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<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Functional expression of <italic>TcoAT1</italic> reveals it to be a P1-type nucleoside transporter with no capacity for diminazene uptake<sup><xref ref-type="fn" rid="d32e303">☆</xref></sup></title><author><name sortKey="Munday, Jane C" sort="Munday, Jane C" uniqKey="Munday J" first="Jane C." last="Munday">Jane C. Munday</name><affiliation><nlm:aff id="aff1">Wellcome Trust Centre for Molecular Parasitology, University of Glasgow, Glasgow, United Kingdom</nlm:aff></affiliation><affiliation><nlm:aff id="aff2">Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, United Kingdom</nlm:aff></affiliation></author><author><name sortKey="Rojas L Pez, Karla E" sort="Rojas L Pez, Karla E" uniqKey="Rojas L Pez K" first="Karla E." last="Rojas L Pez">Karla E. Rojas L Pez</name><affiliation><nlm:aff id="aff2">Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, United Kingdom</nlm:aff></affiliation></author><author><name sortKey="Eze, Anthonius A" sort="Eze, Anthonius A" uniqKey="Eze A" first="Anthonius A." last="Eze">Anthonius A. Eze</name><affiliation><nlm:aff id="aff2">Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, United Kingdom</nlm:aff></affiliation></author><author><name sortKey="Delespaux, Vincent" sort="Delespaux, Vincent" uniqKey="Delespaux V" first="Vincent" last="Delespaux">Vincent Delespaux</name><affiliation><nlm:aff id="aff3">Institute of Tropical Medicine, Nationalestraat 155, B-2000 Antwerp, Belgium</nlm:aff></affiliation></author><author><name sortKey="Van Den Abbeele, Jan" sort="Van Den Abbeele, Jan" uniqKey="Van Den Abbeele J" first="Jan" last="Van Den Abbeele">Jan Van Den Abbeele</name><affiliation><nlm:aff id="aff3">Institute of Tropical Medicine, Nationalestraat 155, B-2000 Antwerp, Belgium</nlm:aff></affiliation></author><author><name sortKey="Rowan, Tim" sort="Rowan, Tim" uniqKey="Rowan T" first="Tim" last="Rowan">Tim Rowan</name><affiliation><nlm:aff id="aff4">GALVmed, Pentlands Science Park, Bush Loan, Edinburgh, United Kingdom</nlm:aff></affiliation></author><author><name sortKey="Barrett, Michael P" sort="Barrett, Michael P" uniqKey="Barrett M" first="Michael P." last="Barrett">Michael P. Barrett</name><affiliation><nlm:aff id="aff1">Wellcome Trust Centre for Molecular Parasitology, University of Glasgow, Glasgow, United Kingdom</nlm:aff></affiliation><affiliation><nlm:aff id="aff2">Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, United Kingdom</nlm:aff></affiliation></author><author><name sortKey="Morrison, Liam J" sort="Morrison, Liam J" uniqKey="Morrison L" first="Liam J." last="Morrison">Liam J. Morrison</name><affiliation><nlm:aff id="aff1">Wellcome Trust Centre for Molecular Parasitology, University of Glasgow, Glasgow, United Kingdom</nlm:aff></affiliation><affiliation><nlm:aff id="aff2">Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, United Kingdom</nlm:aff></affiliation><affiliation><nlm:aff id="aff5">Roslin Institute, University of Edinburgh, Easter Bush, Midlothian, United Kingdom</nlm:aff></affiliation></author><author><name sortKey="De Koning, Harry P" sort="De Koning, Harry P" uniqKey="De Koning H" first="Harry P." last="De Koning">Harry P. De Koning</name><affiliation><nlm:aff id="aff2">Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, United Kingdom</nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">24533295</idno><idno type="pmc">3862423</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3862423</idno><idno type="RBID">PMC:3862423</idno><idno type="doi">10.1016/j.ijpddr.2013.01.004</idno><date when="2013">2013</date><idno type="wicri:Area/Pmc/Corpus">000379</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Functional expression of <italic>TcoAT1</italic> reveals it to be a P1-type nucleoside transporter with no capacity for diminazene uptake<sup><xref ref-type="fn" rid="d32e303">☆</xref></sup></title><author><name sortKey="Munday, Jane C" sort="Munday, Jane C" uniqKey="Munday J" first="Jane C." last="Munday">Jane C. Munday</name><affiliation><nlm:aff id="aff1">Wellcome Trust Centre for Molecular Parasitology, University of Glasgow, Glasgow, United Kingdom</nlm:aff></affiliation><affiliation><nlm:aff id="aff2">Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, United Kingdom</nlm:aff></affiliation></author><author><name sortKey="Rojas L Pez, Karla E" sort="Rojas L Pez, Karla E" uniqKey="Rojas L Pez K" first="Karla E." last="Rojas L Pez">Karla E. Rojas L Pez</name><affiliation><nlm:aff id="aff2">Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, United Kingdom</nlm:aff></affiliation></author><author><name sortKey="Eze, Anthonius A" sort="Eze, Anthonius A" uniqKey="Eze A" first="Anthonius A." last="Eze">Anthonius A. Eze</name><affiliation><nlm:aff id="aff2">Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, United Kingdom</nlm:aff></affiliation></author><author><name sortKey="Delespaux, Vincent" sort="Delespaux, Vincent" uniqKey="Delespaux V" first="Vincent" last="Delespaux">Vincent Delespaux</name><affiliation><nlm:aff id="aff3">Institute of Tropical Medicine, Nationalestraat 155, B-2000 Antwerp, Belgium</nlm:aff></affiliation></author><author><name sortKey="Van Den Abbeele, Jan" sort="Van Den Abbeele, Jan" uniqKey="Van Den Abbeele J" first="Jan" last="Van Den Abbeele">Jan Van Den Abbeele</name><affiliation><nlm:aff id="aff3">Institute of Tropical Medicine, Nationalestraat 155, B-2000 Antwerp, Belgium</nlm:aff></affiliation></author><author><name sortKey="Rowan, Tim" sort="Rowan, Tim" uniqKey="Rowan T" first="Tim" last="Rowan">Tim Rowan</name><affiliation><nlm:aff id="aff4">GALVmed, Pentlands Science Park, Bush Loan, Edinburgh, United Kingdom</nlm:aff></affiliation></author><author><name sortKey="Barrett, Michael P" sort="Barrett, Michael P" uniqKey="Barrett M" first="Michael P." last="Barrett">Michael P. Barrett</name><affiliation><nlm:aff id="aff1">Wellcome Trust Centre for Molecular Parasitology, University of Glasgow, Glasgow, United Kingdom</nlm:aff></affiliation><affiliation><nlm:aff id="aff2">Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, United Kingdom</nlm:aff></affiliation></author><author><name sortKey="Morrison, Liam J" sort="Morrison, Liam J" uniqKey="Morrison L" first="Liam J." last="Morrison">Liam J. Morrison</name><affiliation><nlm:aff id="aff1">Wellcome Trust Centre for Molecular Parasitology, University of Glasgow, Glasgow, United Kingdom</nlm:aff></affiliation><affiliation><nlm:aff id="aff2">Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, United Kingdom</nlm:aff></affiliation><affiliation><nlm:aff id="aff5">Roslin Institute, University of Edinburgh, Easter Bush, Midlothian, United Kingdom</nlm:aff></affiliation></author><author><name sortKey="De Koning, Harry P" sort="De Koning, Harry P" uniqKey="De Koning H" first="Harry P." last="De Koning">Harry P. De Koning</name><affiliation><nlm:aff id="aff2">Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, United Kingdom</nlm:aff></affiliation></author></analytic><series><title level="j">International Journal for Parasitology, Drugs and Drug Resistance</title><idno type="eISSN">2211-3207</idno><imprint><date when="2013">2013</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><sec><title>Graphical abstract</title><p><fig id="f0040" position="anchor"><graphic xlink:href="fx2"></graphic></fig></p></sec><sec><title>Highlights</title><p>► Diminazene transporter in <italic>Trypanosoma congolense</italic> has been proposed to be TcoAT1. ► Here, <italic>TcoAT1</italic> was cloned and functionally expressed in <italic>Trypanosoma brucei</italic>. ► TcoAT1 did not mediate the uptake of diminazene, only of purine nucleosides. ► Expression of TcoAT1 did not alter drug sensitivity in trypanosomes. ► We conclude that TcoAT1 is a transporter for purine nucleosides, not for diminazene.