Comparative genomics of drug resistance in Trypanosoma brucei rhodesiense
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Trypanosoma brucei rhodesiense is one of the causative agents of human sleeping sickness, a fatal disease that is transmitted by tsetse flies and restricted to Sub-Saharan Africa. Here we investigate two independent lines of T. b. rhodesiense that have been selected with the drugs melarsoprol and pentamidine over the course of 2 years, until they exhibited stable cross-resistance to an unprecedented degree. We apply comparative genomics and transcriptomics to identify the underlying mutations. Only few mutations have become fixed during selection. Three genes were affected by mutations in both lines: the aminopurine transporter AT1, the aquaporin AQP2, and the RNA-binding protein UBP1. The melarsoprol-selected line carried a large deletion including the adenosine transporter gene AT1, whereas the pentamidine-selected line carried a heterozygous point mutation in AT1, G430R, which rendered the transporter non-functional. Both resistant lines had lost AQP2, and both lines carried the same point mutation, R131L, in the RNA-binding motif of UBP1. The finding that concomitant deletion of the known resistance genes AT1 and AQP2 in T. b. brucei failed to phenocopy the high levels of resistance of the T. b. rhodesiense mutants indicated a possible role of UBP1 in melarsoprol–pentamidine cross-resistance. However, homozygous in situ expression of UBP1-Leu131 in T. b. brucei did not affect the sensitivity to melarsoprol or pentamidine.
KeywordsAfrican trypanosomes Aquaporin RNA-binding protein Purine permease Pentamidine Melarsoprol
Spliced leader trapping
Adenosine transporter 1
Uridine-rich binding protein 1
Variant surface glycoprotein
Human African trypanosomiasis (HAT, also known as sleeping sickness) is a fatal disease caused by Trypanosoma brucei rhodesiense and T. b. gambiense in East- and West-Africa, respectively. These protozoan parasites are transmitted by tsetse flies and proliferate extracellularly in the bloodstream and lymph of their mammalian hosts, evading the adaptive immune response through antigenic variation of their variant surface glycoprotein (VSG) coat. Ultimately the trypanosomes also infest the cerebrospinal fluid, causing the ‘sleeping sickness’ syndrome of infected patients. Trypanosoma brucei has an approximate haploid genome size of 35 Mb, which can vary up to 25 % . Excluding the kinetoplast (i.e., mitochondrial) DNA leaves a nuclear core genome of about 26 Mb, divided into 11 megabase-sized chromosomes, where the vast majority of the predicted >9000 protein-coding genes are located . The treatment of HAT relies on just five drugs. Patients in the first, hemolymphatic stage are treated with suramin (T. b. rhodesiense) or pentamidine (T. b. gambiense). In the second stage, when the trypanosomes have invaded the central nervous system, melarsoprol or nifurtimox-eflornithine combination therapy (NECT; only for T. b. gambiense) are used . These drugs are outdated, impractical, and suffer from severe adverse effects. Melarsoprol, in particular, causes unacceptable toxicity . Furthermore, melarsoprol treatment failure rates of up to 30 % have been reported throughout sub-Saharan Africa [5, 6, 7, 8]. New drugs that are safe and orally available are presently in clinical development . Meanwhile, it is essential to sustain the current drugs in spite of their shortcomings, which requires an understanding of the mechanisms of drug resistance. This will also help avoid cross-resistance between current treatments and those in development.
The molecular mechanisms of drug resistance have predominantly been studied in T. b. brucei, which is non-pathogenic to humans and widely used as a model in molecular parasitology. A phenomenon that has been repeatedly observed is melarsoprol–pentamidine cross-resistance (MPXR), i.e., trypanosomes selected for resistance with a melaminophenyl arsenical turned out to be cross-resistant to pentamidine and vice versa [10, 11, 12]. This phenomenon was attributed to the finding that the uptake of melarsoprol and pentamidine into the trypanosomes is mediated by the same set of transporters: the aminopurine permease P2 [13, 14], encoded by the gene AT1, and a high-affinity pentamidine transport activity designated as HAPT1 [15, 16] recently shown to correspond to the aquagylceroporin AQP2 [17, 18]. Mutations in these transporters were described from drug-resistant T. brucei ssp. isolates from the field [19, 20, 21]. In the lab, MPXR was phenocopied by reverse genetics. Homozygous deletion of either AT1 or AQP2 resulted in resistance to both melarsoprol and pentamidine. However, the obtained resistance factors were only between 2 and 3 for melarsoprol and pentamidine in AT1 null trypanosomes [22, 23], respectively, and 2 for melarsoprol and 15 for pentamidine in AQP2 null mutants .
