Elucidating the pleiotropic effects of clpC deletion in Clostridioides difficile: proteomic and phenotypic insights

Clostridioides difficile is a major cause of antibiotic-associated diarrhea and a significant public health threat. During its infectious cycle, C. difficile encounters various stresses within the gastrointestinal tract. Clp proteases play a crucial role in bacterial stress responses and protein homeostasis. In this study, we investigated the role of the Clp ATPase, ClpC, in C. difficile physiology using genetic, phenotypic, and proteomic analyses. Deletion of clpC reduced heat shock survival but did not affect growth or stationary phase survival under non-stress conditions. Comparative proteomics revealed that ClpC influences the abundance of proteins involved in sporulation, motility, metabolism, and cell wall biosynthesis. The ΔclpC mutant exhibited faster sporulation and increased motility compared to the parental strain. Peptidoglycan quantification showed a significant increase in the ΔclpC mutant, suggesting ClpC's involvement in cell wall homeostasis. The mutant also displayed altered sensitivity to cell wall-targeting antibiotics. Unlike in other bacteria, ClpC did not control the level of MurA, a key enzyme in peptidoglycan precursor synthesis. Instead, the SEDS protein RodA, a transglycosylase involved in peptidoglycan polymerization, accumulated in the ΔclpC mutant. Our findings highlight the pleiotropic role of ClpC in C. difficile, particularly in sporulation, motility, and cell wall metabolism, likely through the degradation of key proteins. Understanding the molecular mechanisms of ClpC-mediated proteolysis in C. difficile stress responses and virulence may provide insights for the development of novel strategies to combat this important pathogen.