Function of Neospora caninum dense granule protein 7 in innate immunity in mice

摘要

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 (Delta NcGRA7) and a NcGRA7 complementary strain (i Delta NcGRA7) to study their function. We found that Delta NcGRA7 exhibited slower growth in vitro and weakened virulence in mice compared with Nc1 and i Delta NcGRA7. All parasite strains can stimulate host immune cells to produce IFN-gamma, and the amount of IFN-gamma production stimulated by Nc1 was significantly higher than that stimulated by Delta NcGRA7. The transcription levels of the cellular immune factors GBP1, GBP2, IRGa6, and IRGb6 were significantly higher after stimulation with Delta NcGRA7 parasites than after stimulation with Nc1. Furthermore, Delta NcGRA7 infection resulted in greater IRGa6 recruitment to the PVM than Nc1 infection. Delta NcGRA7 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.