Philippine Journal of Science 148 (S1): 219-233, Special Issue on ISSN 0031 - 7683 Date Received: 21 Mar 2019

Genome-wide Analysis for Variants in Philippine Trypanosoma evansi Isolates with Varying Drug Resistance Profiles

Jose Enrico H. Lazaro1§, Neil Andrew D. Bascos1,3§, Francis A. Tablizo3§, Nancy S. Abes2, Renlyn Ivy DG. Paynaganan1, Michelle A. Miguel2, Hector M. Espiritu2, Mary Rose D. Uy2, Claro N. Mingala2, and Cynthia P. Saloma1,3*

1National Institute of Molecular Biology and , College of Science, University of the Diliman, Quezon City 1101 Philippines 2Philippine Carabao Center, Department of Agriculture, Science City of Munoz, Nueva Ecija 3120 Philippines 3Philippine Genome Center, University of the Philippines, Diliman, Quezon City 1101 Philippines §These authors contributed equally to this work

Surra, a parasitic disease transmitted by hematophagous flies and caused by Trypanosoma evansi, affects many domesticated animals – including water buffaloes, camels, horses, pigs, dogs, and other carnivores – throughout the world. When left untreated, this disease can cause anemia, significant loss of weight, abortion, and death in affected animals. Among Philippine isolates of T. evansi, variability has been reported in terms of virulence as well as response to drug treatment. In this study, trypanosoma-positive blood was obtained from 15 Philippine water buffalo samples from different sites in the country. The collected T. evansi strains were propagated in mice then subjected to in vivo virulence, in vitro drug sensitivity testing, and whole genome . One strain (O14) was found to be highly virulent in vivo, and was found to be resistant to three commonly used drugs [i.e., isometamidium chloride (IC), diminazene diaceturate (DD), and melarsamine hydrochloride (CY for Cymelarsan®)] in vitro. This highly resistant sample was compared with two less-virulent strains using genome-wide analysis of single nucleotide polymorphisms (SNPs) and short insertions and deletions (indels) relative to the reference strain STIB 805. Variant analysis between O14 and the less virulent strains (M4 and C117) identified a number of distinctive SNPs, many of which corroborate previous data. Genes with relatively high copy numbers were observed in mutation hotspots. These included genes that code for variant surface glycoproteins (VSGs), expression site-associated genes (ESAGs), retrotransposon hot spot (RHS) proteins, and leucine rich repeat proteins. Notable mutations were also predicted from genes coding for membrane transporters and cysteine peptidases, as well as those involved in RNA degradation. The whole genome sequences acquired from the Philippine isolates (O14, M4, and C117) vary greatly from the reference strain (STIB 805). These WGS data serve as a good resource for the discovery of genetic and phenotypic features that may be translated to effective treatment strategies, relevant to the Philippine setting.

*Corresponding Author: [email protected] [email protected]

219 Special Issue on Genomics Lazaro et al.: Genome-wide Analysis for Philippine T. evansi Variants

INTRODUCTION design. A similar strategy has been employed to generate protective antibodies against the parasites that cause Blood parasitic diseases caused by vector-borne malaria (Martin 2004). The observed features in these protozoa gravely affect livestock production in many genomes provide leads on possible avenues for more developing countries. Surra, a parasitic disease caused efficient drug therapies and potential vaccine targets that by Trypanosoma evansi, affects many domesticated would be relevant for the prevention of surra in Philippine animal species including buffaloes, camels, and horses domesticated animals. in Asia, Africa, and Central and South America. Surra is transmitted by hematophagous flies (e.g., Tabanids and Stomoxys) that act as mechanical vectors of the disease. Infected individuals exhibit highly variable clinical MATERIALS AND METHODS effects, depending on the host and their geographical area (Desquesnes et al. 2013). IACUC Permit for Animal Studies Buffaloes are cryptic hosts of T. evansi. Although infected All procedures on the use of mice for trypanosome buffaloes do not exhibit common symptoms of the disease propagation and antibiotic drug testing were approved (e.g., weakness, edema, pyrexia), infection is known to by the UP Diliman Institutional Animal Care and Use induce still births and abortions in these animals (Luckins Committee (UPD IACUC) under Protocol Permit No. 1988, Reid 2002, Dobson et al. 2009). The latter results in a PAF-NIMBB-2015-02. significant loss in productivity for the Philippine livestock sector. Demographic and epidemic models suggest a net benefit loss of PhP 7 million per year for villages under Sample Collection and Propagation moderate/high prevalence of surra (Dargantes et al. 2009, Blood samples infected with parasites were obtained Dobson et al. 2009). The Philippines has suffered from from the PCC in two forms: cryopreserved in liquid several surra outbreaks in the past decades. Manuel (1998) nitrogen and extracted from mice infected with parasites. reported that in a span of three years (1994–1996), a total Cryopreserved blood samples were thawed and then of 1,151 deaths (985 carabaos, 71 cattle, and 95 horses) inoculated into mice via intraperitoneal injections. Blood have been documented all over the country. From 1999 parasite examination was done everyday to monitor to 2001, at least six municipalities experienced similar parasite numbers. Parasites were collected once peak serious outbreaks of surra in Central and Western Visayas parasitemia was observed (usually on Day 3) by cardiac – with mortality reaching 35% (Dargantes et al. 2009). puncture, washed, resuspended in buffer, and either The high mortality rate associated with local trypanosomal used immediately for in vivo and in vitro testing or for outbreaks suggests increased virulence in the Philippine subsequent cryopreservation. Methods were done as strains of T. evansi. described previously (Tavares et al. 2011). Current disease control strategies are principally based In Vivo Virulence and In Vitro Drug Sensitivity Assays on the use of trypanocides and preventive management To propagate parasites, ICR mice were inoculated with methods to protect animals from infection (Desquesnes et cryopreserved parasites via intraperitoneal injection. ICR al. 2013). Three drugs are used by the Philippine Carabao mice are outbred mice named after the Institute of Cancer Center (PCC) for treating surra cases in the Philippines Research, USA, where the mouse strain was developed. – namely IC, DD, and CY. However, treatment failure Parasitemia was monitored daily by tail blood wet mount. had been observed with their application on the field Once peak parasitemia was observed (> 60 parasites per (Mungube et al. 2012). These occurrences suggest the view at 40X magnification), mice were anaesthetized and development of resistance in some strains of T. evansi blood was collected via cardiac puncture. Collected blood for these commonly used drugs. was mixed with 4:6 phosphate buffered saline with 1% This study investigated Philippine T. evansi isolates with glucose (PBSG, pH 7.2) in a 1:1 ratio. The mixture was varied drug resistance and virulence profiles in the mouse centrifuged for 30 min at 8 x g in 4 °C. The buffy coat was model. Whole genome sequences of these Philippine carefully collected and applied into equilibrated DEAE isolates were acquired and compared in search of genetic resin-packed mini columns in the same buffer. Once all markers that could be correlated with virulence and drug parasites were eluted, their concentrations within the response. Large scale structural variations were found samples were estimated using a hemocytometer. Parasites in genes of resistant strains, some of which have been were maintained in culture media (25 mM HEPES, 1 previously implicated in virulence. These include VSGs g/L glucose, 2.2 g/L NaHCO3, 10 mL/L 100x MEM and flagellar proteins. Conserved regions in the encoded non-essential amino acids, 0.2 mM 2-mercaptoethanol, cellular structures serve as potential targets for vaccine 2 mM Na-pyruvate, 0.1 mM hypoxanthine, 0.016 mM