</p></sec></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Al Salabi, M I" uniqKey="Al Salabi M">M.I. 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<front><journal-meta><journal-id journal-id-type="nlm-ta">Int J Parasitol Drugs Drug Resist</journal-id><journal-id journal-id-type="iso-abbrev">Int J Parasitol Drugs Drug Resist</journal-id><journal-title-group><journal-title>International Journal for Parasitology, Drugs and Drug Resistance</journal-title></journal-title-group><issn pub-type="epub">2211-3207</issn><publisher><publisher-name>Elsevier</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">24533295</article-id><article-id pub-id-type="pmc">3862423</article-id><article-id pub-id-type="publisher-id">S2211-3207(13)00005-5</article-id><article-id pub-id-type="doi">10.1016/j.ijpddr.2013.01.004</article-id><article-categories><subj-group subj-group-type="heading"><subject>Article</subject></subj-group></article-categories><title-group><article-title>Functional expression of <italic>TcoAT1</italic> reveals it to be a P1-type nucleoside transporter with no capacity for diminazene uptake<sup><xref ref-type="fn" rid="d32e303">☆</xref></sup></article-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Munday</surname><given-names>Jane C.</given-names></name><xref rid="aff1" ref-type="aff">a</xref><xref rid="aff2" ref-type="aff">b</xref></contrib><contrib contrib-type="author"><name><surname>Rojas López</surname><given-names>Karla E.</given-names></name><xref rid="aff2" ref-type="aff">b</xref></contrib><contrib contrib-type="author"><name><surname>Eze</surname><given-names>Anthonius A.</given-names></name><xref rid="aff2" ref-type="aff">b</xref></contrib><contrib contrib-type="author"><name><surname>Delespaux</surname><given-names>Vincent</given-names></name><xref rid="aff3" ref-type="aff">c</xref></contrib><contrib contrib-type="author"><name><surname>Van Den Abbeele</surname><given-names>Jan</given-names></name><xref rid="aff3" ref-type="aff">c</xref></contrib><contrib contrib-type="author"><name><surname>Rowan</surname><given-names>Tim</given-names></name><xref rid="aff4" ref-type="aff">d</xref></contrib><contrib contrib-type="author"><name><surname>Barrett</surname><given-names>Michael P.</given-names></name><xref rid="aff1" ref-type="aff">a</xref><xref rid="aff2" ref-type="aff">b</xref></contrib><contrib contrib-type="author"><name><surname>Morrison</surname><given-names>Liam J.</given-names></name><xref rid="aff1" ref-type="aff">a</xref><xref rid="aff2" ref-type="aff">b</xref><xref rid="aff5" ref-type="aff">e</xref></contrib><contrib contrib-type="author"><name><surname>de Koning</surname><given-names>Harry P.</given-names></name><email>Harry.de-Koning@glasgow.ac.uk</email><xref rid="aff2" ref-type="aff">b</xref><xref rid="cor1" ref-type="corresp">⁎</xref></contrib></contrib-group><aff id="aff1"><label>a</label>Wellcome Trust Centre for Molecular Parasitology, University of Glasgow, Glasgow, United Kingdom</aff><aff id="aff2"><label>b</label>Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, United Kingdom</aff><aff id="aff3"><label>c</label>Institute of Tropical Medicine, Nationalestraat 155, B-2000 Antwerp, Belgium</aff><aff id="aff4"><label>d</label>GALVmed, Pentlands Science Park, Bush Loan, Edinburgh, United Kingdom</aff><aff id="aff5"><label>e</label>Roslin Institute, University of Edinburgh, Easter Bush, Midlothian, United Kingdom</aff><author-notes><corresp id="cor1"><label>⁎</label>Corresponding author. Address: Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, 120 University Place, Glasgow G12 8TA, United Kingdom. Tel.: +44 141 3303753; fax: +44 141 3306900. <email>Harry.de-Koning@glasgow.ac.uk</email></corresp></author-notes><pub-date pub-type="pmc-release"><day>10</day><month>2</month><year>2013</year></pub-date><pmc-comment> PMC Release delay is 0 months and 0 days and was
based on .</pmc-comment>
<pub-date pub-type="epub"><day>10</day><month>2</month><year>2013</year></pub-date><pub-date pub-type="collection"><month>12</month><year>2013</year></pub-date><volume>3</volume><fpage>69</fpage><lpage>76</lpage><history><date date-type="received"><day>14</day><month>12</month><year>2012</year></date><date date-type="rev-recd"><day>19</day><month>1</month><year>2013</year></date><date date-type="accepted"><day>23</day><month>1</month><year>2013</year></date></history><permissions><copyright-statement>© 2013 Published by Elsevier Ltd on behalf of Australian Society for Parasitology.</copyright-statement><copyright-year>2013</copyright-year><copyright-holder>Australian Society for Parasitology</copyright-holder><license license-type="open-access" xlink:href="http://creativecommons.org/licenses/by-nc-sa/3.0/"><license-p>This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike License, which permits non-commercial use, distribution, and reproduction in any medium, provided the original author and source are credited.</license-p></license></permissions><abstract abstract-type="graphical"><sec><title>Graphical abstract</title><p><fig id="f0040" position="anchor"><graphic xlink:href="fx2"></graphic></fig></p></sec><sec><title>Highlights</title><p>► Diminazene transporter in <italic>Trypanosoma congolense</italic> has been proposed to be TcoAT1. ► Here, <italic>TcoAT1</italic> was cloned and functionally expressed in <italic>Trypanosoma brucei</italic>. ► TcoAT1 did not mediate the uptake of diminazene, only of purine nucleosides. ► Expression of TcoAT1 did not alter drug sensitivity in trypanosomes. ► We conclude that TcoAT1 is a transporter for purine nucleosides, not for diminazene.