The requirement for co-germinants during Clostridium difficile spore germination is influenced by mutations in yabG and cspA

Clostridium difficile spore germination is critical for the transmission of disease. C. difficile spores germinate in response to cholic acid derivatives, such as taurocholate (TA), and amino acids, such as glycine or alanine. Although the receptor with which bile acids are recognized (germinant receptor) is known, the amino acid co-germinant receptor has remained elusive. Here, we used EMS mutagenesis to generate mutants with altered requirements for the amino acid co-germinant, similar to the strategy we used previously to identify the bile acid germinant receptor, CspC. Surprisingly, we identified strains that do not require co-germinants, and the mutant spores germinated in response to TA alone. Upon sequencing these mutants, we identified different mutations in yabG. In C. difficile, yabG expression is required for the processing of key germination components to their mature forms (e.g., CspBA to CspB and CspA). A defined yabG mutant exacerbated the EMS mutant phenotype. Building upon this work, we found that small deletions in cspA resulted in spores that germinated in the presence of TA alone without the requirement of a co-germinant. cspA encodes a pseudoprotease that was previously shown to be important for incorporation of the CspC germinant receptor. Herein, our study builds upon the role of CspA during C. difficile spore germination by providing evidence that CspA is important for recognition of co-germinants during C. difficile spore germination. Our work suggests that two pseudoproteases (CspC and CspA) likely function as the C. difficile germinant receptors. Author summary Germination by C. difficile spores is one of the very first steps in the pathogenesis of this organism. The transition from the metabolically dormant spore form to the actively-growing, toxin-producing vegetative form is initiated by certain host-derived bile acids and amino acid signals. Despite near universal conservation in endospore-forming bacteria of the Ger-type germinant receptors, C. difficile and related organisms do not encode these proteins. In prior work, we identified the C. difficile bile acid germinant receptor as the CspC pseudoprotease. In this manuscript, we implicate the CspA pseudoprotease as the C. difficile co-germinant receptor. C. difficile cspA is encoded as a translational fusion to cspB. The resulting CspBA protein is processed post-translationally by the YabG protease. Inactivation of yabG resulted in strains whose spores no longer responded to amino acids or divalent cations as co-germinants and germinated in response to bile acid alone. Building upon this, we found that small deletions in the cspA portion of cspBA resulted in spores that could germinate in response to bile acids alone. Our results suggest that two pseudoproteases regulate C. difficile spore germination and provide further evidence that C. difficile spore germination proceeds through a novel spore germination pathway.