Development of a Dual-Fluorescent-Reporter System in Clostridioides difficile Reveals a Division of Labor between Virulence and Transmission Gene Expression
M. Lauren Donnelly, Shailab Shrestha, John W. Ribis, Pola Kuhn, Maria Krasilnikov, Carolina Alves Feliciano, Aimee Shen
Abstract
Clostridioides difficile is an important nosocomial pathogen that causes severe diarrhea by producing toxins and transmits disease by producing spores. While both processes are crucial for C. difficile disease, only a subset of cells express toxins and/or undergo sporulation. Whether C. difficile coordinates the subset of cells inducing these energy-expensive processes remains unknown. To address this question, we developed a dual-fluorescent-reporter system coupled with an automated image analysis pipeline to rapidly compare the expression of two genes of interest across thousands of cells. Using this system, we discovered that certain growth conditions, particularly growth on agar plates, induce a "division of labor" between toxin and sporulation gene expression. Since C. difficile exhibits phenotypic heterogeneity for numerous vital cellular processes, this novel dual-reporter system will enable future studies aimed at understanding how C. difficile coordinates various subpopulations throughout its infectious disease cycle.