220 Special Issue on Genomics Lazaro et al.: Genome-wide Analysis for Philippine T. evansi Variants thymidine, 15% heat inactivated rabbit serum, 1x pen- min) and Qubit™ fluorometric quantitation (Thermo Fisher strep, pH 7.2) until further treatment (Baltz et al. 1985). Scientific) to verify DNA quality and quantity. The identity of the isolates as T. evansi was verified using targeted PCR To measure in vivo virulence, eight-week-old female amplification of the extracted DNA. ICR mice were acclimatized for one week and maintained under standard laboratory conditions. The mice were administered cyclophosphamide to induce immunosuppression. Cyclophosphamide in phosphate Genomes of the three T. evansi isolates were sequenced buffered saline was administered 24 h before inoculation. using the Illumina MiSeq platform (paired-end, 300 About 1 x 105 parasites suspended in culture medium were bp read length). Libraries were prepared using the inoculated by intraperitoneal injection into each mouse at Nextera DNA Library Preparation kit for simultaneous a maximum volume of 0.2 mL. Virulence was measured fragmentation (600 bp) and tagging with barcoded based on the number of days to reach peak parasitemia sequencing adapters. The samples were then pooled and post-inoculation (greater than 60 parasites per field of were run through the Illumina MiSeq sequencer at > view at 40X magnification). 100X coverage at the Philippine Genome Center DNA Sequencing Core Facility. To measure in vitro drug sensitivity, IC, DD, and CY were initially dissolved in dimethyl sulfoxide (DMSO) to produce stock solutions. Aliquots were added to Variant Calling, , and Functional the complete culture media resulting in a final DMSO Analysis concentration of less than 10% in the working solutions. The quality of the raw sequence data was initially assessed This concentration of DMSO was shown to have no effect using the tool FastQC (http://www.bioinformatics. on parasite survival. Twenty-five microliters (25 µL) of the babraham.ac.uk/projects/fastqc/). Quality trimming of working solutions were loaded into the microtiter plates low-quality sequence ends (min quality = 20; min length and were serially diluted two-fold. A volume of 100 µL = 50) was then done using TrimGalore (http://www. of parasites at a density of 2,000 parasites/µL was then .babraham.ac.uk/projects/trim_galore/). The loaded into flat-bottomed microtiter wells. The highest remaining sequences were then subjected to quality filtering final concentrations of the drugs were 2 mg/mL IC, 10 (min quality = 20; % passing bases = 80%) using the tool mg/mL DD, and 40 µg/mL CY. Cultures were exposed to fastq_quality_filter included in the FASTX-Toolkit (http:// the drugs and 10 µL of Alamar Blue for 6 h at 37 °C and hannonlab.cshl.edu/fastx_ toolkit/). A final assessment of the quality control reads was done using FastQC and the 5% CO2 prior to analysis. The assays were performed in triplicate for each drug. pairing of the remaining reads was restored using an in- house script. The quality assessment metrics before and Control setups containing media with parasites and no after quality control are provided in Table I. drug treatments were prepared in parallel with the drug assays. Standard curves for the control samples were The T. evansi sequencing reads were mapped against the prepared by serially diluting two-fold from an initial sequences of the annotated reference T. evansi STIB 805 density of 5,000 parasites/µL in 125 uL in a 96-well strain obtained from TriTrypDB (tritrypdb.org) using the microplate. Alamar Blue dye was added to each well prior Burrows-Wheeler Aligner software (Li and Durbin 2010). The mapping metrics – including the number of mapped to incubation at 37 °C and 5% CO2 for 6 h. reads, the total number of mapped bases, and average The analysis was done using a Varioskan Flash mapping coverage – can be seen in Table II. Multimode Reader. One hundred microliters (100 µL) of well contents from treatment and control samples To determine single nucleotide variants and short indels, were transferred into black flat-bottom microplates the best practices workflow suggested by GATK version for fluorescence readings. Fluorescence excitation and 3.7 (McKenna et al. 2010) was implemented. Briefly, emission were done at 530 nm and 590 nm, respectively. the mapping files were sorted according to reference coordinates using the tool SortSam from PICARD Tools Median inhibitory concentration (IC50) was calculated using Compusyn (www.combosyn.com) (http://broadinstitute.github.io/picard). Duplicate reads were marked using the MarkDuplicates tool of PICARD (https://broadinstitute.github.io/picard/). Genomic DNA Extraction from T. evansi Isolates Read groups were added to each of the mapping files Parasites isolated and purified using the DEAE columns were using the PICARD tool AddOrReplaceReadGroups. pelleted and subjected to DNA extraction. The QIAGEN Realignment of mapped reads around indel regions was DNA Mini kit was used following the manufacturer’s done using the RealignerTargetCreator and IndelRealigner instructions. The extracted genomic DNA was then tools in GATK. Finally, variants were called using the subjected to gel electrophoresis (1% agarose, 100V for 30

