Electrostatic interactions between the CTX phage minor coat protein and the bacterial host receptor TolA drive the pathogenic conversion of Vibrio cholerae
2017; Elsevier BV; Volume: 292; Issue: 33 Linguagem: Inglês
10.1074/jbc.m117.786061
ISSN1083-351X
AutoresLaetitia Houot, Romain Navarro, Matthieu Nouailler, Denis Duché, Françoise Guerlesquin, Roland Lloubès,
Tópico(s)Bacterial Genetics and Biotechnology
ResumoVibrio cholerae is a natural inhabitant of aquatic environments and converts to a pathogen upon infection by a filamentous phage, CTXΦ, that transmits the cholera toxin-encoding genes. This toxigenic conversion of V. cholerae has evident implication in both genome plasticity and epidemic risk, but the early stages of the infection have not been thoroughly studied. CTXΦ transit across the bacterial periplasm requires binding between the minor coat protein named pIII and a bacterial inner-membrane receptor, TolA, which is part of the conserved Tol-Pal molecular motor. To gain insight into the TolA–pIII complex, we developed a bacterial two-hybrid approach, named Oxi-BTH, suited for studying the interactions between disulfide bond-folded proteins in the bacterial cytoplasm of an Escherichia coli reporter strain. We found that two of the four disulfide bonds of pIII are required for its interaction with TolA. By combining Oxi-BTH assays, NMR, and genetic studies, we also demonstrate that two intermolecular salt bridges between TolA and pIII provide the driving forces of the complex interaction. Moreover, we show that TolA residue Arg-325 involved in one of the two salt bridges is critical for proper functioning of the Tol-Pal system. Our results imply that to prevent host evasion, CTXΦ uses an infection strategy that targets a highly conserved protein of Gram-negative bacteria essential for the fitness of V. cholerae in its natural environment. Vibrio cholerae is a natural inhabitant of aquatic environments and converts to a pathogen upon infection by a filamentous phage, CTXΦ, that transmits the cholera toxin-encoding genes. This toxigenic conversion of V. cholerae has evident implication in both genome plasticity and epidemic risk, but the early stages of the infection have not been thoroughly studied. CTXΦ transit across the bacterial periplasm requires binding between the minor coat protein named pIII and a bacterial inner-membrane receptor, TolA, which is part of the conserved Tol-Pal molecular motor. To gain insight into the TolA–pIII complex, we developed a bacterial two-hybrid approach, named Oxi-BTH, suited for studying the interactions between disulfide bond-folded proteins in the bacterial cytoplasm of an Escherichia coli reporter strain. We found that two of the four disulfide bonds of pIII are required for its interaction with TolA. By combining Oxi-BTH assays, NMR, and genetic studies, we also demonstrate that two intermolecular salt bridges between TolA and pIII provide the driving forces of the complex interaction. Moreover, we show that TolA residue Arg-325 involved in one of the two salt bridges is critical for proper functioning of the Tol-Pal system. Our results imply that to prevent host evasion, CTXΦ uses an infection strategy that targets a highly conserved protein of Gram-negative bacteria essential for the fitness of V. cholerae in its natural environment. Electrostatic interactions between the CTX phage minor coat protein and the bacterial host receptor TolA drive the pathogenic conversion of Vibrio cholerae.Journal of Biological ChemistryVol. 