</p></sec></abstract><abstract><p>It has long been established that the <italic>Trypanosoma brucei</italic> TbAT1/P2 aminopurine transporter is involved in the uptake of diamidine and arsenical drugs including pentamidine, diminazene aceturate and melarsoprol. Accordingly, it was proposed that the closest <italic>Trypanosoma congolense</italic> paralogue, <italic>TcoAT1</italic>, might perform the same function in this parasite, and an apparent correlation between a Single Nucleotide Polymorphism (SNP) in that gene and diminazene tolerance was reported for the strains examined. Here, we report the functional cloning and expression of <italic>TcoAT1</italic> and show that in fact it is the syntenic homologue of another <italic>T. brucei</italic> gene of the same Equilibrative Nucleoside Transporter (ENT) family: TbNT10. The <italic>T. congolense</italic> genome does not seem to contain a syntenic equivalent to <italic>TbAT1</italic>. Two <italic>TcoAT1</italic> alleles, differentiated by three independent SNPs, were expressed in the <italic>T. brucei</italic> clone B48, a <italic>TbAT1</italic>-null strain that further lacks the High Affinity Pentamidine Transporter (HAPT1); <italic>TbAT1</italic> was also expressed as a control. The <italic>TbAT1</italic> and <italic>TcoAT1</italic> transporters were functional and increased sensitivity to cytotoxic nucleoside analogues. However, only <italic>TbAT1</italic> increased sensitivity to diamidines and to cymelarsan. Uptake of [<sup>3</sup>H]-diminazene was detectable only in the B48 cells expressing <italic>TbAT1</italic> but not <italic>TcoAT1</italic>, whereas uptake of [<sup>3</sup>H]-inosine was increased by both <italic>TcoAT1</italic> alleles but not by <italic>TbAT1</italic>. Uptake of [<sup>3</sup>H]-adenosine was increased by all three ENT genes. We conclude that TcoAT1 is a P1-type purine nucleoside transporter and the syntenic equivalent to the previously characterised TbNT10; it does not mediate diminazene uptake and is therefore unlikely to play a role in diminazene resistance in <italic>T. congolense</italic>.</p></abstract><kwd-group><title>Keywords</title><kwd><italic>Trypanosoma congolense</italic></kwd><kwd>Diminazene aceturate</kwd><kwd>Drug resistance</kwd><kwd>TcoAT1</kwd></kwd-group></article-meta></front><body><sec id="s0005"><label>1</label><title>Introduction</title><p>The most common drug to treat animal trypanosomiasis in sub-Saharan Africa is the diamidine compound diminazene aceturate (DA) and the effective treatment of livestock with trypanocides remains of the highest importance for farmers in the tsetse belt where an estimated 46–62 million head of cattle are at risk of trypanosomiasis (<xref rid="b0235" ref-type="bibr">Swallow, 2000</xref>). Control of the disease is largely either by vector control or chemotherapy, of which an estimated 35 million doses are administered annually (<xref rid="b0135" ref-type="bibr">Geerts and Holmes, 1998</xref>). However, the effectiveness of DA and the only other common drug for trypanosomiasis, isometamidium chloride, are threatened by drug resistance (<xref rid="b0140 b0115" ref-type="bibr">Geerts et al., 2001; Delespaux et al., 2008a</xref>). In order to manage the situation effectively, it is necessary to understand the resistance mechanism and devise predictive tests that distinguish between drug sensitive and resistant populations (<xref rid="b0115" ref-type="bibr">Delespaux et al., 2008a</xref>).</p><p>The diamidine trypanocides were developed in the late 1930s (<xref rid="b0175" ref-type="bibr">Lourie and Yorke, 1939</xref>) and as early as 1944 Hawking reported that aromatic diamidines are actively accumulated by live but not by dead <italic>Trypanosoma brucei</italic> to intracellular concentrations up to three orders of magnitude higher than the extracellular concentration (<xref rid="b0155" ref-type="bibr">Hawking, 1944</xref>). He proposed that the massive drug accumulation, not observed in the surrounding blood cells, was the basis of the selective trypanocidal action. A similar accumulation, apparently energy dependent, has also been reported for DA (<xref rid="b0145 b0095" ref-type="bibr">Girgis-Takla and James, 1974; De Koning et al., 2004</xref>) and for pentamidine (<xref rid="b0065" ref-type="bibr">Damper and Patton, 1976</xref>), a related diamidine drug used to treat human African trypanosomiasis (<xref rid="b0105" ref-type="bibr">Delespaux and De Koning, 2007</xref>). Dependent on the extracellular concentration, about 50% of the uptake of pentamidine in <italic>T. b. brucei</italic> is mediated by the TbAT1/P2 transporter (<xref rid="b0045 b0085 b0020" ref-type="bibr">Carter et al., 1995; De Koning and Jarvis, 2001; Bray et al., 2003</xref>). The rest is transported by a High Affinity Pentamidine Transporter (HAPT1) and a Low Affinity Pentamidine Transporter (LAPT1) (<xref rid="b0070 b0075" ref-type="bibr">De Koning, 2001, 2008</xref>). However, the uptake of DA is almost exclusively through TbAT1/P2 (<xref rid="b0095" ref-type="bibr">De Koning et al., 2004</xref>), with only a very minor contribution from HAPT1 (<xref rid="b0245" ref-type="bibr">Teka et al., 2011</xref>). The most likely explanation for the differences in DA and pentamidine transport is the flexibility of the linker chain between the pentamidine benzamidine ends, allowing the molecule to assume many different conformations whereas the diminazene structure is rigid, locking the benzamidine moieties in a fixed position.</p><p>There is abundant evidence that diamidine resistance in <italic>T. brucei</italic> complex species is linked to loss of transport (<xref rid="b0105" ref-type="bibr">Delespaux and De Koning, 2007</xref>). A stilbamidine-resistant <italic>T. b. rhodesiense</italic> strain was deficient in accumulation of the drug (<xref rid="b0130" ref-type="bibr">Fulton and Grant, 1955</xref>). Loss of TbAT1/P2 and HAPT1 gives a high pentamidine resistance phenotype in <italic>T. b. brucei</italic> (<xref rid="b0025" ref-type="bibr">Bridges et al., 2007</xref>) and loss of the P2 aminopurine transport activity alone is sufficient to give substantial DA resistance in <italic>T. b. brucei</italic> (<xref rid="b0195 b0095" ref-type="bibr">Matovu et al., 2003; De Koning et al., 2004</xref>), <italic>Trypanosoma equiperdum</italic> (<xref rid="b0010" ref-type="bibr">Barrett et al., 1995</xref>) and <italic>Trypanosoma evansi</italic> (<xref rid="b0255" ref-type="bibr">Witola et al., 2004</xref>). However, the most important trypanosomatid pathogen for livestock in sub-Saharan Africa is <italic>Trypanosoma congolense</italic> and it is important to establish whether the same DA resistance model applies for this parasite. Additionally, the need for novel chemotherapeutic and chemoprophylactic tools for trypanosomiasis is grave, and given the range of diamidines available for development, understanding any mechanism of resistance will aid the exploitation of new members of this class of compounds. The most likely <italic>TbAT1</italic> orthologue in the <italic>T. congolense</italic> genome was identified as TcIL3000.5.2500 and it was named <italic>TcoAT1</italic> (<xref rid="b0110" ref-type="bibr">Delespaux et al., 2006</xref>). A recent in-depth analysis of <italic>Trypanosoma</italic> genomes confirmed that it is closely related (<xref rid="b0170" ref-type="bibr">Jackson, 2012</xref>). A Single Strand Conformation Polymorphism (SSCP) technique was used to try and establish whether <italic>TcoAT1</italic> polymorphisms might be associated with a diminazene sensitivity phenotype (<xref rid="b0110" ref-type="bibr">Delespaux et al., 2006</xref>), using a single dose mouse test (<xref rid="b0125" ref-type="bibr">Eisler et al., 2001</xref>) to report on resistance. This analysis found a strong correlation between SSCP pattern and drug sensitivity: out of 26 <italic>T. congolense</italic> strains 14 DA-sensitive and 9 resistant strains were correctly predicted using 20 mg/kg. The remaining 3 strains were predicted to be resistant by SSCP pattern, but were classified as sensitive by the mouse test, albeit with some infections relapsing, particularly at 5 mg/kg DA (<xref rid="b0110" ref-type="bibr">Delespaux et al., 2006</xref>). It was concluded that TcoAT1 was likely to be equivalent to the AT1 transporters in <italic>T. brucei</italic>, <italic>T. equiperdum</italic> and <italic>T. evansi</italic> and this seemed confirmed with the detection of an Ile306Val polymorphism in some of the <italic>TcoAT1</italic> alleles cloned from intermediate and highly resistant strains. The presence of this SNP was investigated by Restriction Fragment Length Polymorphism (RFLP) in all 26 strains and correlated perfectly with the observed resistance phenotype (<xref rid="b0110" ref-type="bibr">Delespaux et al., 2006</xref>); polymorphisms in <italic>TbAT1</italic> have been similarly associated with failure to transport melaminophenyl arsenicals (<xref rid="b0185 b0190" ref-type="bibr">Mäser et al., 1999; Matovu et al., 2001</xref>).</p><p>A further study on 11 <italic>T. congolense</italic> strains collected in Cameroon (<xref rid="b0180" ref-type="bibr">Mamoudou et al., 2008</xref>) and 12 strains collected in Ethiopia (<xref rid="b0200" ref-type="bibr">Moti et al., 2012</xref>) showed all strains to be DA-resistant by the single-dose mouse test and all the Cameroonian and only six of the Ethiopian being classified as ‘resistant’ by <italic>TcoAT1</italic> PCR–RFLP. However, these studies did not contain any ‘sensitive strains’ and thus did not in themselves rule out the trivial explanation that the <italic>TcoAT1</italic> PCR–RFLP simply identified the dominant polymorphism in <italic>T. congolense</italic> strains of the regions investigated. The observation that the RFLP-identified SNP was also common in <italic>T. congolense</italic> isolated from wildlife in areas with no history of prior trypanocide use (<xref rid="b0055" ref-type="bibr">Chitanga et al., 2011</xref>) does appear to show that this is a common polymorphism and unlikely to be the result of drug pressure.</p><p>Therefore, the similarity between <italic>TcoAT1</italic> and <italic>TbAT1</italic>, and the apparent association with DA-resistance in <italic>T. congolense</italic> are tempered by the lack of total correlation. Moreover, no functional expression or characterization has been performed for this member of Equilibrative Nucleoside Transporter (ENT) family. In order to provide a definitive test on whether TcoAT1 is involved in diamidine transport, and whether <italic>TcoAT1</italic> mutations are involved in resistance, we have expressed two separate <italic>TcoAT1</italic> alleles (‘sensitive’ and ‘resistant’) as well as <italic>TbAT1</italic>, in a well-characterised DA-resistant <italic>TbAT1</italic> null strain of <italic>T. b. brucei</italic> (<xref rid="b0025" ref-type="bibr">Bridges et al., 2007</xref>).</p></sec><sec sec-type="materials|methods" id="s0010"><label>2</label><title>Materials and methods</title><sec id="s0015"><label>2.1</label><title>Trypanosome strains and culturing</title><p>Bloodstream-form <italic>T. b. brucei</italic> strain B48 and its derivatives were maintained as previously described (<xref rid="b0025" ref-type="bibr">Bridges et al., 2007</xref>). The B48 strain is derived from the Lister 427 strain, but lacks the <italic>T. brucei AT-1</italic> gene and the high affinity pentamidine transporter (<xref rid="b0025" ref-type="bibr">Bridges et al., 2007</xref>).</p></sec><sec id="s0020"><label>2.2</label><title>Plasmid construction and transfection</title><p>The full <italic>TcoAT-1</italic> (TcIL3000_9_2500) gene was amplified from the sensitive <italic>T. congolense</italic> strain TRT12 (<xref rid="b0110" ref-type="bibr">Delespaux et al., 2006</xref>) and the resistant strain Alick 339 (<xref rid="b0120" ref-type="bibr">Delespaux et al., 2008b</xref>) (both isolated from cattle in Zambia), using the high-fidelity proof-reading polymerase Phusion (Finnzymes), with forward primer 5′-GGGCCCATGCTCGGTTTTGAATCC-3′ and reverse primer 5′-AGATCTTTACCACTCTGGCAGGGCCT-3′. The amplicons were ligated into the pGEM-T Easy sub-cloning vector (Promega) and sequenced using standard procedures.</p><p>The <italic>TcoAT1</italic> gene from the sensitive and resistant strains was then ligated into the expression vector pHD1336 (<xref rid="b0015" ref-type="bibr">Biebinger et al., 1997</xref>), to give pHDK45 and pHDK46 respectively. The <italic>TbAT1</italic> gene from <italic>T. brucei</italic> (Tb927.5.286b) was generated by high-fidelity PCR using Lister 427 strain gDNA, with forward primer 5′- GGGCCCATGCTCGGGTTTGACTCA-3′ and reverse primer 5′- GGATCCCTACTTGGGAAGCCCCTC-3′, and ligated into the pHD1336 expression vector to give pHDK44. All three constructs were linearised with <italic>Not</italic>I prior to transfection; B48 parasites were washed into Human T Cell Solution for transfection using the desired cassette with an Amaxa Nucleofector as described (<xref rid="b0035" ref-type="bibr">Burkard et al., 2007</xref>). Transfectants were grown and cloned out, by limiting dilution, in standard HMI-11 (<xref rid="b0165" ref-type="bibr">Hirumi and Hirumi, 1989</xref>) containing 5 μg ml<sup>−1</sup> blasticidin S.</p></sec><sec id="s0025"><label>2.3</label><title>Drug sensitivity assays</title><p>Drug sensitivities were assessed using the dye resazurin (Alamar blue) using a protocol adapted from <xref rid="b0210" ref-type="bibr">Räz et al. (1997)</xref>, as described (<xref rid="b0150" ref-type="bibr">Gould et al., 2008</xref>). Briefly, drugs were serially-diluted in 100 μl of complete HMI-11 media across two rows of a 96 well plate. Cultures of bloodstream-form trypanosomes were diluted to 2 × 10<sup>5</sup> cells/ml in complete HMI-11, and 100 μl was added to all wells. Plates were incubated for 48 h at 37 °C/5% CO<sub>2</sub>, prior to the addition of 20 μl of 5 mM resazurin sodium salt (Sigma–Aldrich) in PBS, pH7.4. Plates were incubated for a further 24 h in the same conditions, before fluorescence was measured using a FLUOstar Optima fluorimeter (BMG Labtech). Fifty percent inhibitory concentrations (IC<sub>50</sub>) were calculated using the sigmoidal curve algorithm of Prism 5 (GraphPad). Experiments were performed independently at least four times.