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. 2016 Mar;71(3):625-34.
doi: 10.1093/jac/dkv376. Epub 2015 Nov 17.

Nitroheterocyclic drug resistance mechanisms in Trypanosoma brucei

Susan Wyllie  1 , Bernardo J Foth  2 , Anna Kelner  1 , Antoaneta Y Sokolova  1 , Matthew Berriman  2 , Alan H Fairlamb  3
Affiliations

Nitroheterocyclic drug resistance mechanisms in Trypanosoma brucei

Susan Wyllie et al. J Antimicrob Chemother. 2016 Mar.

Abstract

Objectives: The objective of this study was to identify the mechanisms of resistance to nifurtimox and fexinidazole in African trypanosomes.

Methods: Bloodstream-form Trypanosoma brucei were selected for resistance to nifurtimox and fexinidazole by stepwise exposure to increasing drug concentrations. Clones were subjected to WGS to identify putative resistance genes. Transgenic parasites modulating expression of genes of interest were generated and drug susceptibility phenotypes determined.

Results: Nifurtimox-resistant (NfxR) and fexinidazole-resistant (FxR) parasites shared reciprocal cross-resistance suggestive of a common mechanism of action. Previously, a type I nitroreductase (NTR) has been implicated in nitro drug activation. WGS of resistant clones revealed that NfxR parasites had lost >100 kb from one copy of chromosome 7, rendering them hemizygous for NTR as well as over 30 other genes. FxR parasites retained both copies of NTR, but lost >70 kb downstream of one NTR allele, decreasing NTR transcription by half. A single knockout line of NTR displayed 1.6- and 1.9-fold resistance to nifurtimox and fexinidazole, respectively. Since NfxR and FxR parasites are ∼6- and 20-fold resistant to nifurtimox and fexinidazole, respectively, additional factors must be involved. Overexpression and knockout studies ruled out a role for a putative oxidoreductase (Tb927.7.7410) and a hypothetical gene (Tb927.1.1050), previously identified in a genome-scale RNAi screen.

Conclusions: NTR was confirmed as a key resistance determinant, either by loss of one gene copy or loss of gene expression. Further work is required to identify which of the many dozens of SNPs identified in the drug-resistant cell lines contribute to the overall resistance phenotype.

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Figures

Figure 1.
Figure 1.
Generation and analysis of NTR single gene knockout cell lines. (a) A schematic representation of NTR gene deletion in the knockout T. brucei cell lines SKOHYG (top) and SKOPAC (bottom). Arrows indicate the position of PCR primer pairs 5′UTR_−595_F and 3′UTR_+600_R (outer pair) and 5′UTR_−107_F and 3′UTR_+152_R (inner pair). (b) Agarose gel electrophoresis of PCR products generated with the primer pairs indicated in (a). The template genomic DNA was derived from WT T. brucei, SKOHYG clone 1 and SKOPAC clone 1.
Figure 2.
Figure 2.
Hemizygous regions in the drug-resistant parasite lines identified by WGS. The read coverage plots show the relative read depth (analysed in windows of 800 bp width) by plotting the log2-based ratio of the read depth observed in a given window over the average read depth of the corresponding chromosome. A log2-based relative read depth of −1 therefore indicates a read coverage that is 0.5-fold that of the average read depth, which may be due to the loss of one of the two alleles in that region (hemizygosity). Note the variable but highly reproducible (across the different parasite lines) nature of the read coverage, which is caused by differences in sequence read mapping efficiency across the genome especially in regions of repetitive sequence. The location of genes along the chromosomes is indicated below the graphs. (a) The plot shows the 3′-end (the ‘right-hand side’) of the chromosome core region of chromosome 7. A 50% reduction in read depth is apparent for the three replicate NfxR lines for more than 100 kb starting from around genomic position 2 065 600 and similarly for the three replicate FxR lines starting from around genomic position 2 104 500. The genomic variants listed in Table S3 confirm the expected concomitant loss of heterozygosity in this region. The hemizygous region observed in the NfxR lines includes NTR, whereas the hemizygous region in the FxR lines does not, although it still apparently overlaps the polycistronic transcription unit in this region. (b) A 5.8 kb hemizygous region on chromosome 1 observed only in the three replicate NfxR lines.
Figure 3.
Figure 3.
Overexpression and single gene knockout of a putative oxidoreductase encoded by Tb927.7.7410 in bloodstream trypanosomes. (a) Overexpression of the HA-tagged protein encoded by Tb927.7.7410 was induced by the addition of tetracycline 72 h prior to analysis. Cell lysates (1 × 10 parasites in each lane) were separated by SDS–PAGE and analysed by western blotting with a rabbit anti-HA antibody. Lysates were derived from WT cells (lane 1) and cells transfected with pLEW82-7410-HA3 (clone 1 in lane 2 and clone 2 in lane 3). Arrows indicate the position of Tb927.7.7410. (b) Schematic representation of the ORF of Tb927.7.7410 flanked by 5′UTR and 3′UTR regions. Successfully replacing a single copy of this gene with HYG allows a PCR fragment of 1.5 kb to be generated by specific primers designed to the 5′UTR (outside of the gene replacement construct) and HYG. (c) Agarose gel electrophoresis of PCR products generated with the primer pairs indicated in (b). The template genomic DNA was derived from WT T. brucei (lane 1), Tb927.7.7410SKO clone 1 (lane 2) and NTR/Tb927.7.7410SKO (lane 3). (d) EC50 values of 1.6 ± 0.03, 1.5 ± 0.03, 3.4 ± 0.06 and 3.1 ± 0.04 μM were determined for nifurtimox against WT (open circles), Tb927.7.7410SKO (filled circles), NTRSKO (open squares) and Tb927.7.7410/NTRSKO (filled squares) trypanosomes, respectively.
Figure 4.
Figure 4.
Generation of a Tb927.1.1050 single gene knockout in NTR SKOHYG cells. (a) The black bars represent the 5′UTR region upstream of the ORF of Tb927.1.1050 used as a probe in Southern blot analysis. SalI sites with expected fragment sizes are shown. The endogenous Tb927.1.1050 gene contains a SalI site, which results in a 2 kb band. Successful replacement of one allelic copy of the endogenous gene with HYG, which does not contain a SalI site, results in a 4.5 kb fragment. (b) Southern blot analysis of SalI-digested genomic DNA (∼5 μg) from T. brucei NTR SKOPAC cells (lane 1), NTR SKOPAC background and Tb927.1.1050 SKOHYG clone 1 cells (lane 2) and NTR SKOPAC background and Tb927.1.1050 SKOHYG clone 2 cells (lane 3). The 5′UTR of the gene Tb927.1.1050 was used as a probe. A faint non-specific band can be seen at ∼3 kb in all three lanes.
Figure 5.
Figure 5.
Venn diagram of overlapping gene ‘hits’ from RIT-seq screening of nifurtimox in T. brucei and of genes with high-confidence SNPs from WGS of NfxR and FxR trypanosomes. See Table S4 for further details.
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