Bile salts act as effective protein-unfolding agents and instigators of disulfide stress in vivo.

Commensal and pathogenic bacteria must deal with many different stress conditions to survive in and colonize the human gastrointestinal tract. One major challenge that bacteria encounter in the gut is the high concentration of bile salts, which not only aid in food absorption but also act as effective physiological antimicrobials. The mechanism by which bile salts limit bacterial growth is still largely unknown. Here, we show that bile salts cause widespread protein unfolding and aggregation, affecting many essential proteins. Simultaneously, the bacterial cytosol becomes highly oxidizing, indicative of disulfide stress. Strains defective in reducing oxidative thiol modifications, restoring redox homeostasis, or preventing irreversible protein aggregation under disulfide stress conditions are sensitive to bile salt treatment. Surprisingly, cholate and deoxycholate, two of the most abundant and very closely related physiological bile salts, vary substantially in their destabilizing effects on proteins in vitro and cause protein unfolding of different subsets of proteins in vivo. Our results provide a potential mechanistic explanation for the antimicrobial effects of bile salts, help explain the beneficial effects of bile salt mixtures, and suggest that we have identified a physiological source of protein-unfolding disulfide stress conditions in bacteria.