背景:根尖丛寄生虫东方巴贝虫,中国水牛巴贝斯虫病的病原体,在中国中部和南部很普遍,每年造成巨大的经济损失。目前,没有有效的疫苗或药物来对抗这种疾病。据报道,牛巴贝斯虫和恶性疟原虫具有一个含有可被膦霉素抑制的甲基赤藓糖醇磷酸(MEP)途径的原生质体,这表明该途径可以作为筛选新药的药物靶标。然而,它在B.Orientalis中仍然未知。
方法:根据小巴贝斯虫和牛巴贝斯虫的7个MEP通路基因设计引物。这些基因被克隆,测序和分析。前两个酶基因的开放阅读框(ORFs),1-脱氧-D-木酮糖5-磷酸合酶(BoDXS)和1-脱氧-D-木酮糖5-磷酸还原异构酶(BoDXR),将其克隆到pET-32a表达载体中并表达为Trx-tag融合蛋白。产生兔抗rBoDXS和兔抗rBoDXR抗体。进行Western印迹以鉴定东方芽孢杆菌中的BoDXS和BoDXR的天然蛋白质。使用福司霉素和香叶基香叶醇进行抑制测定和挽救测定。分别,在东方芽孢杆菌的体外培养中。
结果:东方芽孢杆菌MEP途径的七个酶基因(DXS,DXR,IspD,IspE,IspF,IspG和IspH)被克隆和测序,全长2094、1554、1344、1521、654、1932和1056bp,分别。BoDXS和BoDXR表达为Trx标签融合蛋白,大小为95和67kDa,分别。Western印迹鉴定了天然BoDXS的77kDa条带和天然BoDXR的49kDa条带。药物测定结果表明,膦霉素能抑制侧柏的生长,和香叶基香叶醇可以逆转膦霉素的作用。
结论:东方巴贝虫具有类异戊二烯生物合成途径,这可能是控制和治疗巴贝西虫病的潜在药物靶标。考虑到膦霉素的高价格和不稳定性,进一步的研究应集中在稳定和廉价药物的筛选上。
BACKGROUND: The apicomplexan parasite Babesia orientalis, the causative agent of water buffalo babesiosis in
China, is widespread in central and south
China, resulting in a huge economic loss annually. Currently, there is no effective vaccine or drug against this disease. Babesia bovis and Plasmodium falciparum were reported to possess an apicoplast which contains the methylerythritol phosphate (MEP) pathway inhibitable by fosmidomycin, suggesting that the pathway could serve as a drug target for screening new drugs. However, it remains unknown in B. orientalis.
METHODS: Primers were designed according to the seven MEP pathway genes of Babesia microti and Babesia bovis. The genes were cloned, sequenced and analyzed. The open reading frames (ORFs) of the first two enzyme genes, 1-deoxy-D-xylulose 5-phosphate synthase (BoDXS) and 1-Deoxy-D-xylulose 5-phosphate reductoisomerase (BoDXR), were cloned into the pET-32a expression vector and expressed as a Trx-tag fusion protein. Rabbit anti-rBoDXS and rabbit anti-rBoDXR antibodies were generated. Western blot was performed to identify the native proteins of BoDXS and BoDXR in B. orientalis. Fosmidomycin and geranylgeraniol were used for inhibition assay and rescue assay, respectively, in the in vitro cultivation of B. orientalis.
RESULTS: The seven enzyme genes of the B. orientalis MEP pathway (DXS, DXR, IspD, IspE, IspF, IspG and IspH) were cloned and sequenced, with a full length of 2094, 1554, 1344, 1521, 654, 1932 and 1056 bp, respectively. BoDXS and BoDXR were expressed as Trx-tag fusion proteins, with a size of 95 and 67 kDa, respectively. Western blot identified a 77 kDa band for the native BoDXS and a 49 kDa band for the native BoDXR. The drug assay results showed that fosmidomycin could inhibit the growth of B. orientalis, and geranylgeraniol could reverse the effect of fosmidomycin.
CONCLUSIONS: Babesia orientalis has the
isoprenoid biosynthesis pathway, which could be a potential drug target for controlling and curing babesiosis. Considering the high price and instability of fosmidomycin, further studies should focus on the screening of stable and cheap drugs.