Proteins detected by SBP3 immunoprecipitation.

Babesia bovis, an apicomplexan intraerythrocytic protozoan parasite, causes serious economic loss to cattle industries around the world. Infection with this parasite leads to accumulation of infected red blood cells (iRBCs) in the brain microvasculature that results in severe clinical complications known as cerebral babesiosis. Throughout its growth within iRBCs, the parasite exports various proteins to the iRBCs that lead to the formation of protrusions known as “ridges” on the surface of iRBCs, which serve as sites for cytoadhesion to endothelial cells. Spherical body proteins (SBPs; proteins secreted from spherical bodies, which are organelles specific to Piroplasmida) are exported into iRBCs, and four proteins (SBP1-4) have been reported to date. In this study, we elucidated the function of SBP3 using an inducible gene knockdown (KD) system. Localization of SBP3 was assessed by immunofluorescence assay, and only partial colocalization was detected between SBP3 and SBP4 inside the iRBCs. In contrast, colocalization was observed with VESA-1, which is a major parasite ligand responsible for the cytoadhesion. Immunoelectron microscopy confirmed localization of SBP3 at the ridges. SBP3 KD was performed using the glmS system, and effective KD was confirmed by Western blotting, immunofluorescence assay, and RNA-seq analysis. The SBP3 KD parasites showed severe growth defect suggesting its importance for parasite survival in the iRBCs. VESA-1 on the surface of iRBCs was scarcely detected in SBP3 KD parasites, whereas SBP4 was still detected in the iRBCs. Moreover, abolition of ridges on the iRBCs and reduction of iRBCs cytoadhesion to the bovine brain endothelial cells were observed in SBP3 KD parasites. Immunoprecipitation followed by mass spectrometry analysis detected the host Band 3 multiprotein complex, suggesting an association of SBP3 with iRBC cytoskeleton proteins. Taken together, this study revealed the vital role of SBP3 in ridge formation and its significance in the pathogenesis of cerebral babesiosis.牛巴贝斯虫（Babesia bovis）是一类顶复门红细胞内原生动物寄生虫，对全球养牛业造成严重经济损失。该寄生虫感染会导致受感染红细胞（infected red blood cells, iRBCs）在脑微血管内聚集，进而引发以严重临床并发症为特征的脑型巴贝斯虫病。在受感染红细胞内的整个生长周期中，该寄生虫会向宿主红细胞分泌多种蛋白质，最终在受感染红细胞表面形成被称为“凸起（ridges）”的突起结构，该结构可作为寄生虫与内皮细胞发生细胞黏附的位点。球形体蛋白（spherical body proteins, SBPs）指由梨形虫目（Piroplasmida）特有的细胞器——球形体分泌的蛋白质，此类蛋白会被转运至受感染红细胞中，目前已报道存在4种此类蛋白（SBP1-4）。本研究借助诱导型基因敲低（knockdown, KD）系统解析了SBP3的生物学功能。通过免疫荧光测定（immunofluorescence assay）对SBP3的亚细胞定位进行评估，结果显示在受感染红细胞内部，SBP3与SBP4仅存在部分共定位现象。与之相反，SBP3与负责介导细胞黏附的主要寄生虫配体VESA-1呈现明显共定位。免疫电子显微镜（immunoelectron microscopy）进一步证实，SBP3定位于上述凸起结构之上。本研究采用glmS系统开展SBP3基因敲低实验，通过蛋白质免疫印迹（Western blotting）、免疫荧光测定及RNA测序（RNA-seq）分析验证了敲低的有效性。SBP3敲低的寄生虫株表现出显著的生长缺陷，提示SBP3对寄生虫在受感染红细胞内的存活具有关键作用。在SBP3敲低的寄生虫中，受感染红细胞表面的VESA-1几乎无法被检测到，而SBP4仍可在受感染红细胞中稳定检出。此外，在SBP3敲低的寄生虫中，受感染红细胞表面的凸起结构完全消失，且受感染红细胞与牛脑内皮细胞的细胞黏附能力显著降低。通过免疫沉淀（immunoprecipitation）联合质谱（mass spectrometry）分析，本研究检测到了宿主3型带多蛋白复合物（Band 3 multiprotein complex），提示SBP3与受感染红细胞的细胞骨架蛋白存在相互作用关联。综上，本研究揭示了SBP3在受感染红细胞凸起结构形成中的核心作用，以及其在脑型巴贝斯虫病发病机制中的重要意义。