刚地弓形虫致密颗粒蛋白 3 通过激活 PERK 通路促进内质网应激诱导的细胞凋亡。
Toxoplasma gondii dense granule protein 3 promotes endoplasmic reticulum stress-induced apoptosis by activating the PERK pathway.
机构信息
Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Anhui Medical University, Hefei, 230032, China.
The Research Center for Infectious Diseases, School of Basic Medical Sciences, Anhui Medical University, Hefei, 230032, China.
出版信息
Parasit Vectors. 2022 Aug 2;15(1):276. doi: 10.1186/s13071-022-05394-5.
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-GRA3 was constructed by inserting Chinese 1 Wh6 GRA3 (GRA3) cDNA into a plasmid encoding the enhanced GFP. Mouse neuro2a (N2a) cells were transfected with either pEGFP or pEGFP-GRA3 (GRA3) 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 GRA3 transfection, and the effect of the inhibitors on GRA3-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 GRA3 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.
CONCLUSION
GRA3 induces neural cell apoptosis via the ER stress signaling pathway, which could play a role in toxoplasmic encephalitis.
背景
刚地弓形虫是一种感染包括人类在内的哺乳动物的神经滋养单细胞寄生虫。刚地弓形虫中枢神经系统感染可导致弓形体脑炎。刚地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 已被鉴定为有助于刚地弓形虫毒力的分泌蛋白之一；然而，其机制仍不清楚。
方法
使用定量实时聚合酶链反应（qRT-PCR）检测 RH、ME49、Wh3 和 Wh6 株中 GRA3 基因的表达。通过将中国 1 型 Wh6 GRA3（GRA3）cDNA 插入编码增强 GFP 的质粒中构建 pEGFP-GRA3。将 pEGFP 或 pEGFP-GRA3（GRA3）转染到小鼠神经母细胞瘤（N2a）细胞中并孵育 24-36 小时。通过流式细胞术和免疫印迹法测定 N2a 细胞凋亡和 ER 应激相关蛋白。此外，在 GRA3 转染前用 GSK2656157（PERK 抑制剂）和 Z-ATAD-FMK（caspase-12 抑制剂）预处理 N2a 细胞，并研究抑制剂对 GRA3 诱导的 ER 应激和细胞凋亡的影响。
结果
与强毒力的 I 型 RH 株和中国 1 型 Wh3 株相比，低毒力的 II 型 ME49 株和中国 1 型 Wh6 株的 GRA3 基因表达更高。与对照载体相比，GRA3 质粒转染诱导神经元凋亡，并增加 GRP78、p-PERK、cleaved caspase-12、cleaved caspase-3 和 CHOP 的表达。GSK2656157 和 Z-ATAD-FMK 预处理可减少 N2a 细胞凋亡，同样，显著降低 ER 应激和凋亡相关蛋白水平。
结论
GRA3 通过内质网应激信号通路诱导神经细胞凋亡，这可能在弓形体脑炎中起作用。
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