Hoane Jessica S, Carruthers Vernon B, Striepen Boris, Morrison David P, Entzeroth Rolf, Howe Daniel K
Department of Veterinary Science, University of Kentucky, 108 Gluck Equine Research Center, Lexington, KY 40546-0099, USA.
Int J Parasitol. 2003 Jul;33(7):671-9. doi: 10.1016/s0020-7519(03)00031-6.
Sarcocystis neurona, an apicomplexan parasite, is the primary causative agent of equine protozoal myeloencephalitis. Like other members of the Apicomplexa, S. neurona zoites possess secretory organelles that contain proteins necessary for host cell invasion and intracellular survival. From a collection of S. neurona expressed sequence tags, we identified a sequence encoding a putative microneme protein based on similarity to Toxoplasma gondii MIC10 (TgMIC10). Pairwise sequence alignments of SnMIC10 to TgMIC10 and NcMIC10 from Neospora caninum revealed approximately 33% identity to both orthologues. The open reading frame of the S. neurona gene encodes a 255 amino acid protein with a predicted 39-residue signal peptide. Like TgMIC10 and NcMIC10, SnMIC10 is predicted to be hydrophilic, highly alpha-helical in structure, and devoid of identifiable adhesive domains. Antibodies raised against recombinant SnMIC10 recognised a protein band with an apparent molecular weight of 24 kDa in Western blots of S. neurona merozoites, consistent with the size predicted for SnMIC10. In vitro secretion assays demonstrated that this protein is secreted by extracellular merozoites in a temperature-dependent manner. Indirect immunofluorescence analysis of SnMIC10 showed a polar labelling pattern, which is consistent with the apical position of the micronemes, and immunoelectron microscopy provided definitive localisation of the protein to these secretory organelles. Further analysis of SnMIC10 in intracellular parasites revealed that expression of this protein is temporally regulated during endopolygeny, supporting the view that micronemes are only needed during host cell invasion. Collectively, the data indicate that SnMIC10 is a microneme protein that is part of the excreted/secreted antigen fraction of S. neurona. Identification and characterisation of additional S. neurona microneme antigens and comparisons to orthologues in other Apicomplexa could provide further insight into the functions that these proteins serve during invasion of host cells.
肉孢子虫属神经元种（Sarcocystis neurona）是一种顶复门寄生虫，是马属动物原虫性脑脊髓炎的主要病原体。与顶复门的其他成员一样，肉孢子虫属神经元种的子孢子拥有分泌细胞器，其中含有宿主细胞入侵和细胞内存活所必需的蛋白质。从肉孢子虫属神经元种表达序列标签的集合中，我们基于与刚地弓形虫MIC10（TgMIC10）的相似性，鉴定出一个编码假定微线体蛋白的序列。将SnMIC10与来自犬新孢子虫（Neospora caninum）的TgMIC10和NcMIC10进行成对序列比对，发现与这两个直系同源物的同一性约为33%。肉孢子虫属神经元种基因的开放阅读框编码一个255个氨基酸的蛋白质，带有一个预测的39个残基的信号肽。与TgMIC10和NcMIC10一样，SnMIC10预计是亲水性的，结构上高度α螺旋，且没有可识别的粘附结构域。针对重组SnMIC10产生的抗体在肉孢子虫属神经元种裂殖子的免疫印迹中识别出一条表观分子量为24 kDa的蛋白带，与预测的SnMIC10大小一致。体外分泌试验表明，该蛋白由细胞外裂殖子以温度依赖性方式分泌。对SnMIC10的间接免疫荧光分析显示出极性标记模式，这与微线体的顶端位置一致，免疫电子显微镜确定了该蛋白在这些分泌细胞器中的定位。对细胞内寄生虫中SnMIC10的进一步分析表明，该蛋白的表达在内生增殖过程中受到时间调控，支持微线体仅在宿主细胞入侵期间才需要的观点。总体而言，数据表明SnMIC10是一种微线体蛋白，是肉孢子虫属神经元种排泄/分泌抗原部分的一部分。鉴定和表征其他肉孢子虫属神经元种微线体抗原，并与其他顶复门的直系同源物进行比较，可能会进一步深入了解这些蛋白在宿主细胞入侵过程中所起的作用。
