Pseudoprotease-mediated regulation of germinant sensing in Clostridioides difficile - ABSTRACT Clostridioides difficile infections begin when its metabolically dormant spores encounter germinants in the vertebrate gut and initiate the process of germination. This process is essential for C. difficile to produce the vegetative cells that will release toxins and cause disease. Interestingly, distinct mechanisms control C. difficile germinant signaling relative to most spore-forming bacteria. First, C. difficile spore germination requires two distinct signals: a bile acid germinant signal must be combined with amino acid and/or Ca2+ co-germinant signals. Second, C. difficile lacks the transmembrane germinant receptors conserved in almost all spore-forming receptors and instead uses two soluble pseudoproteases, CspA and CspC, to sense co-germinants and germinants, respectively. CspA and CspC then go on to activate the CspB protease, which in turn activates a lytic enzyme responsible for degrading the protective cortex layer of the spore. While CspA and CspC clearly play key roles in regulating the initiation of germination, precisely how they integrate germinant and co-germinant signals is poorly understood. We recently discovered that CspA forms a homodimer while CspC and CspA form a stable heterodimer which is preferred over the CspA homodimer. We solved the crystal structure of the CspA homodimer and the CspA:CspC heterodimer and identified residues that are predicted to regulate these interactions. Given that we previously showed that CspA is required for CspC to be stably incorporated into mature spores, we propose that the CspA:CspC heterodimer is the inactive signaling complex found in dormant spores and that germinants and co-germinants destabilize the heterodimer. Disruption of the CspA:CspC heterodimer likely promotes CspA homodimerization and frees CspC to bind and activate CspB, which activates downstream germination events. In this proposal, I will test this “Partner-Swap” model by identifying residues that mediate CspA homodimer and CspA:CspC heterodimer binding and determine the impact of mutating these residues on C. difficile germination. Using a combination of biochemical, genetic, and cytological analyses, I will also determine the effect of germinants and co-germinants on the stability of the CspA:CspC heterodimer. Collectively, these analyses will reveal how these critical proteins regulate the initiation of C. difficile germination and advance our understanding of pseudoenzymes and their responsibility in the regulation of key cellular processes.