293Issue 19PreviewVOLUME 292 (2017) PAGES 13584–13598 Full-Text PDF Open Access Phage/bacterium interaction is one of the driven forces for gene acquisition and bacterial host adaptation to their environment, and it has been frequently associated with increased virulence of the bacterial host. A striking example of this parasitism-dependent adaptation is Vibrio cholerae, a bacterial natural inhabitant of estuaries, and the causative agent of epidemic disease cholera. Although there are more than 200 O-antigen serogroups described, only two have been reported to cause the pandemic disease cholera, the O1 and O139 serotypes, due to the production of two essential virulence factors, the toxin co-regulated pilus (TCP) 3The abbreviations used are: TCP, toxin co-regulated pilus; IPTG, isopropyl 1-thio-β-d-galactopyranoside; CT, cholera toxin; PDB, Protein Data Bank; OM, outer membrane; IM, inner membrane; BACTH, bacterial two-hybrid; DOC, deoxycholate; HSQC, heteronuclear single quantum coherence; Sm, streptomycin; Cm, chloramphenicol. and the cholera toxin (CT). Interestingly, the genes ctxAB encoding the enterotoxin CT are not carried by the core genome of the bacterium but can be acquired after infection by a lysogenic bacteriophage known as CTXΦ (1.Waldor M.K. Mekalanos J.J. Lysogenic conversion by a filamentous phage encoding cholera toxin.Science. 1996; 272: 1910-1914Crossref PubMed Scopus (1388) Google Scholar). Once infected, the bacterium produces CT and assembles new phage particles (carrying the ctxAB genes) that will be secreted in the environment, and it may convert non-pathogenic V. cholerae cells to pathogenicity. Most of the knowledge on CTXΦ infection have been extrapolated from the canonical model of Escherichia coli F-pilus-specific coliphages Ff (including f1Φ, fdΦ, and M13Φ). CTXΦ and FfΦ both belong to the genus Inovirus, which are filamentous particles containing a circular single-stranded DNA genome. The genome of inoviruses includes about 10 genes and is generally organized in a conserved modular structure in which functionally related genes are grouped together (2.Mai-Prochnow A. Hui J.G. Kjelleberg S. Rakonjac J. McDougald D. Rice S.A. Big things in small packages: the genetics of filamentous phage and effects on fitness of their host.FEMS Microbiol. Rev. 2015; 39: 465-487Crossref PubMed Scopus (95) Google Scholar, 3.Rakonjac J. Bennett N.J. Spagnuolo J. Gagic D. Russel M. Filamentous bacteriophage: biology, phage display and nanotechnology applications.Curr. Issues Mol. Biol. 2011; 13: 51-76PubMed Google Scholar). FfΦ and CTXΦ binding and uptake into the host cell rely primarily on the minor coat protein pIII located at the distal tip of the phage and present at three to five copies. Although there is no sequence conservation between pIIIFf and pIIICTX, both proteins are composed of three distinct domains separated by two low-complexity regions that serve as linkers. Although the N-terminal (N1) and the central domains (N2) are exposed at the capsid surface, the C-terminal domain (N3) anchors the pIII protein to the phage particle through hydrophobic interactions (4.Holliger P. Riechmann L. A conserved infection pathway for filamentous bacteriophages is suggested by the structure of the membrane penetration domain of the minor coat protein g3p from phage fd.Structure. 1997; 5: 265-275Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 5.Heilpern A.J. Waldor M.K. pIIICTX, a predicted CTX minor coat protein, can expand the host range of coliphage fd to include Vibrio cholerae.J. Bacteriol. 2003; 185: 1037-1044Crossref PubMed Scopus (57) Google Scholar, 6.Heilpern A.J. Waldor M.K. CTXΦ infection of Vibrio cholerae requires the tolQRA gene products.J. Bacteriol. 