A family of small proteins connects c-di-GMP-signaling with sporulation in the human pathogen Clostridioides difficile

Cyclic diguanosine monophosphate (c-di-GMP) is a ubiquitous bacterial second messenger that coordinates lifestyle transitions, virulence, and developmental processes. 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 as previously unrecognized effectors of c-di-GMP signaling. Their expression is controlled by class I c-di-GMP-responsive riboswitches. 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 member, 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. Our work establishes these small proteins as critical effectors linking c-di-GMP signaling to developmental output in C. difficile. Their redundancy, conservation, and integration into riboswitch- and RgaR-mediated regulatory pathways highlight their central role in spore formation, a process essential for pathogen persistence and transmission. These findings expand the repertoire of second-messenger effectors in bacterial physiology.