Infections with the microaerophilic protozoan parasite Trichomonas vaginalis are commonly treated with
metronidazole, a
5-nitroimidazole drug.
Metronidazole is selectively toxic to microaerophiles and anaerobes because reduction at the drug's nitro group, which is a precondition for toxicity, occurs only quantitatively in these organisms. In our previous work we identified the
flavin enzyme thioredoxin reductase as an electron donor to
5-nitroimidazole drugs in T. vaginalis and observed that highly
metronidazole-resistant cell lines lack
thioredoxin reductase and
flavin reductase activities. In this study we added the
flavin inhibitor
diphenyleneiodonium (DPI) to T. vaginalis cultures in order to test our hypothesis that
metronidazole reduction is catalyzed by
flavin enzymes, e.g.
thioredoxin reductase, and intracellular free
flavins. Indeed, within hours, DPI rendered T. vaginalis insensitive to
metronidazole concentrations as high as 1mM and prevented the formation of
metronidazole adducts with
proteins.
Thioredoxin reductase activity was absent from DPI-treated cells and
flavin reductase activity was sharply decreased. In addition, DPI-treated cells also upregulated the expression of
antioxidant enzymes, i.e.
thioredoxin peroxidases and
superoxide dismutases, and displayed a fundamentally altered metabolism caused by inactivation of
pyruvate:ferredoxin oxidoreductase (PFOR) and concomitant upregulation of
lactate dehydrogenase (LDH) activity. Thus, the disruption of the cellular
flavin metabolism by DPI mediated metabolic steps which are similar to that of cells with
metronidazole resistance induced in vitro. Finally, we present direct evidence that the increased expression of
antioxidant enzymes is dispensable for acquiring resistance to
metronidazole.