Benznidazole-resistance in Trypanosoma cruzi is a readily acquired trait that can arise independently in a single population

Abstract

Benznidazole is the frontline drug used against Trypanosoma cruzi, the causative agent of Chagas disease. However, treatment failures are often reported. Here, we demonstrate that independently acquired mutations in the gene encoding a mitochondrial nitroreductase (TcNTR) can give rise to distinct drug-resistant clones within a single population. Following selection of benznidazole-resistant parasites, all clones examined had lost one of the chromosomes containing the TcNTR gene. Sequence analysis of the remaining TcNTR allele revealed 3 distinct mutant genes in different resistant clones. Expression studies showed that these mutant proteins were unable to activate benznidazole. This correlated with loss of flavin mononucleotide binding. The drug-resistant phenotype could be reversed by transfection with wild-type TcNTR. These results identify TcNTR as a central player in acquired resistance to benznidazole. They also demonstrate that T. cruzi has a propensity to undergo genetic changes that can lead to drug resistance, a finding that has implications for future therapeutic strategies.

Figure 1.

Properties of Trypanosoma cruzi clones derived by benznidazole selection. A Median inhibitory concentration (IC₅₀) of benznidazole for resistant parasites (61R noncloned population and clones 1–6) and parental (61S) cells (Materials and Methods). B, The 61R cells are cross-resistant to the other nitroheterocycles nifurtimox and nitrofurazone. C, T. cruzi chromosomal DNA separated by contour-clamped homogenous field electrophoresis and hybridized with a TcNTR gene probe. Left panel, ethidium bromide–stained gel; right panel, autoradiograph of gel after Southern blotting. Lane 1, parental 61S; lane 2, 61R clone 3; lane 3, 61R (noncloned population). D, Reintroduction of an active copy of TcNTR into benznidazole-resistant parasites (61R clone 2), using the pTEX vector reverses the drug-resistance phenotype. Growth inhibition data are the mean (±SD) of 3 experiments. An autoradiograph (right) shows BamHI-digested DNA from parental 61R clone 2 (lanes 1 and 2) and pTEX-TcNTR–transformed cells (lanes 3 and 4) hybridized with a TcNTR gene probe.

Figure 2.

Mutations in TcNTR from benznidazole-resistant Trypanosoma cruzi. The TcNTR schematic identifies the amino terminal extension (excluded from recombinant proteins) and the location of putative flavin mononucleotide (FMN)–binding regions inferred by analogy with Escherichia coli nfsB [30]. Full-length copies of TcNTR from 61R resistant clones were amplified and sequenced. Differences in the amino acid sequence as compared to the parental TcNTR (61S) were restricted to a single region and are highlighted in red. Several 61S clones were sequenced, but no differences were identified. The sequence in this region of 61S TcNTR (residues 112–162) is identical to that in the genome strain CL Brener (GenBank accession no. XP_810645). The corresponding CL Brener TcNTR residues are numbered 110–160 because of an insertion or deletion in the amino terminal domain. Mutations in the corresponding region of E. coli nfsB that confer nitrofurantoin resistance are indicated by asterisks [31].

Figure 3.

Biochemical analysis of TcNTR from benznidazole (BNZ)-sensitive and -resistant Trypanosoma cruzi. A, Purification of recombinant TcNTR. Upper image, wild-type (WT; 61S) enzyme; lower, clone 4. Protein expression was induced by isopropyl-β-D-thiogalactopyranoside (Materials and Methods), and a clarified fraction (lane 1) was loaded onto a nickel–nitriloacetic acid column and the flow-through collected (lane 2). The column was washed with 50 mM and then 100 mM imidazole (lanes 3 and 4) and the recombinant protein eluted with 500 mM imidazole, 1% triton X-100 (lane 5). The 32-kDa TcNTR band is highlighted by an arrow. Recombinant protein from each of the resistant clones was purified in a similar manner. B, TcNTR activity was monitored (on the basis of absorbance [Abs] at a wavelength of 340 nm) by following oxidation of nicotinamide adenine dinucleotide, reduced (NADH; 100 μM), in the presence of WT or mutant (P125L, clone 4) enzyme (0.2 μg) and BNZ (100 μM). C, Activity (v) of the WT enzyme (nmol NADH per min per mg) was established by this assay with a fixed concentration of NADH (100 μM) in the presence of different levels of BNZ (10–100 μM). D, Fluorescence (excitation λ = 450 nm; emission λ = 535 nm) of the TcNTR cofactor (WT and P125L) and flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) controls (in arbitrary units) under acidic and neutral conditions (Materials and Methods). E, Activity of WT and mutant TcNTRs.

Figure 4.

Loss of 1 copy of TcNTR does not reduce infectivity. Targeted disruption of TcNTR in 61S epimastigotes was achieved using a construct that confers blasticidin resistance (Supplementary Figure 1). A, TcNTR heterozygotes are benznidazole resistant. 61S TcNTR homozygotes (+/+) and heterozygotes (+/−) were tested to establish their median inhibitory concentration (IC₅₀). Data are the mean (±SD) of 3 experiments. B, Heterozygotes are not deficient in infectivity. Cell-derived trypomastigotes were added to an L6 cell monolayer at a ratio of 1:5 cells to parasites. Infected cells were counted after 72 hours, (experiment performed in triplicate, with data presented mean ± SD). C, Heterozygotes are not deficient in their ability to replicate in mammalian cells. Infections were performed and monitored as described above. D, Infection of Vero cells with 61S sensitive and 61R resistant parasites, stained with Giemsa: (1) 61S; (2) 61R clone 3; (3) 61R clone 4; (4) 61R clone 6. Arrows indicate intracellular amastigotes 48 hours after infection. E, Benznidazole-resistant clones are deficient in their ability to infect Vero cells. Values shown are from 5 experiments (P < .05). (F) Benznidazole-resistant clones are less able to replicate in Vero cells. Values shown are from 5 experiments (P < .05).