Mechanistic insights into metronidazole activation and antiprotozoal action in anaerobic parasites

Metronidazole (MTZ) is a nitroimidazole compound widely used as a drug for the treatment of infections caused by anaerobic and microaerophilic pathogens, including Entamoeba histolytica, Giardia lamblia, Trichomonas vaginalis, and Helicobacter pylori. The drug is administered as a prodrug that requires intracellular reduction to generate cytotoxic nitro radicals that damage DNA and other cellular macromolecules. Four major and an additional but overlapping pathway exclusively in Trichomonas have been described. In protozoan parasites Entamoeba, Giardia, and Trichomonas, resistance is often associated with alterations in PFOR and ferredoxin-mediated metabolic pathways responsible for drug activation. Studies have shown that enzymes such as oxygen-insensitive nitroreductases (NTRs) activate MTZ, whereas nitroimidazole reductases (NIMs) can convert the drug into non-toxic derivatives, thereby contributing to resistance phenotypes in Entamoeba. Recent advances indicate the activation of an alternative pathway involving MTZ reduction, and activation by the malic enzyme. Additionally, transcriptomic analyses of resistant Giardia lines reveal changes in the redox metabolism, oxidative stress response, and energy metabolism that may reduce drug activation or detoxify reactive intermediates.Recently, our research team has reported that the MTZ-associated oxidative stress induces the activation of the Apoptosis-inducing Factor, and nucleophagy, which, together, contribute to the trophozoite death in E. histolytica. Effective treatment strategies are also significantly intensifying, with increasing reports of MTZ resistance. On the other hand, in Helicobacter pylori, resistance frequently arises through mutations or inactivation of nitroreductase-encoding genes such as rdxA and frxA, which prevent the conversion of MTZ into its active form. Other mechanisms, including increased oxidative stress and cellular efflux systems, may also contribute to reduced susceptibility. Overall, anaerobic and microaerophilic pathogens are involved in the continuous process of developing newer strategies to defeat the antimicrobial activity of MTZ as a part of co-evolution. Understanding these molecular mechanisms is essential for improving therapeutic strategies and finding new drug targets as well as developing new nitroimidazole derivatives or combination therapies. Therefore, we attempt to make a comprehensive review on the mechanisms of MTZ activation, cell death mode(s) triggered by MTZ, as well as the pathogenic strategies to develop resistance against this drug, including all the recent advances.