2000; 182: 1739-1747Crossref PubMed Scopus (89) Google Scholar). Filamentous phage infection of the bacterial host is seen as a sequential two-step process. First, phage recruitment occurs upon specific binding between the phage capsid pIII-N2 domain and a primary receptor exposed at the surface of the cell host, the conjugative F pilus for E. coli (3.Rakonjac J. Bennett N.J. Spagnuolo J. Gagic D. Russel M. Filamentous bacteriophage: biology, phage display and nanotechnology applications.Curr. Issues Mol. Biol. 2011; 13: 51-76PubMed Google Scholar, 7.Deng L.-W. Perham R.N. Delineating the site of interaction on the pIII protein of filamentous bacteriophage fd with the F-pilus of Escherichia coli.J. Mol. Biol. 2002; 319: 603-614Crossref PubMed Scopus (45) Google Scholar) and the TCP for V. cholerae (1.Waldor M.K. Mekalanos J.J. Lysogenic conversion by a filamentous phage encoding cholera toxin.Science. 1996; 272: 1910-1914Crossref PubMed Scopus (1388) Google Scholar, 5.Heilpern A.J. Waldor M.K. pIIICTX, a predicted CTX minor coat protein, can expand the host range of coliphage fd to include Vibrio cholerae.J. Bacteriol. 2003; 185: 1037-1044Crossref PubMed Scopus (57) Google Scholar). In E. coli, ATP-dependent retraction of the F pilus brings the phage in contact with the cell envelope, promoting its transport across the outer membrane (OM) by an unknown mechanism. Then, pIII must partially unfold to separate N1 and N2 domains (8.Lubkowski J. Hennecke F. Plückthun A. Wlodawer A. Filamentous phage infection: crystal structure of g3p in complex with its coreceptor, the C-terminal domain of TolA.Structure. 1999; 7: 711-722Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). This event is crucial in the infection process as it unmasks the pIII-N1 domain for subsequent binding to a second receptor, the TolAEc protein located in the cell envelope (8.Lubkowski J. Hennecke F. Plückthun A. Wlodawer A. Filamentous phage infection: crystal structure of g3p in complex with its coreceptor, the C-terminal domain of TolA.Structure. 1999; 7: 711-722Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, 9.Lubkowski J. Hennecke F. Plückthun A. Wlodawer A. The structural basis of phage display elucidated by the crystal structure of the N-terminal domains of g3p.Nat. Struct. Biol. 1998; 5: 140-147Crossref PubMed Scopus (106) Google Scholar, 10.Pommier S. Gavioli M. Cascales E. Lloubès R. Tol-dependent macromolecule import through the Escherichia coli cell envelope requires the presence of an exposed TolA binding motif.J. Bacteriol. 2005; 187: 7526-7534Crossref PubMed Scopus (20) Google Scholar, 11.Riechmann L. Holliger P. The C-terminal domain of TolA is the coreceptor for filamentous phage infection of E. coli.Cell. 1997; 90: 351-360Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar, 12.Deprez C. Lloubès R. Gavioli M. Marion D. Guerlesquin F. Blanchard L. Solution structure of the E. coli TolA C-terminal domain reveals conformational changes upon binding to the phage g3p N-terminal domain.J. Mol. Biol. 2005; 346: 1047-1057Crossref PubMed Scopus (57) Google Scholar). In E. coli, it has been proposed that the pIII-N1Ff/TolAIIIEc interaction triggers conformational modifications permitting the pIII-N3 domain to form a pore in the bacterial IM, allowing the subsequent phage DNA injection into the cell cytoplasm (13.Bennett N.J. Rakonjac J. Unlocking of the filamentous bacteriophage virion during infection is mediated by the C domain of pIII.J. Mol. Biol. 2006; 356: 266-273Crossref PubMed Scopus (27) Google Scholar). The nature of the force driving the DNA out of the capsid remains unknown. In V. cholerae, TCP retraction seems central to the phage infection process, as TCP production alone is not sufficient to allow CTXΦ uptake (14.Ng D. Harn T. Altindal T. Kolappan S. Marles J.M. Lala R. Spielman I. Gao Y. Hauke C.A. Kovacikova G. Verjee Z. Taylor R.K. Biais N. Craig L. The Vibrio cholerae minor Pilin TcpB initiates assembly and retraction of the toxin-coregulated pilus.PLoS Pathog. 