Title: Secretion of the Intimin Passenger Domain Is Driven by Protein Folding
Abstract: Intimin is an essential adhesin of attaching and effacing organisms such as entropathogenic Escherichia coli. It is also the prototype of type Ve secretion or inverse autotransport, where the extracellular C-terminal region or passenger is exported with the help of an N-terminal transmembrane β-barrel domain. We recently reported a stalled secretion intermediate of intimin, where the passenger is located in the periplasm but the β-barrel is already inserted into the membrane. Stalling of this mutant is due to the insertion of an epitope tag at the very N terminus of the passenger. Here, we examined how this insertion disrupts autotransport and found that it causes misfolding of the N-terminal immunoglobulin (Ig)-like domain D00. We could also stall the secretion by making an internal deletion in D00, and introducing the epitope tag into the second Ig-like domain, D0, also resulted in reduced passenger secretion. In contrast to many classical autotransporters, where a proximal folding core in the passenger is required for secretion, the D00 domain is dispensable, as the passenger of an intimin mutant lacking D00 entirely is efficiently exported. Furthermore, the D00 domain is slightly less stable than the D0 and D1 domains, unfolding at ∼200 piconewtons (pN) compared with ∼250 pN for D0 and D1 domains as measured by atomic force microscopy. Our results support a model where the secretion of the passenger is driven by sequential folding of the extracellular Ig-like domains, leading to vectorial transport of the passenger domain across the outer membrane in an N to C direction. Intimin is an essential adhesin of attaching and effacing organisms such as entropathogenic Escherichia coli. It is also the prototype of type Ve secretion or inverse autotransport, where the extracellular C-terminal region or passenger is exported with the help of an N-terminal transmembrane β-barrel domain. We recently reported a stalled secretion intermediate of intimin, where the passenger is located in the periplasm but the β-barrel is already inserted into the membrane. Stalling of this mutant is due to the insertion of an epitope tag at the very N terminus of the passenger. Here, we examined how this insertion disrupts autotransport and found that it causes misfolding of the N-terminal immunoglobulin (Ig)-like domain D00. We could also stall the secretion by making an internal deletion in D00, and introducing the epitope tag into the second Ig-like domain, D0, also resulted in reduced passenger secretion. In contrast to many classical autotransporters, where a proximal folding core in the passenger is required for secretion, the D00 domain is dispensable, as the passenger of an intimin mutant lacking D00 entirely is efficiently exported. Furthermore, the D00 domain is slightly less stable than the D0 and D1 domains, unfolding at ∼200 piconewtons (pN) compared with ∼250 pN for D0 and D1 domains as measured by atomic force microscopy. Our results support a model where the secretion of the passenger is driven by sequential folding of the extracellular Ig-like domains, leading to vectorial transport of the passenger domain across the outer membrane in an N to C direction. Intimin is a well known virulence factor of attaching and effacing pathogens, such as enterohemorrhagic and enteropathogenic Escherichia coli (EPEC), 3The abbreviations used are: EPEC, enteropathogenic E. coli; AFM, atomic force microscopy; BAM, β-barrel assembly machinery; HA, hemagglutinin; Ig, immunoglobulin; LB, lysogeny broth; TAM, translocation and assembly machinery; pN, piconewtons. 3The abbreviations used are: EPEC, enteropathogenic E. coli; AFM, atomic force microscopy; BAM, β-barrel assembly machinery; HA, hemagglutinin; Ig, immunoglobulin; LB, lysogeny broth; TAM, translocation and assembly machinery; pN, piconewtons. Citrobacter rodentium, and Hafnia alvei (1Schmidt M.A. LEEways: tales of EPEC, ATEC, and EHEC.Cell. Microbiol. 