Here we investigate two clonal drug-resistant lines of T. b. rhodesiense that exhibit markedly higher levels of MPXR than observed after deletion of either AT1 or AQP2. The lines T. b. rhodesiense STIB900-M and STIB900-P had been selected in vitro from their drug-susceptible parent T. b. rhodesiense STIB900 by continuous in vitro exposure to increasing concentrations of melarsoprol and pentamidine, respectively, over a period of 24 months . Finally, both lines exhibited a high level of MPXR with in vitro resistance factors up to 80 (the resistance factor was defined as IC50 of the selected line divided by the IC50 of the drug-sensitive parent). This phenotype was stable in the absence of drug pressure and after passage through mice. An initial genotypic characterization demonstrated that AT1 had been lost in STIB900-M but was still present in STIB900-P . Evidently, given the high level of drug resistance, further mutations must be involved. We have performed whole genome sequencing and RNA-Seq of the parental T. b. rhodesiense STIB900 and its resistant derivatives STIB900-M and STIB900-P, aiming to elucidate the molecular mechanisms underlying the unprecedented level of MPXR by comparative genomics and transcriptomics.
Materials and methods
T. b. rhodesiense lines
Trypanosoma brucei rhodesiense STIB900 is a derivative of STIB704, isolated from a male patient at St. Francis Hospital in Ifakara, Tanzania, in 1981. After several passages in rodents and a cyclic passage through a tsetse fly (Glossina morsitans morsitans), a cloned population was adapted to axenic growth. T. b. rhodesiense STIB900-M and STIB900-P had been selected independently in vitro for resistance to melarsoprol and pentamidine, respectively . Bloodstream-form trypanosomes were propagated in vitro as described in  and adapted from . Cells were counted with the CASY® Cell Counter system (Roche). Large numbers of trypanosomes for DNA isolation were obtained by inoculating female Naval Medical Research Institute (NMRI) mice (Harlan Laboratories) with 106 trypanosomes. At peak parasitemia, the trypanosomes were harvested and separated from the blood cells on DEAE-cellulose columns .
In vitro drug sensitivity
For all the STIB900 lines drug sensitivities were determined with the Alamar blue assay . T. b. brucei B48 drug sensitivities were determined as described using a slightly modified protocol . The SoftMax Pro software and Prism 5 (GraphPad) were used to calculate 50 % inhibitory concentrations (IC50) by non-linear regression fitting to a sigmoidal dose–response curve. All assays were performed at least three times independently, each in duplicate or triplicate. The following compounds were tested, each individually: melarsoprol (Sanofi-Aventis/WHO), eflornithine (Sanofi-Aventis), DB75 (Immtech), fexinidazole (DNDi, Geneva), nifurtimox (WHO, Geneva), suramin (Bayer), pentamidine isethionate, diminazene aceturate, phenylarsine oxide, aminopurinol, cordycepin, adenosine arabinoside, and tubercidin (Sigma). Statistical tests were performed with GraphPad Prism 5.0.
Isolation of nucleic acids
Genomic DNA was isolated by phenol/chloroform extraction from bloodstream-form trypanosomes propagated in mice. To check for contamination with mouse DNA we performed PCR with primers for mouse glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) and mouse cDNA as a positive control. For each T. b. rhodesiense line, about 60 µg of genomic DNA was prepared for sequencing. Total RNA was isolated from exponentially growing cultures of trypanosomes (106 cells/ml) with TRIzol (Life technologies). Equal amounts of total RNA were pooled from three independent isolations, and from each pooled sample 12 µg were used for sequencing.
