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Crystal Structure of the VgrG1 Actin Cross-linking Domain of the Vibrio cholerae Type VI Secretion System

  • Eric Durand
    Footnotes
    Affiliations
    Aix-Marseille Université, Architecture et Fonction des Macromolécules Biologiques, UMR 7257, Campus de Luminy, Case 932, 13288 Marseille Cedex 09, France

    CNRS, Architecture et Fonction des Macromolécules Biologiques, UMR 7257, Campus de Luminy, Case 932, 13288 Marseille Cedex 09, France
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  • Estelle Derrez
    Footnotes
    Affiliations
    Aix-Marseille Université, Architecture et Fonction des Macromolécules Biologiques, UMR 7257, Campus de Luminy, Case 932, 13288 Marseille Cedex 09, France

    CNRS, Architecture et Fonction des Macromolécules Biologiques, UMR 7257, Campus de Luminy, Case 932, 13288 Marseille Cedex 09, France
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  • Gilles Audoly
    Footnotes
    Affiliations
    Unité des Rickettsies URMITE, UMR CNRS 6236, IRD 198, Faculté de Médecine la Timone, 27 Boulevard Jean Moulin, 13385 Marseille Cedex 05, France
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  • Silvia Spinelli
    Affiliations
    Aix-Marseille Université, Architecture et Fonction des Macromolécules Biologiques, UMR 7257, Campus de Luminy, Case 932, 13288 Marseille Cedex 09, France

    CNRS, Architecture et Fonction des Macromolécules Biologiques, UMR 7257, Campus de Luminy, Case 932, 13288 Marseille Cedex 09, France
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  • Miguel Ortiz-Lombardia
    Affiliations
    Aix-Marseille Université, Architecture et Fonction des Macromolécules Biologiques, UMR 7257, Campus de Luminy, Case 932, 13288 Marseille Cedex 09, France

    CNRS, Architecture et Fonction des Macromolécules Biologiques, UMR 7257, Campus de Luminy, Case 932, 13288 Marseille Cedex 09, France
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  • Didier Raoult
    Affiliations
    Unité des Rickettsies URMITE, UMR CNRS 6236, IRD 198, Faculté de Médecine la Timone, 27 Boulevard Jean Moulin, 13385 Marseille Cedex 05, France
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  • Eric Cascales
    Footnotes
    Affiliations
    Laboratoire d'Ingénierie des Systèmes Macromoléculaires, UMR7255, Institut de Microbiologie de la Méditerranée, CNRS, Aix-Marseille Université, 31 Chemin Joseph Aiguier, 13402 Marseille Cedex 20, France
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  • Christian Cambillau
    Correspondence
    Work in the laboratory of this author was supported by CNRS, Université de la Méditerranée, and grants from the Marseille-Nice Génopole, IBiSA, and the Fondation de la Recherche Médicale (FRM DEQ2011-0421282). To whom correspondence should be addressed: AFMB, CNRS UMR7257, Aix-Marseille Université, 163 Ave. de Luminy, Case 932, 13288 Marseille Cedex 09, France. Tel.: 33-491825590; Fax: 33-491266720
    Affiliations
    Aix-Marseille Université, Architecture et Fonction des Macromolécules Biologiques, UMR 7257, Campus de Luminy, Case 932, 13288 Marseille Cedex 09, France

    CNRS, Architecture et Fonction des Macromolécules Biologiques, UMR 7257, Campus de Luminy, Case 932, 13288 Marseille Cedex 09, France
    Search for articles by this author
  • Author Footnotes
    This article contains supplemental Table S1 and Figs. S1–S4.
    1 These authors contributed equally to this work.
    2 Supported by Fondation pour la Recherche Médicale Postdoctoral Fellowship SPF20101221116.
    3 Work in the laboratory of this author was supported by CNRS and funded by Agence Nationale de la Recherche Grant ANR-10-JCJC-1303-03.
Open AccessPublished:August 16, 2012DOI:https://doi.org/10.1074/jbc.M112.390153
      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.

