Abstract：
Neospora caninum is an obligate intracellular parasite that infects a wide range of mammalian species, and particularly causes abortions in cattle and nervous system dysfunction in dogs. Dense granule proteins (GRAs) are thought to play an important role in the mediation of host-parasite interactions and facilitating parasitism. However, a large number of potential GRAs remain uncharacterized, and the functions of most of the identified GRAs have not been elucidated. Previously, we screened a large number GRAs including NcGRA27 and NcGRA61 using the proximity-dependent biotin identification (BioID) technique. Here, we identified a novel GRA protein NcGRA85 and used C-terminal endogenous gene tagging to determine its localization at the parasitophorous vacuole (PV) in the tachyzoite. We successfully disrupted three gra genes (NcGRA27, NcGRA61 and NcGRA85) of N. caninum NC1 strain using CRISPR-Cas9-mediated homologous recombination and phenotyped the single knockout strain. The NcGRA61 and NcGRA85 genes were not essential for parasite replication and growth in vitro and for virulence during infection of mice, as observed by replication assays, plaque assays and in vitro virulence assays in mice. Deletion of the NcGRA27 gene in the NC1 strain reduced the in vitro replication and growth of the parasite, as well as the pathogenicity of the NC1 strain in mice. In summary, our findings provide a basis for in-depth studies of N. caninum pathogenesis and demonstrate the importance of NcGRA27 in parasite growth and virulence, most likely a new virulence factor of N. caninum.
摘要：
犬新孢子虫是一种专性细胞内寄生虫，感染多种哺乳动物物种，特别是导致牛流产和狗的神经系统功能障碍。致密颗粒蛋白（GRAs）被认为在介导宿主-寄生虫相互作用和促进寄生中起重要作用。然而,大量潜在的GRA仍未表征，大多数已确定的GRA的功能尚未阐明。以前,我们使用邻近依赖生物素鉴定(BioID)技术筛选了大量GRAs,包括NcGRA27和NcGRA61.这里,我们鉴定了一种新的GRA蛋白NcGRA85，并使用C端内源性基因标记来确定其在速殖子中的寄生虫液泡（PV）的定位。我们使用CRISPR-Cas9介导的同源重组成功地破坏了犬NC1菌株的三个gra基因（NcGRA27，NcGRA61和NcGRA85），并对单个敲除菌株进行了表型分析。NcGRA61和NcGRA85基因对于寄生虫体外复制和生长以及小鼠感染期间的毒力不是必需的，如复制试验所观察到的，小鼠的菌斑测定和体外毒力测定。NC1菌株中NcGRA27基因的缺失减少了寄生虫的体外复制和生长，以及NC1菌株对小鼠的致病性。总之,我们的研究结果提供了一个基础，深入的研究犬的发病机制，并证明了NcGRA27在寄生虫生长和毒力的重要性，很可能是一种新的犬齿毒力因子。
