National Animal Protozoa Laboratory, College of Veterinary Medicine, China Agricultural University, Beijing, 100193, People's Republic of China.
Key Laboratory of Animal Epidemiology of the Ministry of Agriculture, College of Veterinary Medicine, China Agricultural University, Beijing, 100193, People's Republic of China.
Parasitol Res. 2021 Jan;120(1):197-207. doi: 10.1007/s00436-020-06961-4. Epub 2020 Nov 8.
The intracellular parasite Neospora caninum can parasitize all nucleated cells of the host. Dense granule proteins (GRAs) secreted by dense granules are an important material involved in the formation of parasitophorous vacuoles (PVs), which facilitate parasite survival and replication in host cells. Due to the secretory and immune properties of NcGRA7, it is considered to be a promising serodiagnosis marker and an effective neosporosis vaccine candidate. However, the intracellular regulatory mechanisms involved in NcGRA7-induced host responses have rarely been examined. Here, we used the CRISPR/Cas9 genome editing system to obtain a NcGRA7 knockout strain (ΔNcGRA7) and a NcGRA7 complementary strain (iΔNcGRA7) to study their function. We found that ΔNcGRA7 exhibited slower growth in vitro and weakened virulence in mice compared with Nc1 and iΔNcGRA7. All parasite strains can stimulate host immune cells to produce IFN-γ, and the amount of IFN-γ production stimulated by Nc1 was significantly higher than that stimulated by ΔNcGRA7. The transcription levels of the cellular immune factors GBP1, GBP2, IRGa6, and IRGb6 were significantly higher after stimulation with ΔNcGRA7 parasites than after stimulation with Nc1. Furthermore, ΔNcGRA7 infection resulted in greater IRGa6 recruitment to the PVM than Nc1 infection. ΔNcGRA7 parasites were more easily cleared by macrophages than Nc1 parasites. Collectively, these results showed that NcGRA7 plays an important role in regulating the immune factors of mice and the aggregation of IRGa6 at the PVM, which affects the pathogenicity of N. caninum.
细胞内寄生虫刚地弓形虫能够寄生宿主的所有有核细胞。致密颗粒分泌的致密颗粒蛋白（GRAs）是形成寄生空泡（PVs）的重要物质，有助于寄生虫在宿主细胞中的存活和复制。由于 NcGRA7 的分泌和免疫特性，它被认为是一种很有前途的血清学诊断标记物和有效的新孢子虫病疫苗候选物。然而，NcGRA7 诱导宿主反应的细胞内调节机制很少被研究。在这里，我们使用 CRISPR/Cas9 基因组编辑系统获得了 NcGRA7 敲除株（ΔNcGRA7）和 NcGRA7 互补株（iΔNcGRA7），以研究它们的功能。我们发现，与 Nc1 和 iΔNcGRA7 相比，ΔNcGRA7 在体外生长较慢，在小鼠体内毒力减弱。所有寄生虫株都能刺激宿主免疫细胞产生 IFN-γ，Nc1 刺激产生的 IFN-γ量明显高于 ΔNcGRA7。与 Nc1 刺激相比，ΔNcGRA7 寄生虫刺激后细胞免疫因子 GBP1、GBP2、IRGa6 和 IRGb6 的转录水平显著升高。与 Nc1 感染相比，ΔNcGRA7 感染导致更多的 IRGa6 募集到 PVM。与 Nc1 寄生虫相比，ΔNcGRA7 寄生虫更容易被巨噬细胞清除。综上所述，这些结果表明 NcGRA7 在调节小鼠的免疫因子和 IRGa6 在 PVM 上的聚集方面发挥重要作用，从而影响刚地弓形虫的致病性。
