A cell surface transporter mediates phenanthridine resistance in African trypanosomes

A cell surface transporter mediates phenanthridine resistance in African trypanosomes | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article A cell surface transporter mediates phenanthridine resistance in African trypanosomes Pieter C. Steketee, Marzuq A. Ungogo, Edith Paxton, Ella Maria Rogerson, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8903249/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 10 You are reading this latest preprint version Abstract African animal trypanosomosis poses a significant threat to livestock health and agricultural productivity across sub-Saharan Africa. Isometamidium chloride is the only available drug that is both prophylactic and curative. Despite sustained reports of resistance since the 1970s, a definitive molecular mechanism of resistance remains unresolved in the clinically relevant pathogen species Trypanosoma congolense . In this study, the role of a putative drug/metabolite transporter protein, TcoDMT, was validated via the analysis of in vitro -derived mutants, showing that expression levels of this protein correlated strongly with isometamidium sensitivity. Functional analyses revealed that the protein is a cell surface phenanthridine transporter and, notably, copy number variation correlates with Isometamidium sensitivity in T. congolense field isolates. This study validates, for the first time, a plasma membrane transporter with a defined role in phenanthridine action and resistance, advancing our understanding of drug resistance mechanisms in parasitic protists, and informing strategies to combat animal trypanosomosis. Health sciences/Diseases Biological sciences/Microbiology Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction African trypanosomes are extracellular protozoan parasites that cause both human and livestock disease. Human African Trypanosomiasis (HAT) is prevalent across sub-Saharan Africa, with case numbers decreasing to < 1,000 annually in recent years, the disease is currently targeted for elimination of transmission by the WHO 1 . In stark contrast, African Animal Trypanosomosis (AAT) remains a major hindrance to the development of sustainable agriculture in the 37 sub-Saharan African countries where the disease is endemic 2 . Although affecting multiple livestock species, the burden of AAT primarily falls on cattle, and is estimated to result in more than three million cattle deaths annually, with ~ 90 million at risk, leading to yearly losses of ~ $ 4.5 billion 3 . The three main species of clinical relevance for AAT are Trypanosoma congolense , T. vivax and to a lesser extent, T. brucei . The three species exhibit significant divergence in their biology, including at the genome 4 , transcriptome 5 , proteome 6 and metabolome 7 level, as well as in infection phenotypes 8 . However, disease symptoms are largely indistinguishable, and mixed infection is common 9 . There are no vaccines to prevent AAT, although a vaccine target was recently identified for T. vivax 10 . Tsetse control is hindered by lack of sustainable infrastructure, leaving chemotherapy as the dominant form of AAT control. Three trypanocides are used in sub-Saharan Africa: the diamidine diminazene aceturate (DA) and the phenanthridines isometamidium chloride (ISM) and homidium bromide (ethidium bromide; EtB). ISM and DA are in widespread use, and although EtB is no longer recommended, it is still sold and used in some areas 11 . ISM is currently the only drug used for both prophylaxis and curative treatment of AAT; prophylaxis being enabled by the long half-life of ISM in the host 9 . ISM is derived from the coupling of EtB with amidinobenzenediazonium chloride, the latter derived from DA, and is marketed as a mixture of four compounds 12 , all of which are bioactive - likely through a shared mechanism 13 . ISM mode of action studies have predominantly focused on the lab-adapted T. brucei where it has been hypothesised that ISM interacts with the kinetoplast DNA (kDNA; trypanosome mitochondrial DNA), with strong affinity for double stranded DNA in particular 14 , 15 . This interaction results in linearization of the kDNA minicircles, with inhibition of a kinetoplast-specific topoisomerase II enzyme proposed as a possible mechanism of action 16 . Unlike ISM, its parental phenanthridine compound (EtB) distributes throughout the trypanosome cell 14 , although it likewise kills trypanosomes by specific interactions with kDNA, in addition to the inhibition of nuclear DNA replication 17 . The mechanisms governing ISM transport into the cytoplasm and mitochondrion remain unresolved. It is hypothesised that transport into the cytoplasm occurs via facilitated diffusion owing to its positive charge 18 , and reduced cytosolic ISM accumulation, as well as increased efflux have been posited as mechanisms of resistance in T. brucei 19 . A possible role in ISM uptake has been proposed for the aminopurine TbAT1/P2 transporter, and although ISM-resistant field isolates of T. brucei exhibited mutations in the TbAT1 gene 20 , in vitro uptake studies suggest that the role of TbAT1/P2 is minor 21 . ISM entry in the mitochondrion is thought to rely on the mitochondrial membrane potential (ΔΨ m ), established by the trypanosome F 0 F 1 -ATPase 18 , 19 , which functions in reverse to the mammalian ATPase 22 . Agents that depolarise ΔΨ m have been shown to reduce ISM accumulation into the mitochondrion 18 , 19 . Furthermore, loss of the kinetoplast results in ISM resistance in T. brucei 19 . However, ISM remains active against animal trypanosome species lacking a kinetoplast (dyskinetoplastic), albeit with a significantly increased dose, suggesting the presence of other cellular targets 23 . A genome-scale RNAi study in T. brucei demonstrated that disruption of vacuolar ATPase (vATPase) was also capable of generating ISM resistance, reflecting the connection between vATPase and mitochondrial F 0 F 1 -ATPase 24 . There is limited data on ISM modes of action and resistance in T. congolense , but it can be predicted that mechanisms are likely to differ from T. brucei based on metabolic divergence. An increased reliance upon diverse mitochondrial activity (for example, substrate-level phosphorylation and an expanded electron transport chain) likely explains why dyskinetoplasty has not been reported in T. congolense . However, similar to T. brucei , ΔΨ m correlates closely with ISM sensitivity in T. congolense genome-reference strain IL3000, which exhibits both a reduced ΔΨ m and increased ISM resistance, compared with other strains 18 . T. congolense lacks a TbAT1 orthologue 25 and to date no ISM transporter has been experimentally validated for either cytoplasmic or mitochondrial uptake. Earlier studies using field isolates showed that ISM resistance correlated with mutations in a number of T. congolense genes, including the topoisomerase II enzyme and an ATP-binding cassette (ABC) transporter 26 . However, correlation with resistance was inconsistent, and a definitive mechanism of ISM resistance has remained elusive 27 , 28 . A recent study of in vivo generated ISM-resistant T. congolense revealed putative determinants via copy number variation (CNV), including ABC transporters, topoisomerase II and a drug/metabolite transporter (TcoDMT) 29 . Expression analysis demonstrated that TcoDMT was downregulated in resistant cells, consistent with a potential role in ISM resistance 29 . However, in the absence of functional analyses, the extent to which any of these genetic determinants contribute to ISM resistance has remained unknown. In this study, the role of TcoDMT in ISM-resistance in T. congolense and T. brucei was functionally evaluated. Results TcoDMT expression is directly correlated to ISM sensitivity The recent development of a genetic toolkit for T. congolense has enabled manipulation of genes and their functional characterisation, including potential roles in drug mode of action or resistance 30 , 31 , 32 . These advances were exploited to genetically manipulate TcoDMT (gene ID: TcIL3000.A.H_000575200), a single-copy gene in the genome of the reference strain IL3000, which, although exhibiting notable resistance to ISM compared to other strains used in vivo , is the only strain currently amenable to culture. Over-expressor (TcoDMT OE ) and double knock-out (TcoDMT dKO ) cell lines were generated, the latter achieved via sequential allelic replacement to also produce a TcoDMT single allele knock-out (hemizygous) intermediate (TcoDMT sKO ). TcoDMT expression was elevated in TcoDMT OE cells (5.27 ± 1.09-fold vs control) and completely ablated in TcoDMT dKO cells (Fig. 1 A), whilst qPCR analysis of genomic DNA confirmed expected gene copy number in the four cell lines tested (Fig. 1 B). TcoDMT overexpression did not affect cell growth (doubling time, DT: 12.5 hr vs 12.3 hr in WT cells; Fig. 1 C), but a defect resulting in significantly increased doubling time (DT: 15.6 hr vs 12.3 hr in WT cells; P = 1.20 × 10 − 5 , t -test) was observed in TcoDMT dKO (Fig. 1 C), independent of the presence of selective antibiotics (Fig S1 A). Therefore, ablation of TcoDMT expression in T. congolense leads to a fitness cost in vitro . Drug sensitivity analyses revealed a 27-fold reduction in ISM EC 50 in TcoDMT OE ( P = 1.25 × 10 − 8 , t -test; Table 1 ; Fig. 1 D). In contrast, a 3.14-fold increase in EC 50 was observed in TcoDMT dKO compared to WT cells ( P = 4.12 × 10 − 9 ; Table 1 ; Fig. 1 D), indicating increased ISM resistance in dKO mutants. Thus, in vitro , TcoDMT expression levels correlate linearly with ISM sensitivity in T. congolense (r 2 = 0.99; Fig. 1 E). Minor, but not significant, changes in ISM resistance were observed in TcoDMT sKO , and this line was therefore not included in downstream analyses (Fig S1 B). Knock-down of TcoDMT was also attempted using a T. congolense IL3000 single-marker cell line (TcoSM) that constitutively expresses T7 RNA polymerase and Tet Repressor 30 . However, RNAi penetrance is low in T. congolense 7 , 30 , and only 20–40% reduction in TcoDMT mRNA levels was observed, which did not affect ISM sensitivity (Fig S2 ). The RNAi and TcoDMT sKO data jointly suggest that near complete ablation of TcoDMT expression was required for increased ISM resistance in the in vitro T. congolense strain IL3000. Table 1 DMT mutant EC 50 s for ISM, DA and EtB. A summary of drug sensitivity data for the T. congolense and T. brucei DMT mutants described in this study. Fold changes and P-values are in relation to WT parental cells, or in the case of T. brucei RNAi and Cas9, in relation to uninduced controls. Abbreviations: Ctrl: control; FC: fold change; P-val: P-value Species Cell line ISM DA EtB EC 50 (nM) FC vs ctrl P-val vs ctrl EC 50 (nM) FC vs ctrl P-val vs ctrl EC 50 (nM) FC vs ctrl P-val vs ctrl T. congolense WT 6.88 ± 0.39 - - 216.4 ± 14.5 - - 6.7 ± 0.5 - - T. congolense TcoDMT OE 0.25 ± 0.08 0.04 1.25 × 10 − 8 212.4 ± 18.5 0.98 > 0.05 0.3 ± 0.1 0.04 2.07 × 10 − 7 T. congolense TcoDMT dKO 21.59 ± 1.14 3.14 4.12 × 10 − 9 208.1 ± 4.1 0.96 > 0.05 26.9 ± 0.8 4.01 2.25 × 10 − 11 T. brucei WT 141.78 ± 26.69 - - 80.77 ± 8.56 - - 1,343.3 ± 43.8 - - T. brucei TbDMT OE 9.21 ± 2.6 0.10 2.57× 10 − 4 79.42 ± 16.39 0.85 > 0.05 760.7 ± 23.0 0.57 2.27 × 10 − 5 T. brucei Tb-TcoDMT OE 9.20 ± 0.21 0.06 0.02 79.40 ± 16.40 1.02 > 0.05 Not tested - - T. brucei TbDMT RNAi (uninduced) 289.77 ± 5.71 - - 68.50 ± 1.89 - 859 ± 23.9 - - T. brucei TbDMT RNAi (induced) 337.25 ± 18.06 1.16 0.03 54.27 ± 2.18 0.79 0.001 761.6 ± 15.7 0.89 0.007 T. brucei TbDMT Cas-9 dKO (uninduced) 573.10 ± 66.32 - 149.55 ± 13.21 - Not tested - - T. brucei TbDMT Cas-9 dKO (induced) 650.04 ± 43.99 1.13 > 0.05 167.48 ± 21.78 1.12 > 0.05 Not tested - - Given that ISM consists of an EtB base coupled to the m -amidinobenzenediazonium chloride moiety derived from DA 33 , sensitivities for all T. congolense cell lines to EtB and DA were determined in parallel (Fig. 1 D). Whilst sensitivity or resistance to EtB was altered to approximately the same extent in the TcoDMT mutants, when compared to changes in ISM sensitivity ( P = 2.01 × 10 − 7 and 0.03 for WT vs TcoDMT OE and WT vs DMT dKO , respectively; Table 1 ; Fig. 1 D), DA sensitivity DA remained unchanged, irrespective of changes in TcoDMT expression (Table 1 ; Fig. 1 D). Thus, the association of TcoDMT with ISM sensitivity is due to interactions with the EtB moiety of the ISM molecule, and TcoDMT does not confer sensitivity/resistance to DA. TcoDMT is a cell surface protein To assess the cellular location of TcoDMT, the gene was endogenously-locus tagged at its N-terminus using codon-optimised superfolder GFP (sfGFP) 34 , and the resulting cell line analysed by fluorescence microscopy. The sfGFP signal was relatively weak, albeit with detectable accumulation at the cell surface (Fig. 2 A, S3 A). Analysis of pixel intensity supported the hypothesis that TcoDMT is a cell surface protein (Fig S3 B). To increase TcoDMT expression levels for improved visualisation, an N-terminally tagged ectopic copy was inserted into the tubulin locus (analogous to the TcoDMT OE line). The TcoDMT OE−sfGP line exhibited clear accumulation of sfGFP at the cell surface (Fig. 2 A, S3 A), thus confirming that TcoDMT is a plasma membrane protein. Previous studies have reported a correlation between mitochondrial membrane potential (ΔΨ m ) and ISM sensitivity in T. brucei 19 , and a putative link in T. congolense 18 . Whilst a cell surface transporter is unlikely to impact upon ΔΨ m , we nevertheless assessed ΔΨ m in the TcoDMT mutants (Fig. 2 B). Untreated TcoDMT OE exhibited a slight, but not significant ( P > 0.05) reduction in ΔΨ m (0.74 ± 0.1 arbitrary units [AU], compared with 1.00 ± 0.1 AU in untreated WT cells; Fig. 2 B). While ISM treatment of T. brucei results in a rapid ΔΨ m reduction 19 , no effect was observed after a two-hour ISM treatment of T. congolense WT or mutant cells (Fig. 2 B). Notably, T. congolense IL3000 exhibits a lower ΔΨ m compared to ISM-sensitive T. congolense strains 18 . Nevertheless, treating all cell lines with valinomycin, a mitochondrial membrane depolariser, resulted in a > 50% reduction in ΔΨ m (Fig. 2 B); troglitazone, known to hyperpolarise ΔΨ m in T. brucei 35 , had no effect. These data suggest that TcoDMT plays no active role in ΔΨ m maintenance. TcoDMT is a putative EamA-like transporter encoding 10 transmembrane domains and also harbouring an SLC35F, family 5 (SLC35F5) domain (Fig. 2 C). The human orthologue of SLC35F5 is an orphan transporter implicated in anti-cancer chemotherapeutics resistance 36 , 37 . The predicted T. brucei orthologue, TbDMT (Gene ID: Tb927.8.1460), was previously predicted as a nucleotide sugar transporter (TbNST)-7, where TbNST1-4 were experimentally characterised as functional nucleotide sugar transporters, while TbNST7 was not functionally chracterised 38 . Phylogenetic analysis confirmed that TcoDMT and TbDMT are closely related to the F5 subgroup of the SLC35 superfamily, and are syntenic orthologues (Fig S4 A). A DMT orthologue was also identified in T. vivax (Gene ID: TvY486_0800880) and Leishmania major (gene ID: LmjF.07.0400; Fig S4 A). However, no orthologues were identified in T. cruzi , nor in the intracellular apicomplexans Toxoplasma gondii and Plasmodium falciparum . A ColabFold 39 TcoDMT model was shown to be most similar to predicted models of SLC35F5 proteins from T. brucei , L. major , animals and yeast (Fig S4 B-D, Table S1 ). An untargeted metabolomics approach was carried out to determine whether changes in abundance to any metabolite could be identified in TcoDMT OE and TcoDMT dKO when compared to WT cells (Fig S5). However, in spite of several differences appearing in metabolites between WT and TcoDMT OE lines, no clear cut substrate specificity could be attributed using this approach, leaving the normal physiological role of TcoDMT unresolved at this time (Fig S5). TcoDMT is an ISM transporter To date, no ISM transporters have been identified in African trypanosomes. To determine whether TcoDMT could transport ISM, uptake assays were carried out using radioactive ( 14 C)-ISM (Fig. 2 D), as well as taking advantage of the intrinsic fluorescence of ISM (which is dependent on kDNA binding) to analyse mitochondrial uptake (Fig. 2 E). No statistical difference was found in the uptake of 1 mM 14 C-ISM between TcoDMT dKO and WT cells, where uptake was minor and remained flat over 15.5 min (Fig. 2 D). However, the uptake of 14 C-ISM in DMT OE was rapid (significantly increased in 30 s; P < 0.05 for all time points) and plateaued within 3.5 min, indicating a rapid equilibration between the extracellular and cytosolic ISM concentrations (Fig. 2 D). At 16 min, TcoDMT OE accumulated approximately 22-fold higher ISM than WT control. These findings were validated using fluorescence-based uptake, which depends on the binding of the drug to the trypanosome kinetoplast 15 , 19 (Fig. 2 E). A reduction in ISM uptake was observed in DMT dKO , which reached 59.3 ± 5.1% at 30 min compared to 93.4 ± 10.8% in WT cells (Fig. 2 E). Fluorescence-based uptake reached a plateau, indicative of an equilibrative transporter mechanism, after 20 minutes in all cell lines (Fig. 2 E), and although the equilibrium was reached sooner in TcoDMT OE compared to WT, the difference was not statistically significant ( P > 0.05) at any single time point. Fluorescence microscopy showed that the ISM-derived fluorescent signal was dependent on uptake into the kinetoplast in all samples, including TcoDMT dKO cells (Fig. 2 F & 2 E). DMT plays a role in ISM sensitivity in T. congolense , but apparently not T. brucei TcoDMT shares 59.7% protein sequence identity with TbDMT, and 50.4% with the T. vivax syntenic orthologue (Tv486_0800880; Fig S6). TbDMT was not identified to be not essential in RNAi screens 40 , and nor was TbDMT an identified determinant of ISM resistance in a T. brucei genome-wide RNAi screen carried out under ISM selection 24 , suggesting that the role of DMT in ISM resistance may be particular to T. congolense . To investigate this further, a TbDMT overexpression line (TbDMT OE ), a T. brucei mutant expressing TcoDMT from the tubulin locus (Tb TcoDMT−OE ), as well as gene knock-down (TbDMT RNAi ) and knock-out (inducible CRISPR/cas9; TbDMT dKO ) mutants were generated, in order to assess any role of DMT in ISM-resistance in T. brucei (Fig. 3 ). Overexpression of TbDMT (Fig. 3 A) resulted in a 22-fold increase in ISM sensitivity ( P = 0.0176; Table 1 ; Fig. 3 C), with no significant differences in DA sensitivity ( P = 0.95; Table 1 ; Fig. 3 C). However, TbDMT OE cells only showed a 1.8-fold increase in sensitivity to EtB ( P = 0.0003; Table 1 ; Fig. 3 C). Additionally, RNAi-mediated knockdown of TbDMT (~ 75% reduction in mRNA, Fig. 3 D) did not affect parasite growth (Fig. 3 E), nor sensitivity to either DA or ISM (Fig. 3 F). To rule out the possible reversal of induced knockdown over the period of drug assay, two independent