Mechanisms of metronidazole resistance in T. vaginalis

The anaerobic/microaerophilic parasite Trichomonas vaginalis is a causative agent of vaginitis in women and urethritis in men (trichomoniasis). Trichomoniasis is the most frequent curable, non-viral, sexually transmitted disease (STM) worldwide with an estimated number of 170 million cases per year. The treatment of T. vaginalis infections is almost exclusively based on therapy with metronidazole or other 5-nitroimidazole drugs. In fact, metronidazole is active against practically all anaerobic/microaerophilic pathogens, and therefore belongs to the 100 most often prescribed drugs worldwide. The specific activity of 5-nitroimidazoles against anaerobes and microaerophiles is caused by the necessity of reduction at the drugs` nitro group in order for the toxic metabolite to be formed. This readily occurs under low oxygen tensions but less so, or not at all, under aerobic conditions. In our previous work on metronidazole action in T. vaginalis we identified the flavin enzyme thioredoxin reductase as a reducer of nitroimidazole drugs. In a cell line with high-level metronidazole resistance induced in vitro, we found thioredoxin reductase to be expressed but almost inactive. Activity could be restored in enzymatic assays upon addition of the cofactor flavin adenine dinucleotide (FAD). In addition, we found that metronidazole-resistent cells cannot reduce free flavins such as riboflavin, flavin mononucleotide (FMN) and FAD. Thus, we concluded that reduction of metronidazole is flavin-mediated and that metronidazole resistance is caused by a severe impairment of the cellular flavin metabolism which results in less drug reduced. Indeed, direct inhibition of flavin- linked pathways, including thioredoxin reductase, by the flavin inhibitor diphenylneiodonium (DPI) almost instantly renders T. vaginalis highly resistant to metronidazole. It will, therefore, be the major aim of this project to further delineate the interdependence of flavin metabolism and metronidazole reduction and to deepen our understanding of metronidazole resistance. To this end, the role of thioredoxin reductase in metronidazole reduction and T. vaginalis physiology will be directly addressed by knocking out the respective gene by targeted gene replacement. In addition, concentrations of riboflavin, FMN, and FAD will be determined in normal and metronidazole-resistant T. vaginalis. Finally, those enzymes that are involved in flavin synthesis and flavin metabolism will be studied at the biochemical and genetic level in metronidazole-susceptible and resistant cells.

It was the major aim of this research project to identify and characterise factors involved in the development of metronidazole resistance in the microaerophilic protist parasite Trichomonas vaginalis. Despite the highly reliable and effective use of metronidazole as standard drug in the treatment of T. vaginalis infections throughout the past 50 years, metronidazole-resistant strains are commonly reported by practitioners. In fact, trichomoniasis is one of the most frequent sexually-transmitted diseases worldwide, and one of the most prevalent parasitoses in general, rendering metronidazole resistance highly problematic. Previous studies, in part conducted by the principal investigator of this project, suggested the existence of two distinct pathways underlying metronidazole resistance: 1, impaired reduction of the nitro group of the drug, a prerequisite of metronidazoles toxicity, or 2, increased intracellular oxygen levels which promote detoxification of metronidazole by reoxidation. In course of this project it could be shown that the first pathway only contributes to metronidazole resistance induced in the laboratory. In contrast, metronidazole-resistant laboratory strains and metronidazole-resitant clinical isolates both demonstrated impaired oxygen scavenging pathways, consequently leading to increased intracellular oxygen levels. Further, the enzyme whose reduced activity is responsible for the previously reported increased intracellular oxygen levels was isolated, identified, and characterised. This enzyme, flavin reductase, reduces free flavins (i.e. FMN, FAD, and riboflavin) using NADPH as an oxidant. Reduced flavins, in turn, rapidly react with molecular oxygen to form hydrogen peroxide. Flavin reductase had either a clearly impaired or no activity at all in metronidazole-resistant T. vaginalis. Re-introduction of an episomal and actively transcribed flavin reductase gene into a highly-metronidazole-resistant clinical isolate almost completely restored metronidazole sensitivity. Interestingly, no close homologues of flavin reductase could be identified in the genomes of other parasites, although it could be shown that also metronidazole-resistant Giardia lamblia, another microaerophilic protist parasite, showed impaired reduction of free flavins. Therefore it is likely that impaired flavin reduction is a hallmark of metronidazole resistance in a wider range of pathogens. These insights might have relevant impact on the treatment and diagnosis of microaerophilic/anaerobic infections in the future.