      Introduction

      The life-threatening disease cholera is caused by the Gram-negative bacterium Vibrio cholerae. The main symptom of cholera is severe, profuse watery diarrhea. Humans are usually poisoned by absorbing water contaminated by the stools of infected people associated with poor sanitation (
      • Charles R.C.
      • Ryan E.T.
      Cholera in the 21st century.
      ,
      • Piarroux R.
      • Barrais R.
      • Faucher B.
      • Haus R.
      • Piarroux M.
      • Gaudart J.
      • Magloire R.
      • Raoult D.
      Understanding the cholera epidemic, Haiti.
      ). Cholera pandemics have spread around the world, and the most recent outbreak started in Haiti a few months after the 2010 earthquake (
      • Piarroux R.
      • Barrais R.
      • Faucher B.
      • Haus R.
      • Piarroux M.
      • Gaudart J.
      • Magloire R.
      • Raoult D.
      Understanding the cholera epidemic, Haiti.
      ,
      • Weil A.A.
      • Ivers L.C.
      • Harris J.B.
      Cholera. Lessons from Haiti and beyond.
      ). During its life in water, V. cholerae has to resist predation by amoebas. It also kills competing bacteria to colonize the niche and target human host cells (
      • Nelson E.J.
      • Harris J.B.
      • Morris Jr., J.G.
      • Calderwood S.B.
      • Camilli A.
      Cholera transmission. The host, pathogen, and bacteriophage dynamic.
      ). A large repertoire of toxins and virulence factors has been linked to V. cholerae pathogenesis. The O1 and O139 V. cholerae serogroups produce an enterotoxin, the cholera toxin, which is internalized and induces ADP-ribosylation of G protein. The resulting constitutive activation of adenylate cyclase provokes massive loss of water and electrolytes (
      • Sanchez J.
      • Holmgren J.
      Cholera toxin. A foe and a friend.
      ).
      A second toxin is RtxA, the prototype of the multifunctional autoprocessing repats-in-toxins (MARTX)
      The abbreviations used are: MARTX
      multifunctional autoprocessing repeats-in-toxins
      ACD
      actin cross-linking domain
      AMP-PNP
      5′-adenylyl-β,γ-imidodiphosphate
      BisTris
      2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol
      GCS
      γ-glutamylcysteine synthetase.
      family (
      • Satchell K.J.
      Structure and function of MARTX toxins and other large repetitive RTX proteins.
      ). Upon secretion by a dedicated Type I secretion system, the catalytic actin cross-linking domain (ACD) of RtxA is delivered into host cells, where it mediates the covalent cross-linking of G-actin, leading to cytoskeleton rearrangements and cell rounding (
      • Sheahan K.L.
      • Cordero C.L.
      • Satchell K.J.
      Identification of a domain within the multifunctional Vibrio cholerae RTX toxin that covalently cross-links actin.
      ). ACDs are enzyme ligases that catalyze isopeptide bond formation between residues Glu-270 and Lys-50 of two actin monomers (
      • Satchell K.J.
      Structure and function of MARTX toxins and other large repetitive RTX proteins.
      ). By curbing actin assembly and dynamics, RtxA prevents phagocytosis and bacterial engulfment (
      • Satchell K.J.
      Structure and function of MARTX toxins and other large repetitive RTX proteins.
      ,
      • Satchell K.J.
      Actin cross-linking toxins of Gram-negative bacteria.
      ). MARTX toxins carrying an ACD are found in several Vibrio species as well as closely related pathogens, such as Aeromonas (
      • Satchell K.J.
      Actin cross-linking toxins of Gram-negative bacteria.
      ).
      Interestingly, the ACD domain is also found combined with the V. cholerae VgrG1 protein (
      • Sheahan K.L.
      • Cordero C.L.
      • Satchell K.J.
      Identification of a domain within the multifunctional Vibrio cholerae RTX toxin that covalently cross-links actin.
      ,
      • Pukatzki S.
      • Ma A.T.
      • Sturtevant D.
      • Krastins B.
      • Sarracino D.
      • Nelson W.C.
      • Heidelberg J.F.
      • Mekalanos J.J.
      Identification of a conserved bacterial protein secretion system in Vibrio cholerae using the Dictyostelium host model system.
      ). VgrG is a core component of the Type VI secretion system, a versatile macromolecular machine dedicated to the secretion of toxins toward eukaryotic or prokaryotic target cells (
      • Cascales E.
      The type VI secretion toolkit.
      ,
      • Schwarz S.
      • Hood R.D.
      • Mougous J.D.
      What is type VI secretion doing in all those bugs?.
      ,
      • Schwarz S.
      • West T.E.
      • Boyer F.
      • Chiang W.C.
      • Carl M.A.
      • Hood R.D.
      • Rohmer L.
      • Tolker-Nielsen T.
      • Skerrett S.J.
      • Mougous J.D.
