Deoxycholate induces proline provision from Escherichia coli to enhance Clostridioides difficile virulence

Clostridioides difficile infection (CDI) is a refractory enteritis. A key indicator of successful treatment and microbial restoration is the elevation of intestinal deoxycholate (DCA) levels. Nevertheless, a small proportion of patients experience disease recurrence despite this apparent metabolic recovery, the underlying mechanisms of which remain poorly understood. We investigated the ecological dynamics of Clostridioides difficile (CD) and Escherichia coli (EC) under DCA exposure, revealing that DCA exacerbates the severity of co-infection. Specifically, DCA promotes the formation of a robust and dense symbiotic biofilm that confers a substantial survival advantage to CD. Through a combination of in vitro co-culture and validation in a murine infection model, we demonstrated that EC produces substantial quantities of L-proline in response to DCA. This amino acid is converted to D-proline by the proline racemase and subsequently catabolized by the D-proline reductase in CD, thereby enhancing its environmental fitness and pathogenicity within the gut. The indispensable nature of this tripartite interaction was further corroborated using additional EC or CD strains, including isogenic mutants deficient in D-proline reductase, in subsequent murine infection models. These experiments collectively confirm that EC, proline, and the D-proline reductase are all essential components mediating the DCA-induced exacerbation of CDI. In summary, our study elucidates a mechanism by which a DCA environment promotes the exacerbation of CDI through a metabolic cross-feeding interaction between CD and EC. Specifically, EC-derived L-proline serves as a critical nutrient that enhances the fitness and pathogenicity of CD. This finding provides a plausible explanation for the paradoxical phenomenon of CDI recurrence in patients exhibiting elevated intestinal DCA levels.