; 286(27):24407-16. · 4.65 Impact Factor
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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.
    Acta Crystallographica Section D Biological Crystallography 02/2011; 67(Pt 2):124-30. · 12.67 Impact Factor
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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.
    BMC Structural Biology 01/2011; 11:4. · 2.10 Impact Factor
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    ABSTRACT: Type IV secretion (T4S) systems are involved in several secretion processes, including secretion of virulence factors, such as toxins or transforming molecules, or bacterial conjugation whereby two mating bacteria exchange genetic material. T4S systems are generally composed of 12 protein components, three of which, termed VirB4, VirB11, and VirD4, are ATPases. VirB4 is the largest protein of the T4S system, is known to play a central role, and interacts with many other T4S system proteins. In this study, we have biochemically characterized the protein TraB, a VirB4 homologue from the pKM101 conjugation T4S system. We demonstrated that TraB is a modular protein, composed of two domains, both able to bind DNA in a non-sequence-specific manner. Surprisingly, both TraB N- and C-terminal domains can bind ATP, revealing a new degenerated nucleotide-binding site in the TraB N-terminal domain. TraB purified from the membrane forms stable dimers and is unable to hydrolyze ATP while, when purified from the soluble fraction, TraB can form hexamers capable of hydrolyzing ATP. Remarkably, both the N- and C-terminal domains display ATP-hydrolyzing activity. These properties define a new class of VirB4 proteins.
    Journal of bacteriology 02/2010; 192(9):2315-23. · 3.94 Impact Factor
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    ABSTRACT: Gram-negative bacteria have evolved diverse secretion systems/machineries to translocate substrates across the cell envelope. These various machineries fulfil a wide variety of functions but are also essential for pathogenic bacteria to infect human or plant cells. Secretion systems, of which there are seven, utilize one of two secretion mechanisms: (i) the one-step mechanism, whereby substrates are translocated directly from the bacterial-cytoplasm to the extracellular medium or into the eukaryotic-target cell; (ii) the two-step mechanism, whereby substrates are first translocated across the bacterial-inner membrane; once in the periplasm, substrates are targeted to one of the secretion systems that mediate the transport across the outer membrane and the release outside the bacterial cell. This review describes in details the main structural features of these secretion systems. Structural biology offers the possibility to understand the molecular mechanisms at play in the various secretion systems. It also helps to design specifically drugs that can block these machineries and thus attenuate the virulence of pathogenic bacteria.
    Infectious disorders drug targets. 11/2009; 9(5):518-47.
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    ABSTRACT: Gram-negative bacteria use the sophisticated type II secretion system (T2SS) to secrete a large number of exoproteins into the extracellular environment. Five proteins of the T2SS, the pseudopilins GspG-H-I-J-K, are proposed to assemble into a pseudopilus involved in the extrusion of the substrate through the outer membrane channel. Recent structural data have suggested that the three pseudopilins GspI-J-K are organized in a trimeric complex located at the tip of the GspG-containing pseudopilus. In the present work we combined two biochemical techniques to investigate the protein-protein interaction network between the five Pseudomonas aeruginosa Xcp pseudopilins. The soluble domains of XcpT-U-V-W-X (respectively homologous to GspG-H-I-J-K) were purified, and the interactions were tested by surface plasmon resonance and affinity co-purification in all possible combinations. We found an XcpV(I)-W(J)-X(K) complex, which demonstrates that the crystallized trimeric complex also exists in the P. aeruginosa T2SS. Interestingly, our systematic approach revealed an additional and yet uncharacterized interaction between XcpU(H) and XcpW(J). This observation suggested the existence of a quaternary, rather than ternary, complex (XcpU(H)-V(I)-W(J)-X(K)) at the tip of the pseudopilus. The assembly of this quaternary complex was further demonstrated by co-purification using affinity chromatography. Moreover, by testing various combinations of pseudopilins by surface plasmon resonance and affinity chromatography, we were able to dissect the different possible successive steps occurring during the formation of the quaternary complex. We propose a model in which XcpV(I) is the nucleator that first binds XcpX(K) and XcpW(J) at different sites. Then the ternary complex recruits XcpU(H) through a direct interaction with XcpW(J).
    Journal of Biological Chemistry 10/2009; 284(50):34580-9. · 4.65 Impact Factor
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    ABSTRACT: The bacterial type II protein secretion (T2S) and type IV piliation (T4P) systems share several common features. In particular, it is well established that the T2S system requires the function of a pilus-like structure, called pseudopilus, which is built upon assembly of pilin-like subunits, called pseudopilins. Pilins and pseudopilins have a hydrophobic N-terminal region, which precedes an extended hydrophilic C-terminal region. In the case of pilins, it was shown that oligomerisation and formation of helical fibers, takes place through interaction between the hydrophobic domains. XcpT, is the most abundant protein of the Pseudomonas aeruginosa T2S, and was proposed to be the main component in the pseudopilus. In this study we present the high-resolution NMR structure of the hydrophilic domain of XcpT (XcpTp). XcpTp is lacking the C-terminal disulfide bridged "D" domain found in type IV pilins and likely involved in receptor binding. This is in agreement with the idea that the XcpT-containing pseudopilus is required for protein secretion and not for bacterial attachment. Interestingly, by solving the 3D structure of XcpTp we revealed that the previously called alphabeta-loop pilin region is in fact highly conserved among major type II pseudopilins and constitutes a specific consensus motif for identifying major pseudopilins, which belong to this family.
    Journal of Structural Biology 09/2009; 169(1):75-80. · 3.36 Impact Factor

Publication Stats

370 Citations
107.45 Total Impact Points

Institutions

  • 2011–2014
    • Aix-Marseille Université
      • • Unité de Recherche d'Architecture et Fonction des Macromolécules Biologiques (UMR 7257 AFMB)
      • • Institut de Microbiologie de la Méditerranée (FR 3479 IMM)
      Marsiglia, Provence-Alpes-Côte d'Azur, France
  • 2010–2011
    • The Institute of Structural and Molecular Biology
      Londinium, England, United Kingdom
  • 2003–2011
    • French National Centre for Scientific Research
      • Institut de Microbiologie de la Méditerranée
      Lutetia Parisorum, Île-de-France, France
  • 2009
    • Birkbeck, University of London
      • Institute of Structural and Molecular Biology
      London, ENG, United Kingdom
    • Architecture et Fonction des Macromolécules Biologiques
      Marsiglia, Provence-Alpes-Côte d'Azur, France