The Gram-positive bacterium Clostridioides difficile is a primary cause of hospital-acquired diarrhea, threatening both immunocompromised and healthy individuals. An important aspect of elucidating mechanisms that drive C. difficile persistence and virulence relies on developing a more complete understanding of sporulation. C. difficile sporulation is the single determinant of transmission and complicates treatment and prevention due to the chemical and physical resilience of spores. Hence, the identification of potentially druggable targets that significantly attenuate sporulation is important. In this report, we describe the impact of the loss of caseinolytic protease P (ClpP) isoforms in C. difficile strain 630 on sporulation phenotypes. Using CRISPR-Cas9 nickase mediated genome editing, stop codons were inserted early in the coding sequence for clpP1 and clpP2 to generate C. difficile mutants that no longer produced ClpP1 or ClpP2. The data show that these genetic modifications lead to altered sporulation phenotypes, germination efficiencies, and cytotoxicity. Comparative proteome profiling of C. difficile 630 WT and clpP mutants reveals potential proteolytic targets of ClpP that are involved in sporulation. These analyses further reveal the potential for preferred co-chaperone interactions for each ClpP isoform. Taken together, our results demonstrate that ClpP, a promising target in other Gram-positive pathogens, holds promise as an anti-sporulation target in C. difficile.
