Flagellum and toxin phase variation impacts intestinal colonization and disease development in a mouse model of <i>Clostridioides difficile</i> infection

Abstract

<i>Clostridioides difficile</i> is a major nosocomial pathogen that can cause severe, toxin-mediated diarrhea and pseudomembranous colitis. Recent work has shown that <i>C. difficile</i> exhibits heterogeneity in swimming motility and toxin production <i>in vitro</i> through phase variation by site-specific DNA recombination. The recombinase RecV reversibly inverts the flagellar switch sequence upstream of the <i>flgB</i> operon, leading to the ON/OFF expression of flagellum and toxin genes. How this phenomenon impacts <i>C. difficile</i> virulence <i>in vivo</i> remains unknown. We identified mutations in the right inverted repeat that reduced or prevented flagellar switch inversion by RecV. We introduced these mutations into <i>C. difficile</i> R20291 to create strains with the flagellar switch "locked" in either the ON or OFF orientation. These mutants exhibited a loss of flagellum and toxin phase variation during growth <i>in vitro</i>, yielding precisely modified mutants suitable for assessing virulence <i>in vivo</i>. In a hamster model of acute <i>C. difficile</i> infection, the phase-locked ON mutant caused greater toxin accumulation than the phase-locked OFF mutant but did not differ significantly in the ability to cause acute disease symptoms. In contrast, in a mouse model, preventing flagellum and toxin phase variation affected the ability of <i>C. difficile</i> to colonize the intestinal tract and to elicit weight loss, which is attributable to differences in toxin production during infection. These results show that the ability of <i>C. difficile</i> to phase vary flagella and toxins influences colonization and disease development and suggest that the phenotypic variants generated by flagellar switch inversion have distinct capacities for causing disease.