Clostridium difficile is a Gram-positive bacterium that causes a toxin-mediated disease, typically in individuals whose normal intestinal flora has been compromised by antibiotic therapy. C. difficile is naturally resistant to many antibiotics and produces spores that can withstand harsh environmental conditions and many disinfectants, making the infection difficult to clear and easy to spread. The infection begins when spores from the environment are ingested and germinate upon exposure to taurocholate and glycine in the digestive tract. This germination process is required to initiate infection and thus represents a good target for the development of novel therapeutics. Although spore germination is necessary for disease transmission, the molecular mechanisms regulating this process are poorly understood. Germination relies on sensing a germinant and triggering degradation of the cortex layer of the spore, which is important for spore resistance. Once the cortex is degraded, the spore can undergo outgrowth to a vegetative cell and secrete toxins to cause disease symptoms. There are several discrete steps to the proteolytic cascade that ultimately lead to cortex hydrolysis. First, the pseudoprotease CspC acts as a germinant receptor for the bile salt taurocholate; CspC then relays this signal to the subtilisin-like serine protease, CspB. CspB is required for efficient cleavage and activation of the cortex hydrolase. SleC. Upon proteolytic activation of SleC, cortex hydrolysis can proceed, which allows subsequent outgrowth. To better understand the mechanistic basis of the germination process, we solved the 1.6 Å structure of the required germination protease, CspB, from C. perfringens (a related pathogen). This structure revealed that CspB is comprised of three domains: an associated prodomain, a subtilase domain, and a jellyroll domain. Our work significantly advanced our understanding of the proteolytic cascade that leads to germination; in particular the structure and function of the CspB protease, and the role of its three domains. We have described the four domains of the cortex hydrolase, SleC, and how they contribute to the activity of SleC. We have recently obtained diffraction-quality crystals of the pseudoprotease, CspC, from an organism more closely related to C. difficile, C. bifermentans. Our latest work, focusing on the germination receptor, CspC, has brought us closer to a three-dimensional structure of this protein, which will likely reveal how it binds ligands and functions in germination.

Lay Abstract / Prostate cancer (PrCa) is a leading cause of cancer-related death in men worldwide. Standard treatments for unresected PrCa include radiation therapy, androgen deprivation or anti-androgen therapies. However, many patients develop resistance to these therapies indicating a need for a new treatment that can enhance their therapeutic efficacy. Our studies show a promising new strategy to achieve this task using canagliflozin, an FDA-approved medication currently used to treat diabetes, kidney and heart failure. We found that canagliflozin interrupts cellular energy production, inhibits biosynthetic pathways and improves the efficacy of radiotherapy in radiation naïve and radio-resistant human PrCa models. In addition, we found that canagliflozin has promising anti-androgen activity by reducing the protein levels and the activity of androgen receptor (AR) in PrCa cells and tumors both alone and in combination with commercially available anti-androgens. These promising findings suggest that canagliflozin could be used in combination with current standard-of-care therapies for the treatment of PrCa. Given that canagliflozin is an approved medication, tolerated well by diabetic and non-diabatic individuals, the work presented here generates a basis for rapid investigation of this new treatment in early phase clinical trials. / Thesis / Doctor of Philosophy (PhD)