Oxidative protein folding pathways in Gram-positive Actinobacteria

Disulfide bonds are important for the stability of many secreted proteins. These covalent linkages, which result from the oxidation of neighboring cysteine (Cys) residues, are often rate-limiting steps for protein folding and maturation. Disulfide bond formation is restricted to extracellular oxidizing compartments like the eukaryotic endoplasmic reticulum and Gram-negative bacterial periplasm. Protein oxidation has been well-studied in these organisms, but largely ignored in Gram-positive bacteria. Due to the absence of an outer membrane, these organisms are thought to lack compartments in which to catalyze oxidative protein folding. This thesis reveals that Gram-positive Actinobacteria use disulfide bond formation to help fold secreted proteins in the exoplasm. Using the assembly of adhesive pili as a marker for disulfide bond formation in A. oris and C. diphtheriae, we found that protein oxidation is catalyzed by the membrane-bound MdbA. In A. oris, MdbA activity is maintained by VKOR, which is absent in C. diphtheriae. MdbA-catalyzed disulfide bond formation is required for the production of multiple virulence factors including diphtheria toxin. Therefore, mutations targeting mdbA have profound consequences for pathogenesis. A. oris mutants are defective in biofilm growth, while C. diphtheriae exhibits attenuated virulence in an animal model. A major difference between disulfide bond forming enzymes expressed by Gram-negative and Actinobacteria is also revealed. Unlike the Gram-negative DsbA, MdbA is important for viability. The depletion of A. oris mdbA, and deletion of C. diphtheriae mdbA are associated with growth and division defects. We provide evidence that these phenotypes result because secreted growth factors like PBPs fail to form disulfide bonds. Remarkably, the deletion of C. diphtheriae mdbA selects for a suppressor mutation that causes the overexpression of an oxidoreductase named TsdA. In summary, this thesis shows that disulfide bond formation is a major pathway used by Gram-positive Actinobacteria to help fold secreted proteins. This work provides a better understanding of how proteins are folded within the Gram-positive exoplasm, and offers important considerations for developing antibacterial drugs that target oxidative folding pathways