2010; 12: 1544-1552Crossref PubMed Scopus (117) Google Scholar). Intimin is an adhesin that mediates tight attachment to epithelial cells, which ultimately leads to the effacement of microvilli and formation of actin pedestals on the host cell. Remarkably, the main receptor for intimin is not a host cell receptor but a bacterial protein called Tir for translocated intimin receptor. This protein is secreted by the pathogens directly into the host cells via a type III secretion system (2Kenny B. DeVinney R. Stein M. Reinscheid D.J. Frey E.A. Finlay B.B. Enteropathogenic E. coli (EPEC) transfers its receptor for intimate adherence into mammalian cells.Cell. 1997; 91: 511-520Abstract Full Text Full Text PDF PubMed Scopus (995) Google Scholar). Intimin consists of three functional components: an extracellular domain, a transmembrane domain, and a small periplasmically located domain. The adhesive, extracellular region (referred to as the passenger) of intimin is an extended, rod-like structure (Fig. 1A). The crystal structure of the intimin C terminus shows three domains: two immunoglobulin (Ig)-like domains (D1–2) capped by a C-type lectin-like domain (D3) (3Luo Y. Frey E.A. Pfuetzner R.A. Creagh A.L. Knoechel D.G. Haynes C.A. Finlay B.B. Strynadka N.C. Crystal structure of enteropathogenic Escherichia coli intimin-receptor complex.Nature. 2000; 405: 1073-1077Crossref PubMed Scopus (248) Google Scholar). The Ig-like domain D2 and the lectin-like domain D3 together form the Tir binding region (4Kelly G. Prasannan S. Daniell S. Fleming K. Frankel G. Dougan G. Connerton I. Matthews S. Structure of the cell-adhesion fragment of intimin from enteropathogenic Escherichia coli.Nat. Struct. Biol. 1999; 6: 313-318Crossref PubMed Scopus (139) Google Scholar). In addition, a third Ig-like domain (D0) was predicted at the N terminus of the passenger. Later, an additional domain at the very N terminus of the extracellular domain has been identified, referred to as the D00 domain, although its fold was not conclusively predicted (5Fairman J.W. Dautin N. Wojtowicz D. Liu W. Noinaj N. Barnard T.J. Udho E. Przytycka T.M. Cherezov V. Buchanan S.K. Crystal structures of the outer membrane domain of intimin and invasin from enterohemorrhagic E. coli and enteropathogenic Y. pseudotuberculosis.Structure. 2012; 20: 1233-1243Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). Intimin is anchored in the outer membrane by a 12-stranded β-barrel domain (5Fairman J.W. Dautin N. Wojtowicz D. Liu W. Noinaj N. Barnard T.J. Udho E. Przytycka T.M. Cherezov V. Buchanan S.K. Crystal structures of the outer membrane domain of intimin and invasin from enterohemorrhagic E. coli and enteropathogenic Y. pseudotuberculosis.Structure. 2012; 20: 1233-1243Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). In addition, intimin contains a short N-terminal periplasmic region containing a lysin motif, which mediates dimerization and binding to peptidoglycan at low pH (6Leo J.C. Oberhettinger P. Chaubey M. Schütz M. Kühner D. Bertsche U. Schwarz H. Götz F. Autenrieth I.B. Coles M. Linke D. The intimin periplasmic domain mediates dimerisation and binding to peptidoglycan.Mol. Microbiol. 2015; 95: 80-100Crossref PubMed Scopus (26) Google Scholar). As a surface-exposed protein, the intimin passenger is secreted across the outer membrane to the cell surface. This is accomplished by an autotransport process, termed type Ve secretion (7Leo J.C. Grin I. Linke D. Type V secretion: mechanism(s) of autotransport through the bacterial outer membrane.Philos. Trans. R Soc. Lond. B. Biol. Sci. 2012; 367: 1088-1101Crossref PubMed Scopus (174) Google Scholar, 8Oberhettinger P. Schütz M. Leo J.C. Heinz N. Berger J. Autenrieth I.B. Linke D. intimin and invasin export their C-terminus to the bacterial cell surface using an inverse mechanism compared to classical autotransport.PLoS ONE. 2012; 7: e47069Crossref PubMed Scopus (45) Google Scholar). Intimin is the prototype of type Ve secretion, also known as inverse autotransport (9Leo J.C. Oberhettinger P. Schütz M. Linke D. The inverse autotransporter family: intimin, invasin, and related proteins.Int. J. Med. Microbiol. 2015; 305: 276-282Crossref PubMed Scopus (51) Google Scholar). In the current model for type Ve secretion, the β-barrel domain acts as a secretion pore through which the passenger domain is transported via a hairpin intermediate, such that the N terminus of the passenger is the first to reach the extracellular space (5Fairman J.W. Dautin N. Wojtowicz D. Liu W. Noinaj N. Barnard T.J. Udho E. Przytycka T.M. Cherezov V. Buchanan S.K. Crystal structures of the outer membrane domain of intimin and invasin from enterohemorrhagic E. coli and enteropathogenic Y. pseudotuberculosis.Structure. 