<i>Enterococcus faecalis</i> modulates phase variation in <i>Clostridioides difficile</i>

To adapt and persist in the gastrointestinal tract, many enteric pathogens, including <i>Clostridioides difficile</i>, employ strategies such as phase variation to generate phenotypically heterogeneous populations. Notably, the role of the gut microbiota and polymicrobial interactions in shaping population heterogeneity of invading pathogens has not been explored. Here, we show that <i>Enterococcus faecalis</i>, an opportunistic pathogen that thrives in the inflamed gut during <i>C. difficile</i> infection, can impact the phase-variable CmrRST signal transduction system in <i>C. difficile</i>. The CmrRST system controls multiple phenotypes, including colony morphology, cell elongation, and cell chaining in <i>C. difficile</i>. Here, we describe how interactions between <i>E. faecalis</i> and <i>C. difficile</i> on solid media lead to a marked shift in <i>C. difficile</i> phenotypes associated with phase variation of CmrRST. Specifically, <i>E. faecalis</i> drives a switch of the <i>C. difficile</i> population to the <i>cmr-</i>ON state, leading to chaining and a rough colony morphology. This phenomenon is most pronounced with <i>E. faecalis</i>, as other enterococcal species and select Gram-negative enteric bacteria do not show a similar effect. These results suggest that the composition of the polymicrobial environment in the gut is critical to influencing <i>C. difficile</i> population heterogeneity. Our findings shed light on the complex role that microbial ecology and polymicrobial interactions can have in the phenotypic heterogeneity of invading pathogens.IMPORTANCE<i>Clostridioides difficile</i> is an enteric pathogen with critical implications for public health. The microbial ecosystem in which <i>C. difficile</i> resides shapes the behavior and fitness of <i>C. difficile</i>; however, the mechanisms underlying these interactions are not well defined. Here, we demonstrate that <i>Enterococcus faecalis</i>, an opportunistic pathogen known to co-colonize the gut with <i>C. difficile</i>, influences phase variation and downstream growth phenotypes in <i>C. difficile</i>. This phenomenon represents a new paradigm by which co-residing bacteria can modulate phase variation dynamics in <i>C. difficile</i> or other enteric pathogens. Understanding factors that influence <i>C. difficile</i> behavior may elucidate new therapeutic strategies, especially in complex polymicrobial infections.