PDF(Pubmed)
Abstract：
Tropical theileriosis is a fatal leukemic-like disease of cattle caused by the tick-transmitted protozoan parasite Theileria annulata. The economics of cattle meat and milk production is severely affected by theileriosis in endemic areas. The hydroxynaphtoquinone buparvaquone (BPQ) is the only available drug currently used to treat clinical theileriosis, whilst BPQ resistance is emerging and spreading in endemic areas. Here, we chronically exposed T. annulata-transformed macrophages in vitro to BPQ and monitored the emergence of drug-resistant parasites. Surviving parasites revealed a significant increase in BPQ IC50 compared to the wild type parasites. Drug resistant parasites from two independent cloned lines had an identical single mutation, M128I, in the gene coding for T. annulata cytochrome B (Tacytb). This in vitro generated mutation has not been reported in resistant field isolates previously, but is reminiscent of the methionine to isoleucine mutation in atovaquone-resistant Plasmodium and Babesia. The M128I mutation did not appear to exert any deleterious effect on parasite fitness (proliferation and differentiation to merozoites). To gain insight into whether drug-resistance could have resulted from altered drug binding to TaCytB we generated in silico a 3D-model of wild type TaCytB and docked BPQ to the predicted 3D-structure. Potential binding sites cluster in four areas of the protein structure including the Q01 site. The bound drug in the Q01 site is expected to pack against an alpha helix, which included M128, suggesting that the change in amino acid in this position may alter drug-binding. The in vitro generated BPQ resistant T. annulata is a useful tool to determine the contribution of the various predicted docking sites to BPQ resistance and will also allow testing novel drugs against theileriosis for their potential to overcome BPQ resistance.
摘要：
热带Theileriosis是由tick传播的原生动物寄生虫Theileriaannulata引起的致命的牛白血病样疾病。在流行地区，牛肉和牛奶的生产经济受到严重影响。羟基萘醌-丁巴伐醌（BPQ）是目前用于治疗临床Theileriosis的唯一可用药物，而BPQ抗性正在流行地区出现并蔓延。这里,我们在体外长期暴露于BPQ并监测耐药寄生虫的出现。与野生型寄生虫相比，存活的寄生虫显示BPQIC50显着增加。来自两个独立克隆系的耐药寄生虫具有相同的单突变，M128I,在编码环抱毛囊细胞色素B(Tacytb)的基因中。这种体外产生的突变以前在抗性领域分离株中没有报道过，但让人联想到甲硫氨酸到异亮氨酸突变在抗阿托夫酮的疟原虫和巴贝虫。M128I突变似乎对寄生虫适应性(增殖和分化为裂殖子)没有任何有害作用。为了深入了解耐药性是否可能是由于药物与TaCytB的结合改变而引起的，我们在计算机上生成了野生型TaCytB的3D模型，并将BPQ与预测的3D结构对接。潜在的结合位点聚集在包括Q01位点的蛋白质结构的四个区域中。预计Q01位点的结合药物会与α螺旋相反，其中包括M128，这表明该位置的氨基酸变化可能会改变药物结合。体外产生的BPQ抗性T.annulata是确定各种预测的对接位点对BPQ抗性的贡献的有用工具，并且还将允许测试针对Theleriosis的新型药物克服BPQ抗性的潜力。
