Evaluation of the basic functions of six calcium-dependent protein kinases in Toxoplasma gondii using CRISPR-Cas9 system
Evaluation of the basic functions of six calcium-dependent protein kinases in Toxoplasma gondii using CRISPR-Cas9 system
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利用CRISPR-Cas9系统评价弓形虫中6种钙依赖性蛋白激酶的基本功能
DOI：
10.1007/s00436-015-4791-6
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发表时间：
2016-02-01
影响因子：
2
通讯作者：
Zhu, Xing-Quan
中科院分区：
文献类型：
--
作者：
Wang, Jin-Lei;Huang, Si-Yang;Zhu, Xing-Quan
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被证明是针对弓形虫化疗的有希望的药物靶点，因为它们在弓形虫中发挥重要作用并且不存在于宿主中。本研究的目的是研究弓形虫中六种钙依赖性蛋白激酶（CDPK4、CDPK4A、CDPK5、CDPK6、CDPK8 和 CDPK9）的功能，以评估它们是否适合设计为药物靶点。我们利用CRISPR-Cas9系统，通过在6个内源CDPK位点的指导RNA靶向区域插入DHFR*，成功破坏了6个CDPK基因，并成功获得了6个敲除（KO）-CDPK菌株。分别通过噬菌斑测定、侵袭、流出、复制和毒力测定来评估六种菌株的生物学特性。结果表明，6株KO-CDPK菌株与野生型菌株的毒力和裂解周期（包括入侵、外出和复制）没有显着差异。结论是这六种CDPK对于弓形虫裂解周期不是必需的，也不是小鼠的毒力因子，这表明这六种CDPK可能参与弓形虫的其他功能。
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.