Zheng Jun, Cheng Ziying, Jia Honglin, Zheng Yonghui
Harbin Veterinary Research Institute, CAAS-Michigan State University Joint Laboratory of Innate Immunity, State Key Laboratory of Veterinary Biotechnology, Chinese Academy of Agricultural Sciences, Maduan Street 427, Nangang District, Harbin 150001, P. R. China.
Sci Rep. 2016 Sep 28;6:34448. doi: 10.1038/srep34448.
Aminopeptidases have emerged as new promising drug targets for the development of novel anti-parasitic drugs. An aspartyl aminopeptidase-like gene has been identified in the Toxoplasma gondii genome (TgAAP), although its function remains unknown. In this study, we characterized TgAAP and performed functional analysis of the gene product. Firstly, we expressed a functional recombinant TgAAP (rTgAAP) protein in Escherichia coli, and found that it required metal ions for activity and showed a substrate preference for N-terminal acidic amino acids Glu and Asp. Then, we evaluated the function and drug target potential of TgAAP using the CRISPR/Cas9 knockout system. Western blotting demonstrated the deletion of TgAAP in the knockout strain. Indirect immunofluorescence analysis showed that TgAAP was localized in the cytoplasm of the wild-type parasite, but was not expressed in the knockout strain. Phenotype analysis revealed that TgAAP knockout inhibited the attachment/invasion, replication, and substrate-specific activity in T. gondii. Finally, the activity of drug CID 23724194, previously described as targeting Plasmodium and malarial parasite AAP, was tested against rTgAAP and the parasite. Overall, TgAAP knockout affected the growth of T. gondii but did not completely abolish parasite replication and growth. Therefore, TgAAP may comprise a useful adjunct drug target of T. gondii.
氨肽酶已成为新型抗寄生虫药物开发中颇具潜力的新药物靶点。在刚地弓形虫基因组中已鉴定出一个天冬氨酰氨肽酶样基因（TgAAP），但其功能仍不清楚。在本研究中，我们对TgAAP进行了表征，并对该基因产物进行了功能分析。首先，我们在大肠杆菌中表达了一种具有功能的重组TgAAP（rTgAAP）蛋白，发现它的活性需要金属离子，并且对N端酸性氨基酸谷氨酸和天冬氨酸表现出底物偏好性。然后，我们使用CRISPR/Cas9敲除系统评估了TgAAP的功能和药物靶点潜力。蛋白质免疫印迹法证明了敲除菌株中TgAAP的缺失。间接免疫荧光分析表明，TgAAP定位于野生型寄生虫的细胞质中，但在敲除菌株中不表达。表型分析显示，TgAAP敲除抑制了刚地弓形虫的附着/入侵、复制和底物特异性活性。最后，测试了先前描述为靶向疟原虫和疟原虫AAP的药物CID 23724194对rTgAAP和该寄生虫的活性。总体而言，TgAAP敲除影响了刚地弓形虫的生长，但并未完全消除寄生虫的复制和生长。因此，TgAAP可能是刚地弓形虫一个有用的辅助药物靶点。