2016; 12: e1006109Crossref PubMed Scopus (44) Google Scholar). Although TCP parasitism facilitates the introduction of CTXΦ into the host cell, subsequent phage binding to TolAVc appears to be the limiting step of the infection process (5.Heilpern A.J. Waldor M.K. pIIICTX, a predicted CTX minor coat protein, can expand the host range of coliphage fd to include Vibrio cholerae.J. Bacteriol. 2003; 185: 1037-1044Crossref PubMed Scopus (57) Google Scholar, 6.Heilpern A.J. Waldor M.K. CTXΦ infection of Vibrio cholerae requires the tolQRA gene products.J. Bacteriol. 2000; 182: 1739-1747Crossref PubMed Scopus (89) Google Scholar, 15.Ford C.G. Kolappan S. Phan H.T. Waldor M.K. Winther-Larsen H.C. Craig L. Crystal structures of a CTX pIII domain unbound and in complex with a Vibrio cholerae TolA domain reveal novel interaction interfaces.J. Biol. Chem. 2012; 287: 36258-36272Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar). Thus, Heilpern and Waldor (5.Heilpern A.J. Waldor M.K. pIIICTX, a predicted CTX minor coat protein, can expand the host range of coliphage fd to include Vibrio cholerae.J. Bacteriol. 2003; 185: 1037-1044Crossref PubMed Scopus (57) Google Scholar) have shown that a chimeric fd phage displaying the pIII-N1CTX domain fused to the pIII-N3fd domain can successfully infect V. cholerae cells. This demonstrates that the pIII-N1CTX domain displayed at the tip of the capsid is critical and sufficient to ensure host-specific recognition in a TCP-independent manner (5.Heilpern A.J. Waldor M.K. pIIICTX, a predicted CTX minor coat protein, can expand the host range of coliphage fd to include Vibrio cholerae.J. Bacteriol. 2003; 185: 1037-1044Crossref PubMed Scopus (57) Google Scholar). TolA is the central protein of the Tol-Pal cell envelope system, which is highly conserved in Gram-negative bacteria (16.Lloubès R. Cascales E. Walburger A. Bouveret E. Lazdunski C. Bernadac A. Journet L. The Tol-Pal proteins of the Escherichia coli cell envelope: an energized system required for outer membrane integrity?.Res. Microbiol. 2001; 152: 523-529Crossref PubMed Scopus (144) Google Scholar, 17.Sturgis J.N. Organisation and evolution of the tol-pal gene cluster.J. Mol. Microbiol. Biotechnol. 2001; 3: 113-122PubMed Google Scholar). In addition to TolA, the Tol-Pal complex is composed of two IM proteins, TolQ and TolR, of the OM lipoprotein Pal and of the periplasmic protein TolB. In several species, including E. coli and V. cholerae, two additional proteins complete the system, the periplasmic protein CpoB (previously YbgF) (18.Gray A.N. Egan A.J. Van't Veer I.L. Verheul J. Colavin A. Koumoutsi A. Biboy J. Altelaar A.M. Damen M.J. Huang K.C. Simorre J.P. Breukink E. den Blaauwen T. Typas A. Gross C.A. Vollmer W. Coordination of peptidoglycan synthesis and outer membrane constriction during Escherichia coli cell division.Elife. 2015; 4: e07118Crossref Scopus (131) Google Scholar) and the cytoplasmic thioesterase YbgC (19.Gully D. Bouveret E. A protein network for phospholipid synthesis uncovered by a variant of the tandem affinity purification method in Escherichia coli.Proteomics. 2006; 6: 282-293Crossref PubMed Scopus (66) Google Scholar). The Tol-Pal complex is suspected to function as a nanomachine, using the proton-motive force of the IM to generate movements and to transfer energy to OM proteins. Multiple interactions connecting the different components of the Tol-Pal system have been identified. TolA, TolQ, and TolR form a complex anchored in the IM. The TolA protein extends in the periplasm thanks to a long α2-helix (TolAII domain), whereas its globular C-terminal domain (TolAIII) interacts with TolB, and with Pal in the presence of proton-motive force (20.Cascales E. Gavioli M. Sturgis J.N. Lloubès R. Proton motive force drives the interaction of the inner membrane TolA and outer membrane pal proteins in Escherichia coli.Mol. Microbiol. 