Evaluation of the basic functions of six calcium-dependent protein kinases in Toxoplasma gondii using CRISPR-Cas9 system

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• 作者：Jin-Lei Wang ; Si-Yang Huang ; Ting-Ting Li ; Kai Chen ; Hong-Rui Ning…
• 关键词：Toxoplasma gondii ; Calcium ; dependent protein kinases (CDPKs) ; CRISPR ; Cas9 system ; Gene functions
• 刊名：Parasitology Research
• 出版年：2016
• 出版时间：February 2016
• 年：2016
• 卷：115
• 期：2
• 页码：697-702
• 全文大小：3,368 KB
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• 作者单位：Jin-Lei Wang (1)
  Si-Yang Huang (1) (2)
  Ting-Ting Li (1)
  Kai Chen (1)
  Hong-Rui Ning (1)
  Xing-Quan Zhu (1) (2)
  
  1. State Key Laboratory of Veterinary Etiological Biology, Key Laboratory of Veterinary Parasitology of Gansu Province, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, Gansu Province, 730046, People’s Republic of China
  2. Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, Jiangsu Province, 225009, People’s Republic of China
  
• 刊物类别：Biomedical and Life Sciences
• 刊物主题：Biomedicine
  Medical Microbiology
  Microbiology
  Immunology
  
• 出版者：Springer Berlin / Heidelberg
• ISSN：1432-1955

文摘

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. Keywords Toxoplasma gondii Calcium-dependent protein kinases (CDPKs) CRISPR-Cas9 system Gene functions