221 Special Issue on Genomics Lazaro et al.: Genome-wide Analysis for Philippine T. evansi Variants

HaplotypeCaller tool of GATK. The single nucleotide RESULTS AND DISCUSSION variants and short indels were annotated using SNPEff (Cingolani et al. 2012). In Vivo Virulence and in Vitro Drug Sensitivity Assay Genes implicated in the variations were then used as input in Mice for sequence homology search against the non-redundant A total of 15 strains were tested for virulence in vivo and protein database of the National Center for Biotechnology drug sensitivity in vitro. Assessment of in vivo virulence Information (NCBI) using the tool DIAMOND (Buchfink was based on daily parasitemia and symptomatology et al. 2015) and subjected to gene ontology analysis using observed in mice. Of these strains, one was highly virulent Blast2GO (Conesa and Götz 2008). Visualization and (+++), four were intermediate (++), and 10 were weakly plotting of the associated ontology terms were done using virulent (+). Strain O14 was the only highly virulent strain, in-house R scripts. consistently killing mice in a 24-h period (Table 1).

Further investigations on the functional significance of IC50 served as an additional indicator of virulence. The the variants observed only in resistant samples were IC50 values could be compared for experiments carried done through the sequence and structural alignments. out under the same conditions of dose and exposure. Prior was primarily done using the Basic experiments to establish optimal exposure conditions Local Alignment Search Tool (BLAST) (Altschul et al. showed that all isolates began to decrease in number after 1990) of NCBI. Structural analysis was done by searching 10–12 h in culture in the absence of drug, although there the protein structures available in the Protein Data are still a few viable parasites at 24 h. Doses of all three Bank (PDB; www.pdb.org) for matches to the observed drugs were optimized to effect a sigmoidal dose-response mutations in the resistant strains. In cases where no PDB over a 6-h period to ensure that parasites have not adapted matches were found, structures from UniProt (www. to exposure in possibly varying ways. .org) were used instead. Predictions made for the Kumar et al. (2015) found that parasites could remain structural significance of the SNPs were based on the viable between 48 h and 72 h depending on media. The location of these mutations in functional domains of the media used in the present study is comparable to medium related proteins. C referred to by Kumar et al. (2015) with parasites able

Table 1. Drug resistance profiles of Philippine T. evansi samples. (+) reached peak parasitemia in 5 days or more; (++) peak parasitemia in 3–4 days; (+++) peak parasitemia in 1–2 days.

IC50 (mg/mL) Phenotypic virulence Strain Location Isometamidium Diminazene Cymelasan chloride diaceturate M4 Mindanao 144 7,194 7 + C117 Cagayan 165 24,889 8 + O14 Nueva Ecija 439 22,478 25 +++ M6 Mindanao 332 15,943 6 ++ C120 Cagayan 134 8,126 18 + C110 Cagayan 137 15,509 6 ++ 5UP 03021 UP Los Baños 220 18,024 4 + 05141 Nueva Ecija 84 20,245 13 ++ M2 Mindanao 95 18,231 5 + C1 Cagayan 252 4,640 4 + M7 Mindanao 217 11,694 11 ++ P75 Nueva Ecija 119 12,341 7 + 2NIZ 11038 Nueva Ecija 356 12,374 4 + M5 Mindanao 131 5,428 2 + TRYPS 3+ Luzon 248 15,907 7 + Highly sensitive + Reaches peak parasitemia at 5 days onward Mildly sensitive ++ Reaches peak parasitemia at 3-4 days Mildly resistant +++ Reaches peak parasitemia at 1-2 days Highly resistant Peak parasitemia: > 60 parasites per field of view at 40x manification

222 Special Issue on Genomics Lazaro et al.: Genome-wide Analysis for Philippine T. evansi Variants to be sustained for 72 h. In medium C, parasite numbers The three strains were from provinces with a high could go down within 24 h then increase to remain viable prevalence of surra. They were selected for whole genome for more than 30 d. Using long exposure, Birhanu et al. sequencing at the PGC DSCF and analyzed at the Core (2016) found an IC50 of 7 ng/mL for isometamidium, Facility for Bioinformatics. 18 ng/mL for diminazene diaceturate, and 3 ng/mL for melarsamine hydrochloride for an exposure of 30 d. SNPs and Short Indels in Local T. evansi Isolates Although these IC values are much smaller than those 50 with Varying Drug Resistance Profiles presented here, the relative magnitudes among the three The ­three strains with varying drug resistance profiles were drugs are somewhat preserved. However, as a screen, coded as O14 (resistant to isometamidium, diminazine, Bulus et al. (2016) working on T. brucei, exposed parasites and cymelarsan); C117 (resistant to diminazene only); for 24 h but found it difficult to estimate an IC for 50 and M4 (sensitive to all three drugs). Search for SNPs diminazene diaceturate, although a visual examination and short indels identified a total of 252,376 variants in suggested a value less than 40 μg/mL. 246,813 genomic loci across the three T. evansi samples Hence, relative to other strains in our experiments, O14 (Table 2). Considering the sheer number of sequence had the maximum value of IC50 for two of the three drugs, variations detected, isolates showing drug resistance and an IC50 for diminazene diaceturate comparable to were found to have a substantially higher number of the highest (Figure 1). Two other strains were selected variations (191,528 for C117 and 183,958 for O14) than for comparison – C117 and M4. They were both weakly the drug-sensitive sample M4 (144,021). Although the virulent in vivo. However, C117 had the highest IC50 for majority of the observed variant loci are shared by the diminazene diaceturate but an IC50 for the other two drugs three isolates (96, 212), M4 harbors the least number of that were near the median, giving it an intermediate profile unique variations as well as the least number of variants between the other two strains. The IC50 values obtained shared with another isolate (Figure 2). For variations that for strain M4 were all at or below the median. affect gene-coding regions, the ratio of missense (non-

Figure 1. Distribution of IC50 of 15 strains tested against isometamidium chloride (IC), diminazene diaceturate (DD), and melarsamine hydrochloride (CY). IC50 values are presented in µg/mL. Strain O14 had the highest IC50 values for IC and CY drugs and the second-highest for DD. Strain M4 had IC50 values that were at or below the median for all drugs. Strain C117 had the highest IC50 for DD and had IC50 values close to the median for IC and CY.