</p></sec><sec id="s0030"><label>2.4</label><title>Transport assays</title><p>Uptake assays of [<sup>3</sup>H]-diminazene, [<sup>3</sup>H]-inosine and [<sup>3</sup>H]-adenosine were performed exactly as described (<xref rid="b0250 b0205" ref-type="bibr">Wallace et al., 2002; Natto et al., 2005</xref>). Briefly, trypanosomes from late-log phase cultures were washed into assay buffer (AB; 33 mM HEPES, 98 mM NaCl, 4.6 mM KCl, 0.55 mM CaCl<sub>2</sub>, 0.07 mM MgSO<sub>4</sub>, 5.8 mM NaH<sub>2</sub>PO<sub>4</sub>, 0.3 mM MgCl<sub>2</sub>, 23 mM NaHCO<sub>3</sub>, 14 mM glucose, pH 7.3) and resuspended at 10<sup>8</sup> cells ml<sup>−1</sup>. One hundred microlitres of cell suspension was incubated with either [Ring-<sup>3</sup>H]-DA (Perkin Elmer, 60.7 Ci/mmol), [2,8-<sup>3</sup>H]-adenosine (American Radiolabeled Chemicals Inc, 40 Ci/mmol) or [8-<sup>3</sup>H]-Inosine (American Radiolabeled Chemicals Inc., 20 Ci/mmol) in the presence or absence of unlabeled substrate or other competitive inhibitors. Incubations were stopped by the addition of 1 ml ice-cold unlabeled substrate (1 mM in AB) and centrifugation through oil (13,000×<italic>g</italic>; 1 min). The cell pellet was transferred to a scintillation tube and radioactivity was determined by liquid scintillation counting. The results were plotted to equations for linear or non-linear regression using Prism 5 (GraphPad) after correction for non-specific association of radiolabel with the pellet, as described (<xref rid="b0250" ref-type="bibr">Wallace et al., 2002</xref>).</p></sec></sec><sec sec-type="results" id="s0035"><label>3</label><title>Results</title><sec id="s0040"><label>3.1</label><title>Sequencing of TcoAT1 alleles</title><p>The <italic>TcoAT1</italic> alleles were PCR-amplified from genomic DNA of the TRT12 (‘sensitive’; <xref rid="b0110" ref-type="bibr">Delespaux et al., 2006</xref>) and Alick339 (‘resistant’; <xref rid="b0120" ref-type="bibr">Delespaux et al., 2008b</xref>) <italic>T. congolense</italic> isolates, and cloned into the pGEMT Easy vector prior to sequencing. From each strain, four independent clones were sequenced; sequences were identical within a strain, consistent with homozygosity for <italic>TcoAT1</italic> as originally suggested by the PCR–RFLP (<xref rid="b0110 b0115 b0120" ref-type="bibr">Delespaux et al., 2006; Delespaux et al., 2008a,b</xref>); Alick339 is a cloned strain, previously reported to be homozygous for the ‘resistant’ polymorphism (<xref rid="b0120" ref-type="bibr">Delespaux et al., 2008b</xref>). The nucleotide sequences of <italic>TcoAT1</italic> alleles from a DA ‘sensitive’ and a ‘resistant strain’ were determined and found to differ by 10 SNPs which do not affect the coding sequence, and 3 SNPs which cause amino acid alterations: Met75Thr, Ala262Gly and Val306Ile (<xref rid="s0070" ref-type="sec">Supplementary Fig. S1</xref>). These alleles and the <italic>T. brucei</italic> 427 <italic>TbAT1</italic> gene were cloned into the pHD1336 expression vector and expressed in the DA-resistant <italic>T. brucei</italic> B48 strain. Correct integration of the genes was established by PCR (not shown).</p></sec><sec id="s0045"><label>3.2</label><title>Genomic analysis of ENT genes in African trypanosomes</title><p>The premise that the gene named <italic>TcoAT1</italic> is equivalent to the known diamidine transporter <italic>TbAT1</italic> is based on it being the closest <italic>T. congolense</italic> paralogue identified from a database of unassembled (pre-genome) contigs. However, there is no syntenic equivalent of <italic>TbAT1</italic> (Tb927.5.286b) in the now-assembled <italic>T. congolense</italic> genome (TriTrypDB.org). Indeed, <italic>TcoAT1</italic> (TcIL3000.9.2500) is syntenic with another of the <italic>T. brucei</italic> ENT family genes, Tb09.160.5480 (<xref rid="b0100" ref-type="bibr">De Koning et al., 2005</xref>), which is known as TbNT10 or AT-B. This gene encodes a high affinity purine nucleoside transporter (<xref rid="b0225" ref-type="bibr">Sanchez et al., 2004</xref>) that has been characterised in detail (<xref rid="b0005" ref-type="bibr">Al-Salabi et al., 2007</xref>) and is preferentially expressed in short-stumpy (<xref rid="b0225" ref-type="bibr">Sanchez et al., 2004</xref>) and procyclic (<xref rid="b0005 b0230" ref-type="bibr">Al-Salabi et al., 2007; Spoerri et al., 2007</xref>) forms of <italic>T. brucei</italic>. A syntenic equivalent is also present in the <italic>T. b. gambiense</italic> genome, but not in <italic>Trypanosoma vivax</italic> or <italic>Leishmania</italic> species.</p><p><xref rid="f0005" ref-type="fig">Fig. 1</xref> shows a phylogenetic tree of ENT genes from <italic>T. b. brucei</italic>, <italic>T. congolense</italic>, <italic>Trypanosoma cruzi</italic>, and <italic>T. vivax</italic>, indicating different branches for nucleobase transporters, purine nucleoside transporters (‘P1-type’) and ‘P2-type’ transporters. The diagram clearly shows that all these species have nucleobase transporter genes, several of which have been characterised by heterologous expression (<xref rid="b0030 b0160" ref-type="bibr">Burchmore et al., 2003; Henriques et al., 2003</xref>) and P1-type nucleoside transporters, of which multiple members also have been functionally characterised (<xref rid="b0215 b0220 b0100" ref-type="bibr">Sanchez et al., 1999, 2002; De Koning et al., 2005</xref>), including TbNT10 (<xref rid="b0005 b0230" ref-type="bibr">Al-Salabi et al., 2007; Spoerri et al., 2007</xref>). In the tree, <italic>TcoAT1</italic> groups with the <italic>T. brucei</italic> P1-type transporters, and is closely related to Tb09.160.5480 (TbNT10), as would be predicted from the synteny. Formally the phylogenetic tree does not statistically differentiate between the P1 and P2-type transporters, though as noted above all of the <italic>T. b. brucei</italic> genes have been functionally confirmed as one type or the other. Therefore, the combination of clear synteny (with all the other <italic>T. b. brucei</italic> P1-type transporters being physically located on different chromosomes), and the phylogenetic grouping of <italic>TcoAT1</italic> with the P1-type transporters strongly suggests that <italic>TcoAT1</italic> is the <italic>T. congolense</italic> equivalent of <italic>TbNT10</italic>. In addition, a BLASTp search using the <italic>TcoAT1</italic> protein sequence as the query sequence returned all the P1-type nucleoside transporters as the highest <italic>T. b. brucei</italic> hits by maximum percentage identity, with <italic>TbNT10</italic> having one of the highest scores (67%; range of other P1-type transporters identity is from 67% to 61%). <italic>TbAT1</italic> had a maximum percentage identity of 54% (<xref rid="s0070" ref-type="sec">Supplementary Table S1</xref>). From data currently available, <italic>T. congolense</italic> does not seem to possess any P2-type transporter genes.