inducible TbDMT-Cas9 cell lines were generated using separate sgRNAs as previously described 41 . Tetracycline induction achieved detectable double-strand breaks within 5 days (Fig. 3 G). However, there was no change in growth rate (Fig. 3 H), nor differential sensitivity to DA or ISM between the uninduced cells and TbDMT knock-out mutants (Fig. 3 I). TcoDMT function was further validated through transgenic expression in the T. brucei tubulin locus, confirmed by qPCR (Fig. 3 B). Tb TcoDMT−OE clones showed approximately 45-fold increase in ISM sensitivity ( P = 0.0154; Table 1 , Fig. 3 C), but no significant difference in the sensitivity to DA compared to the WT control (Fig. 3 C). DMT copy number is associated with ISM sensitivity in field isolates To verify whether DMT copy number is associated with ISM sensitivity in the field, publicly available genome sequences 42 of T. congolense field isolates with documented ISM sensitivity phenotypes were analysed (Fig. 4 ; Table S3 ). Resistance was defined if relapse occurred in 80–100% of cases of infected mice treated with 1 mg/kg ISM, where intermediate resistance was defined if relapse occurred in 40–80%, as defined in the original studies (references in Table S3 ). Sequences from 21 isolates were aligned to the T. congolense IL3000 genome sequence (which encodes one DMT gene copy) and average reads per 100 bases (rolling means coverage; rmCoverage) were plotted (Fig. 4 ). Increased read coverage associated with the TcoDMT gene (TcIL3000.A.H_000575200), as well as in an upstream hypothetical ORF (TcIL3000.A.H_000575100), was detected in ISM-sensitive isolates. For isolates classed either as intermediate or fully resistant to ISM, rmCoverage was reduced compared to sensitive isolates, indicating that TcoDMT copy number positively correlates with ISM sensitivity. These data were compared to several other regions of the genome encompassing well characterised single-copy loci in T. congolense IL3000, aldolase ( TcoALD ), fructose-1,6-bisphosphatase ( TcoFBP ) and telomerase reverse transcriptase ( TcoTERT ; Fig S7). Here, no differences in rmCoverage were found between the three T. congolense ISM-sensitivity phenotype groups (Fig S7), suggesting that the observed increased read coverage in the ISM-sensitive isolates was particular to the TcoDMT locus. Discussion Drug treatment failure remains a significant threat to the chemotherapeutic control of AAT, impacting upon animal health and welfare as well as the development of sustainable agriculture in sub-Saharan Africa 43 , 44 , 45 . ISM is the most common chemotherapy alongside DA, and is also used as a prophylactic, heightening the likelihood of drug resistance acquisition by the parasite, especially after over six decades of widespread use 46 . The molecular mechanisms of ISM resistance, especially in the clinically relevant parasite species T. congolense , remain poorly understood, despite multiple reports of resistance leading to reduced ISM accumulation 18 , 19 , 20 , 29 , 47 , 48 Biological understanding of drug resistance mechanisms in T. congolense is lacking. So far, the best characterised putative mechanism for ISM resistance in this important pathogen is reduced ISM accumulation due to reduced ΔΨ m , limiting ISM import into the mitochondrion 18 (similar to observations in T. brucei ). The study of ISM resistance in T. congolense is further hindered by a lack of culturable field isolates, as the main lab strain, IL3000, is inherently ISM resistant and already exhibits a reduced ΔΨm, when compared to sensitive strains 18 . However, a recent study bypassed this challenge by generating resistance via iterative drug treatment of an ISM-sensitive strain (KTT/MSOROM7C1 49 ) during mouse infections 29 . A key finding in this study was the observed variation in copy number of several genes, including a putative drug/metabolite transporter (TcoDMT), an ABC transporter and the mitochondrial type II topoisomerase 29 . The latter was previously implicated in ISM resistance, although subsequent studies have invalidated this hypothesis 27 . TcoDMT gene expression was also reduced in ISM resistant cells, suggesting TcoDMT protein abundance may play a role in ISM resistance 29 . In this study, validating the role of TcoDMT in ISM sensitivity was made possible due to recently-developed genetic tools 30 , 31 enabling the generation of TcoDMT expression mutants. Whilst TcoDMT overexpression led to a significant increase in ISM and EtB sensitivity, loss of TcoDMT expression led to increased ISM and EtB resistance, thus confirming that TcoDMT is a genetic determinant of ISM and EtB resistance in T. congolense . Altered expression of TcoDMT had no impact on sensitivity to DA, suggesting a structure-activity relationship between TcoDMT and the EtB-base moiety of the ISM molecule, as well as supporting previous observations in this species of ISM resistance being associated with high levels of cross-resistance to EtB, but not DA 50 . The small, yet significant increase in resistance in TcoDMT dKO mutants could be explained by the IL3000 strain already being ISM-resistant 18 and TcoDMT being a single-copy gene. The fact that TcoDMT dKO cells retain low levels of ISM retention could indicate that there are additional uptake routes that, at the minimal level of TcoDMT expression, significantly contribute to the residual activity. Indeed, uptake assays and fluorescence microscopy suggest that, even in the absence of TcoDMT, there is some degree of ISM uptake. T. brucei studies have linked mutations in the F 1 -subunit γ of the F 1 F 0 -ATP synthase to ISM resistance, as these mutations allow bloodstream form parasites to compensate for loss of the kinetoplast with a concurrent reduction in ΔΨ m 19,51,52 . In contrast to T. brucei , dyskinetoplasty has not been reported for T. congolense to the best of our knowledge, and this is consistent with elevated mitochondrial activity in the bloodstream form of this species compared to T. brucei , suggesting the kinetoplast is likely to be essential 7 , 53 . Modifying TcoDMT expression had no effect on ΔΨ m , indicating that the ISM resistance mechanism uncovered here operates independently of membrane potential. Instead, the evidence presented here support a role for TcoDMT in cytosolic import of ISM. The protein localises to the cell surface, and uptake assays demonstrate altered drug accumulation when expression is modified. Fluorescence-based assays exploit ISM binding to the kDNA, thereby directly reporting mitochondrial accumulation. TcoDMT dKO cells displayed reduced mitochondrial signal, consistent with impaired drug accumulation. TcoDMT overexpression did not increase steady-state mitochondrial accumulation over 30 minutes, although initial rate of influx appeared enhanced. In contrast, whole cell uptake measured using 14 C-ISM was substantially increased in TcoDMT OE cells, while ablation of TcoDMT expression had no significant effect on 14 C-ISM accumulation. The likely explanation for these data is the consideration that ISM must traverse three membranes - the plasma membrane, and the outer and inner mitochondrial membranes - to reach the kDNA. Two concentration gradients therefore exist: i) that between the extracellular environment and the cytosol, and ii) that between the cytosol and mitochondrial matrix. The following model is therefore proposed (Fig. 5 ), whereby TcoDMT mediates ISM import across the plasma membrane, with the plasma membrane potential (ΔΨ pm ) providing the drive force for the dicationic drug, analogous to the role of ΔΨ m in mitochondrial accumulation. Under WT conditions (Fig. 5 A), ISM uptake proceeds until mitochondrial binding sites are saturated. Increasing TcoDMT (Fig. 5 B) enhances the rate of cytosolic import and thereby accelerates mitochondrial equilibration, but does not substantially alter steady-state accumulation, which is ultimately constrained by downstream steps, including kDNA saturation. Conversely, disruption of TcoDMT (Fig. 5 C) slows cytosolic entry, reducing the rate of mitochondrial accumulation and resulting in a modest loss of sensitivity. Importantly, although active ISM concentrations are in the nM-µM range, the uptake assays were performed with either 1 µM 14 C-ISM of 10 µM ISM by fluorescence, due to detection limits. These experimental conditions will overestimate the contribution of a secondary, low-affinity ISM transporter. At therapeutic concentrations, TcoDMT may contribute proportionally more to uptake if it has a higher affinity than secondary transporters. Similarly, in T. brucei , the principle determinant for pentamidine sensitivity is TbAQP2, first characterised as the High Affinity Pentamidine Transporter. Although TbAT1/P2 exhibits a higher pentamidine transport capacity (V max ) TbAQP2 dominates at therapeutic concentrations owing to its very low K m 54 . Importantly, complete gene loss seems only to be possible in T. congolense under in vitro conditions, as all field isolates appear to possess at minimum one TcoDMT copy – presumably because the observed growth defect reduces fitness of null mutants in vivo , such that low copy number mutants which are resistant to the selective pressure of ISM will be preferentially selected under these conditions. Therefore, is it likely that in the field, TcoDMT CNV is the main driver of resistance, rather than complete loss of expression. Indeed, analysis of genomic data from T. congolense field isolates corroborates this hypothesis, suggesting that TcoDMT is likely to play a role in ISM resistance in a field setting. CNV of genes or chromosomal regions has previously been associated with changes in drug sensitivity in trypanosomatids. For example, CNV in nitroreductase (NTR)-1 correlated with nifurtimox resistance in T. brucei 55 and T. cruzi 56 , and a recent study characterised CNV in trypanosomal serine carboxypeptidases in the acquisition of benzoxaborole resistance 31 . Whilst ISM resistance is likely to arise via multiple independent (or cumulative) mechanisms 19 , 24 , 29 , the correlation of TcoDMT copy number with susceptibility/resistance across multiple field-isolated strains from diverse geographic and temporal origins suggests that TcoDMT CNV is a principal mechanism that is frequently involved in ISM resistance. Whilst DMT has a capacity for both ISM and EtB uptake, this study was not able to experimentally identify the physiological substrate of this transporter. DMT is a homologue of SLC35F5, a class of solute carrier generally known as nucleotide sugar transporters, although the SLC35F subfamily are largely regarded as orphan transporters with substrate-specificity yet to be determined in most cases, although H. sapiens SLC35F5 was recently proposed to carry choline 57 , and queuine and queuosine (SLF35F2 58 ), as well as thiamine (SLC35F3 59 ) have all been proposed as substrates recently. Given its localisation on the cell surface, DMT is unlikely to transport nucleotide sugars (trypanosomes synthesise these in the glycosome 60 , and glycolsylation takes place in the Golgi). In addition, no significant changes in nucleotide sugars were detected using an LC-MS approach, nor were changes in choline or choline phosphate levels detected. Further work is required to identify the natural substrate(s) of TcoDMT. In summary, this study characterises and validates the first defined mechanism of resistance to the widely used trypanocide ISM in the disease relevant species T. congolense , and thereby uncovers a novel eukaryotic phenanthridine transporter. TcoDMT is also the first genetic determinant definitively shown to regulate both ISM and EtB resistance in this parasite species. Furthermore, copy number of this transporter strongly correlates with ISM resistance in field isolates. A logical next step will be to develop and validate assays to monitor resistance in field isolates, likely via RT-qPCR to determine CNV. Should these experiments succeed, DMT will prove a useful marker in both regional and time-series studies to predict or monitor ISM sensitivity in T. congolense isolates and strains, with implications for disease management as well as informing stakeholders how to best maximise chemotherapeutic lifetime. Materials and Methods Parasite culture T. congolense WT and OE/sKO/dKO mutants were grown at 34°C, 5% CO 2 and cultured in HMI-93 61 supplemented with 20% goat serum (Gibco). TcoDMT OE was selected and grown in 0.4 µg/mL blasticidin, TcoDMT sKO in 0.4 µg/mL hygromycin, and TcoDMT dKO in 0.4 µg/mL hygromycin and 0.4 µg/mL puromycin until knockout confirmation, after which the latter could be maintained in the absence of antibiotics. TcoDMT RNAi cells were selected and grown in 0.4 µg/mL puromycin and 0.4 µg/mL G418 (neomycin), and RNAi was induced via the daily addition of 1 µg/mL tetracycline. T. b. brucei Lister 427 WT and OE/dKO mutants, 2T1 and 2T1 T7-Cas9 cell lines were cultured in HMI-11 medium 62 , and maintained at 37°C and 5% CO 2 . TbDMT OE was selected and grown in 5 µg/mL blasticidin, T. brucei 2T1 maintained in 0.5 µg/mL phleomycin and 0.2 µg/mL puromycin 63 and the resulting TbDMT RNAi selected with 0.5 µg/mL phleomycin and 2.5 µg/mL hygromycin after transfection 64 . T. brucei 2T1 T7-Cas9 was grown in 2.5 µg/mL hygromycin and the generated T. brucei DMT-Cas9 cells selected and grown in 2 µg/mL phleomycin following transfection 41 . Tetracycline at 1 µg/mL was added to the culture daily in order to induce RNA interference and double strand break in TbDMT RNAi and TbDMT-Cas9 cell lines, respectively. To determine parasite growth rate, cells were seeded at predetermined density and cell densities determined daily via haemocytometry. T. congolense were detached via pipetting prior to assessing densities. Parasite transfection Both T. congolense and T. brucei were transfected with 10-15 µg linearised construct DNA. Briefly, per transfection, 2 - 4 × 10 7 parasites were centrifuged at 1,500 × g for 10 minutes. The medium supernatant was poured off, leaving ~1 mL. Cells were resuspended and transferred to 1.5 mL eppendorf tubes, and centrifuged for 5 minutes at 1,500 × g . Remaining supernatant was removed, cells washed in phosphate-buffered saline (P­BS; 137 mM NaCl, 3 mM KCl, 10 mM Na 2 HPO 4 , 1.8 mM KH 2 PO 4 ), subsequently resuspended in 100 µL transfection buffer 65 and transferred to an electroporation cuvette along with linearised vector, prior to electroporation using programme Z-001 on the Nucleofector II (Lonza). Cells were then transferred to 20 mL warm medium and allowed to grow. Selective antibiotics were added after 24 hours and cells were simultaneously cloned by diluting (1:50, 1:100 and 1:200) into 96-well plates. Resultant transfectants were isolated after ~7 days and were deemed clonal if <35 wells contained viable cells (based on a Poisson model of 1 cell per well). Parasite genetic manipulation Unless stated otherwise, all cell line constructs were typically generated as follows: amplicons generated by PCR using T. congolense or T. brucei genomic DNA (gDNA) templates were first ligated into the pGEM-T easy vector using T4 DNA ligase (both Promega), prior to excision with construct-specific restriction enzymes (see S4 table for primer sequences and restriction enzyme information). Sanger sequencing was typically performed at this stage. Excised constructs were subsequently ligated into Antarctic phosphatase-treated (NEB) target vector using T4 ligase. Final vectors were linearised with plasmid-specific restriction enzymes prior to transfection into target cells as described above. To generate the TcoDMT OE line, a previously-generated vector targeting the T. congolense tubulin array for constitutive expressionwasused 31 (Table S4). The TcoDMT (gene ID: TcIL3000.A.H_000575200) ORF was amplified using primers containing ClaI and BamHI sites in the forward and reverse primer, respectively (Table S4). The final vector was linearised using AscI prior to electroporation. To generate TcoDMT dKO constructs, sequential allelic replacement was used. Replacement cassettes were generated by amplifying a region of DNA encompassing the 5’-untranslated region (UTR), using forward and reverse primers containing a NotI and XbaI site, respectively. In addition, a region of DNA encompassing the 3’-UTR was amplified using forward and reverse primers containing a NsiI and XhoI site, respectively (Table S4). To ligate the constructs into the final vectors, two rounds of digestion and ligation were carried out, one for each UTR. Two plasmids were generated, each with a different antibiotic resistance cassette (hygromycin and puromycin) flanked by the aforementioned UTRs. Constructs were linearised with NotI and XhoI prior to transfection. For the generation of the TcoDMT RNAi construct, a previously-generated RNAi vector was used 30 . A 503 bp fragment of the TcoDMT gene was amplified from T. congolense IL3000 gDNA using forward and reverse primers flanked by FseI and HindIII sites, respectively (Table S4). The final vector was linearised with NotI prior to electroporation. In order to generate TbDMT OE cells, the TbDMT gene (Tb927.8.1460) was amplified using forward and reverse primers containing XbaI and BamHI restriction sites respectively (Table S4) and the cloned into the pGL2271 31 final vector. The plasmid was linearised with AscI prior to electroporation. TbDMT RNAi stem-loop plasmids were constructed using two-step ligation with TbDMT fragment as described previously 64 . The forward primer contained KpnI and BamHI sites whilst the reverse primer containted ApaI and XbaI sites (Table S4). After excision from pGET-T easy, the gene fragments were cloned into the pRPa iSL vector in two steps. In the first step, construct and vector were digested with KpnI and BamHI and ligated to generate an intermediate RNAi vector 64 . In the second step, the original construct as well as aforementioned intermediate RNAi vector were digested using ApaI and XbaI and ligated together. Constructs were, linearised with AscI and transfected into T. brucei 2T1 cells 63,64 . Inducible CRISPR/Cas9 dKO cell lines of TbDMT were generated using two separate sgRNA pairs as previously described 41 . Potential sgRNAs were identified using the LeishGEdit.net platform and manually selected based on PAM-adjacent GC-content. AGGG and AAAC overhangs were added to the 5’ end of the forward and reverse oligo sequences respectively. Synthesised oligos were annealed by heating to 95°C followed by slowly cooling over 4 hours, and annealed oligos were subsequently ligated into BbsI-digested pT7 sgRNA backbone. Constructs were linearised using NotI prior to electroporation into T. brucei 2T1 T7-Cas9 cells 41 . For N-terminal tagging of DMT protein with superfolder GFP by modification of the endogenous locus, PCR amplicons containing ~750 bp from the 5’-end UTR (untranslated region) and ~750 bp from the N-terminal end of the CDS (coding sequence) of DMT were amplified from T. congolense IL3000 gDNA and cloned together into XbaI-BamHI sites