THE HOST ENVIRONMENT AND THE INTESTINAL MICROBIOTA DRIVE PHASE VARIATION IN CLOSTRIDIOIDES DIFFICILE

Clostridioides difficile is a Gram-positive, spore-forming, obligate anaerobe and is one of the leading agents of nosocomial infections worldwide. C. difficile causes a wide range of symptoms from moderate diarrhea to severe conditions like pseudomembranous colitis. Most strains of C. difficile can form two colony morphotypes: rough and smooth. The rough and smooth colony morphotypes show differences in multiple phenotypes, including cell length and chaining, swimming and surface motility, biofilm production, metabolism, and virulence in the hamster model of C. difficile infection (CDI). Colony morphology undergoes phase variation, a reversible ON/OFF switching process that generates phenotypically heterogeneous populations as a bet-hedging strategy to ensure survival. The phenotypic switch is controlled by the reversible inversion of the ‘cmr switch’, a regulatory DNA sequence upstream of the colony morphology regulator RST (cmrRST) operon. CmrRST is a signal transduction system in which CmrS is the sensor kinase, and CmrR and CmrT are response regulators. The orientation of the cmr switch correlates with transcription of cmrRST and colony morphotype—cmrRST transcription (cmr-ON) leads to rough colonies, while reduced transcription (cmr-OFF) results in smooth colonies. The role of this system in the physiology of C. difficile in complex environments remains to be elucidated. Here, we characterize the impact of the host and intestinal microbiota on CmrRST phase variation. We describe how C. difficile and a second enteric pathogen, E. faecalis, interact on solid media, leading to a marked shift in C. difficile phenotypes associated with CmrRST. E. faecalis drives the C. difficile population to shift to the cmr-ON state, leading to rough colonies and cell chaining. In a mouse model of CDI, we provide evidence that the cmr switch is transiently and niche-specifically inverted during infection. Using co-infection of mice with phenotypically locked mutants, we show that the smooth colony morphotype has greater fitness than the rough morphotype. The host transcriptional response was exacerbated in mice inoculated with the phenotypically locked rough mutant independent of bacterial burden or toxin levels. Altogether, modulation of the phase-variable CmrRST system within the host milieu harboring other enteric bacteria could impact C. difficile fitness and CDI severity.