The CspC:CspA heterodimer transduces germinant and co-germinant signals during Clostridioides difficile spore germination.

The clinically significant pathogen Clostridioides difficile lacks the transmembrane nutrient germinant receptors conserved in almost all spore-forming bacteria. Instead, C. difficile initiates spore germination using a unique mechanism that requires two signals: a bile acid germinant and a co-germinant, which can be either an amino acid or a divalent cation. While two soluble pseudoproteases, CspC and CspA, were initially identified as the germinant and co-germinant receptors, respectively, in C. difficile, we previously identified residues in an unstructured region of CspC that regulate the sensitivity of C. difficile spores to both signals. However, the mechanism by which CspC transduces these signals remained unclear. Here, we demonstrate that CspC forms a stable complex with CspA and determine the crystal structure of the CspC:CspA heterodimer. The structure reveals extensive interactions along the binding interface, including direct interactions between the unstructured region of CspC and CspA. Using structure-function analyses, we identify CspC:CspA interactions that regulate the sensitivity of C. difficile spores to germinant signals and show that CspA regulates the response of C. difficile to not only co-germinant but also germinant signals. While we show that CspA can form a homodimer and determine its crystal structure, CspA homodimerization appears unimportant for C. difficile spore germination. Collectively, our analyses establish the CspC:CspA heterodimer, rather than its individual constituents, as a critical signaling node for sensing both germinant and co-germinant signals. They also suggest a new mechanistic model for how C. difficile transduces germinant signals, which could guide the development of therapeutics against this important pathogen.