Development of a Dual-Fluorescent-Reporter System in Clostridioides difficile Reveals a Division of Labor between Virulence and Transmission Gene Expression

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. ABSTRACT The bacterial pathogen Clostridioides difficile causes gastroenteritis by producing toxins and transmits disease by making resistant spores. Toxin and spore production are energy-expensive processes that are regulated by multiple transcription factors in response to many environmental inputs. While toxin and sporulation genes are both induced in only a subset of C. difficile cells, the relationship between these two subpopulations remains unclear. To address whether C. difficile coordinates the generation of these subpopulations, we developed a dual-transcriptional-reporter system that allows toxin and sporulation gene expression to be simultaneously visualized at the single-cell level using chromosomally encoded mScarlet and mNeonGreen fluorescent transcriptional reporters. We then adapted an automated image analysis pipeline to quantify toxin and sporulation gene expression in thousands of individual cells under different medium conditions and in different genetic backgrounds. These analyses revealed that toxin and sporulation gene expression rarely overlap during growth on agar plates, whereas broth culture increases this overlap. Our results suggest that certain growth conditions promote a “division of labor” between transmission and virulence gene expression, highlighting how environmental inputs influence these subpopulations. Our data further suggest that the RstA transcriptional regulator skews the population to activate sporulation genes rather than toxin genes. Given that recent work has revealed population-wide heterogeneity for numerous cellular processes in C. difficile, we anticipate that our dual-reporter system will be broadly useful for determining the overlap between these subpopulations. IMPORTANCE 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.