A mechanism for cross-resistance to nifurtimox and benznidazole in trypanosomes

Abstract

Nifurtimox and benznidazole are the front-line drugs used to treat Chagas disease, the most important parasitic infection in the Americas. These agents function as prodrugs and must be activated within the parasite to have trypanocidal effects. Despite >40 years of research, the mechanism(s) of action and resistance have remained elusive. Here, we report that in trypanosomes, both drugs are activated by a NADH-dependent, mitochondrially localized, bacterial-like, type I nitroreductase (NTR), and that down-regulation of this explains how resistance may emerge. Loss of a single copy of this gene in Trypanosoma cruzi, either through in vitro drug selection or by targeted gene deletion, is sufficient to cause significant cross-resistance to a wide range of nitroheterocyclic drugs. In Trypanosoma brucei, loss of a single NTR allele confers similar cross-resistance without affecting growth rate or the ability to establish an infection. This potential for drug resistance by a simple mechanism has important implications, because nifurtimox is currently undergoing phase III clinical trials against African trypanosomiasis.

Fig. 1.

Nifurtimox-resistant T. cruzi obtained by in vitro culture are cross-resistant to other nitroheterocyclic drugs and have lost a copy of a TcNTR-containing chromosome. (A) Cumulative cell density of parental (X10/6) and laboratory-generated nifurtimox-resistant (Nif^R) lines in medium containing 10 μM nifurtimox. Two other Nif^R clones analyzed in parallel displayed similar growth properties. (B) The drug concentrations that inhibited T. cruzi growth by 50% were established. The data are means from three experiments ± standard deviation (SD). Differences observed in susceptibility were statistically significant (P < 0.01), as assessed by Student's t test. Rimantadine was used as a drug control. (C) Ethidium bromide-stained CHEFE gel containing genomic DNA from T. cruzi. The asterisk (*) identifies an 800-kbp chromosome present in X10/6 (lane 1), but missing in Nif^R clones (lane 2). M corresponds to yeast DNA markers. (D) Autoradiograph of T. cruzi chromosomal DNA separated by CHEFE and hybridized with TcNTR gene probe (lane order as in B).

Fig. 2.

Biochemical properties of TcNTR. (A) Postulated scheme for the reduction of nitroheterocyclic compounds by NTRs; “red” represents the reduced form of drug, whereas “oxid” represents the oxidized form. (B) TcNTR activity was monitored by following the oxidation of NADPH or NADH (100 μM) in the presence of TcNTR (0.2 μg) and nifurtimox (100 μM). (C) TcNTR activity was monitored after the oxidation of NADH (100 μM) in the presence of TcNTR (0.2 μg) and nifurtimox (0.5–100 μM). All reactions were initiated by the addition of the recombinant enzyme. TcNTR activities are expressed as nanomoles NADH oxidized min⁻¹·mg⁻¹ of enzyme. (D) TcNTR metabolizes a range of nitroheterocyclic compounds.

Fig. 3.

Phenotypic effect of deleting NTR from the T. cruzi genome. (A) T. cruzi null mutants have a growth defect. Mean cell density was determined from three independent epimastigote cultures. TcNTR+/− and TcNTR−/− represent the TcNTR heterozygous and null-mutant cell lines, respectively. (B) Growth inhibitory effect of nitroheterocyclic drugs on NTR-deficient T. cruzi epimastigotes. Data are means from fourexperiments ± SD, and the differences in susceptibility were statistically significant (P < 0.01), as assessed by Student's t test. G418 was used as a drug control.

Fig. 4.

Phenotypic effect of altering the TbNTR copy number in T. brucei. (A) Deletion of one copy of TbNTR confers cross-resistance on T. brucei as judged by IC₅₀. (B) Overexpression of TbNTR (TbNTR^RV) confers hypersensitivity to nitroheterocyclic drugs. Data in A and B are means from four experiments ± SD, and the differences in susceptibility were statistically significant (P < 0.01), as assessed by Student's t test. Melarsoprol was used as a drug control. (C) Cumulative cell density of T. brucei BSF cells grown in the absence or presence of tetracycline. Two other TbNTR^RV−/− clones analyzed in parallel displayed similar growth properties. On the fifth and subsequent days, we observed an outgrowth of viable parasites. This type of reversion is frequently observed in T. brucei (21).

Fig. 5.

Localization of TbNTR in bloodstream form T. brucei. Parasites expressing TbNTR-GFP (green) were costained with DAPI (DNA; blue) and MitoTracker (mitochondrion; red). The cells were examined by confocal microscopy. The pattern of colocalization (yellow) is shown in the merged image. (A) Expression of TbNTR-GFP in a population of cells. (B) Single cell at higher resolution.