Characterization of strain-specific phenotypes associated with knockout of dense granule protein 9 in Toxoplasma gondii
Characterization of strain-specific phenotypes associated with knockout of dense granule protein 9 in Toxoplasma gondii
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DOI：
10.1016/j.molbiopara.2019.01.003
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发表时间：
2019-04-01
影响因子：
1.5
通讯作者：
Xuan, Xuenan
中科院分区：
文献类型：
--
作者：
Guo, Huanping;Gao, Yang;Xuan, Xuenan
弓形虫是一种专性细胞内寄生虫，可侵入哺乳动物的任何有核细胞并引起弓形虫病。致密颗粒蛋白在嗜寄空泡（PV）和T.刚地此外，它们在PV内的特定位置允许它们参与寄生虫和宿主细胞之间的各种相互作用。致密颗粒蛋白9（GRA 9）基因已在T.弓形虫，虽然其在溶解周期中的作用尚不清楚。在本研究中，GRA 9在I型和II型弓形虫寄生虫中的功能被表征。T.目的分析弓形虫GRA 9基因的序列及其表达，并对弓形虫GRA 9基因的衍生物进行分析。使用CRISPR/Cas9系统产生在GRA 9中具有无效突变的弓形虫RH和PLK菌株。采用Vero细胞和BALB/c小鼠分别在体外和体内对野生型、敲除型和补充型GRA 9的表型进行了分析。氨基酸序列比对表明，RH株GRA 9与PLK株相比，有一个氨基酸发生了替换。Western印迹分析显示PLK株的GRA 9表达水平高于RH株。表型分析显示，PLK寄生虫中GRA 9的敲除可抑制斑块形成和PV的排出。通过与PLK菌株GRA 9的同义等位基因互补，PLK Delta GRA 9菌株的空斑形成和排出能力均得以恢复。小鼠实验表明，PLK株GRA 9基因的缺失显著降低了T.刚地。RH株与RHAGRA 9株在表型上无明显差异，但在宿主细胞侵袭能力上存在差异。总的来说，T。GRA 9基因敲除仅影响PLK株的生长和毒力。这些结果表明GRA 9可能以品系特异性方式参与寄生虫在小鼠中的排出和毒力。
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 PLK Delta GRA9 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 RHAGRA9 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.