Oxidative Protein Folding Pathways In Gram-Positive Actinobacteria

<p>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.</p> <p>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 <em>A. oris</em> and <em>C. diphtheriae</em>, we found that protein oxidation is catalyzed by the membrane-bound MdbA. In <em>A. oris</em>, MdbA activity is maintained by VKOR, which is absent in <em>C. diphtheriae</em>. MdbA-catalyzed disulfide bond formation is required for the production of multiple virulence factors including diphtheria toxin. Therefore, mutations targeting <em>mdbA</em> have profound consequences for pathogenesis. <em>A. oris </em>mutants are defective in biofilm growth, while C<em>. diphtheriae </em>exhibits attenuated virulence in an animal model.</p> <p>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 <em>A. oris</em> <em>mdbA, </em>and deletion of <em>C. diphtheriae</em> <em>mdbA</em> 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 <em>C. diphtheriae mdbA</em> selects for a suppressor mutation that causes the overexpression of an oxidoreductase named TsdA.</p> <p>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.</p>

Name thesis:degree_name

Doctor of Philosophy (PhD)

Level thesis:degree_level

Dissertation (PhD)

Year dc:date.available

2015

Author dc:creator

• Robinson, Melissa E

Contributors dc:contributor

• Hung Ton-That
• Ziyin Li
• William Margolin

• Actinomyces
• Corynebacterium
• Actinobacteria
• disulfide bond formation
• pilus assembly
• pathogenesis
• Medicine and Health Sciences

Repository record dc:identifier

https://digitalcommons.library.tmc.edu/utgsbs_dissertations/565

OAI identifier oai:identifier

oai:digitalcommons.library.tmc.edu:utgsbs_dissertations-1606