Spliced leader trapping
Library preparation and RNA-Seq were performed according to the spliced leader trapping (SLT) protocol . This is a modification of the standard Illumina protocol that uses the T. brucei 39 nt spliced leader sequence (which is a peculiarity of trypanosomatids and gets ligated to the 5′ end of every mRNA) for 2nd strand cDNA synthesis and sequencing. Two independent experiments were performed: run 1 on the Genome Analyzer IIx (Illumina) and run 2 on the HiSeq 2000 (Illumina). The Fastq files were read into the Spliced Leader ADDition (SLADD) program  and mapped onto the reference genome sequence of T. brucei TREU927 , using MAQ  with n = 3 and with a read length of ≥24. Multi-mapping reads were separated from single mappers by an alignment quality threshold of 30. Read counts were normalized according to library size and expressed as tags per million reads (TPM). Statistical analysis for differentially expressed genes was performed with the DESeq package in R . DESeq uses a negative binomial distribution and a shrinkage factor for the distribution’s variance. Only mapped reads (raw counts) with a stringent quality score of q < 30 and data from both performed SLT runs were included.
Whole genome sequencing
Whole genome sequencing of T. b. rhodesiense STIB900 was carried out on the Illumina HiSeq 2000 platform. Two times 12,243,924 paired-end reads of 76 b were mapped chromosome-wise to the reference genome T. b. brucei 927 (v5) using MIRA (v3.9.16) . Gene models from the reference genome T. b. brucei 927 were transferred to the assembled STIB900 genome using rapid annotation transfer tool (RATT)  from the post assembly genome improvement toolkit (PAGIT)  package. Whole genome sequencing of T. b. rhodesiense STIB900-M and STIB900-P was carried out on the Genome Sequencer FLX Titanium by Roche/454. Two shotgun runs per line were performed. FASTQ format was extracted from .sff files using ‘SFF converter’ from Galaxy . High-quality (HQ) reads were mapped to the assembled STIB900 genome, indexed with word length 13 and skip step 1, using the program SMALT (ftp.sanger.ac.uk/pub4/resources/software/smalt). Consensus sequence and variants relative to the assembled STIB900 genome were identified with ‘mpileup’ from SAMtools . Ad hoc Perl scripts were used to compare nucleotide variants between the mapped reads of STIB900-M, STIB900-P, and the assembled STIB900 genome. For comparison also STIB900 reads generated on the Roche/454 platform were mapped to the assembled STIB900 genome. SNPs were called if they had a read depth of at least five high-quality bases (DP4 ≥ 5) and a read mapping quality of minimum 20 (mapq ≥20). All identified SNPs, indels, and gene deletions were inspected manually using Artemis . The Roche/454 reads are accessible via the European Nucleotide Archive (http://www.ebi.ac.uk/ena) under accession number PRJEB12780.
PCR amplification and Sanger sequencing
The PCR primers to amplify the 3.2 kb AQP2/AQP3 tandem locus were AQP2/3_F (aagaaggctgaaactccacttg) and AQP2/3_R (tgcactcaaaaacaggaaaaga), annealing at 58 °C. AT1-G430R was amplified from genomic DNA of STIB900-P with primers AT1_F (gaaatccccgtcttttctcac) and AT1_R (atgtgctgagcctttttcctt), annealing at 56 °C. The PCR product was purified on silica-membrane columns (Nucleospin gel and PCR clean up, Macherey–Nagel) and digested with NruI (New England Biolabs), run on a 1.5 % agarose gel and visualized with ethidium bromide. UBP1 and UBP1-R131L were amplified from STIB900 and STIB900-M, respectively, with the primers UBP1_F (ccgctctagatctcaggttccactggcttc) and UBP1_R (ccgcggatcctcatttacgggcaggccgac).
