Investigation of distinct contribution of nitric oxide and each reactive oxygen species in indole-3-propionic-acid-induced apoptosis-like death in Escherichia coli

Aims: Indole-3-propionic acid (IPA) is a natural product from human microbiota, exhibiting diverse biological activities. The study focused on investigating the antibacterial mode of action(s) triggered by IPA in Escherichia coli. Separate influence of nitric oxide (NO) and each reactive oxygen species, including superoxide anion (O₂⁻), hydrogen peroxide (H₂O₂), hydroxyl radical (OH⁻), was specifically analyzed throughout the process. Main methods: The generation of respective reactive oxygen species (ROS), NO, and ONOO⁻ was conducted using flow cytometer using different dyes. Further analysis of separate influences was held based on usage of each scavenger: sodium pyruvate, thiourea, tiron, and L-NAME. Oxidative cell damage was observed through the detection of glutathione depletion and lipid peroxidation. DNA fragmentation and membrane depolarization were observed by TUNEL and DiBAC₄(3) staining agent. Finally, Annexin V/PI and FITC-VAD-FMK were applied to detect apoptosis-like death. Key findings: IPA exhibited antibacterial activity in E. coli through the accumulation of ROS, NO, ONOO⁻, and DNA damage, eventually leading to apoptosis-like death. NO and O₂⁻ exerted the most potent influence on oxidative damage of E. coli, whereas H₂O₂ accounts for the least impact. Moreover, the results reveal the major contribution of ONOO⁻ in IPA-induced apoptosis-like death in E. coli. Significance: This is the first study that introduces the antibacterial activity and apoptosis-like death induced by IPA and suggests the possibility of being an alternative for current antibiotics. Furthermore, the distinct influence of each ROS and NO was analyzed to investigate their contribution to oxidative damage leading to bacterial apoptosis-like death.