2012; 20: 1233-1243Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 8Oberhettinger P. Schütz M. Leo J.C. Heinz N. Berger J. Autenrieth I.B. Linke D. intimin and invasin export their C-terminus to the bacterial cell surface using an inverse mechanism compared to classical autotransport.PLoS ONE. 2012; 7: e47069Crossref PubMed Scopus (45) Google Scholar). Secretion of the entire passenger then proceeds N to C, with the C terminus the last to reach the surface. This model is supported by the crystal structure of the intimin β-barrel domain, where the linker region connecting the passenger and the β-barrel domain is located within the pore of the β-barrel (5Fairman J.W. Dautin N. Wojtowicz D. Liu W. Noinaj N. Barnard T.J. Udho E. Przytycka T.M. Cherezov V. Buchanan S.K. Crystal structures of the outer membrane domain of intimin and invasin from enterohemorrhagic E. coli and enteropathogenic Y. pseudotuberculosis.Structure. 2012; 20: 1233-1243Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). However, outer membrane insertion of intimin is dependent on the β-barrel assembly machinery or BAM complex (10Bodelón G. Marín E. Fernández L.A. Role of periplasmic chaperones and BamA (YaeT/Omp85) in folding and secretion of intimin from enteropathogenic Escherichia coli strains.J. Bacteriol. 2009; 191: 5169-5179Crossref PubMed Scopus (68) Google Scholar), and the BAM complex may also be involved in passenger secretion (11Oberhettinger P. Leo J.C. Linke D. Autenrieth I.B. Schütz M. The inverse autotransporter intimin exports its passenger domain via a hairpin intermediate.J. Biol. Chem. 2015; 290: 1837-1849Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). Additionally, the periplasmic chaperone SurA is required for inverse autotransporter biogenesis (8Oberhettinger P. Schütz M. Leo J.C. Heinz N. Berger J. Autenrieth I.B. Linke D. intimin and invasin export their C-terminus to the bacterial cell surface using an inverse mechanism compared to classical autotransport.PLoS ONE. 2012; 7: e47069Crossref PubMed Scopus (45) Google Scholar, 10Bodelón G. Marín E. Fernández L.A. Role of periplasmic chaperones and BamA (YaeT/Omp85) in folding and secretion of intimin from enteropathogenic Escherichia coli strains.J. Bacteriol. 2009; 191: 5169-5179Crossref PubMed Scopus (68) Google Scholar, 11Oberhettinger P. Leo J.C. Linke D. Autenrieth I.B. Schütz M. The inverse autotransporter intimin exports its passenger domain via a hairpin intermediate.J. Biol. Chem. 2015; 290: 1837-1849Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). Thus, the term “autotransporter” is somewhat misleading but may still be valid for the export of the passenger. This study was prompted by our recent observation that introduction of a double hemagglutinin (HA) tag (sequence GSGYPYDVPDYAGSGYPYDVPDYAGSG) at the N terminus of the passenger domain prevents secretion of the passenger and traps intimin in what is most probably the hairpin intermediate (11Oberhettinger P. Leo J.C. Linke D. Autenrieth I.B. Schütz M. The inverse autotransporter intimin exports its passenger domain via a hairpin intermediate.J. Biol. Chem. 2015; 290: 1837-1849Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). In this mutant the β-barrel domain is fully formed and inserted into the membrane, but the C terminus of the passenger is located in the periplasm (Fig. 1B). The double HA tag of this mutant is in fact located in the beginning of the D00 domain sequence, where it disrupts a predicted β-strand (Fig. 1C). In this study we investigated the effect of this insertion on the D00 domain and its implications for passenger secretion. The C-terminal, membrane-proximal region of several classical autotransporter passengers functions as a highly stable folding core that is necessary for efficient secretion (12Soprova Z. Sauri A. van Ulsen P. Tame J.R. den Blaauwen T. Jong W.S. Luirink J. A conserved aromatic residue in the autochaperone domain of the autotransporter Hbp is critical for initiation of outer membrane translocation.J. Biol. Chem. 2010; 285: 38224-38233Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar13Peterson J.H. Tian P. Ieva R. Dautin N. Bernstein H.D. Secretion of a bacterial virulence factor is driven by the folding of a C-terminal segment.Proc. Natl. Acad. Sci. U.S.A. 2010; 107: 17739-17744Crossref PubMed Scopus (84) Google Scholar, 14Oliver D.C. Huang G. Nodel E. Pleasance S. Fernandez R.C. A conserved region within the Bordetella pertussis autotransporter BrkA is necessary for folding of its passenger domain.Mol. Microbiol. 