downstream of the fluorescent protein ORF in pEnNY0 66 using standard methods (Table S4). In the same step, a NotI linearization site was introduced between the UTR and CDS. For N-terminal tagging of ectopic TcoDMT expression (TcoDMT OE-sfGFP ), the same vector used to generate TcoDMT OE cells was further manipulated via insertion of sGFP at the 5’-end. The sfGFP ORF was amplified from the aforementioned pEnNY0 vector using primers flanked by ClaI sites and ligated into the TcoDMT OE vector digested with the same enzyme. Of note, the resulting vector was transformed into methyltransferase deficient ( dam – / dcm – ; NEB) chemically competent E. coli , as ClaI is methylation-sensitive and the forward primer contained a potential CpG island. The resulting plasmid was linearised using AscI. Quantitative Real-Time PCR Total RNA was extracted using a commercial kit (RNeasy; QIAgen) and a total of 1 µg RNA was reverse-transcribed using High-Capacity cDNA RT kit (Thermo Applied Biosystems). For RNAi studies, RNA samples were obtained from tetracycline-induced and uninduced samples daily for 3 days post-induction 7 . qPCR reactions were carried out with SensiFAST SYBR Hi-ROX mix (Bioline), in a BioRad Thermocycler (Biorad) as described 7 . Normalised transcript level was determined through the ΔΔCt method 67 using TcoTERT and TbTERT as endogenous controls (Table S4) for T. congolense and T. brucei , respectively 29,68 . To calculate TcoDMT copy number from genomic DNA, qPCR reactions were carried out as above using 2 ng gDNA. Expression levels, calculated using the ΔΔCt method, were then multiplied by 2 to account for the diploidy of the T. congolense genome. Alamar Blue Drug Sensitivity Assays Drug sensitivities were determined using the resazurin-based Alamar blue assay as described previously 7,69 . Briefly, two-fold serial dilutions of the test drug including drug-free control were prepared in 100 μL of culture medium in 96-well white opaque flat-bottomed plates (Cell Star, Greiner Bio-one). Parasites at log-phase of growth were harvested and distributed across the wells containing the drug dilution series at 100 μL/well, with final concentrations of 2 × 10 4 and 2.5 × 10 5 cells/mL for T. brucei and T. congolense , respectively. Plates were incubated for 48 h at the relevant growth conditions for each species, after which 20 μL of 125 mg/mL (0.49 mM) resazurin dye in PBS was added to each well, and the plate further incubated for 24 h. The endpoint fluorescence at λ em 544 nm/λ exc 590 nm was determined for each well of the plate using a Cytation 5 imaging reader (BioTek), and the EC 50 of each drug was calculated using non-linear regression fitted to a sigmoidal dose-response curve in GraphPad Prism (v10). Drug Uptake Assays The rate of uptake of ISM was determined using the natural fluorescence of ISM as described 19 . Briefly, 1 ×10 7 cells in assay buffer (33 mM HEPES, 98 mM NaCl, 4.6 mM KCl, 0.55 mM CaCl 2 , 0.07 mM MgSO 4 , 5.8 mM NaH 2 PO 4 , 0.3 mM MgCl 2 , 23 mM NaHCO 3 and 14 mM glucose, pH 7.3) were incubated with 10 µM ISM for an intended duration, followed by centrifugation at 12,500 × g for 1 min to stop the reaction and to separate the cells from the drug mix through an oil layer (7:1 v/v dibutylphthalate/mineral oil; Sigma-Aldrich). The oil and drug mix were removed by capillary suction and 50 μL of a 0.1 N HCl/methanol (1:8 v/v) mixture added to the tube to lyse the cell pellet. Following 1 h incubation at room temperature, samples were transferred to a 96-well plate and the fluorescence determined at λ em 620 nm/λ exc 355 nm using a Cytation 5 imaging reader. Uptake was reported as percentage of the maximum fluorescence in arbitrary units recorded in WT samples 70 . For the determination of radiolabelled ISM uptake, the cell pellet was collected and lysed as described previously for radiolabelled trypanocides 71 . Following incubation of cells with 1 µM [ 14 C]-ISM ( 14 C-Samorin), microcentrifuge tubes were flash frozen in liquid nitrogen, and the cell pellet cut off and transferred to a scintillation vial. Cell pellet was lysed in 2% SDS solution under slow agitation for 30 min and 3 mL of scintillation fluid (Scintilogic U, Lablogic) was subsequently added, followed by further incubation overnight in the dark. Radiation was measured in a 300SL scintillation counter (Hidex) and the rate of uptake per unit time was measured using GraphPad Prism version (v10). Flow cytometry for the Measurement of Mitochondrial Membrane Potential Mitochondrial membrane potential was assessed as described previously, with minor modifications 72 . Cells were harvested and adjusted to 1 ×10 6 cells/mL in 1 mL HMI-93 medium, and samples were either treated with 500 nM ISM for 3 h, 200 nM valinomycin for 1 h, 20 µM troglitazone for 1 h or left untreated. Thereafter, all samples were treated with 0.05 µM Mitotracker Red (Thermo Fisher Scientific) and incubated for 15 minutes at 37°C and 5% CO 2 . Cells were then washed in PBS at 1,500 × g for 10 min, fixed in 4% paraformaldehyde for 5 min, then washed again and resuspended in PBS. Mitotracker Red fluorescence intensity was determined using a Fortessa X20 flow cytometer (BD Bioscience) set at λ em 578 nm/λ exc 599 nm and the data analysed using FlowJo software (BD Bioscience). Native fluorescence microscopy For localisation of tagged DMT by native fluorescence, cells were harvested from mid-log phase cultures, washed twice in PBS and allowed to settle for 5 min onto glass slides at ~2 × 10 7 cells/mL density 30 . Cells were fixed for 5 min in 2% (w/v) formaldehyde, permeabilised in -20°C methanol, re-hydrated in PBS and incubated with 15 ng/mL 4,6-diamidino-2-phenylindole for 5 min, before mounting in 1% (w/v) 1,4-diazabicyclo[2.2.2]octane, 90% (v/v) glycerol, 50 mM sodium phosphate, pH 8.0 30 . Images were captured on an Olympus BX51 microscope equipped with a 100× UPlanApo objective (1.35 NA; Olympus) and Retiga R6 CCD camera (Qimaging) without binning. All fluorescent channel images were captured at equal exposure settings without prior illumination. Images for level comparison were also processed in parallel with the same alterations to minimum and maximum display levels. Image acquisition was controlled by μManager open source software 73 . Processing and analysis were performed in Fiji 74 . To plot pixel intensity profiles of native fluorescence images, a straight line covering 7 µm was drawn through the fluorescent panel. Data was imported into GraphPad Prism and the line was smoothed with the following parameters: Number of neighbours averaged: 5; Order of the smoothing polynomial: 2 nd . For ISM fluorescence, a final concentration of 10 µM ISM was added to cells in PBS at a density of 2 × 10 7 cells/mL for 30 minutes prior to the aforementioned steps. Images were captured on a Zeiss Axio Observer Z.1 microscope equipped with a 63× Plan-Apochromat objective (1.4 NA; Zeiss) and a Axiocam 702 mono (Zeiss). All images were captured and processed as above. TcoDMT read depth in field isolate sequencing data Genomic sequences of 56 T. congolense isolates were obtained from EBI under the accession number PRJEB15251, previously deposited by Tihon and colleagues 42 . Of these, 21 had associated metadata indicating sensitivity to ISM (Table S1). These 21 genome sequences were subsequently aligned to the T. congolense IL3000 2019 reference genome sequence (v51; TriTrypDB) using Hisat2 75 with the following parameters to ensure multimapping reads were aligned only once: “ -- no-spliced-alignment -k 1 ”. Using Samtools 76 , the resulting alignment files were filtered (“ view -bS -q 1 -F 0x100 ”) and sorted (“ sort ”) to generate .bam files. To obtain normalised read depth for each of the 21 alignments, the “bamCoverage” function from the DeepTools suite 77 was employed. The following parameters were used: the output format was bedgraph ( “--outFileFormat “bedgraph” ), a rolling bin size of 100 bp (“ --binSize 100 ”) and RPKM normalisation (“ --normalizeUsing RPKM ”) and only chromosome 8 reads were normalised for analysis of the DMT region ( “--region Tc.A.H.pschr_08 ”). For other loci, the relevant chromosome was selected (chromosomes 11, 9 and 10 for TcoTERT, TcoFBP and TcoALD, respectively). A custom R script was then used to generate rolling mean coverages (rmCoverage) for each bedgraph file. This script made use of the “rollapply” function from the “zoo” package 78 . The “unionbedg” function from the bedtools suite 79 was used to merge all RPKM normalized bedgraph into one file for downstream analyses. To generate figures, the “Gviz” package for R was used 80 . The “ ucscChromosomeNames=FALSE ” option was applied in order to utilise a custom T. congolense genome sequence using publicly available annotation and sequence files (v51; TriTrypDB). Figures were edited in Inkscape v1.2. In silico analysis of TcoDMT The protein sequence of TcoDMT was analysed using InterProScan 81 and domain graphics were generated using R. Colabfold 39 was used to generate a structural predication of TcoDMT, with default parameters. The model with highest rank was uploaded in FoldSeek to search for similar 3D protein structures. Images of overlaid protein structures were downloaded from FoldSeek. Phylogenetics Sequences for all H. sapiens SLC35 proteins were downloaded from Uniprot using the search term “SLC35*”. Previous studies identified eight NSTs in T. brucei 38 (strain TREU 927) and 11 NSTs in T. cruzi (strain CL Brener Non-Esmeraldo-like). The protein sequences for these NSTs were used to identify NSTs in T. congolense and L. major (strain Friedlin) via BLASTp searches in TriTrypDB. To further confirm all NSTs had been identified, the predicted proteomes from the aforementioned parasite species were downloaded from TriTrypDB and compared using Orthofinder 82 . A multiple sequence alignment of NSTs and SLC35 sequences (total of 55) was generated using Clustal Omega Multiple Sequence Aligner 83 with default settings and the Pearson/FASTA output format. Tree construction was carried out using iqtree2 84 with 1,000 bootstrap replicates (“ -B 1000 ”). The resulting tree was annotated and rendered using iTOL 85 . Metabolite extractions and liquid chromatography-mass spectrometry Metabolite extractions were carried out on both cell pellets and culture supernatants. Three replicates for each line were grown for 48 hours to a final density of 2 × 10 6 cells/mL, and 10 8 cells were rapidly quenched to 4°C in a dry ice/ethanol bath. All subsequent steps and incubations were carried out at 4°C. Samples were centrifuged at 1,250 × g for 10 minutes after which, for supernatant samples, 5 µL was taken from each sample and added to 200 µL extraction solvent (chloroform:methanol:water in a 1:3:1 ratio) in 1.5 mL eppendorf tubes. The remaining supernatant was discarded and cells resuspended in 1 mL sterile PBS. After transferring cell pellet samples to 1.5 mL eppendorf tubes, they were centrifuged at 1,250 × g for 10 minutes. Supernatant was discarded and 200 µL extraction solvent (as above) was added to each sample. All samples were then incubated for 1 hour on a thermomixer at 4°C. Samples were centrifuged at 16,060 × g for 10 minutes, and supernatants (~195 µL) transferred to new eppendorf tubes. As controls, 10 µL from each metabolite extraction was added to a new tube. Finally, air in the tubes was displaced using argon gas, and samples stored at -80°C prior to LC-MS analysis. Hydrophobic interaction liquid chromatography (HILIC) was carried out on a Dionex UltiMate 3000 RSLC system (Thermo Fisher) using a ZIC-pHILIC column (150 mm × 4.6 mm, 5 µm column; Merck SeQuant), at the University of Glasgow Shared Research Facility, UK. The column was maintained at 25°C and samples were eluted with a linear gradient (20 mM ammonium carbonate in water and acetonitrile) over 26 minutes at a flow rate of 0.3 mL/min. Injection volume was 10 µL and samples were maintained at 5°C prior to injection. For MS analysis, a Thermo Orbitrap QExactive (Thermo Fisher Scientific) was operated in polarity switching mode. The MS settings were as follows: Resolution: 70,000; AGC: 1e6; m/z range: 70-1,050; Sheath gas: 40; Auxiliary gas: 5; Sweep gas: 1; Probe temperature: 150°C; Capillary temperature: 320°C. For positive mode ionisation: source voltage +3.8 kV, S-Lens RF Level 30.00, S-Lens Voltage 25.00 (V), Skimmer Voltage 15.00 (V), Inject Flatopole Offset 8.00 (V), Bent Flatapole DC 6.00 (V). For negative mode ionisation: source voltage-3.8 kV. A set of authentic standards was run prior to the sample set. Metabolomics analysis Metabolomics data obtained in raw format were converted first to mzXML format and split into positive and negative polarity using msconvert 86 . Files were then converted to peakML format using XCMS, and were further processed using mzMatch 87 . Peak annotation and metabolite identification was carried out using IDEOM 88 . Data were analysed using MetaboAnalyst (v6.0) 89 . Prior to analysis, data were transformed (Log 10 ) and pareto-scaled. Computational methods Statistical analyses were carried out in GraphPad Prism or R 90 . For cartoon graphics (Fig 6), icons were downloaded from Bioicons. These icons were created by Servier (https://smart.servier.com) and are licensed under CC-BY 3.0 Unported (https://creativecommons.org/licenses/by/3.0). Declarations Author Contribution P.S., M.A., C.G., J.vd.A., M.P. and L.M. conceptualised the study. P.S., M.A., E.M.R., H.d.K., C.G., M.P. and L.M. developed the methodology. P.S., M.A., E.P. and E.M.R. performed the investigation. P.S. conducted the formal analysis and curated the data. P.S. wrote the original draft of the manuscript and prepared the visualisations. P.S., M.A., F.M., M.P., H.A., J.vd.A., H.d.K., C.G. and L.M. reviewed and edited the manuscript. L.M. supervised the study. H.A., M.P. and L.M. acquired funding. Data availability All data generated or analysed during this study are included in this published article and its Supplementary Information files. Acknowledgements: P.C.S., M.A.U., E.P., and L.J.M. were supported by funding from the UK Biotechnology and Biological Sciences Research Council (BBSRC; BB/S00243X/1; BB/W000296/1). E.M.R. and C.G. were supported by a BBSRC Project Grant (BB/W005867/1) to CG. P.C.S. is supported by a BBSRC Discovery Fellowship (BB/X009807/7). The Roslin Institute is supported through core funding from the BBSRC (BS/E/D/20002173; BBS/E/RL/230002C). The authors thank Marjorie Bouchier at CEVA Africa for provision of Isometamidium chloride (Veridium), Drs Anna Raper and Arantza Esnal-Zufiaurre (Roslin Bioimaging) for microscopy support, the Mass Spectrometry Facility within the MVLS Shared Research Facilities (University of Glasgow) for provision of metabolomics data and support & assistance, and Prof Sir Michael Ferguson (University of Dundee) and Dr Samuel Duncan (University of Glasgow) for useful discussions regarding nucleotide sugar transporters and metabolism. References WHO. Trypanosomiasis, human African (sleeping sickness).) (2023) Yaro, M., Munyard, K.A., Stear, M.J. & Groth, D.M. Combatting African Animal Trypanosomiasis (AAT) in livestock: The potential role of trypanotolerance. Vet Parasitol 225, 43–52 (2016). Shaw, A.P., Cecchi, G., Wint, G.R., Mattioli, R.C. & Robinson, T.P. Mapping the economic benefits to livestock keepers from intervening against bovine trypanosomosis in Eastern Africa. 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IQ-TREE 2: New Models and Efficient Methods for Phylogenetic Inference in the Genomic Era. Mol Biol Evol 37, 1530–1534 (2020). Letunic, I. & Bork, P. Interactive Tree of Life (iTOL) v6: recent updates to the phylogenetic tree display and annotation tool. Nucleic Acids Res 52, W78-W82 (2024). Chambers, M.C. et al. A cross-platform toolkit for mass spectrometry and proteomics. Nat Biotechnol 30, 918–920 (2012). Scheltema, R.A., Jankevics, A., Jansen, R.C., Swertz, M.A. & Breitling, R. PeakML/mzMatch: a file format, Java library, R library, and tool-chain for mass spectrometry data analysis. Anal Chem 83, 2786–2793 (2011). Creek, D.J., Jankevics, A., Burgess, K.E., Breitling, R. & Barrett, M.P. IDEOM: an Excel interface for analysis of LC-MS-based metabolomics data. Bioinformatics 28, 1048–1049 (2012). Pang, Z. et al. MetaboAnalyst 6.0: towards a unified platform for metabolomics data processing, analysis and interpretation. Nucleic Acids Res 52, W398-W406 (2024). R Core Team. R: A language and environment for statistical computing.). R Foundation for Statistical Computing (2021) Pedelacq, J.D., Cabantous, S., Tran, T., Terwilliger, T.C. & Waldo, G.S. Engineering and characterization of a superfolder green fluorescent protein. Nat Biotechnol 24, 79–88 (2006). Pinder, M. & Authie, E. The appearance of isometamidium resistant Trypanosoma congolense in West Africa. Acta Trop 41, 247–252 (1984). Additional Declarations No competing interests reported. Supplementary Files TableS1FoldSeekoutput.xlsx Table S1: FoldSeek output assessing similarity of TcoDMT predicted structure to other predicted and solved structures. TableS2metabolomicsIdeomoutput.xlsx Table S2: LC-MS metabolomics analysis of T. congolense WT, TcoDMT OE and TcoDMT dKO lines, including both cell pellets and supernatants after 48 hr of in vitro culture. TableS3fieldisolatemetadata.xlsx Table S3: Metadata and references for field isolate samples used in TcoDMT copy number analysis TableS4listofprimers.xlsx Table S4: List of primers used in this study SFigure.docx Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 27 Apr, 2026 Reviews received at journal 17 Apr, 2026 Reviews received at journal 06 Apr, 2026 Reviewers agreed at journal 26 Mar, 2026 Reviewers agreed at journal 26 Mar, 2026 Reviewers agreed at journal 25 Mar, 2026 Reviewers invited by journal 02 Mar, 2026 Editor assigned by journal 02 Mar, 2026 Submission checks completed at journal 21 Feb, 2026 First submitted to journal 17 Feb, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8903249","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":599734071,"identity":"5c7d8ecc-9cbf-4dca-8d97-0cde4a3b060d","order_by":0,"name":"Pieter C. 