Hostettler Isabel, Müller Joachim, Stephens Chad E, Haynes Richard, Hemphill Andrew
Institute for Parasitology, Vetsuisse Faculty, University of Berne, Länggass-Strasse 122, 3012 Bern, Switzerland.
Department of Chemistry and Physics, Augusta State University, Augusta, GA 30904-2200, USA.
Int J Parasitol Drugs Drug Resist. 2014 Sep 19;4(3):201-9. doi: 10.1016/j.ijpddr.2014.09.003. eCollection 2014 Dec.
Intracellular schizonts of the apicomplexans Theileria annulata and Theileria parva immortalize bovine leucocytes thereby causing fatal immunoproliferative diseases. Buparvaquone, a hydroxynaphthoquinone related to parvaquone, is the only drug available against Theileria. The drug is only effective at the onset of infection and emerging resistance underlines the need for identifying alternative compounds. Current drug assays employ monitoring of proliferation of infected cells, with apoptosis of the infected host cell as a read-out, but it is often unclear whether active compounds directly impair the viability of the parasite or primarily induce host cell death. We here report on the development of a quantitative reverse transcriptase real time PCR method based on two Theileria genes, tasp and tap104, which are both expressed in schizonts. Upon in vitro treatment of T. annulata infected bovine monocytes with buparvaquone, TaSP and Tap104 mRNA expression levels significantly decreased in relation to host cell actin already within 4 h of drug exposure, while significant differences in host cell proliferation were detectable only after 48-72 h. TEM revealed marked alterations of the schizont ultrastructure already after 2 h of buparvaquone treatment, while the host cell remained unaffected. Expression of TaSP and Tap104 proteins showed a marked decrease only after 24 h. Therefore, the analysis of expression levels of mRNA coding for TaSP and Tap104 allows to directly measuring impairment of parasite viability. We subsequently applied this method using a series of compounds affecting different targets in other apicomplexan parasites, and show that monitoring of TaSP- and Tap104 mRNA levels constitutes a suitable tool for anti-theilerial drug development.
顶复门寄生虫环形泰勒虫和小泰勒虫的细胞内裂殖体能使牛白细胞永生化，从而引发致命的免疫增殖性疾病。布帕喹酮是一种与帕罗喹酮相关的羟基萘醌，是唯一可用于治疗泰勒虫的药物。该药物仅在感染初期有效，而新出现的耐药性凸显了识别替代化合物的必要性。目前的药物检测方法是监测感染细胞的增殖情况，并以感染宿主细胞的凋亡作为读数，但通常不清楚活性化合物是直接损害寄生虫的活力，还是主要诱导宿主细胞死亡。我们在此报告基于泰勒虫的两个基因tasp和tap104开发的定量逆转录酶实时PCR方法，这两个基因在裂殖体中均有表达。用布帕喹酮对感染环形泰勒虫的牛单核细胞进行体外处理后，在药物暴露4小时内，相对于宿主细胞肌动蛋白，TaSP和Tap104 mRNA表达水平就显著下降，而宿主细胞增殖的显著差异仅在48 - 72小时后才能检测到。透射电子显微镜显示，布帕喹酮处理2小时后，裂殖体超微结构就有明显改变，但宿主细胞未受影响。TaSP和Tap104蛋白的表达仅在24小时后才显著下降。因此，分析编码TaSP和Tap104的mRNA表达水平可以直接测量寄生虫活力的损害情况。我们随后使用一系列影响其他顶复门寄生虫不同靶点的化合物应用了该方法，并表明监测TaSP和Tap104 mRNA水平是抗泰勒虫药物开发的合适工具。
