Baravalle M E, Thompson C, de Echaide S Torioni, Palacios C, Valentini B, Suárez C E, Christensen M Florín, Echaide I
Instituto Nacional de Tecnología Agropecuaria (INTA), Estación Experimental Agropecuaria Rafaela, CP 2300 Rafaela, Santa Fe, Argentina.
Parasitol Int. 2010 Dec;59(4):571-8. doi: 10.1016/j.parint.2010.07.008. Epub 2010 Aug 5.
The apical complex of intracellular hemoparasites contains organelles like micronemes and rhoptries, specialized structures required for adherence and invasion of host cells. Several molecules discharged from rhoptries have been identified from Plasmodium spp., but only a single rhoptry associated protein-1 (RAP-1) has been characterized from Babesia bovis. In silico search of the B. bovis genome allowed to identifying a sequence homologous to the gene that encodes a P. falciparum rhoptry protein PfRhop148. The intron-less 1830 bp novel gene, predicted a 68kDa protein, and it was highly conserved among different B. bovis strains and isolates. The deducted protein from the B. bovis T2Bo strain, named BboRhop68, showed two putative transmembrane domains, at least seven B-cell epitopes, and a well conserved DUF501 super family domain. The bborhop68 gene was amplified, analyzed and compared among different B. bovis strains and isolates showing overall high sequence conservation. A fragment of bborhop68 was expressed as a recombinant fusion protein (rBboRhop68). The mice anti-rBboRhop68 serum identified the novel protein in intraerythrocytic trophozoites and merozoites by WB and ELISA, but not in free merozoites. Sera from naturally and experimentally infected bovines also recognized BboRhop68, suggesting that it is expressed and immunogenic during B. bovis infection. Fluorescence microscopy analysis using anti-rBboRhop68 antibodies showed a rod structure associated to trophozoites and merozoites infected erythrocytes, but this pattern of reactivity was not observed in free merozoites. The BboRhop68 was also not detected in ELISA based on solubilized merozoites. Thus, at least three independent lines of evidence support differential expression of BboRhop68 in intraerythrocytic stages of B. bovis and its possible functional role immediately after B. bovis erythrocyte invasion. The results of this work suggest that BboRhop68 could be considered as a novel additional target for developing improved methods to control bovine babesiosis.
细胞内血寄生虫的顶端复合体包含诸如微线体和棒状体等细胞器，这些是附着和侵入宿主细胞所需的特殊结构。从疟原虫属中已鉴定出几种从棒状体释放的分子，但来自牛巴贝斯虫的只有一种棒状体相关蛋白-1（RAP-1）得到了表征。对牛巴贝斯虫基因组进行电子搜索，得以鉴定出一个与编码恶性疟原虫棒状体蛋白PfRhop148的基因同源的序列。这个无内含子的1830 bp新基因预测编码一个68 kDa的蛋白质，并且在不同的牛巴贝斯虫菌株和分离株中高度保守。从牛巴贝斯虫T2Bo菌株推导的蛋白质，命名为BboRhop68，显示有两个推定的跨膜结构域、至少七个B细胞表位以及一个高度保守的DUF501超家族结构域。对不同的牛巴贝斯虫菌株和分离株进行了bborhop68基因的扩增、分析和比较，结果显示其序列总体高度保守。bborhop68的一个片段被表达为重组融合蛋白（rBboRhop68）。小鼠抗rBboRhop68血清通过蛋白质印迹法（WB）和酶联免疫吸附测定（ELISA）在红细胞内滋养体和裂殖子中鉴定出了这种新蛋白，但在游离裂殖子中未鉴定出。自然感染和实验感染牛的血清也识别BboRhop68，这表明它在牛巴贝斯虫感染期间表达且具有免疫原性。使用抗rBboRhop68抗体的荧光显微镜分析显示，在感染红细胞的滋养体和裂殖子中存在一种棒状结构，但在游离裂殖子中未观察到这种反应模式。基于溶解裂殖子的ELISA也未检测到BboRhop68。因此，至少有三条独立的证据支持BboRhop68在牛巴贝斯虫红细胞内阶段的差异表达及其在牛巴贝斯虫侵入红细胞后可能的功能作用。这项工作的结果表明，BboRhop68可被视为开发改进的牛巴贝斯虫病防控方法的一个新的额外靶点。
