A Disulfide Bond-forming Machine Is Linked to the Sortase-mediated Pilus Assembly Pathway in the Gram-positive Bacterium Actinomyces oris

作者：Reardon Robinson Melissa E; Osipiuk Jerzy; Chang Chungyu; Wu Chenggang; Jooya Neda; Joachimiak Andrzej; Das Asis; Ton That Hung^*

来源：JOURNAL OF BIOLOGICAL CHEMISTRY, 2015, 290(35): 21393-21405.

DOI：10.1074/jbc.M115.672253

• actinobacteria
• crystal structure
• crystallography
• disulfide
• secretion
• Actinomyces
• coaggregation
• oxidative protein folding
• pili
• sortase

Background: Gram-positive bacteria secrete pilins through the Sec translocon in unfolded states. Results: Disruption of pilus disulfide bonds or genetic disruption of oxidoreductase-encoding genes mdbA and vkor abrogates pilus assembly in Actinomyces oris. Conclusion: MdbA and VKOR constitute a disulfide bond-forming machine in A. oris. Significance: Oxidative protein folding may be common in Actinobacteria and an attractive target for antimicrobials.
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 vkor 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.

• 出版日期2015-8-28