2000; 38: 904-915Crossref PubMed Scopus (125) Google Scholar, 21.Cascales E. Lloubès R. Sturgis J.N. The TolQ–TolR proteins energize TolA and share homologies with the flagellar motor proteins MotA–MotB.Mol. Microbiol. 2001; 42: 795-807Crossref PubMed Scopus (160) Google Scholar, 22.Walburger A. Lazdunski C. Corda Y. The Tol/Pal system function requires an interaction between the C-terminal domain of TolA and the N-terminal domain of TolB.Mol. Microbiol. 2002; 44: 695-708Crossref PubMed Scopus (94) Google Scholar). Moreover, Pal interacts with TolB and with the peptidoglycan (23.Cascales E. Lloubès R. Deletion analyses of the peptidoglycan-associated lipoprotein Pal reveals three independent binding sequences including a TolA box: Pal interacts independently with OmpA, TolA and TolB.Mol. Microbiol. 2004; 51: 873-885Crossref PubMed Scopus (71) Google Scholar). Thus, the Tol-Pal system links the IM, the OM, and the peptidoglycan. The system is involved in maintaining OM integrity, conferring pleiotropic phenotypes when one of its genes is mutated: increased sensitivity to detergents, cell filamentation in low and high osmolarity media, and outer membrane hypervesiculation (6.Heilpern A.J. Waldor M.K. CTXΦ infection of Vibrio cholerae requires the tolQRA gene products.J. Bacteriol. 2000; 182: 1739-1747Crossref PubMed Scopus (89) Google Scholar, 16.Lloubès R. Cascales E. Walburger A. Bouveret E. Lazdunski C. Bernadac A. Journet L. The Tol-Pal proteins of the Escherichia coli cell envelope: an energized system required for outer membrane integrity?.Res. Microbiol. 2001; 152: 523-529Crossref PubMed Scopus (144) Google Scholar). In addition, the Tol system is involved in the late stage of cell division corresponding to the OM constriction (25.Gerding M.A. Ogata Y. Pecora N.D. Niki H. de Boer P.A. The trans-envelope Tol-Pal complex is part of the cell division machinery and required for proper outer-membrane invagination during cell constriction in E. coli.Mol. Microbiol. 2007; 63: 1008-1025Crossref PubMed Scopus (275) Google Scholar) and has been found associated with the PBP1B–LpoB complex in E. coli (18.Gray A.N. Egan A.J. Van't Veer I.L. Verheul J. Colavin A. Koumoutsi A. Biboy J. Altelaar A.M. Damen M.J. Huang K.C. Simorre J.P. Breukink E. den Blaauwen T. Typas A. Gross C.A. Vollmer W. Coordination of peptidoglycan synthesis and outer membrane constriction during Escherichia coli cell division.Elife. 2015; 4: e07118Crossref Scopus (131) Google Scholar). It is also required for proper localization of polar factor in Caulobacter crescentus (26.Yeh Y.-C. Comolli L.R. Downing K.H. Shapiro L. McAdams H.H. The caulobacter Tol-Pal complex is essential for outer membrane integrity and the positioning of a polar localization factor.J. Bacteriol. 2010; 192: 4847-4858Crossref PubMed Scopus (84) Google Scholar) and of chemoreceptors in E. coli (27.Santos T.M. Lin T.-Y. Rajendran M. Anderson S.M. Weibel D.B. Polar localization of Escherichia coli chemoreceptors requires an intact Tol-Pal complex: chemoreceptor localization in Escherichia coli.Mol. Microbiol. 