Cysteine enhances oxidative stress of heme-dependent Escherichia coli small colony variants in dairy cows with mastitis.

The heme-dependent small colony variants (HD-SCVs) of Escherichia coli (E. coli) are characterized by a deficiency in active catalase and diminished oxidative stress tolerance, yet they demonstrate prolonged intracellular persistence. However, the response of E. coli HD-SCVs isolated from cows with mastitis to cellular oxidative stress remains poorly understood. This study aimed to induce HD-SCV development from mastitis-causing E. coli using kanamycin and investigate factors influencing extracellular oxidative stress capacity, intracellular reactive oxygen species content, and competitiveness in macrophages through genomic and metabolic methods. The findings from the genomic and metabolomic analyses indicate that frameshift deletion mutations in the hemA gene may result in reduced heme anabolism, thereby potentially contributing to the development of HD-SCVs. Pathways related to cysteine, sulfur and glutathione metabolism, which are implicated in oxidative stress, exhibited significant alterations in HD-SCVs. In comparison, logarithmic-phase SCVs showed decreased cysteine content, intracellular competitiveness, and extracellular oxidative stress levels compared to stable-phase HD-SCVs and wild-type strains. The supplementation of exogenous cysteine significantly increased the survival rate of logarithmic-phase HD-SCVs by 1016.44-fold following hydrogen peroxide treatment and enhanced adherence, invasion, and competitiveness toward macrophages (P < 0.01). In summary, cysteine supplementation enhances the intracellular competitiveness and extracellular oxidative stress response of E. coli HD-SCVs, potentially contributing to their persistent intracellular presence. However, further comprehensive investigation is required to elucidate the specific regulatory mechanisms involved.