Toxoplasma gondii is an obligate intracellular parasite that can invade any nucleated cell of mammals and cause toxoplasmosis. Dense granule proteins play major structural functions within the parasitophorous vacuole (PV) and the cyst wall of T. gondii. Moreover, their particular location within the PV allows them to be involved in various interactions between parasites and the host cells. Dense granule protein 9 (GRA9) gene has been identified in T. gondii, although its role in the lytic cycle remains unclear. In the current study, the function of GRA9 in type I and type II Toxoplasma parasites was characterized. T. gondii GRA9 sequence and its expression were analyzed and derivatives of T. gondii RH and PLK strains with a null mutation in GRA9 were generated using CRISPR/Cas9 system. The phenotypes of GRA9 in wild types, knockout and complemented strains were analyzed in vitro and in vivo using Vero cells and BALB/c mice, respectively. Alignment of the amino acid sequence indicated that RH strain GRA9 contained one amino acid substitution when compared with PLK strain. Western blot analysis revealed that PLK strain had a higher expression level of GRA9 than RH strain. The phenotype analysis revealed that knockout of GRA9 in PLK parasites inhibited the plaque formation and egress from PV. Both the plaque formation and egress ability of PLKΔGRA9 strain were restored by complementation with a synonymous allele of PLK strain GRA9. Mouse experiments demonstrated that loss of GRA9 in PLK strain significantly reduced the pathogenicity of T. gondii. However, there was no phenotypic diferences between RH and RHΔGRA9 strains except the defect in host cell invasion. Overall, T. gondii GRA9 knockout only influenced the growth and virulence of PLK strain. These results indicate that GRA9 may be involved in parasite egress and virulence in mice in a strain-specific manner.

弓形虫是专性的细胞内寄生虫，可侵入哺乳动物的任何有核细胞并引起弓形虫病。致密颗粒蛋白在弓形虫的寄生虫液泡（PV）和囊壁中起主要结构功能。此外，它们在PV中的特定位置使它们能够参与寄生虫与宿主细胞之间的各种相互作用。尽管在溶胞周期中的作用尚不清楚，但已在弓形虫中鉴定出了致密颗粒蛋白9（GRA9）基因。在当前的研究中，表征了GRA9在I型和II型弓形虫寄生虫中的功能。分析弓形虫GRA9序列及其表达，并对弓形虫进行衍生。使用CRISPR / Cas9系统生成GRA9中具有无效突变的RH和PLK菌株。在体外和体内分析了野生型，敲除和互补菌株中GRA9的表型分别使用Vero细胞和BALB / c小鼠。氨基酸序列的比对表明，与PLK菌株相比，RH菌株GRA9含有一个氨基酸取代。Western印迹分析显示PLK菌株比RH菌株具有更高的GRA9表达水平。表型分析表明，在PLK寄生虫中GRA9的敲除抑制了噬菌斑的形成和从PV流出。通过与PLK菌株GRA9的同义等位基因互补，恢复了PLKΔGRA9菌株的噬斑形成和流出能力。小鼠实验表明，PLK菌株中GRA9的缺失显着降低了弓形虫的致病性。但是，除了宿主细胞侵袭的缺陷外，RH和RHΔGRA9菌株之间没有表型差异。总体而言，弓形虫GRA9基因敲除仅影响PLK菌株的生长和毒力。这些结果表明，GRA9可能以菌株特异性方式参与小鼠体内的寄生虫逃逸和致病性。