Plasmid construction, transfection and knock-out generation
The gene encoding the AT1-G430R mutant was amplified by PCR from genomic DNA of STIB900-P and the product was ligated into the expression vector pHD1336  to give pHDK68. The plasmid was verified by Sanger sequencing (Source BioScience, Nottingham, UK) for the presence of the expected mutation and linearized with NotI prior to transfection into T. b. brucei clone B48, which lack the AT1 gene and the high-affinity pentamidine transporter . B48 parasites were washed in Human T Cell Solution for transfection with an Amaxa Nucleofector . Transfectants were cloned by limiting dilution in standard HMI-11 medium  containing 5 µg/ml blasticidin for selection of the positive transfectants. Correct integration of the expression cassettes was tested by PCR.
Trypanosoma brucei rhodesiense 2T1 aqp2/3−/− cells were assembled using a I-SceI meganuclease-based gene-conversion approach. Briefly, blasticidin deaminase (BSD) and neomycin-phosphotransferase (NPT) cassettes were used to replace the AQP2/3 locus. Meganuclease cleavage of the NPT cassette was then used to trigger replacement with, and duplication of, the BSD cassette. AT1 was disrupted in the resulting 2T1 aqp2/3−/− cells by replacing the first allele of the 1392 bp ORF with PCR-amplified NPT containing 100 bp overhangs identical to the UTRs of AT1, followed by selection with 5 µg/ml G418. The second allele was replaced with PAC plus 500 bp AT1 UTR on either end, followed by selection with 0.1 µg/ml puromycin. Homozygous deletion of AT1 was verified by PCR (Figure S1).
Reverse genetics of UBP1
For overexpression of UBP1, the UBP1 and UBP1-R131L PCR products were cloned into pRPaiGFPx  via XbaI and BamHI. T. b. brucei 2T1 cells  were transfected with AscI-digested plasmids in Tb-BSF nucleofection buffer  using the Amaxa nucleofector (Lonza) with program Z-001. Transfectants were cloned by limiting dilution and selected with 2.5 µg/ml hygromycin. PCR and Sanger sequencing confirmed correct integration and sequence of the transgene.
To introduce the mutant UBP1-R131L in 2T1 cells in situ, a plasmid carrying the mutation and a blasticidin resistance gene (BSD) in the 5′ UTR of UBP1, used as a selection marker, was constructed (Supplementary Figure S3A). The synthetic DNA was obtained from GenScript (Piscataway Township, NJ, USA), integrated between the HindIII and BamHI sites of cloning plasmid pUC57. DNA for transfection was prepared by PCR amplification of the insert using primers BLA_UBP1mut_F1 (ttgcattcgctcctttccct) and BLA_UBP1mut_R1 (ccttcagtagtttgttgagg) and subsequent purification as described above. 2T1 cells were transfected with an Amaxa Nucleofector using program Z-001 and clones were obtained as described above. Cells were selected with 5 and 10 µg/ml blasticidin. Correct homozygous integration was verified by PCR (Supplementary Figure S3B) and Sanger sequencing (Fig. 6b).
SDS-PAGE and western blotting
Cells were lysed in NUPAGE® LDS sample buffer (Life Technologies) and samples were loaded on precast 4–12 % Bis–Tris Gradient Gels (NuPAGE Novex®, Life Technologies) in MES running buffer and transferred to nitrocellulose membranes using the iBlot dry-blotting system (Novex®, Life Technologies). Membranes were blocked in 5 % milk in PBS/Tween-20 and incubated with primary antibodies in 5 % milk in TBS/Tween-20 overnight at 4 °C. Membranes were washed and incubated with peroxidase-conjugated secondary antibodies in 5 % milk PBS/Tween-20 for 2 h at room temperature. Blots were developed using the ECL Western Blotting Substrate (Pierce) using a ChemiDoc™ MP Gel Imaging System (Biorad). Primary Antibodies used: rabbit anti-GFP (Abcam, Ab290), mouse anti-BiP (kind gift of Prof A. Schneider). Secondary Antibodies used: goat anti-rabbit (SouthernBiotech: 4050-05), polyclonal rabbit anti-mouse HRP (Dako, Baar, Switzerland).