2014; 92: 985-1004Crossref PubMed Scopus (41) Google Scholar). For both coliphage and CTXΦ, structural studies on isolated protein domains have provided new insights into the complex formed with TolAIII in the bacterial periplasm (Fig. 1). First, the structures demonstrate that although the CTXΦ pIII-N1 and M13Φ pIII-N1 domains have only 15% sequence identity, they are both dominantly composed of β-strands, and multiple disulfide bonds stabilize their structures. On the bacterial side, TolAIIIEc and TolAIIIVc are curved structures mixing α-helices and β-sheets. A high-resolution structure of E. coli TolAIII free in solution has been obtained by heteronuclear NMR (Protein Data Bank PDB code 1S62) (12.Deprez C. Lloubès R. Gavioli M. Marion D. Guerlesquin F. Blanchard L. Solution structure of the E. coli TolA C-terminal domain reveals conformational changes upon binding to the phage g3p N-terminal domain.J. Mol. Biol. 2005; 346: 1047-1057Crossref PubMed Scopus (57) Google Scholar), whereas the TolAIII in complex with coliphage pIII-N1M13 (residues 11–86) has been obtained by X-ray crystallography (PDB code 1Tol). The structure shows that the pIII-N1M13 domain binds the concave side of TolAIIIEc, forming a continuous interprotein β-sheet (8.Lubkowski J. Hennecke F. Plückthun A. Wlodawer A. Filamentous phage infection: crystal structure of g3p in complex with its coreceptor, the C-terminal domain of TolA.Structure. 1999; 7: 711-722Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). In 2012, Ford et al. (15.Ford C.G. Kolappan S. Phan H.T. Waldor M.K. Winther-Larsen H.C. Craig L. Crystal structures of a CTX pIII domain unbound and in complex with a Vibrio cholerae TolA domain reveal novel interaction interfaces.J. Biol. Chem. 2012; 287: 36258-36272Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar) determined the crystal structure of pIII-N1CTX domain alone and in complex with the V. cholerae TolAIII domain (PDB code 4G7X). Surprisingly, the authors showed that pIII-N1CTX binds on the convex face of TolAIIIVc, resulting in a continuous interprotein β-sheet (Fig. 1A). Thus, interaction between the two partners delineates a distinct interface compared with the coliphage model of infection (15.Ford C.G. Kolappan S. Phan H.T. Waldor M.K. Winther-Larsen H.C. Craig L. Crystal structures of a CTX pIII domain unbound and in complex with a Vibrio cholerae TolA domain reveal novel interaction interfaces.J. Biol. Chem. 2012; 287: 36258-36272Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar). Filamentous phages are not the only particles that parasitize the Tol-Pal system to penetrate E. coli cells. Colicins are bacterial toxins comprising various types of lethal activity targeting the IM, the RNA, or the peptidoglycan of its bacterial target. Tol-dependent colicins have been shown to interact with one or more of the Tol proteins during their translocation across the periplasm, showing some similarities with Tol-dependent filamentous phage uptake. In 2012, Li et al. (28.Li C. Zhang Y. Vankemmelbeke M. Hecht O. Aleanizy F.S. Macdonald C. Moore G.R. James R. Penfold C.N. Structural evidence that colicin A protein binds to a novel binding site of TolA protein in Escherichia coli periplasm.J. Biol. Chem. 2012; 287: 19048-19057Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar) demonstrated that a colicin A peptide (residues 53–107) binds on the convex face of TolAIIIEc, forming an intermolecular antiparallel β-sheet. It is puzzling to observe that despite the structural similarities between V. cholerae and E. coli TolAIII domains, the molecular binding interfaces with colicin A, pIIICTX, and pIIIM13 differ, illustrating the versatile functioning of TolA as a periplasmic hub protein. In this study, our goal was to investigate the determinants allowing CTXΦ-specific host selection and periplasm transit in vivo thanks to a new oxidative bacterial two-hybrid approach combined to NMR and in vivo studies. To gain insights into the mechanism of CTXΦ transit through the periplasm, we first analyzed the interaction that occurs between pIII-N1CTX and TolAIIIVc, compared with the pIII-N1M13 and TolAIIIEc interaction, using a bacterial two-hybrid (BACTH) approach. This system relies on the reconstitution of the signaling cAMP transduction cascade in an endogenous adenylate