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Morrison","email":"","orcid":"","institution":"The Roslin Institute, University of Edinburgh","correspondingAuthor":false,"prefix":"","firstName":"Liam","middleName":"J.","lastName":"Morrison","suffix":""}],"badges":[],"createdAt":"2026-02-17 17:23:05","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8903249/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8903249/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":104476089,"identity":"3127f6e7-07e8-4b17-b6c4-1a4d38960ddc","added_by":"auto","created_at":"2026-03-12 08:18:26","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":105539,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGrowth, DMT expression and drug sensitivity in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eT. congolense \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eIL3000 and DMT mutants. \u003c/strong\u003eA) qRT-PCR shows 5-fold increased DMT RNA level in TcoDMT\u003csup\u003eOE\u003c/sup\u003e and loss of signal in TcoDMT\u003csup\u003edKO\u003c/sup\u003e. Transcript level was normalised to TcoTERT mRNA in each sample. Data represents average of biological triplicate determination, each carried out in technical triplicates (error bars as SEM). B) Copy number analysis of TcoDMT in WT, TcoDMT\u003csup\u003eOE\u003c/sup\u003e, TcoDMT\u003csup\u003esKO\u003c/sup\u003e and TcoDMT\u003csup\u003edKO\u003c/sup\u003e mutants. Copy number was analysed via qPCR of genomic DNA and normalised using TcoTERT as endogenous control via the ΔΔCt method. C) Growth curves of IL3000 wild type (WT), TcoDMT\u003csup\u003eOE\u003c/sup\u003e and TcoDMT\u003csup\u003edKO\u003c/sup\u003e reveal growth defect in the DMT\u003csup\u003edKO\u003c/sup\u003e. Cells were seeded at 1 × 10\u003csup\u003e5\u003c/sup\u003e cells/mL and counted by haemocytometer daily for 3 days (error bars indicate SD). D) Alamar blue drug sensitivity assay reveals variable sensitivity to ISM and EtB, but not DA in WT, TcoDMT\u003csup\u003eOE\u003c/sup\u003e and TcoDMT\u003csup\u003edKO ­­\u003c/sup\u003ecells. Data represents average EC\u003csub\u003e50\u003c/sub\u003e of biological triplicate measurement determinations, each carried out in technical duplicates (error bars as SEM). Individual statistics performed by unpaired \u003cem\u003et\u003c/em\u003e-test, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001; ns\u0026nbsp;=\u0026nbsp;not significant. E) Plot showing the correlation between pEC\u003csub\u003e50\u003c/sub\u003e (negative logarithm of the EC\u003csub\u003e50\u003c/sub\u003e) and relative expression values calculated in panel A and D. Grey shaded area indicates 95% confidence interval.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8903249/v1/deef2ac6bf54cad7985bb902.png"},{"id":104780436,"identity":"238276f7-5f34-47b2-bc79-e320356fe21b","added_by":"auto","created_at":"2026-03-17 07:52:55","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":288000,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTcoDMT is a cell surface ISM transporter that does not impact ΔΨ\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003em\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e. \u003c/strong\u003eA) TcoDMT was N-terminally tagged with superfolder (sf)GFP\u003csup\u003e91\u003c/sup\u003e at the endogenous locus and ectopically at the tubulin locus. For both lines, native fluorescence (green, left panel) was observed on the cell periphery, indicative of parasite cell surface. DAPI (cyan) was used to visualize the nuclear and mitochondrial DNA. Three examples are shown for wild-type cells (left), TcoDMT\u003csup\u003esfGFP\u003c/sup\u003e (middle) and TcoDMT\u003csup\u003eOE-sfGFP\u003c/sup\u003e (right). Scale bar represents 5 µm. B) Mitotracker fluorescence intensity in WT, TcoDMT\u003csup\u003eOE\u003c/sup\u003e and TcoDMT\u003csup\u003edKO,\u003c/sup\u003e indicative of mitochondrial uptake of Mitotracker that correlates with ΔΨ\u003csub\u003em\u003c/sub\u003e. Cells were left untreated or treated with 500 nM ISM for 3 h. Cells treated with 20 µM troglitazone or 200 nM valinomycin for 1 h were included as positive and negative control respectively. All samples were incubated with 0.05 µM Mitotracker and fixed prior to detection of fluorescence intensity. The mean of 3 biological replicates was normalized to the untreated WT control (error bars represent SEM). *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.005; ns, not significant by Unpaired Student's T-test compared to the untreated WT control. C) Domain analysis of the TcoDMT protein. The protein sequence of TcoDMT was analysed via InterProScan\u003csup\u003e81\u003c/sup\u003e, highlighting 10 predicted transmembrane domains (light-blue), as well as two predicted EamA-like transporter domains (PF00892; red) and an SCL35, F5 domain as predicted by Panther (dark grey). Also predicted were numerous cytoplasmic (orange) and inner mitochondrial (green) domains. D) Uptake assays with 1 µM \u003csup\u003e14\u003c/sup\u003eC-ISM in IL3000 WT and DMT mutants. Data represent average of biological triplicate determinations, each carried out in technical triplicates (error bars as SEM). E) Fluorescence-based uptake of 10 µM ISM in IL3000 WT and DMT mutants. Data represent average of two biological repeats each carried out in technical triplicates (error bars as SEM). F) Immunofluorescence microscopy analysis of ISM uptake into \u003cem\u003eT. congolense\u003c/em\u003e IL3000 WT and TcoDMT mutant cells. WT, TcoDMT\u003csup\u003edKO\u003c/sup\u003e and TcoDMT\u003csup\u003eOE\u003c/sup\u003e cells were incubated with 10 µM ISM for 30 minutes prior to slide preparation for imaging. For WT and TcoDMT\u003csup\u003eOE\u003c/sup\u003e, ISM localised to the kinetoplast, with staining also observed in the mitochondrion in the latter. Under these brightness settings, no ISM was detected in TcoDMT\u003csup\u003edKO\u003c/sup\u003e cells. G) The same images as in ‘B’, with increased brightness in the ISM channel to highlight that even in TcoDMT\u003csup\u003edKO\u003c/sup\u003e cells, ISM accumulates in the kinetoplast exclusively, albeit as much lower abundance. Scale bar represents 5 µm.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8903249/v1/22125502314661bbe17ae8de.png"},{"id":104808411,"identity":"142a1ecf-76f2-442c-a70d-f4b0e945401d","added_by":"auto","created_at":"2026-03-17 12:37:15","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":170560,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAnalysis of the impact of TbDMT expression on ISM sensitivity.\u003c/strong\u003e A) qPCR analysis of TbDMT gene expression in WT and TbDMT\u003csup\u003eOE\u003c/sup\u003e \u003cem\u003eT. brucei\u003c/em\u003e cell lines shows approximately seven-fold increased expression in TbDMT\u003csup\u003eOE\u003c/sup\u003e. B) qRT-PCR reveals confirms transgenic expression of the TcoDMT gene in \u003cem\u003eT. brucei \u003c/em\u003ecells (Tbb\u003csup\u003eTcoDMT-OE\u003c/sup\u003e). C) Drug sensitivity analysis reveals increased sensitivity to ISM (fifteen-fold) and EtB (two-fold), but not to DA in TbDMT\u003csup\u003eOE\u003c/sup\u003e cells. Additionally, Tbb\u003csup\u003eTcoDMT-OE\u003c/sup\u003e cells also exhibit increased sensitivity to ISM and EtB, but also not to DA. D) qPCR analysis of TbDMT gene expression in TbDMT RNAi cells indicates ~60% and ~75% reduction in DMT mRNA at 24 h and 48 h post-induction via the addition of 1 µg/mL tetracycline, respectively. E) TbDMT RNAi lines display no growth defects \u003cem\u003ein vitro\u003c/em\u003e upon induction. F) Drug sensitivity analysis of TbDMT RNAi cells reveals no significant changes in ISM, EtB or DA sensitivity upon RNAi induction. G) PCR amplification of the TbDMT gene in TbDMT-Cas9 cells confirms knock-out of the TbDMT ORF (~1.2 kB) following tetracycline induction. H) Cas9-mediated knock-out of TbDMT was induced via addition of 1 µg/mL tetracycline. Two different sgRNA sets were assessed (set 1: black lines; set 2: red lines). Growth rates of TbDMT-KO cells remained similar to uninduced cells. I) Cas9-mediated TbDMT knock-out cells exhibit no significant differences in ISM, EtB or DA sensitivity compared to uninduced controls. All qPCR data represents normalised expression relative to WT using TERT as endogenous control, and experiments were carried out with biological triplicates each in technical triplicates (averages shown, error bars as SEM). Cumulative growth rates were determined through daily manual counts of 3 clones (error bars as SEM) seeded at 2×10\u003csup\u003e4\u003c/sup\u003e cells/mL and maintained at the log phase of growth through 1:10 passage.\u0026nbsp; Drug sensitivity data represents average EC\u003csub\u003e50\u003c/sub\u003e after 72 h exposure to drugs of biological triplicate determination each carried out in technical duplicates (error bars as SEM). *, P\u0026lt;0.05; **, P\u0026lt;0.01***, P\u0026lt;0.005; ns, not significant by Unpaired Student's T-test compared to WT control. CRISPR-Cas9-mediated DBS was carried out using 2 separate sgRNA sets, both of which showed similar result.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8903249/v1/14c574b431d534e6e8d2509c.png"},{"id":104780912,"identity":"9c2c96f9-04c5-4989-8ac9-a24be1bd3d28","added_by":"auto","created_at":"2026-03-17 07:54:16","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":214442,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCoverage of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eTcoDMT\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eT. congolense \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003egenome. \u003c/strong\u003eGenomic\u003cstrong\u003e \u003c/strong\u003esequencing data was obtained from previous studies\u003csup\u003e42\u003c/sup\u003e. Of the 56 field isolates sequenced by Tihon and colleagues, \u0026nbsp;21 were previously characterised in terms of ISM resistance (Table S1), and these were taken forward for further analysis. Based on ISM-resistance metadata, samples were divided into three groups based - Sensitive, Intermediate Sensitivity (stemming from two studies\u003csup\u003e26,92\u003c/sup\u003e; Table S1) and Resistant - and the rolling mean coverage (rmCoverage; mean coverage in RPKM at 100 bp intervals) was calculated. \u0026nbsp;rmCoverage for the TcoDMT locus was higher in ISM-sensitive isolates. All sequencing data was aligned to the \u003cem\u003eT.\u003c/em\u003e \u003cem\u003econgolense\u003c/em\u003e IL3000 genome (TriTrypDB, version 51) which contains a single DMT gene copy. S, sensitive; R, resistant; I, intermediate sensitivity. Chromosome and genes in proximity of the TcoDMT locus are shown at the top of the figure (red arrow in the Genome Annotation panel indicates TcoDMT). The second panel shows average rmCoverage (with confidence intervals shown in lighter colour) for the three sample groups, whilst the lower panel shows coverage at 100-bp intervals for each field isolate included in this analysis divided into the three groups.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8903249/v1/73563a59f0a1db3df40ba06d.png"},{"id":104780702,"identity":"2efe35b8-28d2-423e-838c-cbd03e666cb9","added_by":"auto","created_at":"2026-03-17 07:53:39","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":307893,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eProposed mechanism of the role of TcoDMT in ISM sensitivity and resistance. \u003c/strong\u003eA) WT \u003cem\u003eT. congolense\u003c/em\u003e IL3000; B) TcoDMT\u003csup\u003eOE\u003c/sup\u003e; C) TcoDMT\u003csup\u003edKO\u003c/sup\u003e. Under normal conditions, there are two concentration gradients for ISM, that between the extracellular environment and the cytosol, and that between the cytosol and mitochondrial matrix. ISM uptake at the cell surface is mediated by TcoDMT, whilst mitochondrial uptake is mediated via mitochondrial membrane potential. This means that both concentration gradients are maintained until the mitochondrion is saturated. When TcoDMT expression is increased (via CNV, or \u003cem\u003ein vitro\u003c/em\u003e), ISM is imported into the cytosol at higher rates, with mitochondrial uptake becoming the rate limiting step. The mitochondrion therefore becomes saturated at a faster rate. When TcoDMT expression is abolished, cell surface uptake is interrupted, resulting in a knock-on effect of reduced mitochondrial uptake. Schematic generated in Inkscape using graphics from Bioicons.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-8903249/v1/0cc244d6461d108f137d2d92.png"},{"id":104809084,"identity":"c8edf3f9-39c8-41db-9b89-4720b40f6513","added_by":"auto","created_at":"2026-03-17 12:47:21","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2338007,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8903249/v1/144614da-068b-4a66-ace0-decb9e44ad6c.pdf"},{"id":104780713,"identity":"cf00c68b-a8fb-455c-8c56-6656f69b7138","added_by":"auto","created_at":"2026-03-17 07:53:40","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":384684,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTable S1:\u003c/strong\u003e FoldSeek output assessing similarity of TcoDMT predicted structure to other predicted and solved structures.\u003c/p\u003e","description":"","filename":"TableS1FoldSeekoutput.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8903249/v1/d89a2abb495027c7b164f7c5.xlsx"},{"id":104780714,"identity":"03cf7c26-d65f-4acf-9d79-e5fdf910a522","added_by":"auto","created_at":"2026-03-17 07:53:40","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1853184,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTable S2:\u003c/strong\u003e LC-MS metabolomics analysis of \u003cem\u003eT. congolense\u003c/em\u003e WT, TcoDMT\u003csup\u003eOE\u003c/sup\u003e and TcoDMT\u003csup\u003edKO\u003c/sup\u003e lines, including both cell pellets and supernatants after 48 hr of \u003cem\u003ein vitro\u003c/em\u003e culture.\u003c/p\u003e","description":"","filename":"TableS2metabolomicsIdeomoutput.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8903249/v1/7056e77350603c1db8c2e506.xlsx"},{"id":104780287,"identity":"66f62960-05c6-4585-97ad-dff9c644813d","added_by":"auto","created_at":"2026-03-17 07:52:01","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":18032,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTable S3:\u003c/strong\u003e Metadata and references for field isolate samples used in TcoDMT copy number analysis\u003c/p\u003e","description":"","filename":"TableS3fieldisolatemetadata.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8903249/v1/de9bf825f210e05c90c47b3f.xlsx"},{"id":104780493,"identity":"d8fc777f-53ef-49ac-9635-7d51d9b2513c","added_by":"auto","created_at":"2026-03-17 07:53:09","extension":"xlsx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":14180,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTable S4:\u003c/strong\u003e List of primers used in this study\u003c/p\u003e","description":"","filename":"TableS4listofprimers.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8903249/v1/aefa7fc00cc779494ac814b1.xlsx"},{"id":104780851,"identity":"60e40ea5-12c6-4036-be5b-8d281456cdce","added_by":"auto","created_at":"2026-03-17 07:54:06","extension":"docx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":3515506,"visible":true,"origin":"","legend":"","description":"","filename":"SFigure.docx","url":"https://assets-eu.researchsquare.com/files/rs-8903249/v1/827fa6075cc4b45e6bdd524f.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"A cell surface transporter mediates phenanthridine resistance in African trypanosomes","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAfrican trypanosomes are extracellular protozoan parasites that cause both human and livestock disease. Human African Trypanosomiasis (HAT) is prevalent across sub-Saharan Africa, with case numbers decreasing to \u0026lt;\u0026thinsp;1,000 annually in recent years, the disease is currently targeted for elimination of transmission by the WHO\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. In stark contrast, African Animal Trypanosomosis (AAT) remains a major hindrance to the development of sustainable agriculture in the 37 sub-Saharan African countries where the disease is endemic\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Although affecting multiple livestock species, the burden of AAT primarily falls on cattle, and is estimated to result in more than three million cattle deaths annually, with ~\u0026thinsp;90\u0026nbsp;million at risk, leading to yearly losses of ~\u003cspan\u003e$\u003c/span\u003e4.5 billion\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. The three main species of clinical relevance for AAT are \u003cem\u003eTrypanosoma congolense\u003c/em\u003e, \u003cem\u003eT. vivax\u003c/em\u003e and to a lesser extent, \u003cem\u003eT. brucei\u003c/em\u003e. The three species exhibit significant divergence in their biology, including at the genome\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e, transcriptome\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e, proteome\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e and metabolome\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e level, as well as in infection phenotypes\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. However, disease symptoms are largely indistinguishable, and mixed infection is common\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThere are no vaccines to prevent AAT, although a vaccine target was recently identified for \u003cem\u003eT. vivax\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e. Tsetse control is hindered by lack of sustainable infrastructure, leaving chemotherapy as the dominant form of AAT control. Three trypanocides are used in sub-Saharan Africa: the diamidine diminazene aceturate (DA) and the phenanthridines isometamidium chloride (ISM) and homidium bromide (ethidium bromide; EtB). ISM and DA are in widespread use, and although EtB is no longer recommended, it is still sold and used in some areas\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. ISM is currently the only drug used for both prophylaxis and curative treatment of AAT; prophylaxis being enabled by the long half-life of ISM in the host\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. ISM is derived from the coupling of EtB with amidinobenzenediazonium chloride, the latter derived from DA, and is marketed as a mixture of four compounds\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e, all of which are bioactive - likely through a shared mechanism\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eISM mode of action studies have predominantly focused on the lab-adapted \u003cem\u003eT. brucei\u003c/em\u003e where it has been hypothesised that ISM interacts with the kinetoplast DNA (kDNA; trypanosome mitochondrial DNA), with strong affinity for double stranded DNA in particular\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. This interaction results in linearization of the kDNA minicircles, with inhibition of a kinetoplast-specific topoisomerase II enzyme proposed as a possible mechanism of action\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Unlike ISM, its parental phenanthridine compound (EtB) distributes throughout the trypanosome cell\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e, although it likewise kills trypanosomes by specific interactions with kDNA, in addition to the inhibition of nuclear DNA replication\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe mechanisms governing ISM transport into the cytoplasm and mitochondrion remain unresolved. It is hypothesised that transport into the cytoplasm occurs via facilitated diffusion owing to its positive charge\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e, and reduced cytosolic ISM accumulation, as well as increased efflux have been posited as mechanisms of resistance in \u003cem\u003eT. brucei\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e. A possible role in ISM uptake has been proposed for the aminopurine TbAT1/P2 transporter, and although ISM-resistant field isolates of \u003cem\u003eT. brucei\u003c/em\u003e exhibited mutations in the TbAT1 gene\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e, \u003cem\u003ein vitro\u003c/em\u003e uptake studies suggest that the role of TbAT1/P2 is minor\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. ISM entry in the mitochondrion is thought to rely on the mitochondrial membrane potential (ΔΨ\u003csub\u003em\u003c/sub\u003e), established by the trypanosome F\u003csub\u003e0\u003c/sub\u003eF\u003csub\u003e1\u003c/sub\u003e-ATPase\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e, which functions in reverse to the mammalian ATPase\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Agents that depolarise ΔΨ\u003csub\u003em\u003c/sub\u003e have been shown to reduce ISM accumulation into the mitochondrion\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Furthermore, loss of the kinetoplast results in ISM resistance in \u003cem\u003eT. brucei\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e. However, ISM remains active against animal trypanosome species lacking a kinetoplast (dyskinetoplastic), albeit with a significantly increased dose, suggesting the presence of other cellular targets\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. A genome-scale RNAi study in \u003cem\u003eT. brucei\u003c/em\u003e demonstrated that disruption of vacuolar ATPase (vATPase) was also capable of generating ISM resistance, reflecting the connection between vATPase and mitochondrial F\u003csub\u003e0\u003c/sub\u003eF\u003csub\u003e1\u003c/sub\u003e-ATPase\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThere is limited data on ISM modes of action and resistance in \u003cem\u003eT. congolense\u003c/em\u003e, but it can be predicted that mechanisms are likely to differ from \u003cem\u003eT. brucei\u003c/em\u003e based on metabolic divergence. An increased reliance upon diverse mitochondrial activity (for example, substrate-level phosphorylation and an expanded electron transport chain) likely explains why dyskinetoplasty has not been reported in \u003cem\u003eT. congolense\u003c/em\u003e. However, similar to \u003cem\u003eT. brucei\u003c/em\u003e, ΔΨ\u003csub\u003em\u003c/sub\u003e correlates closely with ISM sensitivity in \u003cem\u003eT. congolense\u003c/em\u003e genome-reference strain IL3000, which exhibits both a reduced ΔΨ\u003csub\u003em\u003c/sub\u003e and increased ISM resistance, compared with other strains\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003eT. congolense\u003c/em\u003e lacks a TbAT1 orthologue\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e and to date no ISM transporter has been experimentally validated for either cytoplasmic or mitochondrial uptake.\u003c/p\u003e \u003cp\u003eEarlier studies using field isolates showed that ISM resistance correlated with mutations in a number of \u003cem\u003eT. congolense\u003c/em\u003e genes, including the topoisomerase II enzyme and an ATP-binding cassette (ABC) transporter\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. However, correlation with resistance was inconsistent, and a definitive mechanism of ISM resistance has remained elusive\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e,\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. A recent study of \u003cem\u003ein vivo\u003c/em\u003e generated ISM-resistant \u003cem\u003eT. congolense\u003c/em\u003e revealed putative determinants via copy number variation (CNV), including ABC transporters, topoisomerase II and a drug/metabolite transporter (TcoDMT)\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Expression analysis demonstrated that TcoDMT was downregulated in resistant cells, consistent with a potential role in ISM resistance\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. However, in the absence of functional analyses, the extent to which any of these genetic determinants contribute to ISM resistance has remained unknown. In this study, the role of TcoDMT in ISM-resistance in \u003cem\u003eT. congolense\u003c/em\u003e and \u003cem\u003eT. brucei\u003c/em\u003e was functionally evaluated.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003eTcoDMT expression is directly correlated to ISM sensitivity\u003c/h2\u003e\n \u003cp\u003eThe recent development of a genetic toolkit for \u003cem\u003eT. congolense\u003c/em\u003e has enabled manipulation of genes and their functional characterisation, including potential roles in drug mode of action or resistance\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e30\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e31\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. These advances were exploited to genetically manipulate TcoDMT (gene ID: TcIL3000.A.H_000575200), a single-copy gene in the genome of the reference strain IL3000, which, although exhibiting notable resistance to ISM compared to other strains used \u003cem\u003ein vivo\u003c/em\u003e, is the only strain currently amenable to culture. Over-expressor (TcoDMT\u003csup\u003eOE\u003c/sup\u003e) and double knock-out (TcoDMT\u003csup\u003edKO\u003c/sup\u003e) cell lines were generated, the latter achieved via sequential allelic replacement to also produce a TcoDMT single allele knock-out (hemizygous) intermediate (TcoDMT\u003csup\u003esKO\u003c/sup\u003e).\u003c/p\u003e\n \u003cp\u003eTcoDMT expression was elevated in TcoDMT\u003csup\u003eOE\u003c/sup\u003ecells (5.27\u0026thinsp;\u0026plusmn;\u0026thinsp;1.09-fold vs control) and completely ablated in TcoDMT\u003csup\u003edKO\u003c/sup\u003e cells (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA), whilst qPCR analysis of genomic DNA confirmed expected gene copy number in the four cell lines tested (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB). TcoDMT overexpression did not affect cell growth (doubling time, DT: 12.5 hr vs 12.3 hr in WT cells; Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eC), but a defect resulting in significantly increased doubling time (DT: 15.6 hr vs 12.3 hr in WT cells; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;1.20 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e, \u003cem\u003et\u003c/em\u003e-test) was observed in TcoDMT\u003csup\u003edKO\u003c/sup\u003e (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eC), independent of the presence of selective antibiotics (Fig \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003eA). Therefore, ablation of TcoDMT expression in \u003cem\u003eT. congolense\u003c/em\u003e leads to a fitness cost \u003cem\u003ein vitro\u003c/em\u003e.\u003c/p\u003e\n \u003cp\u003eDrug sensitivity analyses revealed a 27-fold reduction in ISM EC\u003csub\u003e50\u003c/sub\u003e in TcoDMT\u003csup\u003eOE\u003c/sup\u003e (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;1.25 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;8\u003c/sup\u003e, \u003cem\u003et\u003c/em\u003e-test; Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e; Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eD). In contrast, a 3.14-fold increase in EC\u003csub\u003e50\u003c/sub\u003e was observed in TcoDMT\u003csup\u003edKO\u003c/sup\u003e compared to WT cells (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;4.12 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;9\u003c/sup\u003e; Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e; Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eD), indicating increased ISM resistance in dKO mutants. Thus, \u003cem\u003ein vitro\u003c/em\u003e, TcoDMT expression levels correlate linearly with ISM sensitivity in \u003cem\u003eT. congolense\u003c/em\u003e (r\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.99; Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eE). Minor, but not significant, changes in ISM resistance were observed in TcoDMT\u003csup\u003esKO\u003c/sup\u003e, and this line was therefore not included in downstream analyses (Fig \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003eB). Knock-down of TcoDMT was also attempted using a \u003cem\u003eT. congolense\u003c/em\u003e IL3000 single-marker cell line (TcoSM) that constitutively expresses T7 RNA polymerase and Tet Repressor\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. However, RNAi penetrance is low in \u003cem\u003eT. congolense\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cspan class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e, and only 20\u0026ndash;40% reduction in TcoDMT mRNA levels was observed, which did not affect ISM sensitivity (Fig \u003cspan class=\"InternalRef\"\u003eS2\u003c/span\u003e). The RNAi and TcoDMT\u003csup\u003esKO\u003c/sup\u003e data jointly suggest that near complete ablation of TcoDMT expression was required for increased ISM resistance in the \u003cem\u003ein vitro T. congolense\u003c/em\u003e strain IL3000.\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003ctable id=\"Tab1\" border=\"1\" class=\"fr-table-selection-hover\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003e\u003cstrong\u003eDMT mutant EC\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e50\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003es for ISM, DA and EtB.\u003c/strong\u003e A summary of drug sensitivity data for the \u003cem\u003eT. congolense\u003c/em\u003e and \u003cem\u003eT. brucei\u003c/em\u003e DMT mutants described in this study. Fold changes and P-values are in relation to WT parental cells, or in the case of \u003cem\u003eT. brucei\u003c/em\u003e RNAi and Cas9, in relation to uninduced controls. Abbreviations: Ctrl: control; FC: fold change; P-val: P-value\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"11\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eSpecies\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eCell line\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003eISM\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003eDA\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003eEtB\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eEC\u003csub\u003e50\u003c/sub\u003e (nM)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eFC vs ctrl\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eP-val vs ctrl\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eEC\u003csub\u003e50\u003c/sub\u003e (nM)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eFC vs ctrl\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eP-val vs ctrl\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eEC\u003csub\u003e50\u003c/sub\u003e (nM)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eFC vs ctrl\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eP-val vs ctrl\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eT. congolense\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eWT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e6.88\u0026thinsp;\u0026plusmn;\u0026thinsp;0.39\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e216.4\u0026thinsp;\u0026plusmn;\u0026thinsp;14.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eT. congolense\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTcoDMT\u003csup\u003eOE\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.25 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;8\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e212.4\u0026thinsp;\u0026plusmn;\u0026thinsp;18.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.98\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026gt;\u0026thinsp;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.07 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;7\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eT. congolense\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTcoDMT\u003csup\u003edKO\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e21.59\u0026thinsp;\u0026plusmn;\u0026thinsp;1.14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.12 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;9\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e208.1\u0026thinsp;\u0026plusmn;\u0026thinsp;4.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.96\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026gt;\u0026thinsp;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e26.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.25 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;11\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eT. brucei\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eWT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e141.78\u0026thinsp;\u0026plusmn;\u0026thinsp;26.69\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e80.77\u0026thinsp;\u0026plusmn;\u0026thinsp;8.56\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1,343.3\u0026thinsp;\u0026plusmn;\u0026thinsp;43.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eT. brucei\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTbDMT\u003csup\u003eOE\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e9.21\u0026thinsp;\u0026plusmn;\u0026thinsp;2.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.57\u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;4\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e79.42\u0026thinsp;\u0026plusmn;\u0026thinsp;16.39\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.85\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026gt;\u0026thinsp;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e760.7\u0026thinsp;\u0026plusmn;\u0026thinsp;23.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.57\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.27 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eT. brucei\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTb-TcoDMT\u003csup\u003eOE\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e9.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e79.40\u0026thinsp;\u0026plusmn;\u0026thinsp;16.40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026gt;\u0026thinsp;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNot tested\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eT. brucei\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTbDMT RNAi (uninduced)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e289.77\u0026thinsp;\u0026plusmn;\u0026thinsp;5.71\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e68.50\u0026thinsp;\u0026plusmn;\u0026thinsp;1.89\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e859\u0026thinsp;\u0026plusmn;\u0026thinsp;23.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eT. brucei\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTbDMT RNAi (induced)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e337.25\u0026thinsp;\u0026plusmn;\u0026thinsp;18.06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e54.27\u0026thinsp;\u0026plusmn;\u0026thinsp;2.18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.79\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e761.6\u0026thinsp;\u0026plusmn;\u0026thinsp;15.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.89\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.007\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eT. brucei\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTbDMT Cas-9 dKO (uninduced)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e573.10\u0026thinsp;\u0026plusmn;\u0026thinsp;66.32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e149.55\u0026thinsp;\u0026plusmn;\u0026thinsp;13.21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNot tested\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eT. brucei\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTbDMT Cas-9 dKO (induced)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e650.04\u0026thinsp;\u0026plusmn;\u0026thinsp;43.99\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026gt;\u0026thinsp;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e167.48\u0026thinsp;\u0026plusmn;\u0026thinsp;21.78\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026gt;\u0026thinsp;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNot tested\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003eGiven that ISM consists of an EtB base coupled to the \u003cem\u003em\u003c/em\u003e-amidinobenzenediazonium chloride moiety derived from DA\u003csup\u003e33\u003c/sup\u003e, sensitivities for all \u003cem\u003eT. congolense\u003c/em\u003e cell lines to EtB and DA were determined in parallel (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eD). Whilst sensitivity or resistance to EtB was altered to approximately the same extent in the TcoDMT mutants, when compared to changes in ISM sensitivity (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2.01 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;7\u003c/sup\u003e and 0.03 for WT vs TcoDMT\u003csup\u003eOE\u003c/sup\u003e and WT vs DMT\u003csup\u003edKO\u003c/sup\u003e, respectively; Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e; Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eD), DA sensitivity DA remained unchanged, irrespective of changes in TcoDMT expression (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e; Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eD). Thus, the association of TcoDMT with ISM sensitivity is due to interactions with the EtB moiety of the ISM molecule, and TcoDMT does not confer sensitivity/resistance to DA.\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eTcoDMT is a cell surface protein\u003c/h3\u003e\n\u003cp\u003eTo assess the cellular location of TcoDMT, the gene was endogenously-locus tagged at its N-terminus using codon-optimised superfolder GFP (sfGFP)\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e, and the resulting cell line analysed by fluorescence microscopy. The sfGFP signal was relatively weak, albeit with detectable accumulation at the cell surface (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003eS3\u003c/span\u003eA). Analysis of pixel intensity supported the hypothesis that TcoDMT is a cell surface protein (Fig \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eB). To increase TcoDMT expression levels for improved visualisation, an N-terminally tagged ectopic copy was inserted into the tubulin locus (analogous to the TcoDMT\u003csup\u003eOE\u003c/sup\u003e line). The TcoDMT\u003csup\u003eOE\u0026minus;sfGP\u003c/sup\u003e line exhibited clear accumulation of sfGFP at the cell surface (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003eS3\u003c/span\u003eA), thus confirming that TcoDMT is a plasma membrane protein.