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Table 2. Summary of statistics for SNPs and short indels. Variant type Count Percent Single nucleotide 204,843 81.17 Insertions 24,956 9.89 Deletions 22,577 8.95 Number of effects by impact High 13,982 1.19 Moderate 55,280 4.70 Low 30,742 2.61 Modifier 1,075,642 91.49 Number of effects by functional class Missense 53,002 62.06 Nonsense 1,685 1.97 Silent 30,714 35.96 Missense to silent ratio 1.72 Number of effects by region Downstream 464,667 39.52 Exon 99,619 8.47 Intergenic 152,543 12.97 Intron 33 0.003 Splice site acceptor 132 0.01 Splice site donor 118 0.01 Splice site region 134 0.01 Upstream 458,399 38.99 Total number of SNPs and short indels 252,376 Note: A given variant may be counted multiple times for a particular category (e.g., a variant may have a Modifier and Low impact effect and may occur upstream of a gene and downstream of another). Data is taken from the output of the tool SNPEff. synonymous) to silent (synonymous) mutation counts in each of the three isolates all fall around the value 1.7 (Table 2). The occurrence of these non-neutral mutations may be due to their exposure to various environmental pressures, including host responses and drug treatments. Figure 2. Venn diagram for (A) all observed variants and Based on the mutation effects, 13,272 variants involve (B) high impact variants in the T. evansi isolates. significant changes in amino acid properties, stop-loss or Majority of the observed variant loci (96,212) are -gain, and frameshifts that are predicted to have a high shared by the three isolates. However, the drug- impact on the resulting protein product. The majority susceptible isolate M4 harbors the least number (4,226) of these high impact mutations are shared by the of unique variations (16,370) among all samples. three isolates, whereas 1,125 are unique to M4, 1,995 are Furthermore, the drug-resistant samples O14 and unique to O14, and 2,337 are unique to C117. The resistant C117 were found to share considerably more samples (O14 and C117) share mutations in 1,958 genes. variants (39,820) than either of them does with The notable sequence variants are summarized in Tables M4 (C117xM4 – 15,033; O14xM4 – 13,114). A 3–5 and are subsequently discussed. similar trend was observed for the predicted high Gene ontology analysis of the unique variants per isolate, impact variants. as well as those shared by the resistant samples, revealed their association with several ontology terms. The

224 Special Issue on Genomics Lazaro et al.: Genome-wide Analysis for Philippine T. evansi Variants following are the highest GO terms observed in each of the three strains, which may not be directly related to drug three main domains: “integral component of membrane” resistance, points to a conserved mechanism of survival under the cellular component, “macromolecule metabolic that persists independent of the drug resistance properties process” under biological process, and “nucleic acid of a given strain. binding” under molecular function (Figures I–IV). The other observed enriched ontologies are also related to the VSGs and ESAGs are part of polycistronic units known three aforementioned GO domains. However, it is worth as bloodstream expression sites whose differential noting that while many of the GO terms are shared with expression throughout the parasite’s life cycle results in both drug-resistant and susceptible T. evansi strains, the stochastic switching of antigenic VSGs and, consequently, scale of gene counts for the ontology terms in the drug- evasion of the host immune system (Daniels et al. 2010). susceptible M4 strain is substantially lower than those in Having a higher mutation rate for these genes suggests the other two drug-resistant isolates O14 and C117, raising another layer of variability that will further expand the the possibility that a higher number of the relevant genes antigenic landscape of the parasite. suggested by the GO data are affected by high impact In the genome of T. brucei, only a small proportion of the mutations in resistant isolates. annotated VSGs (~7%) appear to encode the necessary The gene ontology terms that were found to be highest features to be fully functional. Other potential VSGs invariant regions have been previously associated with the are marked as either pseudogenes or gene fragments mechanisms for infectivity, disease progression, and drug (Berriman et al. 2005). This observation suggests that resistance in trypanosomes. These may involve variations most of the VSG gene scan harbor more neutral mutations in surface and transporter proteins, accompanied by than genes involved in essential functions. A previous changes in gene expression and high metabolic demands analysis of the T. evansi genome revealed that, on the (Habila et al. 2012). Trypanosomes have a repertoire of average, purifying selection is significantly much weaker VSGs whose sequential expression results in changes in in VSG than in non-VSG genes with mutations in VSGs the parasite’s antigenic pattern, allowing them to evade the to be mostly neutral and do not have substantial functional host’s immune response (Habila et al. 2012). In a study effects. The observed diversity of VSGs may, therefore, on T. brucei rhodesiense, mutations in genes coding for be a consequence of increased recombination and not due adenosine transporter AT1, aquaporin AQP2, and RNA- to selection pressure brought about by the host immune binding protein UBP1 were associated with resistance response (Carnes et al. 2015). against the drugs melarsoprol and pentamidine (Graf et Additional features found in mutation hotspots are other al. 2016). In T. congolense, copy number variations in high copy number genes – including those that code different transporter and transmembrane products were for RHS proteins, leucine-rich repeat proteins, UDP-N- also observed to accompany resistance against the drug acetylglucosamine (UDP-GlcNAc)-dependent glycosyl IC (Tihon et al. 2017). transferase, and receptor-type adenylate cyclase GRESAG 4. Previous studies have looked at some of these proteins Mutation Hotspots in Subtelomeric Regions Rich as possible drug targets to control trypanosomes. For with VSGs instance, synthesis of UDP-GlcNAc was found to be The mutation rates for the three variants were analyzed essential for the growth of the bloodstream form of T. by mapping the occurrence of variance across the brucei (Stokes et al. 2008) and the therapeutic potential different chromosome positions (Figure 3). For all of of an allosteric inhibitor to a step in the UDP-GlcNAc the three isolates, the majority of the mutations occurred biosynthesis has also been described (Urbaniak et al. near the chromosome ends and across the entire length 2013). More recently, RHS proteins were found to be of chromosome 11_02 and 11_03. Second, these involved in RNA polymerase II transcription in T. brucei locations coincide with the distribution of VSG gene and the depletion of these factors impair RNA synthesis loci. Interestingly, chromosomes 11_02 and 11_03 are (Florini et al. 2018). Similar to VSGs, these genes have comprised of genes annotated as VSGs and ESAGs. relatively high copy numbers and may allow for more These genes are known to be mostly concentrated in the neutral mutations without sacrificing function. subtelomeric regions of chromosomes (Carnes et al. 2015) – coinciding with the observed mutation hotspots. These Notable Sequence Variants results corroborate previous observations on the high Considering that isolates O14 and C117 are both highly rate of expansion and mutation of VSGs in the T. evansi resistant to diminazene, mutations shared only by these genome (Carnes et al. 2015). This supports experimental two isolates may provide insights on the development of findings on the hypervariability of the VSG, which has resistance towards the said drug. While diminazene exhibits been related to the parasite’s defense against immunogenic its direct trypanocidal activity by preferentially interacting attack. The observation of VSG hypervariability in all

