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.
authors.affiliation: From the Evolution and Genetics, Department of Biosciences, University of Oslo, 0316 Oslo, Norway.
authors.forename: Jack C
authors.initials: JC
authors.lastname: Leo
authors.affiliation: Interfaculty Institute for Microbiology and Infection Medicine, University Clinics Tübingen, 72076 Tübingen, Germany.
authors.forename: Philipp
authors.initials: P
authors.lastname: Oberhettinger
authors.affiliation: Department of Biotechnology, Graduate School of Engineering, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8603, Japan, and.
authors.forename: Shogo
authors.initials: S
authors.lastname: Yoshimoto
authors.affiliation: From the Evolution and Genetics, Department of Biosciences, University of Oslo, 0316 Oslo, Norway.
authors.forename: D B R K Gupta
authors.initials: DB
authors.lastname: Udatha
authors.affiliation: Centre for Molecular Medicine Norway, 0318 Oslo, Norway.
authors.forename: J Preben
authors.initials: JP
authors.lastname: Morth
authors.affiliation: Interfaculty Institute for Microbiology and Infection Medicine, University Clinics Tübingen, 72076 Tübingen, Germany.
authors.forename: Monika
authors.initials: M
authors.lastname: Schütz
authors.affiliation: Department of Biotechnology, Graduate School of Engineering, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8603, Japan, and.
authors.forename: Katsutoshi
authors.initials: K
authors.lastname: Hori
authors.affiliation: From the Evolution and Genetics, Department of Biosciences, University of Oslo, 0316 Oslo, Norway, dirk.linke@ibv.uio.no.
authors.forename: Dirk
authors.identifier: 0000-0003-3150-6752
authors.initials: D
authors.lastname: Linke
mesh_terms: D018829:Adhesins, Bacterial / Q000235:genetics; D054308:Enteropathogenic Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D008954:Models, Biological; D000072417:Protein Domains; D017510:Protein Folding
chemical_list: D018829:Adhesins, Bacterial; D029968:Escherichia coli Proteins; C073819:eaeA protein, E coli
keywords: atomic force microscopy (AFM); bacterial pathogenesis; immunoglobulin-like domain; protein folding; protein secretion; type V secretion system