\u003c/p\u003e \u003cp\u003ePrevious studies have reported a correlation between mitochondrial membrane potential (ΔΨ\u003csub\u003em\u003c/sub\u003e) and ISM sensitivity in \u003cem\u003eT. brucei\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e, and a putative link in \u003cem\u003eT. congolense\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e. Whilst a cell surface transporter is unlikely to impact upon ΔΨ\u003csub\u003em\u003c/sub\u003e, we nevertheless assessed ΔΨ\u003csub\u003em\u003c/sub\u003e in the TcoDMT mutants (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Untreated TcoDMT\u003csup\u003eOE\u003c/sup\u003e exhibited a slight, but not significant (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05) reduction in ΔΨ\u003csub\u003em\u003c/sub\u003e (0.74\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 arbitrary units [AU], compared with 1.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 AU in untreated WT cells; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). While ISM treatment of \u003cem\u003eT. brucei\u003c/em\u003e results in a rapid ΔΨ\u003csub\u003em\u003c/sub\u003e reduction\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e, no effect was observed after a two-hour ISM treatment of \u003cem\u003eT. congolense\u003c/em\u003e WT or mutant cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Notably, \u003cem\u003eT. congolense\u003c/em\u003e IL3000 exhibits a lower ΔΨ\u003csub\u003em\u003c/sub\u003e compared to ISM-sensitive \u003cem\u003eT. congolense\u003c/em\u003e strains\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Nevertheless, treating all cell lines with valinomycin, a mitochondrial membrane depolariser, resulted in a\u0026thinsp;\u0026gt;\u0026thinsp;50% reduction in ΔΨ\u003csub\u003em\u003c/sub\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB); troglitazone, known to hyperpolarise ΔΨ\u003csub\u003em\u003c/sub\u003e in \u003cem\u003eT. brucei\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e, had no effect. These data suggest that TcoDMT plays no active role in ΔΨ\u003csub\u003em\u003c/sub\u003e maintenance.\u003c/p\u003e \u003cp\u003eTcoDMT is a putative EamA-like transporter encoding 10 transmembrane domains and also harbouring an SLC35F, family 5 (SLC35F5) domain (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). The human orthologue of SLC35F5 is an orphan transporter implicated in anti-cancer chemotherapeutics resistance\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e,\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. The predicted \u003cem\u003eT. brucei\u003c/em\u003e orthologue, TbDMT (Gene ID: Tb927.8.1460), was previously predicted as a nucleotide sugar transporter (TbNST)-7, where TbNST1-4 were experimentally characterised as functional nucleotide sugar transporters, while TbNST7 was not functionally chracterised\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. Phylogenetic analysis confirmed that TcoDMT and TbDMT are closely related to the F5 subgroup of the SLC35 superfamily, and are syntenic orthologues (Fig \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003eA). A DMT orthologue was also identified in \u003cem\u003eT. vivax\u003c/em\u003e (Gene ID: TvY486_0800880) and \u003cem\u003eLeishmania major\u003c/em\u003e (gene ID: LmjF.07.0400; Fig \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003eA). However, no orthologues were identified in \u003cem\u003eT. cruzi\u003c/em\u003e, nor in the intracellular apicomplexans \u003cem\u003eToxoplasma gondii\u003c/em\u003e and \u003cem\u003ePlasmodium falciparum\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eA ColabFold\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e TcoDMT model was shown to be most similar to predicted models of SLC35F5 proteins from \u003cem\u003eT. brucei\u003c/em\u003e, \u003cem\u003eL. major\u003c/em\u003e, animals and yeast (Fig \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003eB-D, Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). An untargeted metabolomics approach was carried out to determine whether changes in abundance to any metabolite could be identified in TcoDMT\u003csup\u003eOE\u003c/sup\u003e and TcoDMT\u003csup\u003edKO\u003c/sup\u003e when compared to WT cells (Fig S5). However, in spite of several differences appearing in metabolites between WT and TcoDMT\u003csup\u003eOE\u003c/sup\u003e lines, no clear cut substrate specificity could be attributed using this approach, leaving the normal physiological role of TcoDMT unresolved at this time (Fig S5).\u003c/p\u003e\n\u003ch3\u003eTcoDMT is an ISM transporter\u003c/h3\u003e\n\u003cp\u003eTo date, no ISM transporters have been identified in African trypanosomes. To determine whether TcoDMT could transport ISM, uptake assays were carried out using radioactive (\u003csup\u003e14\u003c/sup\u003eC)-ISM (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD), as well as taking advantage of the intrinsic fluorescence of ISM (which is dependent on kDNA binding) to analyse mitochondrial uptake (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). No statistical difference was found in the uptake of 1 mM \u003csup\u003e14\u003c/sup\u003eC-ISM between TcoDMT\u003csup\u003edKO\u003c/sup\u003e and WT cells, where uptake was minor and remained flat over 15.5 min (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). However, the uptake of \u003csup\u003e14\u003c/sup\u003eC-ISM in DMT\u003csup\u003eOE\u003c/sup\u003e was rapid (significantly increased in 30 s; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 for all time points) and plateaued within 3.5 min, indicating a rapid equilibration between the extracellular and cytosolic ISM concentrations (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). At 16 min, TcoDMT\u003csup\u003eOE\u003c/sup\u003e accumulated approximately 22-fold higher ISM than WT control. These findings were validated using fluorescence-based uptake, which depends on the binding of the drug to the trypanosome kinetoplast\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). A reduction in ISM uptake was observed in DMT\u003csup\u003edKO\u003c/sup\u003e, which reached 59.3\u0026thinsp;\u0026plusmn;\u0026thinsp;5.1% at 30 min compared to 93.4\u0026thinsp;\u0026plusmn;\u0026thinsp;10.8% in WT cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). Fluorescence-based uptake reached a plateau, indicative of an equilibrative transporter mechanism, after 20 minutes in all cell lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE), and although the equilibrium was reached sooner in TcoDMT\u003csup\u003eOE\u003c/sup\u003e compared to WT, the difference was not statistically significant (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05) at any single time point. Fluorescence microscopy showed that the ISM-derived fluorescent signal was dependent on uptake into the kinetoplast in all samples, including TcoDMT\u003csup\u003edKO\u003c/sup\u003e cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF \u0026amp; \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE).\u003c/p\u003e \u003cp\u003e \u003cb\u003eDMT plays a role in ISM sensitivity in\u003c/b\u003e \u003cb\u003eT. congolense\u003c/b\u003e, \u003cb\u003ebut apparently not\u003c/b\u003e \u003cb\u003eT. brucei\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTcoDMT shares 59.7% protein sequence identity with TbDMT, and 50.4% with the \u003cem\u003eT. vivax\u003c/em\u003e syntenic orthologue (Tv486_0800880; Fig S6). TbDMT was not identified to be not essential in RNAi screens\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e, and nor was TbDMT an identified determinant of ISM resistance in a \u003cem\u003eT. brucei\u003c/em\u003e genome-wide RNAi screen carried out under ISM selection\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e, suggesting that the role of DMT in ISM resistance may be particular to \u003cem\u003eT. congolense\u003c/em\u003e. To investigate this further, a TbDMT overexpression line (TbDMT\u003csup\u003eOE\u003c/sup\u003e), a \u003cem\u003eT. brucei\u003c/em\u003e mutant expressing TcoDMT from the tubulin locus (Tb\u003csup\u003eTcoDMT\u0026minus;OE\u003c/sup\u003e), as well as gene knock-down (TbDMT\u003csup\u003eRNAi\u003c/sup\u003e) and knock-out (inducible CRISPR/cas9; TbDMT\u003csup\u003edKO\u003c/sup\u003e) mutants were generated, in order to assess any role of DMT in ISM-resistance in \u003cem\u003eT. brucei\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eOverexpression of TbDMT (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA) resulted in a 22-fold increase in ISM sensitivity (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0176; Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC), with no significant differences in DA sensitivity (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.95; Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). However, TbDMT\u003csup\u003eOE\u003c/sup\u003e cells only showed a 1.8-fold increase in sensitivity to EtB (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0003; Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Additionally, RNAi-mediated knockdown of TbDMT (~\u0026thinsp;75% reduction in mRNA, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD) did not affect parasite growth (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE), nor sensitivity to either DA or ISM (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). To rule out the possible reversal of induced knockdown over the period of drug assay, two independent inducible TbDMT-Cas9 cell lines were generated using separate sgRNAs as previously described\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. Tetracycline induction achieved detectable double-strand breaks within 5 days (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG). However, there was no change in growth rate (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH), nor differential sensitivity to DA or ISM between the uninduced cells and TbDMT knock-out mutants (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eI). TcoDMT function was further validated through transgenic expression in the \u003cem\u003eT. brucei\u003c/em\u003e tubulin locus, confirmed by qPCR (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Tb\u003csup\u003eTcoDMT\u0026minus;OE\u003c/sup\u003e clones showed approximately 45-fold increase in ISM sensitivity (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0154; Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC), but no significant difference in the sensitivity to DA compared to the WT control (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC).\u003c/p\u003e\n\u003ch3\u003eDMT copy number is associated with ISM sensitivity in field isolates\u003c/h3\u003e\n\u003cp\u003eTo verify whether DMT copy number is associated with ISM sensitivity in the field, publicly available genome sequences\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e of \u003cem\u003eT. congolense\u003c/em\u003e field isolates with documented ISM sensitivity phenotypes were analysed (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e; Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e). Resistance was defined if relapse occurred in 80\u0026ndash;100% of cases of infected mice treated with 1 mg/kg ISM, where intermediate resistance was defined if relapse occurred in 40\u0026ndash;80%, as defined in the original studies (references in Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSequences from 21 isolates were aligned to the \u003cem\u003eT. congolense\u003c/em\u003e IL3000 genome sequence (which encodes one DMT gene copy) and average reads per 100 bases (rolling means coverage; rmCoverage) were plotted (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Increased read coverage associated with the \u003cem\u003eTcoDMT\u003c/em\u003e gene (TcIL3000.A.H_000575200), as well as in an upstream hypothetical ORF (TcIL3000.A.H_000575100), was detected in ISM-sensitive isolates. For isolates classed either as intermediate or fully resistant to ISM, rmCoverage was reduced compared to sensitive isolates, indicating that \u003cem\u003eTcoDMT\u003c/em\u003e copy number positively correlates with ISM sensitivity. These data were compared to several other regions of the genome encompassing well characterised single-copy loci in \u003cem\u003eT. congolense\u003c/em\u003e IL3000, aldolase (\u003cem\u003eTcoALD\u003c/em\u003e), fructose-1,6-bisphosphatase (\u003cem\u003eTcoFBP\u003c/em\u003e) and telomerase reverse transcriptase (\u003cem\u003eTcoTERT\u003c/em\u003e; Fig S7). Here, no differences in rmCoverage were found between the three \u003cem\u003eT. congolense\u003c/em\u003e ISM-sensitivity phenotype groups (Fig S7), suggesting that the observed increased read coverage in the ISM-sensitive isolates was particular to the \u003cem\u003eTcoDMT\u003c/em\u003e locus.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eDrug treatment failure remains a significant threat to the chemotherapeutic control of AAT, impacting upon animal health and welfare as well as the development of sustainable agriculture in sub-Saharan Africa\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e,\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e,\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. ISM is the most common chemotherapy alongside DA, and is also used as a prophylactic, heightening the likelihood of drug resistance acquisition by the parasite, especially after over six decades of widespread use\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. The molecular mechanisms of ISM resistance, especially in the clinically relevant parasite species \u003cem\u003eT. congolense\u003c/em\u003e, remain poorly understood, despite multiple reports of resistance leading to reduced ISM accumulation\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e,\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e,\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eBiological understanding of drug resistance mechanisms in \u003cem\u003eT. congolense\u003c/em\u003e is lacking. So far, the best characterised putative mechanism for ISM resistance in this important pathogen is reduced ISM accumulation due to reduced ΔΨ\u003csub\u003em\u003c/sub\u003e, limiting ISM import into the mitochondrion\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e (similar to observations in \u003cem\u003eT. brucei\u003c/em\u003e). The study of ISM resistance in \u003cem\u003eT. congolense\u003c/em\u003e is further hindered by a lack of culturable field isolates, as the main lab strain, IL3000, is inherently ISM resistant and already exhibits a reduced ΔΨm, when compared to sensitive strains\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. However, a recent study bypassed this challenge by generating resistance via iterative drug treatment of an ISM-sensitive strain (KTT/MSOROM7C1\u003csup\u003e49\u003c/sup\u003e) during mouse infections\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. A key finding in this study was the observed variation in copy number of several genes, including a putative drug/metabolite transporter (TcoDMT), an ABC transporter and the mitochondrial type II topoisomerase\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. The latter was previously implicated in ISM resistance, although subsequent studies have invalidated this hypothesis\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. TcoDMT gene expression was also reduced in ISM resistant cells, suggesting TcoDMT protein abundance may play a role in ISM resistance\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn this study, validating the role of TcoDMT in ISM sensitivity was made possible due to recently-developed genetic tools\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e,\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e enabling the generation of TcoDMT expression mutants. Whilst TcoDMT overexpression led to a significant increase in ISM and EtB sensitivity, loss of TcoDMT expression led to increased ISM and EtB resistance, thus confirming that TcoDMT is a genetic determinant of ISM and EtB resistance in \u003cem\u003eT. congolense\u003c/em\u003e. Altered expression of TcoDMT had no impact on sensitivity to DA, suggesting a structure-activity relationship between TcoDMT and the EtB-base moiety of the ISM molecule, as well as supporting previous observations in this species of ISM resistance being associated with high levels of cross-resistance to EtB, but not DA\u003csup\u003e50\u003c/sup\u003e. The small, yet significant increase in resistance in TcoDMT\u003csup\u003edKO\u003c/sup\u003e mutants could be explained by the IL3000 strain already being ISM-resistant\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e and TcoDMT being a single-copy gene. The fact that TcoDMT\u003csup\u003edKO\u003c/sup\u003e cells retain low levels of ISM retention could indicate that there are additional uptake routes that, at the minimal level of TcoDMT expression, significantly contribute to the residual activity. Indeed, uptake assays and fluorescence microscopy suggest that, even in the absence of TcoDMT, there is some degree of ISM uptake.\u003c/p\u003e \u003cp\u003e \u003cem\u003eT. brucei\u003c/em\u003e studies have linked mutations in the F\u003csub\u003e1\u003c/sub\u003e-subunit γ of the F\u003csub\u003e1\u003c/sub\u003eF\u003csub\u003e0\u003c/sub\u003e-ATP synthase to ISM resistance, as these mutations allow bloodstream form parasites to compensate for loss of the kinetoplast with a concurrent reduction in ΔΨ\u003csub\u003em\u003c/sub\u003e\u003csup\u003e19,51,52\u003c/sup\u003e. In contrast to \u003cem\u003eT. brucei\u003c/em\u003e, dyskinetoplasty has not been reported for \u003cem\u003eT. congolense\u003c/em\u003e to the best of our knowledge, and this is consistent with elevated mitochondrial activity in the bloodstream form of this species compared to \u003cem\u003eT. brucei\u003c/em\u003e, suggesting the kinetoplast is likely to be essential\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e. Modifying TcoDMT expression had no effect on ΔΨ\u003csub\u003em\u003c/sub\u003e, indicating that the ISM resistance mechanism uncovered here operates independently of membrane potential.\u003c/p\u003e \u003cp\u003eInstead, the evidence presented here support a role for TcoDMT in cytosolic import of ISM. The protein localises to the cell surface, and uptake assays demonstrate altered drug accumulation when expression is modified. Fluorescence-based assays exploit ISM binding to the kDNA, thereby directly reporting mitochondrial accumulation. TcoDMT\u003csup\u003edKO\u003c/sup\u003e cells displayed reduced mitochondrial signal, consistent with impaired drug accumulation. TcoDMT overexpression did not increase steady-state mitochondrial accumulation over 30 minutes, although initial rate of influx appeared enhanced. In contrast, whole cell uptake measured using \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003eC-ISM was substantially increased in TcoDMT\u003csup\u003eOE\u003c/sup\u003e cells, while ablation of TcoDMT expression had no significant effect on \u003csup\u003e14\u003c/sup\u003eC-ISM accumulation.