Phenotypic profiling of high-level melarsoprol–pentamidine cross-resistance
In vitro drug sensitivity profiles
IC50 ± standard deviation [nM]
STIB900 vs. 900-P
STIB900 vs. 900-M
STIB900-P vs. 900-M
STIB900-P to 900
STIB900-M to 900
6.0 ± 3.4
84 ± 52
170 ± 63
2.8 ± 0.8
130 ± 69
210 ± 93
3.8 ± 1.5
18 ± 5.4
25 ± 10
3.7 ± 0.9
22 ± 8
64 ± 17
135 ± 62
76 ± 32
125 ± 25
1100 ± 550
1100 ± 460
1500 ± 620
3200 ± 1100
2500 ± 980
6100 ± 2500
5200 ± 1400
1300 ± 640
2700 ± 480
0.46 ± 0.1
2.2 ± 0.54
7.0 ± 1.5
265 ± 108
400 ± 52
650 ± 190
26 ± 6
100 ± 19
4300 ± 1000
1500 ± 190
5400 ± 1100
2000 ± 630
0.52 ± 0.13
0.65 ± 0.11
0.75 ± 0.08
Transcriptomic profiling indicates loss of expression of transporter genes
A reference genome sequence of the T. b. rhodesiense drug-sensitive parent
Before exploring the mutations underlying the strong MPXR phenotype of STIB900-P and STIB900-M by comparative genomics, we had to generate a good-quality draft genome of the susceptible parent T. b. rhodesiense STIB900. Genomic DNA was isolated from bloodstream-form trypanosomes grown in mice. The obtained gDNA was verified to be free of mouse DNA by PCR with primers for mouse GAPDH. Paired-end Illumina reads generated on the HiSeq platform were mapped to the core chromosomes of T. b. brucei TREU927 with an average coverage of 53 fold. The vast majority of gene models (9692 of 9722) were transferred from T. b. brucei TREU927 to the assembled T. b. rhodesiense STIB900 genome, identifying a total of 112,565 high-quality single-nucleotide polymorphisms (SNP) between these two genomes. In protein-coding regions there were 46,453 SNPs, of which 19,575 non-synonymous. As expected, the assembled T. b. rhodesiense STIB900 genome contained the SRA gene (serum resistance-associated; Tb927.9.17380), whose product neutralizes ApoL1, the trypanolytic factor of human serum that protects humans from infection by T. b. brucei. The genome reference strain T. b. brucei TREU927 contains a dysfunctional SRA ortholog [50, 51].
Comparative genomics confirms the loss of transporter genes
STIB900-M vs. STIB900
STIB900-P vs. STIB900
A point mutation that renders TbAT1 non-functional is heterozygous in STIB900-P
Concomitant deletion of AQP2 and AT1 does not phenocopy the high-level MPXR
Testing of the RNA-binding protein UBP1 as a new candidate resistance gene
Cross-resistance of African trypanosomes to melarsoprol and pentamidine (MPXR) is a well-known phenomenon . Here we perform in-depth phenotypic and genotypic profiling of two lab-derived T. b. rhodesiense mutants with most pronounced MPXR phenotypes: STIB900-P, selected with pentamidine, and STIB900-M, selected with melarsoprol. The two independently selected lines exhibited similar—but not identical (Table 1)—resistance profiles, extending MPXR to other diamidines and adenosine analogs, but not to suramin or nifurtimox.
Very few mutations had become fixed in the drug-selected lines in the 2 years’ course of selection (Table 2), and very few genes were differentially expressed in the resistant lines compared to the sensitive parent (Fig. 1). The only striking difference was the complete absence of RNA-Seq reads for AQP2 in STIB900-M and STIB900-P, and for AT1 and neighboring genes in STIB900-M. Both STIB900-M and STIB900-P carried a deletion of the AQP2 locus (Fig. 2). This ‘natural knock-out’ of AQP2 was probably due to homologous recombination with the neighboring, highly similar gene AQP3 (Fig. 2), accompanied by loss of genetic material. Cases of truncation or chimerization of AQP genes in drug-resistant T. brucei had been reported previously [17, 20, 21, 56]. T. b. rhodesiense STIB900-M also carried a large deletion on chromosome five encompassing AT1 and six adjacent genes (Fig. 3), a gene loss possibly facilitated by their telomeric location. STIB900-P had a non-synonymous point mutation in AT1 changing Gly430 to Arg (Fig. 3). Expression of wildtype and G430R-mutant AT1 in a T. b. brucei loss of transport mutant demonstrated that the AT1-G430R did not transport melarsoprol or diamidines (Fig. 4). The finding that STIB900-P is heterozygous for the mutation might explain its milder MPXR phenotype than STIB900-M (Table 1). These results are the best evidence to date that the MPXR models developed in the non-human infective trypanosome T. b. brucei hold true in T. b. rhodesiense, the causative agent of sleeping sickness in East Africa, and that selection for high-level resistance to melarsoprol and pentamidine can lead to loss of both known drug transporters.