      Burkholderia type VI secretion systems have distinct roles in eukaryotic and bacterial cell interactions.
      ,
      • Russell A.B.
      • Hood R.D.
      • Bui N.K.
      • LeRoux M.
      • Vollmer W.
      • Mougous J.D.
      Type VI secretion delivers bacteriolytic effectors to target cells.
      ,
      • Hood R.D.
      • Singh P.
      • Hsu F.
      • Güvener T.
      • Carl M.A.
      • Trinidad R.R.
      • Silverman J.M.
      • Ohlson B.B.
      • Hicks K.G.
      • Plemel R.L.
      • Li M.
      • Schwarz S.
      • Wang W.Y.
      • Merz A.J.
      • Goodlett D.R.
      • Mougous J.D.
      A type VI secretion system of Pseudomonas aeruginosa targets a toxin to bacteria.
      ,
      • Cascales E.
      • Cambillau C.
      Structural biology of type VI secretion systems.
      ). The Type VI secretion system shares structural similarities with tailed bacteriophage, and recent data have demonstrated that a sheath-like structure acts as a contractile machine to deliver the extracellular portion of the secretion apparatus to target cells (
      • Basler M.
      • Pilhofer M.
      • Henderson G.P.
      • Jensen G.J.
      • Mekalanos J.J.
      Type VI secretion requires a dynamic contractile phage tail-like structure.
      ). This extracellular portion is thought to be composed of a tail-like structure formed by the polymerization of the Hcp protein concluded by the VgrG protein. The trimer of the VgrG protein shares similarities with the trimeric gp27-gp5 complex (i.e. the bacteriophage tail spike required to puncture the bacterial cell) (
      • Pukatzki S.
      • Ma A.T.
      • Revel A.T.
      • Sturtevant D.
      • Mekalanos J.J.
      Type VI secretion system translocates a phage tail spike-like protein into target cells where it cross-links actin.
      ,
      • Leiman P.G.
      • Basler M.
      • Ramagopal U.A.
      • Bonanno J.B.
      • Sauder J.M.
      • Pukatzki S.
      • Burley S.K.
      • Almo S.C.
      • Mekalanos J.J.
      Type VI secretion apparatus and phage tail-associated protein complexes share a common evolutionary origin.
      ). A number of VgrG proteins, called specialized VgrGs, carry an additional C-terminal domain (
      • Pukatzki S.
      • McAuley S.B.
      • Miyata S.T.
      The type VI secretion system. Translocation of effectors and effector domains.
      ). By analogy with the bacteriophage spike, it has been proposed that upon host cell puncturing, the C-terminal domain is delivered into the cytosol. Indeed, the C-terminal ACD domain of the V. cholerae VgrG1 protein is translocated into target cells, where it covalently cross-links G-actin (
      • Ma A.T.
      • McAuley S.
      • Pukatzki S.
      • Mekalanos J.J.
      Translocation of a Vibrio cholerae type VI secretion effector requires bacterial endocytosis by host cells.
      ,
      • Satchell K.J.
      Bacterial martyrdom. Phagocytes disabled by type VI secretion after engulfing bacteria.
      ). The resulting cross-linked dimers or polymers are in a conformation not compatible with F-actin filament formation. Therefore, the depletion of the pool of G-actin displaces the equilibrium between F-actin and G-actin toward G-actin and leads to F-actin depolymerization, thus disabling phagocytic functions.
      Despite the critical importance of the MARTX and VgrG1 ACD in bacterial pathogenesis, we still lack structural information to better understand the catalytic mechanism of actin cross-linking. Here, we report the crystal structure and the activity of the V. cholerae VgrG1 ACD. The V-shaped VgrG1 ACD crystal structure harbors an active site composed of five residues, conserved among all MARTX ACDs (
      • Satchell K.J.
      Actin cross-linking toxins of Gram-negative bacteria.
      ). We show that, as for MARTX ACD, the VgrG1 ACD E16Q variant is impaired for in vitro and in vivo actin cross-linking activity. The catalytic site is blocked by the N-terminal segment in the apo-form. Interestingly, this segment is displaced by ATP/ADP and Mg2+/Mn2+ in the holo-forms. We confirm that the purified VgrG1 ACD cross-links G-actin in vitro. This activity requires ATP and Mg2+ or Mn2+. Using microinjection in fibroblasts (
      • Audoly G.
      • Vincentelli R.
      • Edouard S.
      • Georgiades K.
      • Mediannikov O.
      • Gimenez G.
      • Socolovschi C.
      • Mège J.L.
      • Cambillau C.
      • Raoult D.
      Effect of rickettsial toxin VapC on its eukaryotic host.
      ), we demonstrate that less than 100 molecules of VgrG1 ACD suffice to induce F-actin depolymerization. Finally, modeling of the MARTX ACD and comparison with the VgrG1 ACD reveals the complete conservation of the active site and of the probable G-actin binding interface between both molecules.