\u003c/p\u003e \u003cp\u003eThe likely explanation for these data is the consideration that ISM must traverse three membranes - the plasma membrane, and the outer and inner mitochondrial membranes - to reach the kDNA. Two concentration gradients therefore exist: i) that between the extracellular environment and the cytosol, and ii) that between the cytosol and mitochondrial matrix. The following model is therefore proposed (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e), whereby TcoDMT mediates ISM import across the plasma membrane, with the plasma membrane potential (ΔΨ\u003csub\u003epm\u003c/sub\u003e) providing the drive force for the dicationic drug, analogous to the role of ΔΨ\u003csub\u003em\u003c/sub\u003e in mitochondrial accumulation.\u003c/p\u003e \u003cp\u003eUnder WT conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA), ISM uptake proceeds until mitochondrial binding sites are saturated. Increasing TcoDMT (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB) enhances the rate of cytosolic import and thereby accelerates mitochondrial equilibration, but does not substantially alter steady-state accumulation, which is ultimately constrained by downstream steps, including kDNA saturation. Conversely, disruption of TcoDMT (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC) slows cytosolic entry, reducing the rate of mitochondrial accumulation and resulting in a modest loss of sensitivity.\u003c/p\u003e \u003cp\u003eImportantly, although active ISM concentrations are in the nM-\u0026micro;M range, the uptake assays were performed with either 1 \u0026micro;M \u003csup\u003e14\u003c/sup\u003eC-ISM of 10 \u0026micro;M ISM by fluorescence, due to detection limits. These experimental conditions will overestimate the contribution of a secondary, low-affinity ISM transporter. At therapeutic concentrations, TcoDMT may contribute proportionally more to uptake if it has a higher affinity than secondary transporters. Similarly, in \u003cem\u003eT. brucei\u003c/em\u003e, the principle determinant for pentamidine sensitivity is TbAQP2, first characterised as the High Affinity Pentamidine Transporter. Although TbAT1/P2 exhibits a higher pentamidine transport capacity (V\u003csub\u003emax\u003c/sub\u003e) TbAQP2 dominates at therapeutic concentrations owing to its very low K\u003csub\u003em\u003c/sub\u003e\u003csup\u003e54\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eImportantly, complete gene loss seems only to be possible in \u003cem\u003eT. congolense\u003c/em\u003e under \u003cem\u003ein vitro\u003c/em\u003e conditions, as all field isolates appear to possess at minimum one TcoDMT copy \u0026ndash; presumably because the observed growth defect reduces fitness of null mutants \u003cem\u003ein vivo\u003c/em\u003e, such that low copy number mutants which are resistant to the selective pressure of ISM will be preferentially selected under these conditions. Therefore, is it likely that in the field, TcoDMT CNV is the main driver of resistance, rather than complete loss of expression.\u003c/p\u003e \u003cp\u003eIndeed, analysis of genomic data from \u003cem\u003eT. congolense\u003c/em\u003e field isolates corroborates this hypothesis, suggesting that TcoDMT is likely to play a role in ISM resistance in a field setting. CNV of genes or chromosomal regions has previously been associated with changes in drug sensitivity in trypanosomatids. For example, CNV in nitroreductase (NTR)-1 correlated with nifurtimox resistance in \u003cem\u003eT. brucei\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eT. cruzi\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e, and a recent study characterised CNV in trypanosomal serine carboxypeptidases in the acquisition of benzoxaborole resistance\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. Whilst ISM resistance is likely to arise via multiple independent (or cumulative) mechanisms\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e, the correlation of TcoDMT copy number with susceptibility/resistance across multiple field-isolated strains from diverse geographic and temporal origins suggests that TcoDMT CNV is a principal mechanism that is frequently involved in ISM resistance.\u003c/p\u003e \u003cp\u003eWhilst DMT has a capacity for both ISM and EtB uptake, this study was not able to experimentally identify the physiological substrate of this transporter. DMT is a homologue of SLC35F5, a class of solute carrier generally known as nucleotide sugar transporters, although the SLC35F subfamily are largely regarded as orphan transporters with substrate-specificity yet to be determined in most cases, although \u003cem\u003eH. sapiens\u003c/em\u003e SLC35F5 was recently proposed to carry choline\u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e, and queuine and queuosine (SLF35F2\u003csup\u003e58\u003c/sup\u003e), as well as thiamine (SLC35F3\u003csup\u003e59\u003c/sup\u003e) have all been proposed as substrates recently. Given its localisation on the cell surface, DMT is unlikely to transport nucleotide sugars (trypanosomes synthesise these in the glycosome\u003csup\u003e\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e, and glycolsylation takes place in the Golgi). In addition, no significant changes in nucleotide sugars were detected using an LC-MS approach, nor were changes in choline or choline phosphate levels detected. Further work is required to identify the natural substrate(s) of TcoDMT.\u003c/p\u003e \u003cp\u003eIn summary, this study characterises and validates the first defined mechanism of resistance to the widely used trypanocide ISM in the disease relevant species \u003cem\u003eT. congolense\u003c/em\u003e, and thereby uncovers a novel eukaryotic phenanthridine transporter. TcoDMT is also the first genetic determinant definitively shown to regulate both ISM and EtB resistance in this parasite species. Furthermore, copy number of this transporter strongly correlates with ISM resistance in field isolates. A logical next step will be to develop and validate assays to monitor resistance in field isolates, likely via RT-qPCR to determine CNV. Should these experiments succeed, DMT will prove a useful marker in both regional and time-series studies to predict or monitor ISM sensitivity in \u003cem\u003eT. congolense\u003c/em\u003e isolates and strains, with implications for disease management as well as informing stakeholders how to best maximise chemotherapeutic lifetime.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003ch3\u003e\u003cstrong\u003eParasite culture\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003e\u003cem\u003eT. congolense\u003c/em\u003e WT and OE/sKO/dKO mutants\u0026nbsp;were grown at 34°C, 5% CO\u003csub\u003e2\u003c/sub\u003e and cultured in HMI-93\u003csup\u003e61\u003c/sup\u003e supplemented\u0026nbsp;with 20% goat serum (Gibco). TcoDMT\u003csup\u003eOE\u003c/sup\u003e was selected and grown in 0.4 µg/mL blasticidin, TcoDMT\u003csup\u003esKO\u003c/sup\u003e in 0.4 µg/mL hygromycin, and TcoDMT\u003csup\u003edKO\u003c/sup\u003e in 0.4 µg/mL hygromycin and 0.4 µg/mL puromycin until knockout confirmation, after which the latter could be maintained in the absence of antibiotics. TcoDMT\u003csup\u003eRNAi\u003c/sup\u003e cells were selected and grown in 0.4 µg/mL puromycin and 0.4 µg/mL G418 (neomycin), and RNAi was induced via the daily addition of 1 µg/mL tetracycline.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eT. b. brucei\u003c/em\u003e Lister 427 WT and OE/dKO mutants, 2T1 and 2T1\u003csup\u003eT7-Cas9\u003c/sup\u003e cell lines were cultured in HMI-11 medium\u003csup\u003e62\u003c/sup\u003e, and maintained at\u0026nbsp;37°C and 5% CO\u003csub\u003e2\u003c/sub\u003e. TbDMT\u003csup\u003eOE\u003c/sup\u003e was selected and grown in 5 µg/mL blasticidin, \u003cem\u003eT. brucei\u003c/em\u003e 2T1 maintained in 0.5 µg/mL phleomycin and 0.2 µg/mL puromycin\u003csup\u003e63\u003c/sup\u003e and the resulting TbDMT\u003csup\u003eRNAi\u003c/sup\u003e selected with 0.5 µg/mL phleomycin and 2.5 µg/mL hygromycin after transfection\u003csup\u003e64\u003c/sup\u003e. \u003cem\u003eT. brucei\u003c/em\u003e 2T1\u003csup\u003eT7-Cas9\u003c/sup\u003e was grown in 2.5 µg/mL hygromycin and the generated \u003cem\u003eT. brucei\u003c/em\u003e DMT-Cas9 cells selected and grown in 2 µg/mL phleomycin following transfection\u003csup\u003e41\u003c/sup\u003e. Tetracycline at 1 µg/mL was added to the culture daily in order to induce RNA interference and double strand break in TbDMT RNAi and TbDMT-Cas9 cell lines, respectively.\u003c/p\u003e\n\u003cp\u003eTo determine parasite growth rate, cells were seeded at predetermined density and cell densities determined daily via haemocytometry. \u003cem\u003eT. congolense\u003c/em\u003e were detached via pipetting prior to assessing densities.\u003c/p\u003e\n\u003ch3\u003e\u003cstrong\u003eParasite transfection\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eBoth \u003cem\u003eT. congolense\u003c/em\u003e and \u003cem\u003eT. brucei\u003c/em\u003e were transfected with 10-15\u0026nbsp;µg linearised construct DNA. Briefly, per transfection, 2 - 4 × 10\u003csup\u003e7\u003c/sup\u003e parasites were centrifuged at 1,500 × \u003cem\u003eg\u003c/em\u003e for 10 minutes. The medium supernatant was poured off, leaving ~1 mL. Cells were resuspended and transferred to 1.5 mL eppendorf tubes, and centrifuged for 5 minutes at 1,500 × \u003cem\u003eg\u003c/em\u003e. Remaining supernatant was removed, cells washed in phosphate-buffered saline (P­BS; 137 mM NaCl, 3 mM KCl, 10 mM Na\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e, 1.8 mM KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e), subsequently resuspended in 100 µL transfection buffer\u003csup\u003e65\u003c/sup\u003e and transferred to an electroporation cuvette along with linearised vector, prior to electroporation using programme Z-001 on the Nucleofector II (Lonza). Cells were then transferred to 20 mL warm medium and allowed to grow. Selective antibiotics were added after 24 hours and cells were simultaneously cloned by diluting (1:50, 1:100 and 1:200) into 96-well plates. Resultant transfectants were isolated after ~7 days and were deemed clonal if \u0026lt;35 wells contained viable cells (based on a Poisson model of 1 cell per well).\u003c/p\u003e\n\u003ch3\u003e\u003cstrong\u003eParasite genetic manipulation\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eUnless stated otherwise, all cell line constructs were typically generated as follows: amplicons generated by PCR using \u003cem\u003eT. congolense\u003c/em\u003e or \u003cem\u003eT. brucei\u003c/em\u003e genomic DNA (gDNA) templates were first ligated into the pGEM-T easy vector using T4 DNA ligase (both Promega), prior to excision with construct-specific restriction enzymes (see S4 table for primer sequences and restriction enzyme information). Sanger sequencing was typically performed at this stage. Excised constructs were subsequently ligated into Antarctic phosphatase-treated (NEB) target vector using T4 ligase. Final vectors were linearised with plasmid-specific restriction enzymes prior to transfection into target cells as described above.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo generate the TcoDMT\u003csup\u003eOE\u003c/sup\u003e line, a previously-generated vector targeting the \u003cem\u003eT. congolense\u003c/em\u003e tubulin array for constitutive expressionwasused\u003csup\u003e31\u003c/sup\u003e (Table S4). The TcoDMT (gene ID: TcIL3000.A.H_000575200) ORF was amplified using primers containing ClaI and BamHI sites in the forward and reverse primer, respectively (Table S4). The final vector was linearised using AscI prior to electroporation. To generate TcoDMT\u003csup\u003edKO\u003c/sup\u003e constructs, sequential allelic replacement was used. Replacement cassettes were generated by amplifying a region of DNA encompassing the 5’-untranslated region (UTR), using forward and reverse primers containing a NotI and XbaI site, respectively. In addition, a region of DNA encompassing the 3’-UTR was amplified using forward and reverse primers containing a NsiI and XhoI site, respectively (Table S4). \u0026nbsp;To ligate the constructs into the final vectors, two rounds of digestion and ligation were carried out, one for each UTR. Two plasmids were generated, each with a different antibiotic resistance cassette (hygromycin and puromycin) flanked by the aforementioned UTRs. Constructs were linearised with NotI and XhoI prior to transfection. For the generation of the TcoDMT\u003csup\u003eRNAi\u003c/sup\u003e construct, a previously-generated RNAi vector was used\u003csup\u003e30\u003c/sup\u003e. A 503 bp fragment of the TcoDMT gene was amplified from \u003cem\u003eT. congolense\u003c/em\u003e IL3000 gDNA using forward and reverse primers flanked by FseI and HindIII sites, respectively (Table S4). The final vector was linearised with NotI prior to electroporation.\u003c/p\u003e\n\u003cp\u003eIn order to generate TbDMT\u003csup\u003eOE\u0026nbsp;\u003c/sup\u003ecells, the TbDMT gene (Tb927.8.1460) was amplified using forward and reverse primers containing XbaI and BamHI restriction sites respectively (Table S4) and the cloned into the pGL2271\u003csup\u003e31\u003c/sup\u003e final vector. The plasmid was linearised with AscI prior to electroporation. TbDMT RNAi stem-loop plasmids were constructed using two-step ligation with TbDMT fragment as described previously\u003csup\u003e64\u003c/sup\u003e. The forward primer contained KpnI and BamHI sites whilst the reverse primer containted ApaI and XbaI sites (Table S4). After excision from pGET-T easy, the gene fragments were cloned into the pRPa\u003csup\u003eiSL\u003c/sup\u003e vector in two steps. In the first step, construct and vector were digested with KpnI and BamHI and ligated to generate an intermediate RNAi vector\u003csup\u003e64\u003c/sup\u003e. In the second step, the original construct as well as aforementioned intermediate RNAi vector were digested using ApaI and XbaI and ligated together. Constructs were, linearised with AscI and transfected into \u003cem\u003eT. brucei\u003c/em\u003e 2T1 cells \u003csup\u003e63,64\u003c/sup\u003e.\u0026nbsp;Inducible CRISPR/Cas9 dKO cell lines of TbDMT were generated using two separate sgRNA pairs as previously described\u003csup\u003e41\u003c/sup\u003e. Potential sgRNAs were identified using the LeishGEdit.net platform and manually selected based on PAM-adjacent GC-content. AGGG and AAAC overhangs were added to the 5’ end of the forward and reverse oligo sequences respectively. Synthesised oligos were annealed by heating to 95°C followed by slowly cooling over 4 hours, and annealed oligos were subsequently ligated into BbsI-digested pT7\u003csup\u003esgRNA\u003c/sup\u003e backbone. Constructs were linearised using NotI prior to electroporation into \u003cem\u003eT. brucei\u003c/em\u003e 2T1\u003csup\u003eT7-Cas9\u003c/sup\u003e cells\u003csup\u003e41\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFor N-terminal tagging of DMT protein with superfolder GFP by modification of the endogenous locus, PCR amplicons containing ~750 bp from the 5’-end UTR (untranslated region) and ~750 bp from the N-terminal end of the CDS (coding sequence) of DMT were amplified from \u003cem\u003eT.\u0026nbsp;congolense\u003c/em\u003e IL3000 gDNA and cloned together into XbaI-BamHI sites downstream of the fluorescent protein ORF in pEnNY0\u003csup\u003e66\u003c/sup\u003e using standard methods (Table S4). In the same step, a NotI linearization site was introduced between the UTR and CDS. For N-terminal tagging of ectopic TcoDMT expression (TcoDMT\u003csup\u003eOE-sfGFP\u003c/sup\u003e), the same vector used to generate TcoDMT\u003csup\u003eOE\u003c/sup\u003e cells was further manipulated via insertion of sGFP at the 5’-end. The sfGFP ORF was amplified from the aforementioned pEnNY0 vector using primers flanked by ClaI sites and ligated into the TcoDMT\u003csup\u003eOE\u003c/sup\u003e vector digested with the same enzyme. Of note, the resulting vector was transformed into methyltransferase deficient (\u003cem\u003edam\u003csup\u003e–\u003c/sup\u003e\u003c/em\u003e/\u003cem\u003edcm\u003csup\u003e–\u003c/sup\u003e\u003c/em\u003e; NEB) chemically competent \u003cem\u003eE. coli\u003c/em\u003e, as ClaI is methylation-sensitive and the forward primer contained a potential CpG island. The resulting plasmid was linearised using AscI.\u003c/p\u003e\n\u003ch3\u003e\u003cstrong\u003eQuantitative Real-Time PCR\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eTotal RNA was extracted using a commercial kit (RNeasy; QIAgen) and a total of 1 µg RNA was reverse-transcribed using High-Capacity cDNA RT kit (Thermo Applied Biosystems). For RNAi studies, RNA samples were obtained from tetracycline-induced and uninduced samples daily for 3 days post-induction\u003csup\u003e7\u003c/sup\u003e. qPCR reactions were carried out with SensiFAST SYBR Hi-ROX mix (Bioline), in a BioRad Thermocycler (Biorad) as described\u003csup\u003e7\u003c/sup\u003e. Normalised transcript level was determined through the ΔΔCt method\u003csup\u003e67\u003c/sup\u003e using TcoTERT and TbTERT as endogenous controls (Table S4) for \u003cem\u003eT. congolense\u003c/em\u003e and \u003cem\u003eT. brucei\u003c/em\u003e, respectively\u003csup\u003e29,68\u003c/sup\u003e. To calculate TcoDMT copy number from genomic DNA, qPCR reactions were carried out as above using 2 ng gDNA. Expression levels, calculated using the ΔΔCt method, were then multiplied by 2 to account for the diploidy of the \u003cem\u003eT. congolense\u003c/em\u003e genome.\u003c/p\u003e\n\u003ch3\u003e\u003cstrong\u003eAlamar Blue Drug Sensitivity Assays\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eDrug sensitivities were determined using the resazurin-based Alamar blue assay as described previously\u003csup\u003e7,69\u003c/sup\u003e.\u0026nbsp;Briefly, two-fold serial dilutions of the test drug including drug-free control were prepared in 100 μL of culture medium in 96-well white opaque flat-bottomed plates (Cell Star, Greiner Bio-one). Parasites at log-phase of growth were harvested and distributed across the wells containing the drug dilution series at 100\u0026nbsp;μL/well, with final concentrations of 2 × 10\u003csup\u003e4\u003c/sup\u003e and 2.5 × 10\u003csup\u003e5\u003c/sup\u003e cells/mL for \u003cem\u003eT. brucei\u003c/em\u003e and \u003cem\u003eT. congolense\u003c/em\u003e, respectively. Plates were incubated for 48 h at the relevant growth conditions for each species, after which 20 μL of 125 mg/mL (0.49 mM) resazurin dye in PBS was added to each well, and the plate further incubated for 24 h. The endpoint fluorescence at λ\u003csub\u003eem\u003c/sub\u003e544 nm/λ\u003csub\u003eexc\u003c/sub\u003e590 nm was determined for each well of the plate using a Cytation 5 imaging reader (BioTek), and the EC\u003csub\u003e50\u003c/sub\u003e of each drug was calculated using non-linear regression fitted to a sigmoidal dose-response curve in GraphPad Prism (v10).