Enterococcus faecalis modulates phase variation in Clostridioides difficile

To adapt and persist in the gastrointestinal tract, many enteric pathogens, including Clostridioides difficile, 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 Enterococcus faecalis, an opportunistic pathogen that thrives in the inflamed gut during C. difficile infection, can impact the phase variable CmrRST signal transduction system in C. difficile. The CmrRST system controls multiple phenotypes including colony morphology, cell elongation, and cell chaining in C. difficile. Here we describe how interactions between E. faecalis and C. difficile on solid media lead to a marked shift in C. difficile phenotypes associated with phase variation of CmrRST. Specifically, E. faecalis drives a switch of the C. difficile population to the cmr-ON state leading to chaining and a rough colony morphology. This phenomenon preferentially occurs with E. faecalis among the enterococci, as other enterococcal species do not show a similar effect, suggesting that the composition of the polymicrobial environment in the gut is likely critical to shaping C. difficile 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.