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. ImportanceC. difficile infection, characterised by severe diarrhoea and colitis, has a significant impact on healthcare settings globally due to the high rates of recurrence. CDI is closely associated with the gut microbiota status and the use of antibiotics, yet the mechanistic basis of interactions between the causative bacterium C. difficile and individual gut commensal species remains poorly defined. Here, we demonstrate inhibitory effects of Bacteroides species on C. difficile through nutrient competition and a cross-feeding mechanism between these abundant gut commensals and this pathogen which blocks expression of key C. difficile virulence factors. Our findings offer insights into the effective design of microbiota consortia to prevent and treat CDI.