Wang Jin-Lei, Huang Si-Yang, Li Ting-Ting, Chen Kai, Ning Hong-Rui, Zhu Xing-Quan
Parasitol Res. 2016 Feb;115(2):697-702. doi: 10.1007/s00436-015-4791-6.
Toxoplasma gondii, an important protozoan parasite, infects almost all warm-blooded animals and humans. Although treatments in T. gondii are limited by the lack of effective drugs, some calcium-dependent kinases were demonstrated as the promising drug targets to chemotherapy against T. gondii due to their essential roles in T. gondii and absence from their hosts. The objectives of the present study were to investigate the functions of six calcium-dependent protein kinases (CDPK4, CDPK4A, CDPK5, CDPK6, CDPK8, and CDPK9) in T. gondii to assess whether they are suitable for designing as drug targets. We used the CRISPR-Cas9 system to disrupt six CDPK genes successfully by insertion of DHFR* at the guide RNA-targeted region in the six endogenous CDPK loci and successfully obtained the six knockout (KO)-CDPK strains. The biological characteristics of the six strains were evaluated by plaque assays, invasion, egress, replication, and virulence assays, respectively. The results indicated that there was no significant difference between the six KO-CDPK strains and wild-type strain in virulence and the lytic cycle including invasion, egress, and replication. The conclusion was the six CDPKs are not essential for T. gondii lytic cycle and also not virulence factors for mice, suggesting that the six CDPKs may participate in other functions in T. gondii.
刚地弓形虫是一种重要的原生动物寄生虫，几乎感染所有温血动物和人类。尽管针对刚地弓形虫的治疗因缺乏有效药物而受到限制，但一些钙依赖性激酶因其在刚地弓形虫中的关键作用以及在其宿主中不存在，被证明是抗刚地弓形虫化疗的有前景的药物靶点。本研究的目的是调查六种钙依赖性蛋白激酶（CDPK4、CDPK4A、CDPK5、CDPK6、CDPK8和CDPK9）在刚地弓形虫中的功能，以评估它们是否适合被设计为药物靶点。我们使用CRISPR-Cas9系统通过在六个内源性CDPK基因座的引导RNA靶向区域插入DHFR*成功破坏了六个CDPK基因，并成功获得了六个敲除（KO）-CDPK菌株。分别通过噬斑测定、侵袭、逸出、复制和毒力测定评估了这六个菌株的生物学特性。结果表明，六个KO-CDPK菌株与野生型菌株在毒力以及包括侵袭、逸出和复制在内的裂解周期方面没有显著差异。结论是这六种CDPK对刚地弓形虫的裂解周期不是必需的，对小鼠也不是毒力因子，这表明这六种CDPK可能在刚地弓形虫中参与其他功能。