2003; 47: 1367-1383Crossref PubMed Scopus (126) Google Scholar15Junker M. Schuster C.C. McDonnell A.V. Sorg K.A. Finn M.C. Berger B. Clark P.L. Pertactin β-helix folding mechanism suggests common themes for the secretion and folding of autotransporter proteins.Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 4918-4923Crossref PubMed Scopus (167) Google Scholar). This region has earlier been termed an “autochaperone” domain, although this name is no longer recommended (16Drobnak I. Braselmann E. Chaney J.L. Leyton D.L. Bernstein H.D. Lithgow T. Luirink J. Nataro J.P. Clark P.L. Of linkers and autochaperones: An unambiguous nomenclature to identify common and uncommon themes for autotransporter secretion.Mol. Microbiol. 2015; 95: 1-16Crossref PubMed Scopus (25) Google Scholar). By analogy, the N-terminal region of inverse autotransporters has been suggested to play a similar role in the secretion process by promoting protein folding (11Oberhettinger P. Leo J.C. Linke D. Autenrieth I.B. Schütz M. The inverse autotransporter intimin exports its passenger domain via a hairpin intermediate.J. Biol. Chem. 2015; 290: 1837-1849Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar, 17Tsai J.C. Yen M.-R Castillo R. Leyton D.L. Henderson I.R. Saier Jr., M.H. The bacterial intimins and invasins: a large and novel family of secreted proteins.PLoS ONE. 2010; 5: e14403-e14414Crossref PubMed Scopus (44) Google Scholar). We, therefore, set out to test whether the D00 domain acts as a folding core in inverse autotransport. Our results show that disruption of this domain leads to misfolding of D00 and stalling of passenger secretion; however, the D00 domain is not required for passenger secretion, and it does not form a stable folding core. Rather, our results support a model where sequential folding of individual Ig-like domains at the cell surface provides most of the free energy for inverse autotransporter passenger secretion. When investigating the borders of the β-barrel domain of Intimin, Fairman et al. (5Fairman J.W. Dautin N. Wojtowicz D. Liu W. Noinaj N. Barnard T.J. Udho E. Przytycka T.M. Cherezov V. Buchanan S.K. Crystal structures of the outer membrane domain of intimin and invasin from enterohemorrhagic E. coli and enteropathogenic Y. pseudotuberculosis.Structure. 2012; 20: 1233-1243Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar) identified a protease-resistant extracellular fragment that they termed domain D00. This domain was not recognized as either a bacterial Ig-like or a C-type lectin domain by the Pfam database; therefore, Fairman et al. (5Fairman J.W. Dautin N. Wojtowicz D. Liu W. Noinaj N. Barnard T.J. Udho E. Przytycka T.M. Cherezov V. Buchanan S.K. Crystal structures of the outer membrane domain of intimin and invasin from enterohemorrhagic E. coli and enteropathogenic Y. pseudotuberculosis.Structure. 2012; 20: 1233-1243Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar) suggested it might adopt a different fold. However, when we subjected the amino acid sequence of the D00 domain to sensitive homology-based structure prediction using HHPred (18Söding J. Biegert A. Lupas A.N. The HHpred interactive server for protein homology detection and structure prediction.Nucleic Acids Res. 2005; 33: W244-W248Crossref PubMed Scopus (2631) Google Scholar), all the best hits were to Ig-like domains (Fig. 2A). In addition, secondary structure prediction for an alignment of D00 domains from various inverse autotransporters shows seven predicted β-strands, consistent with an Ig-like topology (Fig. 2B). Furthermore, we made a homology model of the D00-D0 domain pair, which clearly shows both adopting an Ig-like fold (Fig. 2C and supplemental File 1). To test our prediction, we produced and purified the D00 domain using recombinant techniques. The circular dichroism (CD) spectrum of the protein shows a broad minimum centered around 212 nm that is typical of β-structured proteins, consistent with our predictions of an Ig-like domain (Fig. 2D).