Amieva Rafael, Coronado Montserrat, Powell Jessica, Arranz-Solís David, Hassan Musa A, Collantes-Fernández Esther, Ortega-Mora Luis Miguel, Horcajo Pilar
SALUVET Group, Animal Health Department, Faculty of Veterinary Sciences, Complutense University of Madrid, Madrid, Spain.
Division of Infection and Immunity, The Roslin Institute, University of Edinburgh, Edinburgh, United Kingdom.
Front Immunol. 2025 Aug 12;16:1617570. doi: 10.3389/fimmu.2025.1617570. eCollection 2025.
is an apicomplexan parasite responsible for bovine neosporosis, a major cause of abortion in cattle worldwide. rhoptry protein 2 (NcROP2) has been identified as an essential factor in host cell invasion and parasitophorous vacuole formation, making it a potential target for disease control strategies.
In this study, we generated knockout () mutants using CRISPR/Cas9 technology to assess their role in parasite virulence.
In a pregnant mouse model, parasites exhibited reduced virulence, as indicated by increased neonatal survival rates and lower parasite burden in the brain and attenuated clinical signs in the dams compared to the wild-type (Nc-Spain7) parental strain. Additionally, the mutants exhibited impaired proliferation and significantly induced the expression of interferon-stimulated genes in bovine monocyte-derived macrophages infected for 60 hours. Transcriptomic analysis further revealed a shift in parasite gene expression, with an upregulation of stress-related and bradyzoite markers. Functional assays confirmed that Δ parasites were less susceptible to IFN-γ-mediated inhibition and displayed an enhanced ability to convert to the semi-dormant bradyzoite stage.
These findings highlight NcROP2 as a key virulence factor involved in immune evasion and parasite proliferation, providing new insights into infection pathogenesis and potential avenues for vaccine development.
是一种顶复门寄生虫，可引发牛新孢子虫病，这是全球牛流产的主要原因。新孢子虫棒状体蛋白2（NcROP2）已被确定为宿主细胞入侵和寄生泡形成的关键因素，使其成为疾病控制策略的潜在靶点。
在本研究中，我们使用CRISPR/Cas9技术构建了NcROP2基因敲除（ΔNcROP2）突变体，以评估其在寄生虫毒力中的作用。
在怀孕小鼠模型中，与野生型（Nc-Spain7）亲代菌株相比，ΔNcROP2寄生虫的毒力降低，表现为新生小鼠存活率提高、脑中寄生虫载量降低以及母鼠临床症状减轻。此外，ΔNcROP2突变体在感染60小时的牛单核细胞衍生巨噬细胞中增殖受损，并显著诱导干扰素刺激基因的表达。转录组分析进一步揭示了寄生虫基因表达的变化，应激相关和缓殖子标志物上调。功能测定证实，ΔNcROP2寄生虫对IFN-γ介导的抑制作用不太敏感，并表现出更强的转化为半休眠缓殖子阶段的能力。
这些发现突出了NcROP2作为参与免疫逃避和寄生虫增殖的关键毒力因子，为新孢子虫感染发病机制及疫苗开发的潜在途径提供了新见解。