cyclase-deficient strain (29.Karimova G. Pidoux J. Ullmann A. Ladant D. A bacterial two-hybrid system based on a reconstituted signal transduction pathway.Proc. Natl. Acad. Sci. U.S.A. 1998; 95: 5752-5756Crossref PubMed Scopus (1213) Google Scholar). The TolAIII domains from V. cholerae and from E. coli were fused to the T18 domain in the pUT18 vector, whereas the pIII-N1 domains from M13 and CTX phages were fused to the T25 domain in the pKT25 vector. Constructs were introduced into the E. coli BTH101 strain, and co-transformants were tested on MacConkey plates. As a control, we first observed that in this assay, the T18-TolAIIIEc construct gives a positive interaction signal with the colicin A N-terminal domain (ColAN) as described previously (10.Pommier S. Gavioli M. Cascales E. Lloubès R. Tol-dependent macromolecule import through the Escherichia coli cell envelope requires the presence of an exposed TolA binding motif.J. Bacteriol. 2005; 187: 7526-7534Crossref PubMed Scopus (20) Google Scholar, 22.Walburger A. Lazdunski C. Corda Y. The Tol/Pal system function requires an interaction between the C-terminal domain of TolA and the N-terminal domain of TolB.Mol. Microbiol. 2002; 44: 695-708Crossref PubMed Scopus (94) Google Scholar, 30.Derouiche R. Zeder-Lutz G. Bénédetti H. Gavioli M. Rigal A. Lazdunski C. Lloubès R. Binding of colicins A and El to purified TolA domains.Microbiology. 1997; 143: 3185-3192Crossref PubMed Scopus (25) Google Scholar), which validated our approach (Fig. 1B, left panel). We also observed that a T18-TolAIIIVc construct is unable to bind T25-ColAN, attesting the specificity for partner recognition between the two bacterial species. Then, we tested the TolAIIIVc and pIII-N1CTX constructs together, but we did not detect interaction between these different domains. We obtained the same negative result when we tried to detect the TolAIIIEc/pIII-N1M13 interaction (Fig. 1B, left panel). We hypothesized that this result could arise from improper folding of disulfide-bonded TolAIII, pIII-N1M13, or pIII-N1CTX domains when expressed in the cell cytoplasm (Fig. 2, A and B). Thus, we envisioned that a bacterial two-hybrid assay in an oxidative environment would allow the proper folding of proteins with disulfide bonds. Several E. coli strains, such as Origami (Novagen) or SHuffle (New England Biolabs), have been engineered to optimize the purification of proteins with disulfide bonds and are commercially available. We chose the SHuffle T7 strain as a chassis because it is deleted for glutaredoxin reductase (gor) and thioredoxin reductase (trxB) genes, allowing disulfide bond formation in the cytoplasm. In addition, this strain expresses a cytoplasmic version of the disulfide bond isomerase DsbC, promoting correct disulfide bond formation and proper oxidative folding of proteins containing multiple cysteines (31.Lobstein J. Emrich C.A. Jeans C. Faulkner M. Riggs P. Berkmen M. SHuffle, a novel Escherichia coli protein expression strain capable of correctly folding disulfide bonded proteins in its cytoplasm.Microb. Cell Fact. 2012; 11: 56Crossref PubMed Scopus (347) Google Scholar). We transduced the cya mutation in the SHuffle strain to make the Oxi-BTH strain (for Oxidative Bacterial Two-Hybrid). The resulting strain was co-transformed with the pUT18 and pKT25 vectors expressing domains of interest. As shown in Fig. 1B (right panel), the Oxi-BTH strain allowed the detection of interaction between TolAIIIVc and pIII-N1CTX, as well as between TolAIIIEc and pIII-N1M13. Thus, we concluded that the Oxi-BTH strain is a powerful tool to apply the BACTH system to the study of disulfide-bonded proteins. Indeed, our data suggest that in both E. coli and V. cholerae, disulfide bond-dependent folding of the