Metronidazole, a 5-nitroimidazole drug, has been the gold standard for several decades in the treatment of infections with microaerophilic protist parasites, including Entamoeba histolytica. For activation, the drug must be chemically reduced, but little is known about the targets of the active metabolites. Applying two-dimensional gel electrophoresis and mass spectrometry, we searched for protein targets in E. histolytica. Of all proteins visualized, only five were found to form adducts with metronidazole metabolites: thioredoxin, thioredoxin reductase, superoxide dismutase, purine nucleoside phosphorylase, and a previously unknown protein. Recombinant thioredoxin reductase carrying the modification displayed reduced enzymatic activity. In treated cells, essential non-protein thiols such as free cysteine were also affected by covalent adduct formation, their levels being drastically reduced. Accordingly, addition of cysteine allowed E. histolytica to survive in the presence of otherwise lethal metronidazole concentrations and reduced protein adduct formation. Finally, we discovered that thioredoxin reductase reduces metronidazole and other nitro compounds, suggesting a new model of metronidazole activation in E. histolytica with a central role for thioredoxin reductase. By reducing metronidazole, the enzyme renders itself and associated thiol-containing proteins vulnerable to adduct formation. Because thioredoxin reductase is a ubiquitous enzyme, similar processes could occur in other eukaryotic or prokaryotic organisms.
The protist parasites Entamoeba histolytica, Trichomonas vaginalis, and Giardia intestinalis grow in environments with low oxygen concentration. Infections with these parasites are commonly treated with metronidazole, a nitroimidazole drug that must be reduced for activation, resulting in several toxic metabolites. We examined the soluble proteome of metronidazole-treated E. histolytica cells for target proteins of these metabolites, applying two-dimensional gel electrophoresis and mass spectrometry. Of about 1,500 proteins visualized, only five formed covalent adducts with metronidazole metabolites, including thioredoxin, thioredoxin reductase, and superoxide dismutase. Metronidazole-bound thioredoxin reductase displayed diminished activity. In addition to these proteins, small thiol molecules, including cysteine, formed adducts with metronidazole. Supplementation with cysteine allowed the cells to survive otherwise lethal metronidazole concentrations. Finally, we discovered that one of the modified proteins, thioredoxin reductase, reduces metronidazole, suggesting a central role for this enzyme with regard to metronidazole toxicity. Taken together, our work reveals a new area of molecular interactions of activated metronidazole with cellular components. Because thioredoxin reductase is a ubiquitous enzyme, similar processes could also occur in other eukaryotic or prokaryotic organisms.
Metronidazole is used for treatment of infections with microaerophilic protist parasites. Here, a new model of metronidazole activation is proposed, with a central role for thioredoxin reductase.