Abstract：
BACKGROUND: Toxoplasma gondii is a neurotropic single-celled parasite that can infect mammals, including humans. Central nervous system infection with T. gondii infection can lead to Toxoplasma encephalitis. Toxoplasma infection can cause endoplasmic reticulum (ER) stress and unfolded protein response (UPR) activation, which ultimately can lead to apoptosis of host cells. The dense granule protein GRA3 has been identified as one of the secretory proteins that contribute to the virulence of T. gondii; however, the mechanism remains enigmatic.
METHODS: The expression of the GRA3 gene in RH, ME49, Wh3, and Wh6 strains was determined using quantitative real-time polymerase chain reaction (qRT-PCR). pEGFP-GRA3Wh6 was constructed by inserting Chinese 1 Wh6 GRA3 (GRA3Wh6) cDNA into a plasmid encoding the enhanced GFP. Mouse neuro2a (N2a) cells were transfected with either pEGFP or pEGFP-GRA3Wh6 (GRA3Wh6) and incubated for 24-36 h. N2a cell apoptosis and ER stress-associated proteins were determined using flow cytometry and immunoblotting. Furthermore, N2a cells were pretreated with GSK2656157 (a PERK inhibitor) and Z-ATAD-FMK (a caspase-12 inhibitor) before GRA3Wh6 transfection, and the effect of the inhibitors on GRA3Wh6-induced ER stress and apoptosis were investigated.
RESULTS: GRA3 gene expression was higher in the less virulent strains of type II ME49 and type Chinese 1 Wh6 strains compared with the virulent strains of type I RH strain and type Chinese 1 Wh3 strain. Transfection with GRA3Wh6 plasmid induced neuronal apoptosis and increased the expression of GRP78, p-PERK, cleaved caspase-12, cleaved caspase-3, and CHOP compared with the control vector. Pretreatment with GSK2656157 and Z-ATAD-FMK decreased apoptosis in N2a cells, and similarly, ER stress- and apoptosis-associated protein levels were significantly decreased.
CONCLUSIONS: GRA3 induces neural cell apoptosis via the ER stress signaling pathway, which could play a role in toxoplasmic encephalitis.
METHODS: The expression of the GRA3 gene in RH, ME49, Wh3, and Wh6 strains was determined using quantitative real-time polymerase chain reaction (qRT-PCR). pEGFP-GRA3Wh6 was constructed by inserting Chinese 1 Wh6 GRA3 (GRA3Wh6) cDNA into a plasmid encoding the enhanced GFP. Mouse neuro2a (N2a) cells were transfected with either pEGFP or pEGFP-GRA3Wh6 (GRA3Wh6) and incubated for 24-36 h. N2a cell apoptosis and ER stress-associated proteins were determined using flow cytometry and immunoblotting. Furthermore, N2a cells were pretreated with GSK2656157 (a PERK inhibitor) and Z-ATAD-FMK (a caspase-12 inhibitor) before GRA3Wh6 transfection, and the effect of the inhibitors on GRA3Wh6-induced ER stress and apoptosis were investigated.
RESULTS: GRA3 gene expression was higher in the less virulent strains of type II ME49 and type Chinese 1 Wh6 strains compared with the virulent strains of type I RH strain and type Chinese 1 Wh3 strain. Transfection with GRA3Wh6 plasmid induced neuronal apoptosis and increased the expression of GRP78, p-PERK, cleaved caspase-12, cleaved caspase-3, and CHOP compared with the control vector. Pretreatment with GSK2656157 and Z-ATAD-FMK decreased apoptosis in N2a cells, and similarly, ER stress- and apoptosis-associated protein levels were significantly decreased.
CONCLUSIONS: GRA3 induces neural cell apoptosis via the ER stress signaling pathway, which could play a role in toxoplasmic encephalitis.
摘要：
背景：弓形虫是一种嗜神经的单细胞寄生虫，可以感染哺乳动物，包括人类。中枢神经系统感染弓形虫感染可导致弓形虫脑炎。弓形虫感染可引起内质网(ER)应激和未折叠蛋白反应(UPR)激活,最终导致宿主细胞凋亡。致密颗粒蛋白GRA3已被确定为有助于弓形虫毒力的分泌蛋白之一；然而，机制仍然是神秘的。
方法：GRA3基因在RH中的表达，使用定量实时聚合酶链反应(qRT-PCR)测定ME49、Wh3和Wh6菌株。pEGFP-GRA3Wh6是通过将中国1Wh6GRA3（GRA3Wh6）cDNA插入编码增强GFP的质粒中而构建的。用pEGFP或pEGFP-GRA3Wh6(GRA3Wh6)转染小鼠神经2a(N2a)细胞并孵育24-36小时。使用流式细胞术和免疫印迹测定N2a细胞凋亡和ER应激相关蛋白。此外，在GRA3Wh6转染之前，用GSK2656157（PERK抑制剂）和Z-ATAD-FMK（caspase-12抑制剂）预处理N2a细胞，并研究了抑制剂对GRA3Wh6诱导的ER应激和细胞凋亡的影响。
结果：GRA3基因在II型ME49和中国1型Wh6毒力较弱的菌株中的表达高于I型RH菌株和中国1型Wh3菌株的毒力菌株。转染GRA3Wh6质粒可诱导神经元凋亡，并增加GRP78，p-PERK的表达，与对照载体相比，裂解的caspase-12，裂解的caspase-3和CHOP。用GSK2656157和Z-ATAD-FMK预处理可降低N2a细胞的凋亡，同样，ER应激和凋亡相关蛋白水平显著降低。
结论：GRA3通过内质网应激信号通路诱导神经细胞凋亡,可能在弓形虫脑炎中发挥作用。
方法：GRA3基因在RH中的表达，使用定量实时聚合酶链反应(qRT-PCR)测定ME49、Wh3和Wh6菌株。pEGFP-GRA3Wh6是通过将中国1Wh6GRA3（GRA3Wh6）cDNA插入编码增强GFP的质粒中而构建的。用pEGFP或pEGFP-GRA3Wh6(GRA3Wh6)转染小鼠神经2a(N2a)细胞并孵育24-36小时。使用流式细胞术和免疫印迹测定N2a细胞凋亡和ER应激相关蛋白。此外，在GRA3Wh6转染之前，用GSK2656157（PERK抑制剂）和Z-ATAD-FMK（caspase-12抑制剂）预处理N2a细胞，并研究了抑制剂对GRA3Wh6诱导的ER应激和细胞凋亡的影响。
结果：GRA3基因在II型ME49和中国1型Wh6毒力较弱的菌株中的表达高于I型RH菌株和中国1型Wh3菌株的毒力菌株。转染GRA3Wh6质粒可诱导神经元凋亡，并增加GRP78，p-PERK的表达，与对照载体相比，裂解的caspase-12，裂解的caspase-3和CHOP。用GSK2656157和Z-ATAD-FMK预处理可降低N2a细胞的凋亡，同样，ER应激和凋亡相关蛋白水平显著降低。
结论：GRA3通过内质网应激信号通路诱导神经细胞凋亡,可能在弓形虫脑炎中发挥作用。
