Cobalamin-mediated protection of Faecalibacterium duncaniae against oxidative stress: Insights from proteomic and membrane fatty acid profiles

Faecalibacterium species are keystone commensals of the human gut, contributing to intestinal homeostasis, immune modulation, and epithelial health. However, their extreme sensitivity to oxygen and reactive oxygen species renders them highly vulnerable during inflammatory conditions, severely limiting their therapeutic application. Understanding the molecular mechanisms underlying their oxidative stress responses is therefore critical for harnessing these bacteria as next-generation probiotics to restore gut health. In this study, we investigated oxidative stress responses in Faecalibacterium duncaniae A2-165 using comprehensive proteomic and membrane fatty acid profiling. We demonstrated that increasing hydrogen peroxide (H₂O₂) concentrations extend the lag phase of growth and affect survival during the first hour of exposure, notably altering the redox potential. Exposure to H₂O₂ triggered a remodeling of the proteome, including detoxification systems, metal transporters, DNA repair systems, transcriptional regulators, and enzymes involved in cobalamin biosynthesis. Complementary RT-qPCR analyses revealed coordinated and time-dependent transcriptional activation of genes involved in oxidative stress response. Remarkably, cobalamin supplementation enhanced bacterial growth, mitigated H₂O₂-induced stress, and lowered superoxide levels in F. duncaniae, highlighting its direct antioxidant activity. By analyzing membrane fatty acid profiles, we showed that cobalamin preserves membrane fluidity, counteracting oxidative stress induced by H₂O₂ in F. duncaniae. These findings reveal the multifaceted strategies employed by F. duncaniae to withstand oxidative stress and provide a foundation for future efforts to optimize its production at industrial scales and its therapeutic potential as a next-generation probiotic.