      DISCUSSION

      In this study, we report the crystal structure of the actin cross-linking domain of an evolved VgrG1 actin cross-linking domain from V. cholerae. Toxins of the MARTX family are important virulence factors because they affect formation of F-actin filament and disable eukaryotic host cells. Both MARTX and VgrG1 domains have a protein ligase activity by catalyzing isopeptide bond formation between two actin monomers (
      • Sheahan K.L.
      • Satchell K.J.
      Inactivation of small Rho GTPases by the multifunctional RTX toxin from Vibrio cholerae.
      ). The in vivo and in vitro activity experiments reported here confirm that the VgrG1 ACD domain is sufficient for G-actin cross-linking. Conversely, the N terminus of VgrG1 might be devoted to puncturing the target cell membrane (
      • Ma A.T.
      • McAuley S.
      • Pukatzki S.
      • Mekalanos J.J.
      Translocation of a Vibrio cholerae type VI secretion effector requires bacterial endocytosis by host cells.
      ), consistent with the function of the homologuous bacteriophage T4 (gp27-gp5)3 tail spike complex (
      • Leiman P.G.
      • Basler M.
      • Ramagopal U.A.
      • Bonanno J.B.
      • Sauder J.M.
      • Pukatzki S.
      • Burley S.K.
      • Almo S.C.
      • Mekalanos J.J.
      Type VI secretion apparatus and phage tail-associated protein complexes share a common evolutionary origin.
      ). The ∼100-amino acid linker domain located between the (gp27-gp5)3 domain and the ACD is predicted as naturally disordered (
      • Receveur-Bréchot V.
      • Bourhis J.M.
      • Uversky V.N.
      • Canard B.
      • Longhi S.
      Assessing protein disorder and induced folding.
      ). One may hypothesize that this region is targeted by host cell proteases upon injection to deliver the ACD in the cytosol, a hypothesis that remains to be tested. Our in vivo assays, using a microinjection device, made it possible to quantify VgrG1 ACD activity in fibroblast cells. They indicated that the VgrG1 ACD is active upon microinjection of a small number of ACDs (50 < n < 100), a value compatible with the concomitant firing of 15–30 type 6 secretion machineries.
      The comparison of the crystal structures of the VgrG1 ACD in the presence and absence of ATP shows that ATP binding induces a conformational modification of the N-terminal segment. In the absence of ATP, this fragment is inserted in the cleft between the two arms of the ACD V shape and therefore fills in the active site and stabilizes it. In the presence of ATP, this segment is dislodged from the cleft and displaced toward the left part of the ACD. In this conformation, it (i) liberates the active site and (ii) participates in the stabilization of the ATP molecule in the active site (supplemental Table S1).
      The activity of the ACD domain requires both ATP and a divalent cation (Mg2+ or Mn2+). Mutagenesis studies have been performed by Satchell's group (
      • Satchell K.J.
      Actin cross-linking toxins of Gram-negative bacteria.
      ,
      • Geissler B.
      • Bonebrake A.
      • Sheahan K.L.
      • Walker M.E.
      • Satchell K.J.
      Genetic determination of essential residues of the Vibrio cholerae actin cross-linking domain reveals functional similarity with glutamine synthetases.
      ) on MARTX-ACD. The results indicate that Glu-16 is the only essential residue for activity (Table 2). Mutations of other residues lead to a decrease of the cross-linking activity (Table 2). We confirmed the critical role of the invariant Glu-16 residue in the cross-linking activity, in vitro and in vivo. We showed that in the E16Q structure, only one Mg2+/Mn2+ cation is present in the structure, at a position incompatible with activity. Because Glu-16 is bridging the two Mg2+/Mn2+ cations (Fig. 6), its mutation disrupts the cation position and hence the catalytic activity. In contrast, mutations of Glu-78 or Glu-339 (
      • Satchell K.J.
      Actin cross-linking toxins of Gram-negative bacteria.
      ,
      • Geissler B.
      • Bonebrake A.
      • Sheahan K.L.
      • Walker M.E.
      • Satchell K.J.
      Genetic determination of essential residues of the Vibrio cholerae actin cross-linking domain reveals functional similarity with glutamine synthetases.
      ), which bind only one cation (Fig. 6), Mn2 and Mn1, respectively, lead only to an activity decrease, indicating thus that the cation positions are modified but not enough to cancel catalysis. Some variations in the postions and the nature of the cations seems to be compatible with activity because both Mn2+ and Mg2+ are able to catalyze the phosphate transfer, although with different efficiencies, despite significant differences in their respective positions.
