Mechanisms of in vitro development of resistance to metronidazole in Trichomonas vaginalis

Abstract

Mechanisms of in vitro development of resistance to metronidazole in Trichomonas vaginalisDominique Rasoloson, SB te) pa! nka Van) a! c) ova! , Eva Tomkova! , Jakub Ra! zga, Ivan Hrdy! , Jan Tachezy and Jaroslav Kulda Development of resistance against metronidazole and mechanisms responsible for this process were studied in a sexually transmitted pathogen of humans, Trichomonas vaginalis. Monitoring of changes in metabolism and protein expression that accompanied increasing resistance of strains derived from a common drug-susceptible parent (TV 10-02) showed the multistep character of the process. The aerobic type of resistance known to occur in isolates from patients non-responsive to treatment appeared at the earliest stage, followed by development of the anaerobic type of resistance which was accompanied by gradual loss of hydrogenosomal proteins associated with drug-activating pathways [pyruvate:ferredoxin oxidoreductase (PFOR), hydrogenase, ferredoxin]. Unexpectedly, the loss of PFOR did not result in acquisition of full anaerobic resistance, thus indicating an alternative source of electrons required for the drug activation. These data suggest involvement of the oxidative decarboxylation of malate in hydrogenosomes, catalysed by NAD Mdependent malic enzyme and subsequent transfer of reduced equivalents to the drug via NADH :ferredoxin oxidoreductase and ferredoxin. Accordingly, all components of this pathway were eliminated before the resistance was fully developed. Resistant Trichomonas vaginalis compensated the impaired function of hydrogenosomes by enhanced conversion of pyruvate to lactate in the cytosol. Further analysis of the two key enzymes involved in metronidazole activation by Northern blotting and assay for nascent mRNA showed that the insufficient expression of the PFOR protein results from decreased gene transcription, while down-regulation of malic enzyme is controlled at the mRNA level.

Drug resistance in the sexually transmitted protozoan Trichomonas vaginalis

“…Also working with the Fd gene but this time in T. foetus, Land et al [49] were able to show that transformation of metronidazoleresistant cells with the Fd gene, which was expressed in vivo, resulted in partial reversion to sensitivity of the cells. Kulda and his colleagues [29,50] maintain that PFOR deficiency in T. foetus completely precludes metronidazole activation, while T. vaginalis possesses an additional drug-activating system which must be eliminated before full resistance is achieved. This alternative pathway involves NAD-dependent ME in the oxidative decarboxylation of malate in hydrogenosomes and the subsequent reduction (or activation of metronidazole) via NADH:ferredoxin oxidoreductase and Fd.…”

Development of Drug Resistance in Trichomonas vaginalis and its Overcoming with Natural Products

“…Thus exposition to high oxygen levels lead to cell death as a result of accumulation of toxic oxygen metabolites, which are normally prevented by effective oxygen scavenging processes [59,60]. Through further metabolic changes, the resistance develops to the anaerobic resistance stage [58], which was demonstrated in vitro by prolonged treatment and drug pressure [10]. This stage is oxygen-independent and characterized by advanced gradual loss of PFOR, the key enzyme in the drug activation pathway.…”

“…It is characterized by reduction of oxygen scavenging processes. This results in an increased oxygen concentration inside the parasite impairing the low redox potential necessary for metronidazole activation [58][59][60]. In addition, activated metronidazole can be oxidized back to the inactive parent compound, a phenomenon called futile cycling.…”

“…Metronidazole resistance in T. vaginalis is described by two stages. Whereas these stages have been suggested to represent unrelated types of resistance formation, novel studies demonstrate a gradual transformation from the first stage to the second [58]. The aerobic resistance represents the earliest stage in resistance development, commonly observed in vivo by application of therapeutic levels of the drug [10].…”

“…However, alternative drug activation mechanisms via malic enzyme, NADH:FOR and ferredoxin still serve to activate the drug. To develop high resistance the hydrogenosome gets fully inactivated, realized by decrease and eventual loss of all proteins involved in drug activation, namely PFOR, malic enzyme, NADH:FOR, ferredoxin and Fe-hydrogenase [58,62]. Down-regulation of PFOR and ferredoxin have also been demonstrated to result in resistance formation in Giardia intestinalis, an amitochondriate protozoan parasite of the intestinal tract [63][64][65].…”