Wellcome Trust Centre for Molecular Parasitology, University of Glasgow, Glasgow, United Kingdom ; Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, United Kingdom.
Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, United Kingdom.
Int J Parasitol Drugs Drug Resist. 2013 Feb 10;3:69-76. doi: 10.1016/j.ijpddr.2013.01.004. eCollection 2013 Dec.
It has long been established that the Trypanosoma brucei 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 Trypanosoma congolense paralogue, TcoAT1, 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 TcoAT1 and show that in fact it is the syntenic homologue of another T. brucei gene of the same Equilibrative Nucleoside Transporter (ENT) family: TbNT10. The T. congolense genome does not seem to contain a syntenic equivalent to TbAT1. Two TcoAT1 alleles, differentiated by three independent SNPs, were expressed in the T. brucei clone B48, a TbAT1-null strain that further lacks the High Affinity Pentamidine Transporter (HAPT1); TbAT1 was also expressed as a control. The TbAT1 and TcoAT1 transporters were functional and increased sensitivity to cytotoxic nucleoside analogues. However, only TbAT1 increased sensitivity to diamidines and to cymelarsan. Uptake of [(3)H]-diminazene was detectable only in the B48 cells expressing TbAT1 but not TcoAT1, whereas uptake of [(3)H]-inosine was increased by both TcoAT1 alleles but not by TbAT1. Uptake of [(3)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 T. congolense.
长期以来，人们已经确定布氏锥虫 TbAT1/P2 氨嘌呤转运蛋白参与二脒嗪和砷剂药物的摄取，包括戊二脒、乙酰苯肼和米拉美隆。因此，人们提出，最接近的刚果锥虫 TcoAT1 类似物可能在这种寄生虫中发挥相同的功能，并且在检查的菌株中，该基因中的单核苷酸多态性 (SNP) 与乙酰苯肼耐受性之间存在明显的相关性。在这里，我们报告了 TcoAT1 的功能克隆和表达，并表明事实上它是布氏锥虫同一平衡核苷转运蛋白 (ENT) 家族中另一个基因 TbNT10 的同源物。刚果锥虫基因组似乎不包含与 TbAT1 同源的基因。通过三个独立的 SNP 区分的两种 TcoAT1 等位基因在 T. brucei 克隆 B48 中表达，B48 是一种 TbAT1 缺失株，进一步缺乏高亲和力戊二脒转运蛋白 (HAPT1)；TbAT1 也作为对照表达。TbAT1 和 TcoAT1 转运蛋白均具有功能，并增加了对细胞毒性核苷类似物的敏感性。然而，只有 TbAT1 增加了对二脒嗪和噻咪隆的敏感性。只有在表达 TbAT1 的 B48 细胞中才能检测到 [(3)H]-苯肼的摄取，但不能检测到 TcoAT1 的摄取，而 [(3)H]-肌苷的摄取则被两种 TcoAT1 等位基因增加，但 TbAT1 则没有。所有三种 ENT 基因均可增加 [(3)H]-腺苷的摄取。我们得出结论，TcoAT1 是一种 P1 型嘌呤核苷转运蛋白，是先前表征的 TbNT10 的同源物；它不介导乙酰苯肼摄取，因此不太可能在刚果锥虫的乙酰苯肼耐药中发挥作用。
