Loss of multiple hydrogenosomal proteins associated with organelle metabolism and high-level drug resistance in trichomonads

In trichomonads, metronidazole is activated to its cytotoxic form in a specialized energy-producing organelle called the hydrogenosome. Electron transport components in the organelle, pyruvate:ferredoxin oxidoreductase and ferredoxin, donate a single electron to the drug, converting it to a cytotoxic free radical. Previous biochemical analyses of enzyme activities of highly resistant strains of both Trichomonas vaginalis and Tritrichomonas foetus reveal undetectable activity for pyruvate:ferredoxin oxidoreductase and another hydrogenosomal enzyme, hydrogenase. We have chosen to analyze a highly drug-resistant strain of T. foetus and its parental drug-sensitive strain from which it was derived to study the molecular basis for these enzyme defects. Quantitation of pyruvate:ferredoxin oxidoreductase and ferredoxin levels in sensitive and resistant cells shows a marked reduction of these proteins in the resistant strain. RNA analysis reveals an approximately 60% reduction in pyruvate:ferredoxin oxidoreductase mRNA and 90-98% reduction in mRNA levels encoding hydrogenosomal proteins hydrogenase, ferredoxin, and malic enzyme. We have measured the levels of transcription of these genes and observed 60% reduction of pyruvate:ferredoxin oxidoreductase gene transcription and 85% reduction in malic enzyme gene transcription in the resistant strain. The reduction or absence of these organellar proteins is likely to reduce or eliminate the ability of the cell to activate the drug, giving rise to the highly resistant phenotype. Ultrastructural analysis of thin sections revealed that resistant cells are 20% smaller in size and hydrogenosomes in resistant cells are approximately one-third the size of those in the drug-sensitive parental strain. These data suggest that altered gene expression of multiple hydrogenosomal proteins results in the modification of the organelle and leads to drug resistance. (C) 2001 Academic Press.