Metronidazole resistance in the protozoan parasite Entamoeba histolytica is associated with increased expression of iron-containing superoxide dismutase and peroxiredoxin and decreased expression of ferredoxin 1 and flavin reductase

To obtain insight into the mechanism of metronidazole resistance in the pro tozoan parasite Entamoeba histolytica, amoeba trophozoites were selected in vitro by stepwise exposures to increasing amounts of metronidazole, starti ng with sublethal doses of 4 mu M. Subsequently, amoebae made resistant wer e able to continuously multiply in the presence of a 40 mu M concentration of the drug. In contrast to mechanisms of metronidazole resistance in other protozoan parasites, resistant amoebae did not substantially down-regulate pyruvate:ferredoxin oxidoreductase or up-regulate P-glycoproteins, but exh ibited increased expression of iron-containing superoxide dismutase (Fe-SOD ) and peroxiredoxin and decreased expression of flavin reductase and ferred oxin 1. Episomal transfection and overexpression of the various antioxidant enzymes revealed significant reduction in susceptibility to metronidazole only in those cells overexpressing Fe-SOD, Reduction was highest in transfe cted cells simultaneously overexpressing Fe-SOD and peroxiredoxin, Although induced overexpression of Fe-SOD did not confer metronidazole resistance t o the extent found in drug-selected cells, transfected cells quickly adapte d to constant exposures of otherwise lethal metronidazole concentrations, M oreover, metronidazole selection of transfected amoebae favored retention o f the Fe-SOD-containing plasmid, These results strongly suggest that peroxi redoxin and, in particular, Fe-SOD together with ferredoxin 1 are important components involved in the mechanism of metronidazole resistance in E. his tolytica.