      TABLE 2Residues mutated in MARTX-ACD and the functional result of the mutations
      VgrG1/MARTX-ACDVgrG1MARTXGCS (1VA6)Mutations affecting cross-linking in MARTX-ACD (
      • Pukatzki S.
      • Ma A.T.
      • Sturtevant D.
      • Krastins B.
      • Sarracino D.
      • Nelson W.C.
      • Heidelberg J.F.
      • Mekalanos J.J.
      Identification of a conserved bacterial protein secretion system in Vibrio cholerae using the Dictyostelium host model system.
      ,
      • Geissler B.
      • Bonebrake A.
      • Sheahan K.L.
      • Walker M.E.
      • Satchell K.J.
      Genetic determination of essential residues of the Vibrio cholerae actin cross-linking domain reveals functional similarity with glutamine synthetases.
      )
      Function in ACD
      Glu-167271990Glu-27AbolishedBinds both cations
      Glu-187291992Glu-29DecreasedFar from active site; actin-1 binding?
      Asp-517622025Asp-60DecreasedFar from cations; water-mediated binding?
      Glu-787892052Glu-67DecreasedBinds a cation (Mg2)
      His-109DecreasedFar from cations; actin-1 binding?
      Glu-33910502313Glu-328DecreasedBinds a cation (Mg1)
      Arg-34110522315Arg-330DecreasedBinds SO4; putative carboxyl binding site
      Figure thumbnail gr6
      FIGURE 6Synthetic representation of the three-dimensional structure of the VgrG1 ACD active site residues, cation-conserved sulfate anion, and the β- and γ-phosphates of ATP.
      The mutation of Arg-341 leads also to an activity reduction (
      • Satchell K.J.
      Actin cross-linking toxins of Gram-negative bacteria.
      ,
      • Geissler B.
      • Bonebrake A.
      • Sheahan K.L.
      • Walker M.E.
      • Satchell K.J.
      Genetic determination of essential residues of the Vibrio cholerae actin cross-linking domain reveals functional similarity with glutamine synthetases.
      ). In our structures, Arg-341 binds to a sulfate ion in the active site, close to the ATP γ-phosphate (Fig. 6). We think that this sulfate ion occupies the position where Glu-270 binds in the first transient intermediate (Fig. 4, A and B). Its mutation probably diminishes the stablilization of the intermediate and decreases the enzyme's activity. Mutations of residues Glu-18, Asp-51, and His-109 also lead to an activity decrease. However, these residues do not directly interact with the cations. The only possible explanation for their function would be a significant stabilizing interaction with actins in step 1 or 2.
      It has been shown that actin-binding proteins modulate the cross-linking activity of MARTX-ACD (
      • Kudryashov D.S.
      • Cordero C.L.
      • Reisler E.
      • Satchell K.J.
      Characterization of the enzymatic activity of the actin cross-linking domain from the Vibrio cholerae MARTX Vc toxin.
      ). Profilin, thymosin-β4, and gelsolin are compatible with actin cross-linking activity, whereas cofilin and DNAseI inhibit it. The observation of the available actin·actin-binding protein complexes makes it possible to interpret these results. The first set of proteins bind in a area far from both Glu-270 and Lys-50, whereas DNase I binds the loop carrying Lys-50, thus inhibiting the second step of the reaction, the nucleophilic attack by actin-2 (Fig. S4).
      These results together with consideration of the Glu-270 position in the active site led us to suggest models of the first and second transient intermediates (Fig. 4, B and C). The model of the first transient binary intermediate indicates that accessibility is left for the second G-actin to perform the SN2 reaction. The model of the second transient ternary intermediate indicates that such a compact reacting intermediate is possible and that only the modeled position is realistic (within a small positional uncertainty). Indeed, it is not possible to crystallize such labile species, but we hope that these models may help in the design of mutants aimed at refining them.
      Finally, the structural characterization of the VgrG1 ACD toxin provides unique opportunities to design compounds targeting the active site. The close relationship between the active sites of the MARTX/VgrG1 ACD and that of the Gly-Cys synthetases suggests that sulfoximines might be good candidates to perform this task.

      Acknowledgments

      We thank Laure Journet and members of the E. C. and C. C. Type VI secretion system groups for fruitful discussions and Ella Danloss for encouragement. We gratefully acknowledge the help of Pierre Legrand (Proxima 1) for data collection and the Soleil synchrotron for beamtime allocation. We thank Michel R. Popoff for the kind gift of toxin B of Clostridium difficile.

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