Objectives: Toxoplasmosis is a zoonotic parasitic disease caused by Toxoplasma gondii (T. gondii), which can lead to complications such as encephalitis and ocular toxoplasmosis. The disease becomes more severe when the host's immune system is compromised. Rhoptry proteins are major virulence factors that enable T. gondii to invade host cells. This study aims to construct a T. gondii rhoptry protein 41 (rop41/ROP41) gene knockout strain and preliminarily investigate the biological function of rop41.
Methods: Using CRISPR/Cas9 technology, a specific single-guide RNA (sgRNA) for the target gene was designed and linked to a recombinant plasmid. Homologous fragments were fused with a pyrimethamine resistance gene for selection purposes. The recombinant plasmid and the homologous fragments were electroporated into T. gondii, and PCR identification was performed after drug selection and monoclonal screening. Plaque assays were used to comprehensively assess whether rop41 affected the growth and proliferation of T. gondii in host cells. Invasion and proliferation assays were conducted to evaluate the invasion ability of the knockout strain into host cells and its intracellular proliferation capacity. The STRING database was utilized to construct a protein-protein interaction (PPI) network, and functional enrichment analysis was performed to predict the signaling pathways in which ROP41 might be involved.
Results: The T. gondiirop41 gene knockout strain (RH Δku80Δrop41) was successfully constructed and stably inherited. Plaque assays showed that compared with the parental strain, the number of plaques formed by the rop41 gene knockout strain did not significantly decrease, but the reduction in plaque size was statistically significant (P<0.05). After the rop41 gene was knocked out, the invasion ability of T. gondii was reduced, but there was no statistically significant difference in its proliferation ability (P>0.05). The PPI network revealed that ROP41 was associated with other protein kinases and autophagy-related proteins. Enrichment analysis indicated that proteins interacting with ROP41 may be involved in signal transduction, biosynthesis, metabolism, and autophagy-related pathways and could be components of various kinase complexes and phagocytic vesicles.
Conclusions: The T. gondii RH Δku80Δrop41 strain has been successfully constructed. ROP41 primarily affects the ability of T. gondii to invade host cells and may play a role in signal transduction and autophagy-related pathways between T. gondii and the host.
目的: 弓形虫病是由弓形虫引起的一种人畜共患寄生虫病，可并发脑炎和弓形虫眼病等，机体免疫力低下时病情更为严重。棒状体蛋白是弓形虫感染宿主细胞的主要毒力因子。本研究旨在构建弓形虫棒状体蛋白41(rhoptry protein 41，rop41/ROP41)基因敲除株，初步研究rop41的生物学功能。方法: 基于CRISPR/Cas9技术，设计目的基因的特异性单向导RNA(single-guide RNA，sgRNA)，将sgRNA连接到重组质粒中，同源片段融合乙胺嘧啶抗性基因以便筛选。同时将重组质粒和同源片段电转入弓形虫中，通过药物筛选和单克隆筛选后进行PCR鉴定。利用噬斑实验综合评估rop41是否影响弓形虫在宿主细胞内的生长和增殖能力。入侵实验和增殖实验分别用来评估敲除株的入侵宿主细胞和胞内的增殖能力。通过STRING 数据库构建蛋白质互作(protein-protein interaction，PPI)网络图，并进行功能富集分析，推测ROP41蛋白可能参与的信号通路。结果: 成功构建了弓形虫rop41基因敲除株(RH Δku80Δrop41)，且能稳定遗传。噬斑实验显示：与母本虫株相比，弓形虫rop41基因敲除株形成的空斑数量没有显著减少，但空斑面积的减少差异具有统计学意义(P<0.05)。rop41基因敲除后弓形虫的入侵能力降低，但是增殖能力的变化差异没有统计学意义(P>0.05)。PPI网络图显示ROP41蛋白与其他蛋白质激酶及自噬蛋白质具有相关性，富集分析的结果表明ROP41蛋白的互作蛋白质可能参与了信号转导、生物合成及代谢与自噬相关途径，并可能是多种激酶复合物以及吞噬泡的成分。结论: RH Δku80Δrop41弓形虫虫株构建成功，ROP41蛋白主要影响弓形虫入侵宿主细胞的能力，并可能参与弓形虫与宿主之间的信号转导和自噬相关通路。.
Keywords: Toxoplasma gondii; functional enrichment analysis; gene knockout; rhoptry protein 41.