Defining the role of phenotypic heterogeneity in Clostridioides difficile fitness

SUMMARY ABSTRACT Clostridioides difficile causes antibiotic-associated disease ranging from mild diarrhea to potentially fatal pseudomembranous colitis and is one of the most common causes of nosocomial infections in the U.S. C. difficile disease is largely mediated by the toxins TcdA and TcdB, which damage the intestinal epithelium and elicit inflammation. How C. difficile adapts to the intestinal environment to elicit virulence factor production is unclear. The expression of the genes encoding TcdA and TcdB is linked to expression of genes for flagellum biosynthesis and motility through SigD, a sigma factor encoded in the flgB operon. Factors that affect expression of the flgB operon therefore impact both motility and toxin production. Transcription of the flgB operon is subject to complex regulation. The signaling molecule c-di-GMP inhibits transcription of flgB, resulting in loss of flagella and reduced toxin production. In addition, a reversibly invertible ‘flg switch’ sequence upstream of flgB modulates expression and results in phase variation of flagella and toxins. This phase variation occurs stochastically and enables the generation of a phenotypically heterogeneous population consisting of flagellated, toxigenic bacteria and aflagellate, nontoxigenic bacteria and serves as a bet-hedging strategy to ensure survival. We have shown that flagellum and toxin production is coupled during infection of mice and preventing flg switch inversion alters infection outcomes. Recent work demonstrated that a c-di-GMP hydrolase also undergoes phase variation and modulates flagellar motility. These findings link c-di-GMP signaling and phase variation and point to a previously unknown strategy for coordinated modulation of virulence factors and development of phenotypic heterogeneity. The goal of this project is to define the interplay between the stochastic and regulatory mechanisms underlying the heterogeneous expression of flagella and toxins. We propose to use molecular genetics, single-cell analyses, and an animal model of C. difficile disease to address fundamental questions: Is phase variation of flagella and toxins regulated beyond stochastic inversion of the flg switch? Do certain environments result in coordinated phase variation of flagella and toxins with other factors? What is the impact of flagellum and toxin phase variation on C. difficile fitness during infection? Completion of the proposed studies will provide much needed mechanistic information on how C. difficile controls production of key virulence factors and the impact on C. difficile fitness in the intestinal environment.