</p></sec><sec id="s0050"><label>3.3</label><title>Sensitivity of TcoAT1 and TbAT1-expressing clones to nucleoside analogues and trypanocides</title><p>Functional expression of <italic>TbAT1</italic> and <italic>TcoAT1</italic> in the <italic>Tbb</italic> B48 clonal line was tested using a series of cytotoxic adenosine analogues. <xref rid="f0010" ref-type="fig">Fig. 2</xref> shows that introduction of <italic>TbAT1</italic> into this strain caused a profound increase in sensitivity to tubercidin (7-deazaadenosine; <xref rid="f0010" ref-type="fig">Fig. 2</xref>A), 5′-deoxyadenosine (5′-dAdo; <xref rid="f0010" ref-type="fig">Fig. 2</xref>B) and cordycepin (3′-deoxyadenosine; <xref rid="f0010" ref-type="fig">Fig. 2</xref>C), of 360 (<italic>P</italic> < 0.001), 20 (<italic>P</italic> < 0.01), and 23-fold (<italic>P</italic> < 0.01), respectively (<italic>n</italic> = 5). Expression of <italic>TcoAT1</italic> (‘sensitive allele’; <italic>TcoAT1</italic>S) also significantly increased sensitivity to these adenosine analogues, but by only 2–2.5-fold (all <italic>P</italic> < 0.05; <italic>n</italic> = 5). Sensitivity to phenylarsine oxide (PAO), a lipophilic arsenic compound, was unchanged in all strains (<xref rid="f0010" ref-type="fig">Fig. 2</xref>D).</p><p>The above results show that ENT transporters can be functionally expressed in the B48 strain and influence drug sensitivity. We thus proceeded with testing sensitivity to a number of trypanocides that have previously been shown to be substrates for TbAT1/P2: diminazene, pentamidine and cymelarsan (<xref rid="b0040 b0045 b0080 b0085 b0095" ref-type="bibr">Carter and Fairlamb, 1993; Carter et al., 1995; De Koning and Jarvis, 1999, 2001; De Koning et al., 2004</xref>). As expected, the expression of <italic>TbAT1</italic> in this strain greatly increased sensitivity to these drugs, by 219, 209 and 53-fold, respectively (all <italic>P</italic> < 0.01; <xref rid="f0020" ref-type="fig">Fig. 3</xref>A–C). However, expression of either the ‘sensitive’ or ‘resistant’ allele of <italic>TcoAT1</italic> did not alter drug sensitivity at all (<xref rid="f0015" ref-type="fig">Fig. 3</xref>). Sensitivity to PAO was not significantly different for any of the strains.</p></sec><sec id="s0055"><label>3.4</label><title>Transport of diminazene and of purine nucleosides by TcoAT1- and TbAT1-expressing clones</title><p>The above results appear to show that TcoAT1 is unable to transport any of the diamidine or melaminophenyl arsenical drugs tested, including diminazene. We therefore directly tested the uptake of [<sup>3</sup>H]-diminazene. Introduction of <italic>TbAT1</italic> into B48 trypanosomes greatly increased the rate of [<sup>3</sup>H]-diminazene uptake to 0.123 ± 0.005 pmol (10<sup>7</sup> cells)<sup>−1</sup> s<sup>−1</sup> (<italic>n</italic> = 3; <italic>r</italic><sup>2</sup> = 0.99) compared to 0.0093 ± 0.0046 pmol (10<sup>7</sup> cells)<sup>−1</sup> s<sup>−1</sup> in the empty vector control, whereas expression of <italic>TcoAT1</italic>S had no effect at all on the uptake rate (0.0093 ± 0.0043; <xref rid="f0020" ref-type="fig">Fig. 4</xref>). However, both <italic>TcoAT1</italic> alleles were clearly functionally expressed in B48 as both mediated transport of [<sup>3</sup>H]-inosine and [<sup>3</sup>H]-adenosine. Inosine uptake was highly significantly enhanced in B48 expressing <italic>TcoAT1</italic>S (<italic>P</italic> < 0.001), and to a slightly lesser extent by <italic>TcoAT1</italic>R (<italic>P</italic> < 0.01); in contrast, expression of <italic>TbAT1</italic> did not change [<sup>3</sup>H]-inosine uptake (<xref rid="f0025" ref-type="fig">Fig. 5</xref>A), consistent with the fact that inosine is not a substrate for this transporter (<xref rid="b0040 b0080 b0060" ref-type="bibr">Carter and Fairlamb, 1993; De Koning and Jarvis, 1999; Collar et al., 2009</xref>). Uptake of adenosine was increased (<italic>P</italic> < 0.001) by all three transporter genes, relative to control (<xref rid="f0025" ref-type="fig">Fig. 5</xref>B). The rate of adenosine transport was <italic>TbAT1 </italic>> <italic>TcoAT1</italic>R > <italic>TcoAT1</italic>S > control.</p></sec></sec><sec sec-type="discussion" id="s0060"><label>4</label><title>Discussion</title><p>Animal African Trypanosomiasis (AAT) remains one of the major challenges to livestock farming in sub-Saharan Africa and, as the control of the disease is mostly through chemotherapy with a few decades-old drugs, the large number of reports on drug resistance is a cause of great concern as it threatens the sustainability of agriculture in many areas (<xref rid="b0135 b0235 b0140 b0115" ref-type="bibr">Geerts and Holmes, 1998; Swallow, 2000; Geerts et al., 2001; Delespaux et al., 2008a</xref>). However, it is not clear whether the increased number of reports on diminazene aceturate (DA) treatment failure in cattle reflect a spread of resistant parasites, or simply a more intense focus on the problem with more studies performed in different parts of the continent. It is difficult to establish the full scale of the problem given the infrastructural and geographical challenges, particularly with the difficulties in confirming parasitological cure, prevention of re-infection, and in follow-up of treated animals. A further confounding factor is the use of counterfeit products and the frequent administration of veterinary trypanocides by untrained people. Therefore, the most robust test for resistance has required isolation of the parasite and testing in a standardised mouse-model (<xref rid="b0125 b0180" ref-type="bibr">Eisler et al., 2001; Mamoudou et al., 2008</xref>). However, this approach is expensive and laborious, and involves growth and treatment in a different animal species, a process with its own potential selective bottlenecks.