Combined use of vitamin C and antifolates induces cuproptosis-like bacterial death

Combined use of antimicrobial agents is a key strategy to combat antimicrobial resistance. After several decades of approval, clinical application of the classical combination co-trimoxazole has gradually become limited, mainly due to the prevalence of resistance. Vitamin C is a natural antimicrobial and redox agent. While sporadic studies suggest vitamin C might synergize with antimicrobial drugs, results have been inconsistent and the underlying mechanisms are unclear. Here we show that vitamin C increased bacterial sensitivity to antifolates and protein synthesis inhibitors through different mechanisms, including altered para-aminobenzoic acid and branched chain amino acids biosynthesis. However, vitamin C enhanced the bactericidal effect of those drugs through a common mechanism-inducing cuproptosis-like bacterial death. Treating bacteria with vitamin C caused a transient accumulation of cuprous ions, which could be buffered by the bacterial copper homeostasis systems. However, coadministration of those drugs undermined the bacterial copper homeostasis systems and led to a buildup of cuprous ion, ultimately causing cuproptosis-like bacterial death as characterized by aggregation of lipoylated proteins. The accumulated cuprous ions further released ferrous ions, which stimulated a surge in reactive oxygen species and exacerbated bacterial death by generating excessive DNA damage. This study provides a novel rationale for the combined use of vitamin C and existing antimicrobial drugs.