A thiol‐disulfide oxidoreductase of the Gram‐positive pathogen Corynebacterium diphtheriae is essential for viability, pilus assembly, toxin production and virulence

Abstract

Summary The Gram-positive pathogen Corynebacterium diphtheriae exports through the Sec apparatus many extracellular proteins that include the key virulence factors diphtheria toxin and the adhesive pili. How these proteins attain their native conformations after translocation as unfolded precursors remains elusive. The fact that the majority of these exported proteins contain multiple cysteine residues and that several membrane-bound oxidoreductases are encoded in the corynebacterial genome suggests the existence of an oxidative protein-folding pathway in this organism. Here we show that the shaft pilin SpaA harbors a disulfide bond in vivo and alanine substitution of these cysteines abrogates SpaA polymerization and leads to the secretion of degraded SpaA peptides. We then identified a thiol-disulfide oxidoreductase (MdbA), whose structure exhibits a conserved thioredoxin-like domain with a CPHC active site. Remarkably, deletion of mdbA results in a severe temperature-sensitive cell division phenotype. This mutant also fails to assemble pilus structures and is greatly defective in toxin production. Consistent with these defects, the ΔmdbA mutant is attenuated in a guinea pig model of diphtheritic toxemia. Given its diverse cellular functions in cell division, pilus assembly and toxin production, we propose that MdbA is a component of the general oxidative folding machine in C. diphtheriae.

Disulfide-Bond-Forming Pathways in Gram-Positive Bacteria

“…The failure to form disulfide bonds is associated with the absence of pili and the secretion of degradation products into the culture medium (23,24). This suggests that the covalent linkages are important for the proper folding and/or stability of pilus precursors in the exoplasm.…”

“…Using a combination of genetics, X-ray crystallization, and biochemical methods, the thiol-disulfide oxidoreductase enzyme called MdbA was identified in A. oris and C. diphtheriae (23,24). In these organisms, it was shown that pilin precursors are oxidized by the membrane-bound oxidoreductase.…”

“…tuberculosis Mt-DsbA (23,24). A. oris and C. diphtheriae MdbA enzymes also contain His residues in their CXXC motifs and possess neutral surface potentials near the predicted sites for substrate binding (23,24).…”

“…In these organisms, it was shown that pilin precursors are oxidized by the membrane-bound oxidoreductase. In A. oris, MdbA activity is recycled by a VKOR homologue (23), while C. diphtheriae MdbA may be reoxidized by an unknown factor (24) (Fig. 2).…”

“…Using adhesive pili and diphtheria toxin as model substrates, these bacteria were revealed to possess membrane-localized disulfide-bond-forming systems led by the oxidoreductase MdbA (23,24). Remarkably, mdbA mutants exhibit severe morphological defects, indicating that unlike known Gram-negative Dsb proteins, MdbA is important for growth.…”