A family of small proteins links c-di-GMP riboswitch signaling with sporulation in Clostridioides difficile

Abstract Cyclic diguanosine monophosphate (c-di-GMP) is a second messenger that coordinates lifestyle transitions, virulence, and developmental processes in bacteria. In Clostridioides difficile , elevated c-di-GMP levels inhibit sporulation, but the underlying mechanism remained unclear. Here, we identified a conserved family of small, membrane-associated proteins encoded by c-di-GMP riboswitch-regulated genes. Transcriptomic analyses revealed that c-di-GMP represses these genes, and reporter assays demonstrated riboswitch-dependent transcriptional regulation via premature termination mechanism. Overexpression of a single gene, CD1980.2 , was sufficient to trigger the transcriptional activation of sporulation genes, including sigma factors and their regulons, and to increase spore formation. Conversely, sporulation efficiency decreased proportionally with the number of deleted small-protein genes, and the strain lacking all seven genes displayed a severe sporulation defect, underscoring their cumulative and functionally redundant roles. Elevating c-di-GMP levels in the deletion mutant did not further reduce sporulation, supporting a model in which c-di-GMP inhibits spore formation by repressing the expression of this small-protein family. Thus, our work establishes these small proteins, encoded by c-di-GMP riboswitch-regulated genes, as important regulators of spore formation in C. difficile , a process essential for pathogen persistence and transmission.