Investigation of the factors that regulate sporulation initiation in Clostridioides difficile Público
DiCandia, Michael (Fall 2022)
Abstract
Clostridioides difficile is an anaerobic, Gram-positive pathogen that is responsible for C. difficile infection (CDI). To spread to new hosts, C. difficile must form metabolically-dormant spores. Spo0A is the conserved, master regulator of sporulation in all spore-forming bacteria and must be activated by phosphorylation at a conserved aspartate residue for sporulation to initiate. Though the regulatory proteins that control Spo0A in Bacillus species have been identified, the direct regulators of Spo0A in C. difficile are incompletely defined. To gain insight into the molecular mechanisms that govern sporulation initiation in C. difficile, we performed site-directed mutagenesis of Spo0A and examined the effects on sporulation. As Spo0A shares high sequence similarity between Bacillus subtilis and C. difficile, we chose to mutate conserved Spo0A residues that are functionally important for interaction with sporulation regulatory proteins in B. subtilis. Our data demonstrate that mutation of conserved Spo0A residues significantly impacts sporulation frequency, suggesting that these sites are likewise important for sporulation in C. difficile. Additionally, we sought to define the Spo0A interactome to identify direct Spo0A regulators. In our co-immunoprecipitation experiments, we identified a putative C. difficile ortholog to the Bacillus protein, Spo0E as a Spo0A interacting partner. In Bacillus, Spo0A is directly dephosphorylated by Spo0E to inhibit sporulation. However, functional evidence for Spo0E function in C. difficile and other spore-forming anaerobes is lacking. To determine Spo0E function in C. difficile, we created a spo0E mutant. Mutation of spo0E resulted in increased sporulation, demonstrating Spo0E represses sporulation in C. difficile. Unexpectedly, the spo0E mutant exhibited increased toxin production and motility, providing the first known evidence that Spo0E is involved in physiological processes independent of Spo0A. Accordingly, the spo0E mutant had increased virulence and earlier toxin production in vivo, demonstrating that Spo0E regulates C. difficile pathogenesis. We found that Spo0E repressed motility in B. subtilis, indicating that Spo0E has conserved functionality outside of sporulation. Lastly, we found that putative Spo0E orthologs are broadly conserved, including in non-sporulating bacteria and Archaea, further demonstrating that Spo0E is not simply a repressor of sporulation. Altogether, our findings further our understanding of the factors and mechanisms that impact sporulation initiation and pathogenesis in C. difficile.
Table of Contents
Abstract
Acknowledgments
Table of Contents
List of Tables and Figures
Chapter 1: Introduction (10)
Chapter 2: Identification of functional residues critical for sporulation in Clostridioides difficile (31)
Chapter 3: Discovery of a mechanism linking sporulation, toxin, and motility in Clostridioides difficile (87)
Chapter 4: Discussion (124)
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