Enterocloster citroniae and related gut microbiome species modulate Vibrio cholerae biofilm formation through the production of bioactive small molecules

Cholera is a diarrheal disease that affects millions of people globally. Although the causative agent, Vibrio cholerae, has been extensively studied in isolation, investigation of its interactions with the gut microbiota started relatively recently. We and others previously showed that microbiota-derived metabolites significantly influence V. cholerae behavior. By investigating how an organic extract of human feces affects V. cholerae gene expression, we showed that gut metabolites strongly suppress swimming motility, a trait important for host colonization. Interestingly, extracts of pure cultures of a gut commensal, Enterocloster citroniae, recapitulated this inhibition. Here, we present a comprehensive examination of the effect of small molecules produced by E. citroniae and related species on V. cholerae behavior. We show that E. citroniae small molecules inhibit motility by various V. cholerae strains, and that several phylogenetically related species produce this activity, although the magnitude of the effect varies between strains. Using biofilm formation assays in static and flow conditions, we show that V. cholerae strongly induces biofilm formation in response to E. citroniae metabolites. Transcriptome and reporter analyses showed that several genes involved in the synthesis of an extracellular polysaccharide are induced by E. citroniae metabolites. Finally, we show that V. cholerae interactions with host cells are also modulated by this commensal. These findings advance our understanding of microbiome–pathogen interactions and how commensal bacteria influence V. cholerae virulence through the production of small molecules. In the future, this knowledge may be used to design novel microbiome-based therapeutic approaches to combat cholera and other infections.