Santos-Santiago, J. A. | Search bioRxiv

bioRxiv2026

Phase variable colony morphotypes of Clostridioides difficile elicit distinct host responses during acute infection

Many Clostridioides difficile strains can form two colony morphotypes: rough and smooth. The rough and smooth morphotypes differ in multiple phenotypes, including cell length and chaining, motility, biofilm production, and virulence in the hamster model of C. difficile infection (CDI). Colony morphology undergoes phase variation and is determined by the ON/OFF expression of cmrRST, which encodes a signal transduction system. Here, we test the hypothesis that differences in colony morphology and the associated phenotypes influence pathogenesis and the host response to infection. We first compared the rough and smooth colony variants of wild-type C. difficile in a mouse model of CDI. However, CmrRST phase varied during infection such that the C. difficile populations became indistinguishable in feces and tissues, and no differences in disease were observed in mice inoculated with these variants. We next circumvented phase variation using mutants that form only rough or only smooth colonies. Co-infection of mice with these phenotypically locked strains revealed that the smooth colony mutant has greater fitness than the rough mutant, which is outcompeted during late infection. In addition, NanoString analyses showed a higher number of differentially expressed pro-inflammatory genes and overall higher expression levels in mice infected with the rough colony mutant, independent of bacterial burden and toxin levels. Our results indicate that in a mouse model of CDI, cells from rough colonies are more immunostimulatory during early murine infection, potentially leading to reduced relative fitness compared to cells from smooth colonies. IMPORTANCEClostridioides difficile is a major cause of antibiotic-associated diarrhea and one of the most common hospital-acquired infections. These infections are often recurrent and recalcitrant to antibiotic treatment. Bacteria use diverse mechanisms to adapt to stressful host environments, including the development of different subpopulations to help ensure survival of the whole population. C. difficile produces two different colony types, rough and smooth, that have multiple distinguishing traits. We interrogated the effect of these different forms of C. difficile on pathogenesis and the host immune response. We show that the rough colony phenotype results in a more robust immune response and has reduced fitness during infection compared to the smooth colony phenotype. This work sheds light on the impact of these phenotypic subpopulations on disease, leading to a deeper understanding of C. difficile-host interactions.

microbiology↗

bioRxiv2025

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. IMPORTANCE STATEMENTClostridioides difficile is an enteric pathogen with critical implications for public health. The microbial ecosystem in which C. difficile resides shapes the behavior and fitness of C. difficile; however, the mechanisms underlying these interactions are not well defined. Here, we demonstrate that Enterococcus faecalis, an opportunistic pathogen known to co-colonize the gut with C. difficile, influences phase variation and downstream growth phenotypes in C. difficile. This phenomenon represents a new paradigm by which co-residing bacteria can modulate phase variation dynamics in C. difficile or other enteric pathogens. Understanding factors that influence C. difficile behavior may elucidate new therapeutic strategies, especially in complex polymicrobial infections.

microbiology↗