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Figure 3. Mutation hotspots per chromosome. The x-axis represents base position within the chromosome, binned every 10 kilobases. The y-axis shows variant counts which were plotted per bin. Counts are highlighted with line markers colored yellow for the drug- susceptible sample M4, blue for the diminazene-resistant sample C117, and red for the highly-resistant sample 014. Majority of the observed variation hotspots were found to coincide with the presence of VSGs and ESAGs, which are known to be mostly concentrated in the subtelomeric regions of the chromosomes and have relatively high expansion and mutation rates.

226 Special Issue on Genomics Lazaro et al.: Genome-wide Analysis for Philippine T. evansi Variants with A-T regions in the kinetoplast DNA and affecting host RNA degradation. These include a C to G transversion immune responses (Kuriakose et al. 2012), mutations for resulting in a Pro116Ala change in the NOT1 gene the resistant isolates show the potential involvement of (TevSTIB805.10.1620) at chromosome 10 and a T genes involved in transport and RNA degradation. to C transition leading to a Val76Ala mutation in the Ribonuclease H gene (TevSTIB805.7.5350) at In both the diminazene-resistant isolates O14 and C117, chromosome 7 (Table 3). To the best of our knowledge, a number of mutations were found in an adenosine there are no previous reports implicating the NOT1 and transporter 2 gene (TevSTIB805.2.3490) at chromosome Ribonuclease H genes in drug resistance. We hypothesize 2 and an ATP-binding cassette transporter gene (multidrug that their roles in gene expression and transcriptional resistance protein E; p-glycoprotein; TevSTBI805.4.4670) at regulation allow the parasite to escape the effect of chromosome 4 (Table 3). In particular, an A to G transition diminazene binding to kinetoplast DNA. A possible at position 220 of the adenosine transporter gene results in a mechanism may involve the high binding affinity Met74Val mutation, whereas an A to G transition at position of diminazene to RNA-based G-quadruplexes. This 464 of the p-glycoprotein results in an Asp155Gly change. involves higher binding affinity (103 stronger) compared A nucleoporin gene (TevSTIB805.4.3000) at chromosome 4 to its AT-rich duplex DNA target (Zhou et al. 2014). was also found to harbor a T to C transition at position 3736, Decreased RNA degradation would, therefore, promote which translates to a Tyr1246His amino acid substitution. the sequestration of cytoplasmic diminazene and hinder None of these three point mutations were observed in the the accumulation of irreversible damages towards the diminazene-susceptible isolate (M4). The location of these kinetoplast DNA leading to parasite death. mutations coincides with recent findings on the central role of membrane transporters for trypanosome drug The highly resistant sample O14 is the only isolate resistant sensitivity (Zoltner et al. 2016). A previous study in T. cruzi to the drugs cymelarsan and isometamidium. The unique demonstrated the importance of the p-glycoprotein efflux mutations observed in this isolate suggest the involvement pump in drug resistance development (Campos et al. 2013). of several genes in resistance generation. We found an A to Although the actual effects of the Met74Val, Asp155Gly, G transition at position 1469 (Asn490Ser) of the pteridine and Tyr1246His mutations are yet to be determined, their transporter gene (TevSTIB805.1.2770) in chromosome presence in only the resistant samples O14 and C117 may 1 (Table 3). Interestingly, a pteridine transporter gene facilitate the development of markers for diminazene was shown to contribute to methotrexate resistance in the resistance. trypanosomatid Leishmania tarentolae (Kündig et al. 1999). Another set of mutations shared only by diminazene- We also found a putative cation transporter gene resistant isolates are found in genes that are involved in (TevSTIB805.11_01.9250) at chromosome 11_01 that

