Diminazene resistance in Trypanosoma congolense is not caused by reduced transport capacity but associated with reduced mitochondrial membrane potential
Diminazene resistance in Trypanosoma congolense is not caused by reduced transport capacity but associated with reduced mitochondrial membrane potential
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DOI：
10.1111/mmi.14733
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发表时间：
2021-05-18
影响因子：
3.6
通讯作者：
De Koning, Harry P.
中科院分区：
文献类型：
--
作者：
Carruthers, Lauren, V;Munday, Jane C.;De Koning, Harry P.
刚果锥虫是导致非洲牲畜锥虫病的主要病原体，给发展中经济体造成数十亿美元的损失，并破坏粮食安全。目前常用的只有联胺类抗菌药物和菲的异丙胺类药物，耐药性普遍存在，但人们对其了解甚少。我们在体外诱导了弓形虫IL3000菌株对氮杂环烯的稳定抗性。除类似物DB829和DB75外，与菲类药物、三聚氰胺类砷化合物、恶硼醇类杀菌剂及联胺类杀菌剂均无交叉耐药性。荧光显微镜观察显示，叶酸抑制了DB75的积累。对[H-3]-Dinazene的摄取较慢，亲和力较低，部分但被叶酸和竞争性二胺相互抑制。铜绿假单胞菌叶酸转运蛋白在布氏锥虫中的表达显著增强了该细胞对Dinazene和DB829的敏感性，但对奥博洛尔AN7973的敏感性不明显。然而，[H-3]-减氮烯转运研究、全基因组测序和RNA-SEQ没有发现减氮烯摄取、叶酸转运体序列或表达的重大变化。相反，所有抗性克隆都表现出线粒体膜电位psim的适度降低。我们得出结论，龙须草对氮烯的吸收是通过包括叶酸转运蛋白在内的多种低亲和力机制进行的；虽然抗性与psi m的降低有关，但尚不清楚这是否是产生抗性的主要原因。
Trypanosoma congolense is a principal agent causing livestock trypanosomiasis in Africa, costing developing economies billions of dollars and undermining food security. Only the diamidine diminazene and the phenanthridine isometamidium are regularly used, and resistance is widespread but poorly understood. We induced stable diminazene resistance in T. congolense strain IL3000 in vitro. There was no cross-resistance with the phenanthridine drugs, melaminophenyl arsenicals, oxaborole trypanocides, or with diamidine trypanocides, except the close analogs DB829 and DB75. Fluorescence microscopy showed that accumulation of DB75 was inhibited by folate. Uptake of [H-3]-diminazene was slow with low affinity and partly but reciprocally inhibited by folate and by competing diamidines. Expression of T. congolense folate transporters in diminazene-resistant Trypanosoma brucei brucei significantly sensitized the cells to diminazene and DB829, but not to oxaborole AN7973. However, [H-3]-diminazene transport studies, whole-genome sequencing, and RNA-seq found no major changes in diminazene uptake, folate transporter sequence, or expression. Instead, all resistant clones displayed a moderate reduction in the mitochondrial membrane potential psi m. We conclude that diminazene uptake in T. congolense proceed via multiple low affinity mechanisms including folate transporters; while resistance is associated with a reduction in psi m it is unclear whether this is the primary cause of the resistance.