</p><p>Clearly, a molecular test for a genetic resistance marker, based on an understanding of the resistance mechanism, has the potential to revolutionise our understanding of the spread of drug-resistant parasites (<xref rid="b0110 b0115" ref-type="bibr">Delespaux et al., 2006, 2008a</xref>). The identification of the P2/AT1 transporter as the principal entry route for diminazene, and the demonstration that genetic loss of this transporter led to substantial levels of diminazene resistance in <italic>T. b. brucei</italic> (<xref rid="b0195 b0095" ref-type="bibr">Matovu et al., 2003; De Koning et al., 2004</xref>), <italic>T. equiperdum</italic> (<xref rid="b0010" ref-type="bibr">Barrett et al., 1995</xref>), and <italic>T. evansi</italic> (<xref rid="b0255" ref-type="bibr">Witola et al., 2004</xref>) appeared to have provided an opportunity for a genetic test. The closest <italic>T. congolense</italic> orthologue to P2/AT1 was identified from a contig database and an SNP that statistically associated with reduced diminazene sensitivity was found (<xref rid="b0110" ref-type="bibr">Delespaux et al., 2006</xref>). A PCR–RFLP system developed to identify the resistance-associated SNP in field isolates was implemented to screen for diminazene resistance in various regions of Africa, including Cameroon (<xref rid="b0180" ref-type="bibr">Mamoudou et al., 2008</xref>), Eastern Zambia (<xref rid="b0120" ref-type="bibr">Delespaux et al., 2008b</xref>), the Ghibe river basin in Ethiopia (<xref rid="b0200" ref-type="bibr">Moti et al., 2012</xref>) and game reserves/national parks in Zambia, South Africa and Zimbabwe (<xref rid="b0055" ref-type="bibr">Chitanga et al., 2011</xref>). The latter study revealed the occurrence of the ‘resistant’ PCR–RFLP marker even in regions where the drug had not been used and it has become clear that the polymorphism initially suggested as resistance-associated is common in <italic>T. congolense</italic> regardless of drug exposure, and does not always correlate with high levels of diminazene resistance.</p><p>We have thus undertaken a study to verify (1) whether <italic>TcoAT1</italic> does indeed encode a P2-type transporter, (2) whether <italic>TcoAT1</italic>S confers sensitivity to diminazene and diminazene transport capacity and (3) whether there is any substantial difference in transport phenotype between <italic>TcoAT1</italic>S and <italic>TcoAT1</italic>R. <italic>TcoAT1</italic> alleles were amplified and cloned from known and well-characterised <italic>T. congolense</italic> isolates and verified by sequencing. We found that the <italic>TcoAT1</italic> sequence, while closely related to <italic>TbAT1</italic> and from the same nucleoside/nucleobase transporter gene family, is in fact not the orthologue of <italic>TbAT1</italic> but is the syntenic orthologue of <italic>TbNT10</italic>, a different but well-characterised purine nucleoside transporter of the P1-type. The P1 versus P2 distinction was first defined in <italic>T. brucei</italic> by <xref rid="b0040" ref-type="bibr">Carter and Fairlamb (1993)</xref>, where P1-type adenosine transporters are sensitive to competitive inhibition by inosine, whereas adenosine uptake by P2 is inhibited by adenine. Further characterization showed the P1 transporters, of which the <italic>T. b. brucei</italic> genome contains numerous copies (<xref rid="b0220 b0100" ref-type="bibr">Sanchez et al., 2002; De Koning et al., 2005</xref>), to be high affinity transporters for the purine nucleosides adenosine, guanosine and inosine (<xref rid="b0090 b0080 b0220" ref-type="bibr">De Koning et al., 1998; De Koning and Jarvis, 1999; Sanchez et al., 2002</xref>) whereas P2 transports only the aminopurines, adenosine and adenine (<xref rid="b0040 b0080" ref-type="bibr">Carter and Fairlamb, 1993; De Koning and Jarvis, 1999</xref>). The <italic>T. b. brucei</italic> genome appears to contain only 1 copy of <italic>TbAT1</italic>, which can be knocked out by two rounds of homologous recombination (<xref rid="b0195" ref-type="bibr">Matovu et al., 2003</xref>). Numerous studies have documented the involvement of P2 in uptake of diamidines and melaminophenyl arsenicals (see reviews by <xref rid="b0050" ref-type="bibr">Carter et al. (1999)</xref> and <xref rid="b0075" ref-type="bibr">De Koning (2008)</xref>), for which the transporter displays sub-micromolar affinity (<xref rid="b0080" ref-type="bibr">De Koning and Jarvis, 1999</xref>). In contrast, the P1-type transporters displayed two orders of magnitude less affinity for these trypanocides (<xref rid="b0080" ref-type="bibr">De Koning and Jarvis, 1999</xref>) and have never been implicated in their transport.</p><p>Our observations that <italic>TcoAT1</italic> increases sensitivity to cytotoxic nucleoside analogues but not to diminazene, pentamidine (both diamidines) or cymelarsan (a melaminophenyl arsenical) are consistent with a classification as P1-type transporter. Expression of <italic>TbAT1</italic> in the same system, used as positive control, did increase drug sensitivity dramatically. The strong and saturable capacity for transport of both inosine and adenosine by <italic>TcoAT1</italic> is completely inconsistent with a P2-type activity but a hallmark of P1 (<xref rid="b0040 b0080" ref-type="bibr">Carter and Fairlamb, 1993; De Koning and Jarvis, 1999</xref>).</p><p>In summary, we conclude that <italic>TcoAT1</italic> encodes a P1-type transporter and is not involved in diminazene uptake. It is clear that <italic>TcoAT1</italic> was incorrectly identified as the P2 transporter equivalent of <italic>T. congolense</italic>, and is instead the syntenic orthologue of <italic>TbNT10</italic> and we therefore propose that <italic>TcoAT1</italic> be renamed <italic>TcoNT10</italic>. Whether polymorphisms in <italic>TcoAT1</italic>/<italic>NT10</italic>, like the one identified by the PCR–RFLP (<xref rid="b0110" ref-type="bibr">Delespaux et al., 2006</xref>) could nonetheless be informative with respect to diminazene resistance now seems unlikely given that the transporter itself does not transport diminazene. Several recent papers have found that this allele is present in most <italic>T. congolense</italic> isolates from across Africa (<xref rid="b0120 b0180 b0055 b0200" ref-type="bibr">Delespaux et al., 2008b; Mamoudou et al., 2008; Chitanga et al., 2011; Moti et al., 2012</xref>), leading to the conclusion that either diminazene resistance is near-complete in the <italic>T. congolense</italic> population, or the proposed resistance allele is a common variant of the transporter instead of a determinant of drug sensitivity. The balance of current evidence suggests the latter possibility - a conclusion strengthened by the observation that the polymorphism was found in at least 1 allele of 33 out of 34 isolates from wild-life without a history of drug exposure (<xref rid="b0055" ref-type="bibr">Chitanga et al., 2011</xref>). Nevertheless, we cannot rule out the possibility that a particular <italic>TcoAT1</italic>/<italic>NT10</italic> allele might be associated with DA resistance through genetic linkage to a resistance determinant elsewhere on the same chromosome. 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Parasitol.