Kato Kentaro, Sugi Tatsuki, Takemae Hitoshi, Takano Ryo, Gong Haiyan, Ishiwa Akiko, Horimoto Taisuke, Akashi Hiroomi
National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro, Hokkaido, 080-8555, Japan.
Department of Veterinary Microbiology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo, 113-8657, Japan.
Parasit Vectors. 2016 Jul 21;9(1):405. doi: 10.1186/s13071-016-1676-1.
Toxoplasma gondii is an obligate intracellular parasite of the phylum Apicomplexa and a major pathogen of animals and immunocompromised humans, in whom it causes encephalitis. Understanding the mechanism of tachyzoite invasion is important for the discovery of new drug targets and may serve as a model for the study of other apicomplexan parasites. We previously showed that Plasmodium falciparum expresses a homolog of human calcium calmodulin-dependent protein kinase (CaMK) that is important for host cell invasion. In this study, to identify novel targets for the treatment of Toxoplasma gondii infection (another apicomplexan parasite), we sought to identify a CaMK-like protein in the T. gondii genome and to characterize its role in the life-cycle of this parasite.
An in vitro kinase assay was performed to assess the phosphorylation activities of a novel CaMK-like protein in T. gondii by using purified proteins with various concentrations of calcium, calmodulin antagonists, or T. gondii glideosome proteins. Indirect immunofluorescence microscopy was performed to detect the localization of this protein kinase by using the antibodies against this protein and organellar maker proteins of T. gondii.
We identified a novel CaMK homolog in T. gondii, T. gondii CaMK-related kinase (TgCaMKrk), which exhibits calmodulin-independent autophosphorylation and substrate phosphorylation activity. However, calmodulin antagonists had no effect on its kinase activity. In T. gondii-infected cells, TgCaMKrk localized to the apical ends of extracellular and intracellular tachyzoites. TgCaMKrk phosphorylated TgGAP45 for phosphorylation in vitro.
Our data improve our understanding of T. gondii motility and infection, the interaction between parasite protein kinases and glideosomes, and drug targets for protozoan diseases.
刚地弓形虫是顶复门的专性细胞内寄生虫，是动物和免疫功能低下人群的主要病原体，可导致这些人群发生脑炎。了解速殖子入侵机制对于发现新的药物靶点很重要，并且可作为研究其他顶复门寄生虫的模型。我们之前表明，恶性疟原虫表达一种对宿主细胞入侵很重要的人钙调蛋白依赖性蛋白激酶（CaMK）的同源物。在本研究中，为了确定治疗刚地弓形虫感染（另一种顶复门寄生虫）的新靶点，我们试图在刚地弓形虫基因组中鉴定一种CaMK样蛋白，并表征其在该寄生虫生命周期中的作用。
通过使用具有不同浓度钙、钙调蛋白拮抗剂或刚地弓形虫滑行体蛋白的纯化蛋白，进行体外激酶测定以评估刚地弓形虫中一种新型CaMK样蛋白的磷酸化活性。通过使用针对该蛋白的抗体和刚地弓形虫的细胞器标记蛋白，进行间接免疫荧光显微镜检查以检测该蛋白激酶的定位。
我们在刚地弓形虫中鉴定出一种新型CaMK同源物，即刚地弓形虫CaMK相关激酶（TgCaMKrk），它表现出不依赖钙调蛋白的自磷酸化和底物磷酸化活性。然而，钙调蛋白拮抗剂对其激酶活性没有影响。在刚地弓形虫感染的细胞中，TgCaMKrk定位于细胞外和细胞内速殖子的顶端。TgCaMKrk在体外使TgGAP45磷酸化。
我们的数据增进了我们对刚地弓形虫运动性和感染、寄生虫蛋白激酶与滑行体之间的相互作用以及原生动物疾病药物靶点的理解。
