Surface proteins bound to the cell membrane by glycosylphosphatidylinositol (GPI) anchors are considered essential for the survival of pathogenic protozoans. In the case of the tick-transmitted hemoparasite Babesia bovis, the most virulent causative agent of bovine babesiosis, the GPI-anchored proteome was recently unraveled by an in silico approach. In this work, one of the identified proteins, GASA-1 (GPI-Anchored Surface Antigen-1), was thoroughly characterized. GASA-1 is 179 aa long and has the characteristic features of a GPI-anchored protein, including a signal peptide, a hydrophilic core and a hydrophobic tail that harbors a GPI anchor signal. Transcriptomic analysis shows that it is expressed in pathogenic and attenuated B. bovis strains. Notably, the gasa-1 gene has syntenic counterparts in B. bigemina and B. ovata, which also encode GPI-anchored proteins. This is highly unusual since all piroplasmid GPI-anchored proteins described so far have been found to be species-specific. Sequencing of gasa-1 alleles from B. bovis geographical isolates originating from Argentina, USA, Brazil, Mexico and Australia showed over 98 % identity in both nucleotide and amino acid sequences. A recombinant form of GASA-1 (rGASA-1) was generated in E. coli and anti-rGASA-1 antibodies were raised in mice. Fixed and live immunofluorescence assays showed that GASA-1 is expressed in in vitro cultured B. bovis merozoites and surface-exposed. Moreover, incubation of B. bovis in vitro cultures with anti-GASA-1 antibodies partially, but significantly, reduced erythrocyte invasion, indicating that this protein bears neutralization-sensitive antibody epitopes. Splenocytes of rGASA-1-inoculated mice showed a specific proliferative response when exposed to the recombinant protein, indicating that GASA-1 bears T-cell epitopes. Finally, sera from a group of B. bovis-infected cattle reacted with the recombinant protein, demonstrating that GASA-1 is expressed during natural infection of bovines with B. bovis, and suggesting that it is immunodominant. The high degree of conservation among B. bovis isolates and the presence of syntenic genes in other Babesia species suggest a relevant role of GASA-1 and GASA-1-like proteins for parasite survival, especially considering that, due to their surface location, they are exposed to the selection pressure of the host immune system. The highlighted features of GASA-1 make it an interesting candidate for the development of vaccines against bovine babesiosis.

通过糖基磷脂酰肌醇 (GPI) 锚与细胞膜结合的表面蛋白被认为是致病性原生动物生存所必需的。在蜱传播的血液寄生虫牛巴贝斯虫（牛巴贝斯虫病的最致命病原体）的情况下，GPI 锚定蛋白质组最近通过计算机方法解开。在这项工作中，一种已鉴定的蛋白质 GASA-1（GPI 锚定表面抗原 1）被彻底表征。GASA-1 长 179 个氨基酸，具有 GPI 锚定蛋白的特征，包括信号肽、亲水核心和带有 GPI 锚定信号的疏水尾。转录组学分析表明它在致病性和减毒牛双歧杆菌中表达菌株。值得注意的是，gasa-1基因在B. bigemina和B. ovata 中具有同线对应物，它们也编码 GPI 锚定蛋白。这是非常不寻常的，因为迄今为止描述的所有 piroplasmid GPI 锚定蛋白都被发现是物种特异性的。对源自阿根廷、美国、巴西、墨西哥和澳大利亚的牛双歧杆菌地理分离株的gasa-1等位基因的测序在核苷酸和氨基酸序列上显示出超过 98% 的同一性。在大肠杆菌中产生重组形式的 GASA-1 (rGASA-1) ，并在小鼠中产生抗 rGASA-1 抗体。固定和活体免疫荧光测定表明 GASA-1在体外培养中表达B. bovis裂殖子和表面暴露。此外，用抗 GASA-1 抗体在体外培养牛双歧杆菌可以部分但显着地减少红细胞侵袭，表明该蛋白质具有中和敏感抗体表位。当暴露于重组蛋白时，rGASA-1 接种小鼠的脾细胞显示出特异性增殖反应，表明 GASA-1 带有 T 细胞表位。最后，来自一组感染牛双歧杆菌的牛的血清与重组蛋白反应，证明 GASA-1 在牛用牛双歧杆菌自然感染期间表达，并表明它是免疫显性的。牛双歧杆菌的高度保护分离株和其他巴贝虫物种中同线基因的存在表明 GASA-1 和 GASA-1 样蛋白对寄生虫存活的相关作用，特别是考虑到由于它们的表面位置，它们暴露于宿主的选择压力免疫系统。GASA-1 的突出特点使其成为开发牛巴贝斯虫病疫苗的有趣候选者。