A programmed reset mechanism primes Clostridioides difficile virulence at the onset of infection.

Clostridioides difficile is a major cause of antibiotic-associated diarrhea. This pathogen produces toxins essential for disease and flagella that promote intestinal colonization. Flagella and toxins are coregulated in an ON/OFF manner leading to a heterogeneous population of toxigenic, flagellated cells (flg-ON) and aflagellate cells with attenuated toxin production (flg-OFF). How selective pressures affect ON/OFF switching dynamics and overall population composition is unknown. Here, we use a mouse model of disease to evaluate the ON/OFF makeup of the C. difficile population in vivo. Two key results emerged: (i) Directional inversion of the flg switch to the "ON" orientation occurs during spore germination, and (ii) toxin-induced inflammation enriches for a flg-OFF population during infection. These findings reveal a cycle in which predominantly flg-OFF spores are disseminated into the environment, and spore germination in the next host resets C. difficile to a virulent population of motile, toxigenic cells at infection onset.