Germination is essential for many spore-forming bacteria to initiate disease. Spores of the nosocomial pathogen Clostridioides difficile induce germination when they sense bile acid (germinants) combined with amino acids and/or Ca2+ (co-germinants). C. difficile senses these small molecules using a mechanism that is distinct from previously studied spore-formers because it lacks the transmembrane germinant receptors encoded by all other spore-formers. Instead, C. difficile uses two soluble proteins of the clostridial serine protease family, CspC and CspA, to sense germinant and co-germinant signals. While most Csps are active proteases, C. difficile CspC and CspA are pseudoproteases due to mutations in their catalytic triads. Despite their lack of catalytic activity, the CspC and CspA pseudoproteases control the activity of a related protease, CspB, during germination. While CspC and CspA were identified as the likely bile acid germinant and co-germinant receptors, respectively, through genetic screens, we unexpectedly discovered in the past funding period that CspC not only senses bile acid germinant but also co-germinant signals. These findings raise the question as to how CspC and CspA integrate signals from such different small molecules, especially since we lack biochemical evidence that either of these proteins binds any of these signaling molecules. We recently gained biochemical insight into this question by demonstrating that CspC and CspA directly interact. We also determined that CspA forms a homodimer, in contrast with CspC, by solving CspA’s crystal structure. Intriguingly, when we model CspC onto our unpublished structure of CspA, CspC residues that control the sensitivity of C. difficile spores to germinants and/or co-germinants cluster to the predicted CspC:CspA heterodimerization interface. These data lead us to hypothesize that germinant and co-germinant signals alter the equilibrium between CspC:CspA heterodimers and CspA homodimers. Since CspA is also needed for CspC to be stably incorporated into spores, we propose that a partner-swap mechanism regulates C. difficile spore germination. According to this model, CspC:CspA heterodimerization allows CspC to be stably incorporated into dormant spores. The inactive heterodimer is disrupted upon germinant and co-germinant addition, which triggers germination potentially by promoting CspA homodimerization. We will test this model by evaluating the functional significance of CspC:CspA heterodimerization and CspA homodimerization (Aim 1) and the impact of germinants and co-germinants on these protein:protein interactions (Aim 2). We will also determine the role of CspA’s unstructured prodomain in regulating the equilibrium between CspA homodimers and CspC:CspA heterodimers in the presence of germinant and co-germinant signals (Aim 3). Collectively, these analyses will provide atomic- level insight into how C. difficile spores transduce germinant and co-germinant signals to induce germ...