State Key Laboratory for Zoonotic Diseases, Key Laboratory for Zoonosis Research of the Ministry of Education, Institute of Zoonosis, and College of Veterinary Medicine, Jilin University, Changchun, China.
Virulence. 2024 Dec;15(1):2427844. doi: 10.1080/21505594.2024.2427844. Epub 2024 Nov 28.
is an important protozoan parasite that causes abortion in cattle and nervous system dysfunction in dogs. No effective drugs and vaccines for neosporosis are available. Further elucidation of proteins related to virulence will provide potential candidates for vaccine development against neosporosis. In the present study, c-Myc regulatory protein (NcMYR1) gene knockout strains (ΔNcMYR1-1, ΔNcMYR1-2, and ΔNcMYR1-3) were generated using the CRISPR-Cas9 gene editing system to investigate phenotype changes and the potential of the ΔNcMYR1-1 strain as an attenuated vaccine, and this is the first time of using the CRISPR-Cas9 gene knockout strain as an attenuated vaccine. NcMYR1 was determined to be a cytoplasmic protein in tachyzoites. The deficiency of NcMYR1 decreased the plaque area and the rate of invasion, replication, and egression of the parasites. ΔNcMYR1-1 strain-infected C57BL/6 mice had 100% survival rate, reduced parasite burden, and alleviated pathological changes in tissues compared with those in Nc-1 strain-infected mice. Immunization with ΔNcMYR1-1 tachyzoites increased the productions of cytokines in mice, with a survival rate reaching 80%, and the parasite burdens in the liver and spleen were greatly reduced when challenged with the Nc-1 strain with a lethal dose after 40 days of ΔNcMYR1-1 tachyzoite immunization. ΔNcMYR1 immunization could decrease the abortion rate of female mice from 71.4% to 12.5% and increase the survival rate of pups from 12.5% to 83.3% against the challenge. Above all, NcMYR1 is a virulence factor and the ΔNcMYR1-1 strain could be used as a candidate vaccine against infection and vertical transmission.
刚地弓形虫是一种重要的原生动物寄生虫，可导致牛流产和犬神经系统功能障碍。目前尚无针对刚地弓形虫病的有效药物和疫苗。进一步阐明与毒力相关的蛋白将为刚地弓形虫病疫苗的开发提供潜在的候选疫苗。本研究利用 CRISPR-Cas9 基因编辑系统生成 c-Myc 调节蛋白（NcMYR1）基因敲除株（ΔNcMYR1-1、ΔNcMYR1-2 和 ΔNcMYR1-3），以研究表型变化和ΔNcMYR1-1 株作为减毒疫苗的潜力，这是首次使用 CRISPR-Cas9 基因敲除株作为减毒疫苗。在速殖子中，NcMYR1 被确定为细胞质蛋白。NcMYR1 的缺乏降低了斑块面积和寄生虫的入侵、复制和逸出率。与 Nc-1 株感染的小鼠相比，ΔNcMYR1-1 株感染的 C57BL/6 小鼠的存活率为 100%，寄生虫负荷降低，组织病理学变化减轻。用ΔNcMYR1-1 速殖体免疫小鼠可增加细胞因子的产生，存活率达到 80%，用致死剂量的 Nc-1 株攻毒后 40 天，肝脾寄生虫负荷大大降低。ΔNcMYR1 免疫可将雌性小鼠的流产率从 71.4%降低到 12.5%，并将幼仔的存活率从 12.5%提高到 83.3%。综上所述，NcMYR1 是一种毒力因子，ΔNcMYR1-1 株可作为刚地弓形虫感染和垂直传播的候选疫苗。
