title: Reoxidation of the Thiol-Disulfide Oxidoreductase MdbA by a Bacterial Vitamin K Epoxide Reductase in the Biofilm-Forming Actinobacterium Actinomyces oris.
abstract: Posttranslocational protein folding in the Gram-positive biofilm-forming actinobacteriumActinomyces orisis mediated by a membrane-bound thiol-disulfide oxidoreductase named MdbA, which catalyzes oxidative folding of nascent polypeptides transported by the Sec translocon. Reoxidation of MdbA involves a bacterialvitaminKepoxidereductase (VKOR)-like protein that contains four cysteine residues, C93/C101 and C175/C178, with the latter forming a canonical CXXC thioredoxin-like motif; however, the mechanism of VKOR-mediated reoxidation of MdbA is not known. We present here a topological view of theA. orismembrane-spanning protein VKOR with these four exoplasmic cysteine residues that participate in MdbA reoxidation. Like deletion of the VKOR gene, alanine replacement of individual cysteine residues abrogated polymicrobial interactions and biofilm formation, concomitant with the failure to form adhesive pili on the bacterial surface. Intriguingly, the mutation of the cysteine at position 101 to alanine (C101A mutation) resulted in a high-molecular-weight complex that was positive for MdbA and VKOR by immunoblotting and was absent in other alanine substitution mutants and the C93A C101A double mutation and after treatment with the reducing agent β-mercaptoethanol. Consistent with this observation, affinity purification followed by immunoblotting confirmed this MdbA-VKOR complex in the C101A mutant. Furthermore, ectopic expression of theMycobacterium tuberculosisVKOR analog in theA. orisVKOR deletion (ΔVKOR) mutant rescued its defects, in contrast to the expression ofM. tuberculosisVKOR variants known to be nonfunctional in the disulfide relay that mediates reoxidation of the disulfide bond-forming catalyst DsbA inEscherichia coliAltogether, the results support a model of a disulfide relay, from its start with the pair C93/C101 to the C175-X-X-C178 motif, that is required for MdbA reoxidation and appears to be conserved in members of the classActinobacteriaIMPORTANCEIt has recently been shown in the high-GC Gram-positive bacteria (orActinobacteria)Actinomyces orisandCorynebacterium diphtheriaethat oxidative folding of nascent polypeptides transported by the Sec machinery is catalyzed by a membrane-anchored oxidoreductase named MdbA. InA. oris, reoxidation of MdbA requires a bacterial VKOR-like protein, and yet, how VKOR mediates MdbA reoxidation is unknown. We show here that theA. orismembrane-spanning protein VKOR employs two pairs of exoplasmic cysteine residues, including the canonical CXXC thioredoxinlike motif, to oxidize MdbA via a disulfide relay mechanism. This mechanism of disulfide relay is essential for pilus assembly, polymicrobial interactions, and biofilm formation and appears to be conserved in members of the classActinobacteria, includingMycobacterium tuberculosis.
authors.affiliation: Department of Microbiology and Molecular Genetics, University of Texas Health Science Center, Houston, Texas, USA.
authors.forename: Truc Thanh
authors.initials: TT
authors.lastname: Luong
authors.affiliation: Department of Microbiology and Molecular Genetics, University of Texas Health Science Center, Houston, Texas, USA.|Department of Microbiology & Immunobiology, Harvard Medical School, Boston, Massachusetts, USA.
authors.forename: Melissa E
authors.initials: ME
authors.lastname: Reardon-Robinson
authors.affiliation: Department of Microbiology and Molecular Genetics, University of Texas Health Science Center, Houston, Texas, USA.
authors.forename: Sara D
authors.initials: SD
authors.lastname: Siegel
authors.affiliation: Department of Microbiology and Molecular Genetics, University of Texas Health Science Center, Houston, Texas, USA ton-that.hung@uth.tmc.edu.
authors.forename: Hung
authors.initials: H
authors.lastname: Ton-That
mesh_terms: D000190:Actinomyces / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D000409:Alanine / Q000235:genetics / Q000378:metabolism; D019943:Amino Acid Substitution; D018441:Biofilms / Q000254:growth & development*; D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D004252:DNA Mutational Analysis; D010861:Fimbriae, Bacterial / Q000378:metabolism; D046529:Microscopy, Electron, Transmission; D008954:Models, Biological; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D009169:Mycobacterium tuberculosis / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D001678:Organelle Biogenesis; D010084:Oxidation-Reduction; D011490:Protein Disulfide Reductase (Glutathione) / Q000378:metabolism*; D064417:Vitamin K Epoxide Reductases / Q000235:genetics / Q000378:metabolism*
chemical_list: D050505:Mutant Proteins; D064417:Vitamin K Epoxide Reductases; D011490:Protein Disulfide Reductase (Glutathione); D003545:Cysteine; D000409:Alanine
keywords: Actinobacteria; biofilm formation; disulfide bond formation; oxidative protein folding; oxidoreductases; pilus assembly; polymicrobial interactions; sortase