TolAIII domain and/or the phage minor capsid domain is required to allow binding of the two partners. Conversely, TolAIII Ec is able to interact with the colicin A N-terminal domain, which does not contain cysteines, in both reducing and oxidizing conditions (Fig. 1B). Finally, we did not observe cross-interaction between the two species (i.e. TolAIIIVc/pIII-N1M13 or TolAIIIEc/pIII-N1CTX) despite strong structural conservation between E. coli and V. cholerae TolAIII domains. Because the TolAIIIVc/pIII-N1CTX interaction is only seen in our oxidative two-hybrid assay, we hypothesized that one or more disulfide bonds might be essential for bacterial and phage domain recognition. To test this hypothesis, each disulfide bond (one in TolAIIIVc and four in pIII-N1CTX, Fig. 2, A and B) was sequentially abolished by introducing substitutions of individual cysteine to serine in the BACTH constructs. The resulting mutants were then tested in the Oxi-BTH assay. As shown in Fig. 2C, TolAIIIVc (C292S) construct was still able to interact with pIII-N1CTX. This suggests that disulfide bond formation in TolAIIIVc is not required for CTX phage binding. We then targeted each of the four disulfide bonds present in pIII-N1CTX. None of the individual mutations we performed was able to totally abolish binding to TolAIIIVc. Interestingly, mutation of the second or the third S–S bond (mutations C47S and C75S, respectively) of the phage pIII-N1 domain resulted in a faint interaction signal, suggesting a weaker binding affinity between TolAIIIVc and these two pIII-N1CTX mutants compared with the wild-type construct. In agreement with this observation, a pIII-N1CTX(C47S/C75S) double mutant was not able to interact with TolAIIIVc. These observations are unlikely to result from stability defects of the cysteine variants compared with the native pIII-N1 protein. Indeed, inserting the same mutations on the His-tagged pIII-N1CTX domain expressed from the pIN vector resulted in equivalent expression of the different constructs (supplemental Fig. S1). Moreover, as the pIII-N1CTX/TolAIII interaction is not seen in the regular BACTH assay, it is more likely that pIII-N1CTX folding via its 2nd and 3rd disulfide bonds is critical for TolAIIIVc binding. It has been shown that Ff coliphages require an activation step to become able to infect the host cell. Indeed, in the native conformation of the minor capsid protein pIIIFf, the N2 domain is tightly associated with N1, which buries the phage TolAIII-binding site at the domain interface. Phage activation is processed upon binding of N2 to the primary receptor, the F pilus, which initiates partial unfolding, prolyl cis-to-trans isomerization in the hinge between N1 and N2 and domain disassembly, thereby exposing its binding site for the ultimate receptor TolA (32.Martin A. Schmid F.X. The folding mechanism of a two-domain protein: folding kinetics and domain docking of the gene-3 protein of phage fd.J. Mol. Biol. 2003; 329: 599-610Crossref PubMed Scopus (32) Google Scholar). It has been proposed that the isomerization sets a molecular timer to maintain the binding-active state long enough for the phage to interact with TolA. Conversely, Craig and co-workers (15.Ford C.G. Kolappan S. Phan H.T. Waldor M.K. Winther-Larsen H.C. Craig L. Crystal structures of a CTX pIII domain unbound and in complex with a Vibrio cholerae TolA domain reveal novel interaction interfaces.J. Biol. Chem. 2012; 287: 36258-36272Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar) have suggested that the TolA-binding site on pIII-N1CTX is permanently accessible and does not require initial pilus-induced conformational change. We wondered whether a fusion to the two-hybrid T25 domain would allow us to test the inf
Referência(s)