Table 3. Notable mutations found only in drug-resistant isolates. Gene Nucleotide Amino Acid Chr ChrPos Gene Name Gene ID Pos Change Change Shared by diminazene-resistant samples (C117 and O14) 2 1105712 Adenosine Transporter 2 TevSTIB805.2.3490 220 A -> G Met74Val 4 1202184 P-glycoprotein TevSTBI805.4.4670 464 A ->G Asp155Gly 4 758913 Nucleoporin TevSTIB805.4.3000 3736 T -> C Tyr1246His 10 396672 NOT1 TevSTIB805.10.1620 346 C ->G Pro116Ala 7 1295816 Ribonuclease H TevSTIB805.7.5350 227 T ->C Val76Ala Only in the highly resistant sample (O14) 1 639160 Pteridine transporter TevSTIB805.1.2770 1469 A ->G Asn490Ser 11_01 2414063 Putative cation transporter TevSTIB805.11_01.9250 689 G -> A Arg230Lys 11_01 2414074 Putative cation transporter TevSTIB805.11_01.9250 700 T -> G Ser234Ala 5 438649 64-kDa invariant surface TevSTIB805.5.1560 663 delA Frameshift glycoprotein 11_01 305307 Calpain-like cysteine TevSTIB805.11_01.1120 5264 T -> C Val1755Ala peptidase 6 393111 Metacaspase 3 TevSTIB805.6.1000 161 A ->G Lys54Arg 6 393114 Metacaspase 3 TevSTIB805.6.1000 158 C ->G Pro53Arg

227 Special Issue on Genomics Lazaro et al.: Genome-wide Analysis for Philippine T. evansi Variants was observed to harbor two mutations, a G to A transition effects difficult. The results of these are shown in Table at position 689 (Arg230Lys), and a T to G transversion III. It must be noted, however, that this search protocol did at position 700 (Ser234Ala) (Table 3). While direct links yield a match of high query coverage (97%) and similarity between cation transporters and drug resistance have (87.3%) for the mutated metacaspase protein. Mutations been suggested, we hypothesize that these transporters P53R and K54R were observed to only occur in the influence drug uptake through the modulation of metal co- highly resistant sample O14. Both of these mutations are factor concentration. Enzymes, such as those involved in predicted to occur near the catalytic triad of the enzyme, drug interactions, often require metal co-factors (Wittung- possibly increasing efficiency for processing metacaspase Stafshede 2002), making the activity of cation transporters 4 – an enzyme related to parasite virulence – and survival crucial in the development of resistance. in the bloodstream (Proto et al. 2011). We further found a single nucleotide deletion at position A second protocol for assigning reference proteins for 663 of a 64-kDa invariant surface glycoprotein (ISG) gene structural comparisons was also utilized. This protocol (TevSTIB805.5.1580) in chromosome 5 (Table 3). ISGs, used a direct search of the PDB for structures that were particularly ISG75, were implicated in the cellular uptake similar or related to the expected products of the mutated of the trypanocidal drug suramin (Zoltner et al. 2016). genes. The names of the mutated genes were used as Apart from membrane transporters, unique mutations were keywords in the search for related structures, and the also found in two cysteine peptidase genes of isolate O14. top hits in PDB were evaluated for their similarity. For These were for metacaspase MCA3 (TevSTIB805.6.1000) example, a keyword search for Metacaspase 2 reveals in chromosome 6 and a calpain-like cysteine peptidase the presence of an archived structure for Metacaspase (TevSTIB805.11_01.1120) in chromosome 11_01 (Table 3 that may be evaluated. The locations of the observed 3). Point mutations in two consecutive codons were found mutations were mapped to the domains of the matched in the metacaspase gene (including an A to G transition) proteins to predict their possible effects. In the absence resulting in a Lys54Arg change, and a C to G transversion of available reference structures in the PDB, predicted leading to a Pro53Arg amino acid substitution. For protein structures in UniProt were used as references. The the calpain-like cysteine peptidase, a T to C transition results of these are shown in Table 4. was found at position 5264, resulting in a Val1755Ala mutation. A number of studies have demonstrated the The predicted effects of the SNPs related altered transport importance of cysteine peptidases in bloodstream forms of molecules through the plasma membrane and the nuclear of T. brucei (Helms 2006, Troeberg et al. 1999). These membrane to the observed drug resistance in the C117 and have also been found to be crucial for the replication of O14 samples. Interestingly, the highly resistant sample the kinetoplast (Grewal et al. 2016), differentiation and (O14) was observed to have additional modifications in virulence of the bloodstream form (Santos et al. 2007), the processing of enzymes (i.e., Metacaspase 4) essential and even crossing the blood-brain barrier (Nikolskaia et al. for parasite survival in the bloodstream. It must be noted 2008). Mutations in cysteine peptidases that are possibly that while some matched proteins (e.g., from Trypanosoma associated with resistance recommend caution about this brucei) had a sequence identity of 86%, the average class of peptidase as possible therapeutic targets unless sequence identities observed for the matches made by combined with other drugs with different modes of action this search was 28.1%. This highlights the need for more (Branquinha et al. 2013, Roy et al. 2010). structural studies in the identified variant proteins for more accurate analyses of their functional significance. To further investigate the functional significance of the discussed variants, two search protocols were employed. The first used the BLAST algorithm of NCBI to find the best matches to the mutated sequences. The BLAST CONCLUSIONS algorithm predicts similarities between query sequences This study utilized whole-genome sequencing to compare and entries within the database on the basis of sequence three T. evansi isolates from the Philippines with varying identity. This provides an unbiased method of locating levels of drug resistance. Through WGS, regions that potential reference proteins without restricting the search can differentiate strains as related to their drug resistance to proteins which are “known” to be related to the query. have been identified and highlight the value of a genomic The average sequence identity for the matches with this approach to design regions that could target specific protocol was 45.9% – with average query coverage of strains. From the analysis of genomic variants, several 32.4%. The query coverage spanned values from 2% to were found to be unique to either single-drug-resistant 97% and, unfortunately, mutations from the sequenced or multidrug-resistant strains. Affected genes in the samples were mostly found in areas that were outside the drug-resistant strains linked genes for transport (e.g., for aligned sequences, making predictions on their functional adenosine, pteridine, glycoproteins, etc.); proteases; and

