title: A Disulfide Bond-forming Machine Is Linked to the Sortase-mediated Pilus Assembly Pathway in the Gram-positive Bacterium Actinomyces oris.
abstract: Export of cell surface pilins in Gram-positive bacteria likely occurs by the translocation of unfolded precursor polypeptides; however, how the unfolded pilins gain their native conformation is presently unknown. Here, we present physiological studies to demonstrate that the FimA pilin of Actinomyces oris contains two disulfide bonds. Alanine substitution of cysteine residues forming the C-terminal disulfide bridge abrogates pilus assembly, in turn eliminating biofilm formation and polymicrobial interaction. Transposon mutagenesis of A. oris yielded a mutant defective in adherence to Streptococcus oralis, and revealed the essential role of a vitamin K epoxide reductase (VKOR) gene in pilus assembly. Targeted deletion of vkor results in the same defects, which are rescued by ectopic expression of VKOR, but not a mutant containing an alanine substitution in its conserved CXXC motif. Depletion of mdbA, which encodes a membrane-bound thiol-disulfide oxidoreductase, abrogates pilus assembly and alters cell morphology. Remarkably, overexpression of MdbA or a counterpart from Corynebacterium diphtheriae, rescues the Δvkor mutant. By alkylation assays, we demonstrate that VKOR is required for MdbA reoxidation. Furthermore, crystallographic studies reveal that A. oris MdbA harbors a thioredoxin-like fold with the conserved CXXC active site. Consistently, each MdbA enzyme catalyzes proper disulfide bond formation within FimA in vitro that requires the catalytic CXXC motif. Because the majority of signal peptide-containing proteins encoded by A. oris possess multiple Cys residues, we propose that MdbA and VKOR constitute a major folding machine for the secretome of this organism. This oxidative protein folding pathway may be a common feature in Actinobacteria.
authors.affiliation: From the Department of Microbiology and Molecular Genetics, University of Texas Health Science Center, Houston, Texas 77030.
authors.forename: Melissa E
authors.initials: ME
authors.lastname: Reardon-Robinson
authors.affiliation: the Department of Biosciences, Midwest Center for Structural Genomics, and the Department of Biosciences, Structural Biology Center, Argonne National Laboratory, Argonne, Illinois 60439, and.
authors.forename: Jerzy
authors.initials: J
authors.lastname: Osipiuk
authors.affiliation: From the Department of Microbiology and Molecular Genetics, University of Texas Health Science Center, Houston, Texas 77030.
authors.forename: Chungyu
authors.initials: C
authors.lastname: Chang
authors.affiliation: From the Department of Microbiology and Molecular Genetics, University of Texas Health Science Center, Houston, Texas 77030.
authors.forename: Chenggang
authors.initials: C
authors.lastname: Wu
authors.affiliation: From the Department of Microbiology and Molecular Genetics, University of Texas Health Science Center, Houston, Texas 77030.
authors.forename: Neda
authors.initials: N
authors.lastname: Jooya
authors.affiliation: the Department of Biosciences, Midwest Center for Structural Genomics, and the Department of Biosciences, Structural Biology Center, Argonne National Laboratory, Argonne, Illinois 60439, and.
authors.forename: Andrzej
authors.initials: A
authors.lastname: Joachimiak
authors.affiliation: the Department of Molecular Biology and Biophysics, University of Connecticut Health Center, Farmington, Connecticut 06030.
authors.forename: Asis
authors.initials: A
authors.lastname: Das
authors.affiliation: From the Department of Microbiology and Molecular Genetics, University of Texas Health Science Center, Houston, Texas 77030, ton-that.hung@uth.tmc.edu.
authors.forename: Hung
authors.initials: H
authors.lastname: Ton-That
mesh_terms: D000190:Actinomyces / Q000737:chemistry / Q000166:cytology / Q000235:genetics / Q000502:physiology*; D000196:Actinomycosis / Q000382:microbiology; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018441:Biofilms / Q000187:drug effects; D018360:Crystallography, X-Ray; D004220:Disulfides / Q000737:chemistry / Q000378:metabolism; D036781:Fimbriae Proteins / Q000737:chemistry / Q000378:metabolism*; D010861:Fimbriae, Bacterial / Q000737:chemistry / Q000378:metabolism*; D017353:Gene Deletion; D006801:Humans; D056265:Microbial Interactions; D008958:Models, Molecular; D011487:Protein Conformation; D011490:Protein Disulfide Reductase (Glutathione) / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017510:Protein Folding; D064417:Vitamin K Epoxide Reductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D004220:Disulfides; D036781:Fimbriae Proteins; D064417:Vitamin K Epoxide Reductases; D011490:Protein Disulfide Reductase (Glutathione)
keywords: Actinomyces; actinobacteria; coaggregation; crystal structure; crystallography; disulfide; oxidative protein folding; pili; secretion; sortase