Bacteroides -driven metabolic remodelling suppresses Clostridioides difficile toxin expression in mixed biofilm communities

Clostridioides difficile is a major cause of hospital-associated diarrhoea worldwide. The intricate interactions between C. difficile and the resident gut microbiota play a crucial role in determining the outcome of C. difficile infection (CDI), although the molecular mechanisms underlying many C. difficile -commensal interactions are not understood. Here we show that selected Bacteroides species can inhibit C. difficile growth within mixed biofilms. A transcriptomic analysis of C. difficile-Bacteroides biofilms showed significant metabolic shifts, with distinct changes in carbohydrate and amino acid metabolism and, interestingly, a downregulation of C. difficile toxin gene expression. A significant reduction in C. difficile toxin production was evident in C. difficile - Bacteroides cocultures, irrespective of the extent of C. difficile growth inhibition. Notably, Stickland fermentation of proline, which is known to repress toxin synthesis, was upregulated in C. difficile , while proline synthesis was induced in the cocultured species B. vulgatus and B. dorei . Furthermore, upregulation of proline reductase pathways and consequent toxin repression were evident within a synthetic 9-species gut commensal biofilm community containing multiple Bacteroides spp. Thus, leveraging multiomics approaches, we demonstrate a potential cross-feeding mechanism where proline produced by B. dorei and B. vulgatus is utilised by C. difficile through Stickland fermentation to drive toxin repression. Our study reveals a new mechanism of microbiota-mediated control of a key virulence factor involved in C. difficile pathogenesis while enabling pathogen co-existence within a polymicrobial commensal community.