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Table 4. Protein domain locations of notable mutations in drug-resistant isolates. Reference proteins are based on Top PDB result. In the absence of available reference structures in the PDB, predicted structures in UniProt were used to infer the structural significance of the observed SNPs. UniProt entries were chosen based on the first matching structures that contained annotations of the documented SNP positions. *Predictions were based on unreviewed predicted structures. Amino acid Location Predicted effect Reference structure Chr. Chr. Pos. Gene name change (PDBID)

Shared by diminazene-resistant samples (C117 and O14) 2 1105712 Adenosine Met74Val Scaffold domain; IH1 helix Stabilization of Concentrative Transporter 2 scaffold domain Nucleotide Transporter Maps to 5L26 (Gly80) association (5L26) with the plasma 19.2% identity membrane 4 1202184 P-glycoprotein Asp155Gly Maps to a region before the *May modulate P-glycoprotein (membrane- N-terminus of the structure in the function of (4Q9H) embedded PDB (4Q9H). this membrane- 24.0% identity transporter) embedded Uniprot Predicted Structure: transporter C9ZMP2-TRYB9 (Unreviewed) Predicts the mutation to occur in a transmembrane helix (AA 138–160) 4 758913 Nucleoporin Tyr1246His Nucleoporin tail region Stabilization of Nucleoporin Nic96 Maps to 2RFO (Ser 780) the Nuclear Pore (2RFO) Complex (NPC) 21.2% identity 10 396672 NOT1 Pro116Ala Maps to a region before the *May modulate N-terminal domain of N-terminus of the structure in the poly-A yeast Not1 PDB (4B8B). specific (4B8B) Uniprot Predicted Structure: ribonuclease 19.2% identity A8DY82-DROME activity of this (Unreviewed) enzyme Predicts the mutation to occur in a disordered region of the protein (AA 70–123). AA 116 occurs in a polyampholyte region (AA 108–123) that succeeds a polar region of this structure (AA 70–107). 7 1295816 Ribonuclease H Val76Ala Maps to a region just before *May modulate PDB Match: the N-terminus of the the ribonuclease E. coli Ribonuclease H structure in PDB (2RN2) activity of this (2RN2) enzyme. A 31.0% identity Uniprot Structure: K69A mutation Related protein, Ribonuclease near the affected Uniprot Match: H2 region has been Ribonuclease H2 O75792-RNH2A-HUMAN documented to PDBID:3P56 (Reviewed) strongly reduce 19.2% identity Predicts the mutation to occur enzyme activity in the 4th helix (AA 73–84). (Figiel et al. 2011) Only in the highly resistant sample (O14)

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Table 4 continuation . . . .

1 639160 Pteridine Asn490Ser No Structure Available in the *May modulate No structure available transporter PDB. pteridine transporter Uniprot Predicted function Structure: Q38A66-TRYB2 (Unreviewed) Predicts the mutation to occur in a transmembrane helix (AA 473-494) 11_01 2414063 Putative cation Arg230Lys Central Pore Modulation of CorA Mg+2 Transporter transporter Maps to 2HN2 (R1232) Cation transport (2HN2) 18.2% identity 11_01 2414074 Putative cation Ser234Ala Central Pore Modulation of CorA Mg+2 Transporter transporter Maps to 2HN2 (S1236) Cation transport (2HN2) 18.2% identity 5 438649 64-kDa Frameshift: C-terminal helix Truncation of Trypanosoma brucei invariant surface Truncation Maps to 5VTL (A205) protein product. metacyclic invariant glycoprotein (434aa  224aa) surface protein (5VTL) 24.4% identity 11_01 305307 Calpain- Val1755Ala Outside available structures May modulate PDB match like cysteine in PDB the protease Homo sapiens peptidase activity of this m-Calpain form II Uniprot Structure: enzyme (1KFU) Related protein, Calpain-type 21.4% Identity Cysteine Protease, DEK1 to L subunit; Q8RVL2-DEK1-ARATH 21.7% identity (Reviewed) to S subunit Predicts the mutation to occur in the cytoplasmic domain (AA 1072-2151). The Val1755Ala mutation is located near a predicted active site (AA 1761) 6 393114 Metacaspase 3 Pro53Arg N-terminal region; external; Processing of Trypanosoma brucei near catalytic dyad Metacaspase Metacaspase 2 4; required (4AFV) Maps to 4AFV (R30) for parasite 85.9% identity survival in the bloodstream 6 393111 Metacaspase 3 Lys54Arg N-terminal region; external; Processing of Trypanosoma brucei near catalytic dyad Metacaspase Metacaspase 2 4; required (4AFV) Maps to 4AFV (R31) for parasite 85.9% identity T. brucei metacaspase 2 survival in the structure bloodstream

