Identification of Functional Spo0A Residues Critical for Sporulation in Clostridioides Difficile

Clostridioides difficile is an anaerobic, Gram-positive pathogen that is responsible for C. difficile infection (CDI). To survive in the environment and spread to new hosts, C. difficile must form metabolically dormant spores. The formation of spores requires activation of the transcription factor Spo0A, which is the master regulator of sporulation in all endospore-forming bacteria. Though the sporulation initiation pathway has been delineated in the Bacilli, including the model spore-former Bacillus subtilis, the direct regulators of Spo0A in C. difficile remain undefined. C. difficile Spo0A shares highly conserved protein interaction regions with the B. subtilis sporulation proteins Spo0F and Spo0A, although many of the interacting factors present in B. subtilis are not encoded in C. difficile. To determine if comparable Spo0A residues are important for C. difficile sporulation initiation, site-directed mutagenesis was performed at conserved receiver domain residues and the effects on sporulation were examined. Mutation of residues important for homodimerization and interaction with positive and negative regulators of B. subtilis Spo0A and Spo0F impacted C. difficile Spo0A function. The data also demonstrated that mutation of many additional conserved residues altered C. difficile Spo0A activity, even when the corresponding Bacillus interacting proteins are not apparent in the C. difficile genome. Finally, the conserved aspartate residue at position 56 of C. difficile Spo0A was determined to be the phosphorylation site that is necessary for Spo0A activation. The finding that Spo0A interacting motifs maintain functionality suggests that C. difficile Spo0A interacts with yet unidentified proteins that regulate its activity and control spore formation.