\u003c/p\u003e\n\u003ch3\u003e\u003cstrong\u003eDrug Uptake Assays\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eThe rate of uptake of ISM was determined using the natural fluorescence of ISM as described\u003csup\u003e19\u003c/sup\u003e. Briefly,\u0026nbsp;1 ×10\u003csup\u003e7\u0026nbsp;\u003c/sup\u003ecells in assay buffer (33 mM HEPES, 98 mM NaCl, 4.6 mM KCl, 0.55 mM CaCl\u003csub\u003e2\u003c/sub\u003e, 0.07 mM MgSO\u003csub\u003e4\u003c/sub\u003e, 5.8 mM NaH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, 0.3 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 23 mM NaHCO\u003csub\u003e3\u003c/sub\u003e and 14 mM glucose, pH 7.3) were incubated with 10 µM ISM\u0026nbsp;for an intended duration, followed by centrifugation at 12,500 × \u003cem\u003eg\u003c/em\u003e for 1 min to stop the reaction and to separate the cells from the drug mix through an oil layer (7:1 v/v dibutylphthalate/mineral oil; Sigma-Aldrich). The oil and drug mix were removed by capillary suction and 50 μL of a 0.1 N HCl/methanol (1:8 v/v) mixture added to the tube to lyse the cell pellet. Following 1 h incubation at room temperature, samples were transferred to a 96-well plate and the fluorescence determined at λ\u003csub\u003eem\u003c/sub\u003e620 nm/λ\u003csub\u003eexc\u003c/sub\u003e355 nm using a Cytation 5 imaging reader. Uptake was reported as percentage of the maximum fluorescence in arbitrary units recorded in WT samples\u003csup\u003e70\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eFor the determination of radiolabelled ISM uptake, the cell pellet was collected and lysed as described previously for radiolabelled trypanocides\u003csup\u003e71\u003c/sup\u003e. Following incubation of cells with 1 µM [\u003csup\u003e14\u003c/sup\u003eC]-ISM (\u003csup\u003e14\u003c/sup\u003eC-Samorin), microcentrifuge tubes were flash frozen in liquid nitrogen, and the cell pellet cut off and transferred to a scintillation vial. Cell pellet was lysed in 2% SDS solution under slow agitation for 30 min and 3 mL of scintillation fluid (Scintilogic U, Lablogic) was subsequently added, followed by further incubation overnight in the dark. Radiation was measured in a 300SL scintillation counter (Hidex) and the rate of uptake per unit time was measured using GraphPad Prism version (v10).\u003c/p\u003e\n\u003ch3\u003e\u003cstrong\u003eFlow cytometry for the Measurement of Mitochondrial Membrane Potential\u0026nbsp;\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eMitochondrial membrane potential was assessed as described previously, with minor modifications\u003csup\u003e72\u003c/sup\u003e. Cells were harvested and adjusted to\u0026nbsp;1 ×10\u003csup\u003e6\u003c/sup\u003e cells/mL in 1 mL HMI-93 medium, and samples were either treated with 500 nM ISM for 3 h, 200 nM valinomycin for 1 h, 20 µM troglitazone for 1 h or left untreated. Thereafter, all samples were treated with\u0026nbsp;0.05 µM Mitotracker Red (Thermo Fisher Scientific) and incubated for 15 minutes at\u0026nbsp;37°C and 5% CO\u003csub\u003e2\u003c/sub\u003e. Cells were then washed in PBS at 1,500 × \u003cem\u003eg\u003c/em\u003e for 10 min, fixed in 4% paraformaldehyde for 5 min, then washed again and resuspended in PBS. Mitotracker Red fluorescence intensity was determined using a Fortessa X20 flow cytometer (BD Bioscience) set at\u0026nbsp;λ\u003csub\u003eem\u003c/sub\u003e578 nm/λ\u003csub\u003eexc\u003c/sub\u003e599 nm\u0026nbsp;and the data analysed using FlowJo software (BD Bioscience).\u0026nbsp;\u003c/p\u003e\n\u003ch3\u003e\u003cstrong\u003eNative fluorescence microscopy\u0026nbsp;\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eFor localisation of tagged DMT by native fluorescence, cells were harvested from mid-log phase cultures, washed twice in PBS and allowed to settle for 5 min onto glass slides at ~2\u0026nbsp;×\u0026nbsp;10\u003csup\u003e7\u003c/sup\u003e cells/mL density\u003csup\u003e30\u003c/sup\u003e. Cells were fixed for 5 min in 2% (w/v) formaldehyde, permeabilised in -20°C methanol, re-hydrated in PBS and incubated with 15 ng/mL 4,6-diamidino-2-phenylindole for 5 min, before mounting in 1% (w/v) 1,4-diazabicyclo[2.2.2]octane, 90% (v/v) glycerol, 50 mM sodium phosphate, pH 8.0\u003csup\u003e30\u003c/sup\u003e. Images were captured on an Olympus BX51 microscope equipped with a 100× UPlanApo objective (1.35 NA; Olympus) and Retiga R6 CCD camera (Qimaging) without binning. All fluorescent channel images were captured at equal exposure settings without prior illumination. Images for level comparison were also processed in parallel with the same alterations to minimum and maximum display levels. Image acquisition was controlled by μManager open source software\u003csup\u003e73\u003c/sup\u003e. Processing and analysis were performed in Fiji\u003csup\u003e74\u003c/sup\u003e. To plot pixel intensity profiles of native fluorescence images, a straight line covering 7 µm was drawn through the fluorescent panel. Data was imported into GraphPad Prism and the line was smoothed with the following parameters: Number of neighbours averaged: 5; Order of the smoothing polynomial: 2\u003csup\u003end\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eFor ISM fluorescence, a final concentration of 10 µM ISM was added to cells in PBS at a density of 2 × 10\u003csup\u003e7\u003c/sup\u003e cells/mL for 30 minutes prior to the aforementioned steps. Images were captured on a Zeiss Axio Observer Z.1 microscope equipped with a 63× Plan-Apochromat objective (1.4 NA; Zeiss) and a Axiocam 702 mono (Zeiss). All images were captured and processed as above.\u003c/p\u003e\n\u003ch3\u003e\u003cstrong\u003eTcoDMT read depth\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ein field isolate sequencing data\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eGenomic sequences of 56 \u003cem\u003eT. congolense\u003c/em\u003e isolates were obtained from EBI under the accession number PRJEB15251, previously deposited by Tihon and colleagues\u003csup\u003e42\u003c/sup\u003e. Of these, 21 had associated metadata indicating sensitivity to ISM (Table S1). These 21 genome sequences were subsequently aligned to the \u003cem\u003eT. congolense\u003c/em\u003e IL3000 2019 reference genome sequence (v51; TriTrypDB) using Hisat2\u003csup\u003e75\u003c/sup\u003e with the following parameters to ensure multimapping reads were aligned only once: “\u003cem\u003e-- no-spliced-alignment -k 1\u003c/em\u003e”. Using Samtools\u003csup\u003e76\u003c/sup\u003e, the resulting alignment files were filtered (“\u003cem\u003eview -bS -q 1 -F 0x100\u003c/em\u003e”) and sorted (“\u003cem\u003esort\u003c/em\u003e”) to generate .bam files.\u003c/p\u003e\n\u003cp\u003eTo obtain normalised read depth for each of the 21 alignments, the “bamCoverage” function from the DeepTools suite\u003csup\u003e77\u003c/sup\u003ewas employed. The following parameters were used: the output format was bedgraph (\u003cem\u003e“--outFileFormat “bedgraph”\u003c/em\u003e), a rolling bin size of 100 bp (“\u003cem\u003e--binSize 100\u003c/em\u003e”) and RPKM normalisation (“\u003cem\u003e--normalizeUsing RPKM\u003c/em\u003e”) and only chromosome 8 reads were normalised for analysis of the DMT region (\u003cem\u003e“--region Tc.A.H.pschr_08\u003c/em\u003e”). For other loci, the relevant chromosome was selected (chromosomes 11, 9 and 10 for TcoTERT, TcoFBP and TcoALD, respectively). A custom R script was then used to generate rolling mean coverages (rmCoverage) for each bedgraph file. This script made use of the “rollapply” function from the “zoo” package\u003csup\u003e78\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe “unionbedg” function from the bedtools suite\u003csup\u003e79\u003c/sup\u003e was used to merge all RPKM normalized bedgraph into one file for downstream analyses. To generate figures, the “Gviz” package for R was used\u003csup\u003e80\u003c/sup\u003e. The “\u003cem\u003eucscChromosomeNames=FALSE\u003c/em\u003e” option was applied in order to utilise a custom \u003cem\u003eT. congolense\u003c/em\u003e genome sequence using publicly available annotation and sequence files (v51; TriTrypDB). Figures were edited in Inkscape v1.2.\u003c/p\u003e\n\u003ch3\u003e\u003cstrong\u003e\u003cem\u003eIn silico\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;analysis of TcoDMT\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eThe protein sequence of TcoDMT was analysed using InterProScan\u003csup\u003e81\u003c/sup\u003e and domain graphics were generated using R. Colabfold\u003csup\u003e39\u003c/sup\u003e was used to generate a structural predication of TcoDMT, with default parameters. The model with highest rank was uploaded in FoldSeek to search for similar 3D protein structures. Images of overlaid protein structures were downloaded from FoldSeek.\u003c/p\u003e\n\u003ch3\u003e\u003cstrong\u003ePhylogenetics\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eSequences for all \u003cem\u003eH. sapiens\u003c/em\u003e SLC35 proteins were downloaded from Uniprot using the search term “SLC35*”. Previous studies identified eight NSTs in \u003cem\u003eT. brucei\u003c/em\u003e\u003cem\u003e\u003csup\u003e38\u003c/sup\u003e\u003c/em\u003e (strain TREU 927) and 11 NSTs in \u003cem\u003eT. cruzi\u0026nbsp;\u003c/em\u003e(strain CL Brener Non-Esmeraldo-like). The protein sequences for these NSTs were used to identify NSTs in \u003cem\u003eT. congolense\u003c/em\u003e and \u003cem\u003eL. major\u003c/em\u003e (strain Friedlin) via BLASTp searches in TriTrypDB. To further confirm all NSTs had been identified, the predicted proteomes from the aforementioned parasite species were downloaded from TriTrypDB and compared using Orthofinder\u003csup\u003e82\u003c/sup\u003e. A multiple sequence alignment of NSTs and SLC35 sequences (total of 55) was generated using Clustal Omega Multiple Sequence Aligner\u003csup\u003e83\u003c/sup\u003e with default settings and the Pearson/FASTA output format. Tree construction was carried out using iqtree2\u003csup\u003e84\u003c/sup\u003e with 1,000 bootstrap replicates (“\u003cem\u003e-B 1000\u003c/em\u003e”). The resulting tree was annotated and rendered using iTOL\u003csup\u003e85\u003c/sup\u003e.\u003c/p\u003e\n\u003ch3\u003e\u003cstrong\u003eMetabolite extractions and liquid chromatography-mass spectrometry\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eMetabolite extractions were carried out on both cell pellets and culture supernatants. Three replicates for each line were grown for 48 hours to a final density of 2\u0026nbsp;×\u0026nbsp;10\u003csup\u003e6\u003c/sup\u003e cells/mL, and 10\u003csup\u003e8\u003c/sup\u003e cells were rapidly quenched to 4°C in a dry ice/ethanol bath. All subsequent steps and incubations were carried out at 4°C. Samples were centrifuged at 1,250 × \u003cem\u003eg\u003c/em\u003e for 10 minutes after which, for supernatant samples, 5 µL was taken from each sample and added to 200 µL extraction solvent (chloroform:methanol:water in a 1:3:1 ratio) in 1.5 mL eppendorf tubes. The remaining supernatant was discarded and cells resuspended in 1 mL sterile PBS. After transferring cell pellet samples to 1.5 mL eppendorf tubes, they were centrifuged at 1,250 × \u003cem\u003eg\u0026nbsp;\u003c/em\u003efor 10 minutes. Supernatant was discarded and 200 µL extraction solvent (as above) was added to each sample. All samples were then incubated for 1 hour on a thermomixer at 4°C. Samples were centrifuged at 16,060 × \u003cem\u003eg\u003c/em\u003e for 10 minutes, and supernatants (~195 µL) transferred to new eppendorf tubes. As controls, 10 µL from each metabolite extraction was added to a new tube. Finally, air in the tubes was displaced using argon gas, and samples stored at -80°C prior to LC-MS analysis.\u003c/p\u003e\n\u003cp\u003eHydrophobic interaction liquid chromatography (HILIC) was carried out on a Dionex UltiMate 3000 RSLC system (Thermo Fisher) using a ZIC-pHILIC column (150 mm × 4.6 mm, 5 µm column; Merck SeQuant), at the University of Glasgow Shared Research Facility, UK. The column was maintained at 25°C and samples were eluted with a linear gradient (20 mM ammonium carbonate in water and acetonitrile) over 26 minutes at a flow rate of 0.3 mL/min. Injection volume was 10 µL and samples were maintained at 5°C prior to injection. For MS analysis, a Thermo Orbitrap QExactive (Thermo Fisher Scientific) was operated in polarity switching mode. The MS settings were as follows: Resolution: 70,000; AGC: 1e6; m/z range: 70-1,050; Sheath gas: 40; Auxiliary gas: 5; Sweep gas: 1; Probe temperature: 150°C; Capillary temperature: 320°C.\u0026nbsp;For positive mode ionisation: source voltage +3.8 kV, S-Lens RF Level 30.00, S-Lens Voltage 25.00 (V), Skimmer Voltage 15.00 (V), Inject Flatopole Offset 8.00 (V), Bent Flatapole DC 6.00 (V). For negative mode ionisation: source voltage-3.8 kV.\u0026nbsp;A set of authentic standards was run prior to the sample set.\u003c/p\u003e\n\u003ch3\u003e\u003cstrong\u003eMetabolomics analysis\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eMetabolomics data obtained in raw format were converted first to mzXML format and split into positive and negative polarity using msconvert\u003csup\u003e86\u003c/sup\u003e. Files were then converted to peakML format using XCMS, and were further processed using mzMatch\u003csup\u003e87\u003c/sup\u003e. Peak annotation and metabolite identification was carried out using IDEOM\u003csup\u003e88\u003c/sup\u003e. Data were analysed using MetaboAnalyst (v6.0)\u003csup\u003e89\u003c/sup\u003e. Prior to analysis, data were transformed (Log\u003csub\u003e10\u003c/sub\u003e) and pareto-scaled.\u0026nbsp;\u003c/p\u003e\n\u003ch3\u003e\u003cstrong\u003eComputational methods\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eStatistical analyses were carried out in GraphPad Prism or R\u003csup\u003e90\u003c/sup\u003e. For cartoon graphics (Fig 6), icons were downloaded from Bioicons. These icons were created by Servier (https://smart.servier.com) and are licensed under CC-BY 3.0 Unported (https://creativecommons.org/licenses/by/3.0).\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eP.S., M.A., C.G., J.vd.A., M.P. and L.M. conceptualised the study. P.S., M.A., E.M.R., H.d.K., C.G., M.P. and L.M. developed the methodology. P.S., M.A., E.P. and E.M.R. performed the investigation. P.S. conducted the formal analysis and curated the data. P.S. wrote the original draft of the manuscript and prepared the visualisations. P.S., M.A., F.M., M.P., H.A., J.vd.A., H.d.K., C.G. and L.M. reviewed and edited the manuscript. L.M. supervised the study. H.A., M.P. and L.M. acquired funding.\u003c/p\u003e\u003ch3\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eAll data generated or analysed during this study are included in this published article and its Supplementary Information files.\u003c/p\u003e\n\u003ch2\u003e\u003cstrong\u003eAcknowledgements:\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eP.C.S., M.A.U., E.P., and L.J.M. were supported by funding from the UK Biotechnology and Biological Sciences Research Council (BBSRC; BB/S00243X/1; BB/W000296/1). E.M.R. and C.G. were supported by a BBSRC Project Grant (BB/W005867/1) to CG. P.C.S. is supported by a BBSRC Discovery Fellowship (BB/X009807/7). The Roslin Institute is supported through core funding from the BBSRC (BS/E/D/20002173; BBS/E/RL/230002C). The authors thank Marjorie Bouchier at CEVA Africa for provision of Isometamidium chloride (Veridium), Drs Anna Raper and Arantza Esnal-Zufiaurre (Roslin Bioimaging) for microscopy support, the Mass Spectrometry Facility within the MVLS Shared Research Facilities (University of Glasgow) for provision of metabolomics data and support \u0026amp; assistance, and Prof Sir Michael Ferguson (University of Dundee) and Dr Samuel Duncan (University of Glasgow) for useful discussions regarding nucleotide sugar transporters and metabolism.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eWHO. Trypanosomiasis, human African (sleeping sickness).) (2023)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYaro, M., Munyard, K.A., Stear, M.J. \u0026amp; Groth, D.M. 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Isometamidium chloride is the only available drug that is both prophylactic and curative. Despite sustained reports of resistance since the 1970s, a definitive molecular mechanism of resistance remains unresolved in the clinically relevant pathogen species \u003cem\u003eTrypanosoma congolense\u003c/em\u003e. In this study, the role of a putative drug/metabolite transporter protein, TcoDMT, was validated via the analysis of \u003cem\u003ein vitro\u003c/em\u003e-derived mutants, showing that expression levels of this protein correlated strongly with isometamidium sensitivity. Functional analyses revealed that the protein is a cell surface phenanthridine transporter and, notably, copy number variation correlates with Isometamidium sensitivity in \u003cem\u003eT. congolense\u003c/em\u003e field isolates. This study validates, for the first time, a plasma membrane transporter with a defined role in phenanthridine action and resistance, advancing our understanding of drug resistance mechanisms in parasitic protists, and informing strategies to combat animal trypanosomosis.\u003c/p\u003e","manuscriptTitle":"A cell surface transporter mediates phenanthridine resistance in African trypanosomes","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-12 08:18:21","doi":"10.21203/rs.3.rs-8903249/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-04-27T11:06:12+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-17T15:20:27+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-06T12:38:08+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"287624239660966083110112919721415336395","date":"2026-03-26T16:16:43+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"234554030949062839367490493637955619852","date":"2026-03-26T14:01:16+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"177696096147363339306893606396258222211","date":"2026-03-25T10:17:52+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-03-03T03:48:30+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-03-02T16:35:23+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-02-21T16:23:21+00:00","index":"","fulltext":""},{"type":"submitted","content":"npj Antimicrobials and Resistance","date":"2026-02-17T17:06:11+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"npj-antimicrobials-and-resistance","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"npjamar","sideBox":"Learn more about [npj Antimicrobials and Resistance](http://www.nature.com/npjamar/)","snPcode":"44259","submissionUrl":"https://submission.springernature.com/new-submission/44259/3","title":"npj Antimicrobials and Resistance","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"NPJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a267639a-b5a1-4cb5-91ac-9ea5d91a4845","owner":[],"postedDate":"March 12th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":63818291,"name":"Health sciences/Diseases"},{"id":63818292,"name":"Biological sciences/Microbiology"}],"tags":[],"updatedAt":"2026-04-30T16:23:27+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-12 08:18:21","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8903249","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8903249","identity":"rs-8903249","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}