To investigate the combined contribution of AQP2 and AT1 to drug sensitivity, we constructed T. b. brucei hetero- and homozygous at1 null mutants in a aqp2/aqp3 null background. The effects of the mutations were additive regarding pentamidine sensitivity, with a maximal resistance factor of 35 for the double null mutant of at1 and aqp2 (Fig. 5). The phenotypes with respect to melarsoprol were less pronounced, as the loss of AT1 did not further increase the melarsoprol resistance of the aqp2 null mutant (Fig. 5). The conclusion from these experiments is that concomitant deletion of AQP2 and AT1 does not completely phenocopy the strong MPXR phenotypes of T. b. rhodesiense STIB900-M and STIB900-P, demonstrating that further genes must be involved. Only one additional gene was affected by a mutation in both resistant lines: the RNA-binding protein UBP1.
Trypanosoma brucei rhodesiense STIB900-M and STIB900-P both carried the mutation R131L in UBP1, which was absent in the parent and must have been acquired by the resistant lines independently. The precise function of UBP1 is unknown, but it has been implicated in regulation of mRNA levels . Based on the alignment to TcUBP1 from T. cruzi, Arg131 of TbUBP1 is predicted to be critical for RNA binding (Fig. 6a), suggesting that Leu131 may impair TbUBP1 function. Complete loss of UBP1 function may be lethal, as indicated by RNAi-mediated knock-down of UBP1 and UBP2 . TcUBP1 is a cytoplasmic protein in epimastigotes of T. cruzi . Intriguingly, TcUBP1 was shown to accumulate in the nucleus when the trypanosomes were under arsenite stress, and mutations affecting RNA binding prevented nuclear accumulation of TcUBP1 . TbUBP2, which shares 73 % global identity with TbUBP1, had appeared as a secondary hit in a genome-wide RNAi screen for pentamidine resistance in T. brucei . While such previous findings may support a possible role of UBP1 in drug resistance, our reverse genetic approaches did not. Non-physiological overexpression of UBP-Leu131-GFP in T. b. brucei bloodstream forms even caused a slight hypersensitivity to pentamidine (Figure S2B). However, this needs to be interpreted cautiously since, in agreement with a previous report , overexpression of the ‘wildtype’ UBP1-GFP fusion protein was lethal (Figure S2A). More physiological in situ expression of the mutant UBP1 in T. b. brucei did not affect the sensitivity to melarsoprol or pentamidine (Fig. 6c). In summary, if the mutation Arg131 to Leu in UBP1 contributes to drug resistance at all, then it does so only in the context of the described loss of AQP2 and/or AT1.
We would like to thank Hansjörg Keller for mouse cDNA and André Schneider for the BiP antibody. We are very grateful to Roche for a 10 GB Sequencing Award that has granted us free access to the Roche-454 platform and to the Emilia Guggenheim-Schnurr Foundation for financial support with spliced leader trapping. This research was supported by the Swiss National Science Foundation, by the MRC and Department for International Development, UK under the MRC/DFID Concordat agreement (grant MR/K000500/1 to DH), and by the Wellcome Trust (Grant 100320/Z/12/Z; Investigator Award to DH). FEG and PL received additional support by the Mathieu Stiftung Basel and the Freiwillige Akademische Gesellschaft Basel. PL was additionally supported by the Emilia Guggenheim-Schnurr Foundation. Support for the STIB900 Illumina data was provided to the NIH R21 Grant AI094615-01 awarded to AC.
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