230 Special Issue on Genomics Lazaro et al.: Genome-wide Analysis for Philippine T. evansi Variants surface glycoproteins with trypanocide resistance. In SANDERS M, SCHOBEL S, SHARP S, SIMMONDS addition, novel mutations observed in the study suggest M, SIMPSON AJ, TALLON L, TURNER CM, TAIT A, the possible involvement of new genes (e.g., for RNA TIVEY AR, VAN AKEN S, WALKER D, WANLESS degradation, cation transport) with drug resistance. A D, WANG S, WHITE B, WHITE O, WHITEHEAD comparison of the single-drug and multi-drug resistant S, WOODWARD J, WORTMAN J, ADAMS MD, strains suggests an additive effect of modulations in EMBLEY TM, GULL K, ULLU E, BARRY JD, cation transport and metacaspase processing for increased FAIRLAMB AH, OPPERDOES F, BARRELL BG, drug resistance. Further investigation of the mechanisms DONELSON JE, HALL N, FRASER CM, MELVILLE through which these genes confer drug resistance will SE, EL-SAYED NM. 2005. The Genome of the African reveal potential targets for disruption, for which novel Trypanosome Trypanosoma brucei. Supplementary therapeutics may be designed. Information. Science 309(5733): 416. BIRHANU H, GEBREHIWOT T, GODDEERIS BM, BÜSCHER P, VAN REET N. 2016. New Trypanosoma evansi Type B Isolates from Ethiopian ACKNOWLEDGMENTS Dromedary Camels. PLoS Neglected Tropical This project was funded by a research grant from the PCC. Diseases10(4):e0004556. We thank the Philippine Genome Center DNA Sequencing BRANQUINHA MH, MARINHO FA, SANGENITO and Bioinformatics Core Facilities for analyzing our LS, OLIVEIRA SS, GONCALVES KC, ENNES- samples. We also acknowledge the help of Ms. Shella VIDAL V, D'AVILA-LEVY CM, SANTOS AL. Badong in facilitating this research project. 2013. Calpains: Potential Targets for Alternative Chemotherapeutic Intervention Against Human Pathogenic Trypanosomatids. Current Medicinal Chemistry 20 (25): 3174–3185. REFERENCES BUCHFINK B, XIE C, HUSON DH. 2015. Fast and ALTSCHUL SF, GISH W, MILLER W, MYERS EW, Sensitive Protein Alignment Using DIAMOND. Nature LIPMAN DJ. 1990. Basic local alignment search tool. Methods 12: 59–60. Journal of Molecular Biology 215(3): 403–410. BULUS T, AHMED AB, ABOI TY, DANBAKI DA. 2016. BALTZ T, BALTZ D, GIROUD C, CROCKETT J. 1985. Determination of IC50 and IC90 values of ethanolic Cultivation in a semi-defined medium of animal extracts of some medicinal plants against Trypanosoma infective forms of Trypanosoma brucei, T. equiperdum, brucei brucei. Archives of Clinical Microbiology 7: 3. T. evansi, T. rhodesiense, and T. gambiense. The EMBO Journal 4(5): 1273–1277. CAMPOS MC, CASTRO-PINTO DB, RIBEIRO GA, BERREDO-PINHO MM, GOMES LH, DA SILVA BERRIMAN M, GHEDIN E, HERTZ-FOWLER C, BELLIENY MS, GOULART CM, ECHEVARRIA A, BLANDIN G, RENAULD H, BARTHOLOMEU LEON LL. 2013. P-Glycoprotein Efflux Pump Plays an DC, LENNARD NJ, CALER E, HAMLIN NE, Important Role in Trypanosoma cruzi Drug Resistance. HAAS B, BÖHME U, HANNICK L, ASLETT Parasitology Research 112(6): 2341–2351. MA, SHALLOM J, MARCELLO L, HOU L, WICKSTEAD B, ALSMARK UC, ARROWSMITH CARNES J, ANUPAMA A, BALMER O, JACKSON A, C, ATKIN RJ, BARRON AJ, BRINGAUD F, LEWIS M, BROWN R, CESTARI I, DESQUESNES BROOKS K, CARRINGTON M, CHEREVACH I, M, GENDRIN C, HERTZ-FOWLER C, IMAMURA CHILLINGWORTH TJ, CHURCHER C, CLARK LN, H, IVENS A, KOŘENÝ L, LAI DH, MACLEOD A, CORTON CH, CRONIN A, DAVIES RM, DOGGETT MCDERMOTT SM, MERRITT C, MONNERAT J, DJIKENG A, FELDBLYUM T, FIELD MC, S, MOON W, MYLER P, PHAN I, RAMASAMY FRASER A, GOODHEAD I, HANCE Z, HARPER G, SIVAM D, LUN ZR, LUKEŠ J, STUART K, D, HARRIS BR, HAUSER H, HOSTETLER J, SCHNAUFER A. 2015.Genome and Phylogenetic IVENS A, JAGELS K, JOHNSON D, JOHNSON Analyses of Trypanosoma Evansi Reveal Extensive J, JONES K, KERHORNOU AX, KOO H, LARKE Similarity to T. brucei and Multiple Independent N, LANDFEAR S, LARKIN C, LEECH V, LINE A, Origins for Dyskinetoplasty. PLoS Neglected Tropical LORD A, MACLEOD A, MOONEY PJ, MOULE Diseases 9(1): e3404. S, MARTIN DM, MORGAN GW, MUNGALL K, CINGOLANI P, PLATTS A, WANG LE L, COON M, NORBERTCZAK H, ORMOND D, PAI G, PEACOCK NGUYEN T, WANG L, LAND SJ, LU X, RUDEN CS, PETERSON J, QUAIL MA, RABBINOWITSCH DM. 2012. A Program for Annotating and Predicting the E, RAJANDREAM MA, REITTER C, SALZBERG SL,

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APPENDIX

Table I. Quality assessment metrics. Initial assessment Final assessment Sample Pair # Final paired Total # of seqs Length %GC Total # of seqs Length %GC O14 1 4,783,175 35–301 44 4,392,053 50–301 44 3,669,087 O14 2 4,783,175 35–301 45 3,812,875 50–301 44 3,669,087 C117 1 6,685,345 35–301 45 6,148,269 50–301 44 5,501,386 C117 2 6,685,345 35–301 45 5,660,139 50–301 44 5,501,386 M4 1 4,356,245 35–301 45 3,986,201 50–301 45 3,130,302 M4 2 4,356,245 35–301 45 3,261,811 50–301 44 3,130,302

Table II. Mapping metrics. Sample # Mapped reads # Mapped bases Ave. mapping depth % Coverage breadth (> 5x) O14 3,103,615 1,089,734,939 42.85 96.71 C117 4,362,439 1,283,185,682 50.46 96.56 M4 2,685,760 982,807,110 38.64 94.24

Figure I. Gene ontology results for genes affected by Figure II. Gene ontology results for genes affected high impact mutations in the highly resistant by high impact mutations in the diminazene- isolate O14. resistant isolate C117.

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Figure III. Gene ontology results for genes affected by high impact mutations in the drug-susceptible isolate M4.

Figure IV. Gene ontology results for genes affected by high impact mutations and shared only by isolates O14 and C117.

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