Germination is essential for the lifecycle of spore-forming obligate anaerobes like the nosocomial pathogen, Clostridioides difficile. Despite the importance of this process, the molecular mechanisms by which bacterial spores physically detect the small molecule germinants that trigger germination remain poorly understood. Furthermore, the mechanism by which C. difficile spores sense and transduce germinant signals is unique among spore-forming bacteria because (i) C. difficile spores respond to bile acid germinants rather than the canonical nutritional germinants (e.g. sugars) used by all spore-formers studied to date, and (ii) C. difficile lacks the transmembrane germinant receptors encoded by almost all other spore-formers. Instead, C. difficile is thought to use a soluble protein, the CspC pseudoprotease, to detect bile acids and trigger a proteolytic signaling cascade that leads to germination. We recently solved the structure of this putative germinant receptor. Our subsequent structure-function analyses challenge the prevailing model that CspC directly senses bile acid germinants and surprisingly revealed that CspC not only integrates signals from bile acid germinants but also from amino acid and calcium co- germinants. Because of these unexpected findings, the proposed studies will address questions such as (i) what are the direct sensors of bile acid germinants in C. difficile spores? and (ii) How does CspC transduce germinant and co-germinant signals? (Co-)germinant sensing remains poorly defined, so our analyses of CspC and identification of bile acid-binding proteins will provide molecular insight into the mechanisms by which C. difficile spores germinate. Furthermore, since spore germination is essential for C. difficile to initiate infection, our studies may help identify novel strategies for preventing the ~224,000 C. difficile infections per year in the US alone. In addition to advancing our understanding of C. difficile germination, this project will provide me with comprehensive academic and career training. My sponsor, Dr.
Aim ee Shen, is committed to teaching me the technical skills to complete the proposed experiments and the academic skills to effectively share my work with the scientific community. As outlined in my training plan, we have set specific goals for training in writing, presentation, teaching, and mentorship that are critical for me to obtain my overall career goal of holding an academic research faculty position. Dr. Shen and I have assembled a group of collaborators with expertise in biochemistry, crystallography, and chemical biology, and I will seize the opportunity to learn numerous experimental approaches from them. The diverse mentorship I will receive from Dr. Shen and my collaborators will ultimately foster my critical thinking skills, technical capabilities, and development into an independent researcher.
Clostridioides difficile is a major nosocomial pathogen that costs the US health care system ~$1 billion to treat each year. Since spore germination is required for C. difficile to initiate infection, elucidating the mechanisms underlying how bile acid germinant and calcium co-germinant activate spore germination could inform the development of strategies for preventing C. difficile disease and recurrence.