Tuesday, April 1, 2025
4/1/2025
Phase variation of virulence factors in Clostridioides difficile - SUMMARY ABSTRACT Clostridioides difficile is a major cause of hospital-acquired intestinal disease, and community-acquired infections not associated with healthcare settings are now common. An estimated 15-30% of patients experience disease recurrence, exacerbating morbidity and increasing the healthcare costs of these infections. Disease ranges from mild diarrhea to potentially fatal pseudomembranous colitis, with disease symptoms primarily driven by TcdA and TcdB, bacterial toxins that damage the intestinal epithelium and elicit inflammation. Recent studies indicate that the toxins liberate nutrients that promote C. difficile growth. Flagella also may contribute to disease development by promoting adherence to intestinal epithelial cells and intestinal colonization. However, flagellar proteins are recognized by the host innate immune system, putting cells that express flagella at risk of clearance. C. difficile must balance the benefits of flagella and toxins with the potential costs of immune recognition. We previously discovered that C. difficile modulates the production of flagella and toxins through phase variation, a means of generating a phenotypically heterogeneous population to help ensure survival in complex and changing environments. Upstream of the flgB flagellar gene operon lies an invertible regulatory sequence flanked by inverted repeats. The orientation of this ‘flg switch’ determines whether the flagellar genes are expressed. Because transcription of the tcdA and tcdB toxin genes is linked to flagellar gene expression, the flg switch also modulates toxin biosynthesis. One orientation of the flg switch corresponds to the ON state characterized by flagellum and toxin biosynthesis, while the inverse, OFF orientation results in non-motile, toxin deficient bacteria. Phase variation thus creates two phenotypic variants where one may be better equipped for survival in different environmental conditions. In the prior funding period, we investigated the mechanisms of flg switch inversion and gene regulation, and the contribution to intestinal colonization and disease. In the current proposal, we present evidence that that the flg switch inverts during infection of mice, indicating that phase variation of flagella and toxins occurs in the intestinal environment. In addition, preliminary studies have identified a host-relevant condition that promotes phase variation through a regulated mechanism. The goals of the current proposal are to identify host cues that regulate phase variation during infection, to define how C. difficile mediates changes in virulence in response to these cues, and to determine the impact of phase variation on disease recurrence. Through the proposed studies, we will gain an understanding of the host-pathogen interactions that drive phenotypic heterogeneity and fitness of C. difficile in vivo. Understanding how C. difficile regulates virulence factors may reveal new strategies for prevention or treatment of infections.
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