A cryptic oxidoreductase safeguards oxidative protein folding in Corynebacterium diphtheriae

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 120; no. 8; p. e2208675120
• Main Authors: Reardon-Robinson, Melissa E, Nguyen, Minh Tan, Sanchez, Belkys C, Osipiuk, Jerzy, Rückert, Christian, Chang, Chungyu, Chen, Bo, Nagvekar, Rahul, Joachimiak, Andrzej, Tauch, Andreas, Das, Asis, Ton-That, Hung
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences 21.02.2023; Proceedings of the National Academy of Sciences
• Subjects: Bacterial Proteins - genetics; Bacterial Proteins - metabolism; BASIC BIOLOGICAL SCIENCES; Biological Sciences; Clonal deletion; Corynebacterium diphtheriae; Corynebacterium diphtheriae - enzymology; Corynebacterium diphtheriae - genetics; diphtheria toxin; disulfide bond; Folding; Genetic analysis; Genetic suppression; gram-positive bacteria; Mutants; Mutation; Oxidative Stress; Oxidoreductase; pili; Pilin; Protein disulfide reductase (glutathione); Protein Disulfide Reductase (Glutathione) - genetics; Protein Disulfide Reductase (Glutathione) - metabolism; Protein Folding; Proteins; Revertants; Substrates; Suppressors; Thermal stress; Toxins; gram-positive bacteria; diphtheria toxin; disulfide bond; pili; Corynebacterium diphtheriae
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.2208675120

In many gram-positive Actinobacteria, including and , the conserved thiol-disulfide oxidoreductase MdbA that catalyzes oxidative folding of exported proteins is essential for bacterial viability by an unidentified mechanism. Intriguingly, in , the deletion of blocks cell growth only at 37 °C but not at 30 °C, suggesting the presence of alternative oxidoreductase enzyme(s). By isolating spontaneous thermotolerant revertants of the mutant at 37 °C, we obtained genetic suppressors, all mapped to a single T-to-G mutation within the promoter region of , causing its elevated expression. Strikingly, increased expression of -via suppressor mutations or a constitutive promoter-rescues the pilus assembly and toxin production defects of this mutant, hence compensating for the loss of . Structural, genetic, and biochemical analyses demonstrated TsdA is a membrane-tethered thiol-disulfide oxidoreductase with a conserved CxxC motif that can substitute for MdbA in mediating oxidative folding of pilin and toxin substrates. Together with our observation that expression is upregulated at nonpermissive temperature (40 °C) in wild-type cells, we posit that TsdA has evolved as a compensatory thiol-disulfide oxidoreductase that safeguards oxidative protein folding in against thermal stress.