</source><volume>107</volume><year>2004</year><fpage>47</fpage><lpage>57</lpage><pub-id pub-id-type="pmid">15208037</pub-id></element-citation></ref></ref-list><sec id="s0070" sec-type="supplementary-material"><label>Appendix A</label><title>Supplementary data</title><p><supplementary-material content-type="local-data" id="m0005"><caption><title>Supplementary data 1</title></caption><media xlink:href="mmc1.pdf"></media></supplementary-material></p></sec><ack><title>Acknowledgements</title><p>The authors gratefully acknowledge funding from the <funding-source id="GS1">Global Alliance for Livestock Veterinary Medicines (GALVmed)</funding-source> and the <funding-source id="GS2">United Kingdom Department for International Development (DFID)</funding-source> for the investigation of diminazene resistance in <italic>Trypanosoma congolense</italic>. This work was supported by the Wellcome Trust. The Wellcome Trust Centre for Molecular Parasitology is supported by core funding from the Wellcome Trust [085349]. L.J.M. is a Royal Society University Research Fellow.</p></ack><fn-group><fn id="d32e303"><label>☆</label><p>This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike License, which permits non-commercial use, distribution, and reproduction in any medium, provided the original author and source are credited.</p></fn></fn-group></back><floats-group><fig id="f0005"><label>Fig. 1</label><caption><p>Phylogenetic tree of ENT genes in trypanosomatid species <italic>T. b. brucei</italic> (red), <italic>T. congolense</italic> (blue), <italic>T. vivax</italic> (green) and <italic>T. cruzi</italic> (black). Separate clusters for nucleobase transporters, P1-type nucleoside transporters and P2-type transporters are indicated with boxes of dashed lines. The positions of <italic>TbAT1</italic> (Tb927.5.286b), <italic>TbNT10</italic> (Tb09.160.5480) and <italic>TcoAT1</italic> (TcIL3000.9.2500) are indicated with arrows. The tree was constructed using the Maximum Likelihood Tree function of MEGA5, with 500 bootstrap replications and, otherwise, the default parameters (<xref rid="b0240" ref-type="bibr">Tamura et al., 2011</xref>). The percentage of trees in which the associated taxa clustered together is shown next to the branches. The scale bar represents 0.5 amino acid substitutions per position. The tree is rooted with a divergent <italic>T. cruzi</italic> sequence (TcCLB.506445.10). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)</p></caption><graphic xlink:href="gr1"></graphic></fig><fig id="f0010"><label>Fig. 2</label><caption><p>Sensitivity of various <italic>T. b. brucei</italic> B48-derived cell lines to adenosine analogues. A, tubercidin; B, 5′-deoxyadenosine; C, cordycepin; D, phenylarsine oxide (arsenical control). The strains were all derived from B48 (<xref rid="b0025" ref-type="bibr">Bridges et al., 2007</xref>), by transfection with the empty vector (EV) pHD1336 or the same vector containing <italic>TbAT1</italic> or <italic>TcoAT1</italic> ‘sensitive variant’ (<italic>TcoAT1</italic>S). Bars show the average and SEM of 5 independent determinations, using the Alamar blue assay. <sup>∗</sup><italic>P</italic> < 0.05; <sup>∗∗</sup>P < 0.01; <sup>∗∗∗</sup>P < 0.001, Student <italic>T</italic>-test, relative to B48 + EV.</p></caption><graphic xlink:href="gr2"></graphic></fig><fig id="f0015"><label>Fig. 3</label><caption><p>Sensitivity of <italic>T. b. brucei</italic> bloodstream forms of various clonal lines to diminazene (A), pentamidine (B), cymelarsan (C) and phenylarsine oxide (D), as determined using standard Alamar blue-based assay. All clonal strains were derived from the B48 line, which is highly resistant to diamidines and melaminophenyl arsenicals (<xref rid="b0025" ref-type="bibr">Bridges et al., 2007</xref>). The B48 strain was transformed with either empty vector pHD1336 (EV), <italic>TbAT1</italic>, <italic>TcoAT1</italic> ‘sensitive variant’ (<italic>TcoAT1</italic>S) or <italic>TcoAT1</italic> ‘resistant variant’ (<italic>TcoAT1</italic>R) and for each two independent clones were tested with identical results; only one is shown. Bar graphs represent the average and standard error of 4–5 independent experiments. <sup>∗∗</sup><italic>P</italic> < 0.01 by unpaired Student’s <italic>T</italic>-test, relative to B48 + EV.</p></caption><graphic xlink:href="gr3"></graphic></fig><fig id="f0020"><label>Fig. 4</label><caption><p>Diminazene uptake in transformed <italic>T. b. brucei</italic> B48. B48 cells transformed with either vector without insert (circles), <italic>TbAT1</italic> (squares) or <italic>TcoAT1</italic>S (up triangles) were incubated with 0.1 μM [<sup>3</sup>H]-diminazene with (open symbols) or without (filled symbols) 1 mM unlabeled diminazene aceturate for up to 5 min as indicated. Symbols represent the average and SEM of three independent experiments, each performed in triplicate. Lines were calculated by linear regression; only uptake of 0.1 μM [<sup>3</sup>H]-diminazene by B48 expressing <italic>TbAT1</italic> was significantly different from zero (<italic>P</italic> < 0.0001, <italic>F</italic>-test; <italic>r</italic><sup>2</sup> > 0.99). Diminazene uptake by cells expressing <italic>TbAT1</italic> was significantly higher than in control cells (B48 with empty vector). <sup>∗∗</sup><italic>P</italic> < 0.01; <sup>∗∗∗</sup><italic>P</italic> < 0.001 (relative to EV control; Student’s <italic>T</italic>-test).</p></caption><graphic xlink:href="gr4"></graphic></fig><fig id="f0025"><label>Fig. 5</label><caption><p>Uptake of (A) inosine and (B) adenosine in transformed <italic>T. b. brucei</italic> B48. B48 cells transformed with either vector without insert (circles), <italic>TbAT1</italic> (squares), <italic>TcoAT1</italic>S (up triangles) or <italic>TcoAT1</italic>R (down triangles) were incubated with 0.1 μM [<sup>3</sup>H]-inosine (A) or 0.1 lM [<sup>3</sup>H]-adenosine (B) in the presence (open symbols and dotted lines) or absence (filled symbols and continuous lines) of 1 mM unlabeled permeant for up to 2 min as indicated. In the presence of 1 mM permeant uptake was inhibited by >99%, showing that uptake was saturable and therefore most likely transporter-mediated. Inosine uptake by cells expressing the <italic>TcoAT1</italic> alleles was significantly higher than in control cells (B48 with empty vector). Data shown are the average and SEM of three independent experiments, each performed in triplicate. <sup>∗</sup><italic>P</italic> < 0.05; <sup>∗∗</sup><italic>P</italic> < 0.01; <sup>∗∗∗</sup><italic>P</italic> < 0.001 (Student’s <italic>T</italic>-test). <italic>Note:</italic> in frame B each of the experimental data points were significantly different from the empty vector control at 20 s, by <italic>P</italic> < 0.01 or better.</p></caption><graphic xlink:href="gr5"></graphic></fig></floats-group></pmc></record>Pour manipuler ce document sous Unix (Dilib)
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