Li Wensheng, Liu Jing, Wang Jing, Fu Yong, Nan Huizhu, Liu Qun
Key Laboratory of Animal Epidemiology and Zoonosis, Ministry of Agriculture, and National Animal Protozoa Laboratory, College of Veterinary Medicine, China Agricultural University, Beijing, China.
Parasitol Res. 2015 Aug;114(8):2893-902. doi: 10.1007/s00436-015-4490-3. Epub 2015 May 10.
Neospora caninum, an Apicomplexa parasite, is the causative agent of neosporosis. As described for other members of Apicomplexa, microneme proteins (MICs) play a key role in attachment and invasion of host cells by N. caninum. Herein we identified N. caninum microneme protein 6 (NcMIC6) that is orthologous to Toxoplasma gondii microneme protein 6 (TgMIC6). The open reading frame of the NcMIC6 gene is 984 bp and encodes a 327 amino acid peptide. Sequence analysis showed that NcMIC6 included a signal peptide, a transmembrane region, three epidermal growth factor-like (EGF) domains, and two low complexity regions. Antibodies raised against recombinant NcMIC6 recognized an approximately 35-kDa native MIC6 protein in Western blots of N. caninum tachyzoites. Immunofluorescence analysis showed that NcMIC6 had a polar labeling pattern, which was consistent with localization of micronemes in the apical region. Pulse invasion assays showed that NcMIC6 translocated from the apical tip to the posterior end of the parasites. Secretion assays demonstrated that NcMIC6 was released into the supernatants. Importantly, it was clearly revealed by co-immunoprecipitation that NcMIC6 formed a complex with other two soluble microneme proteins (NcMIC1 and NcMIC4). In conclusion, identification and characterization of the novel microneme protein NcMIC6 may contribute to understanding how this protein functions during the parasite motility and host cell invasion.
犬新孢子虫是一种顶复门寄生虫，是新孢子虫病的病原体。正如对顶复门其他成员的描述，微线体蛋白（MICs）在犬新孢子虫附着和侵入宿主细胞过程中起关键作用。在此，我们鉴定了与刚地弓形虫微线体蛋白6（TgMIC6）直系同源的犬新孢子虫微线体蛋白6（NcMIC6）。NcMIC6基因的开放阅读框为984 bp，编码一个327个氨基酸的肽段。序列分析表明，NcMIC6包含一个信号肽、一个跨膜区、三个表皮生长因子样（EGF）结构域和两个低复杂性区域。针对重组NcMIC6产生的抗体在犬新孢子虫速殖子的蛋白质免疫印迹中识别出一个约35 kDa的天然MIC6蛋白。免疫荧光分析表明，NcMIC6具有极性标记模式，这与微线体在顶端区域的定位一致。脉冲侵入试验表明，NcMIC6从寄生虫的顶端转移到后端。分泌试验表明，NcMIC6被释放到上清液中。重要的是，免疫共沉淀清楚地显示，NcMIC6与其他两种可溶性微线体蛋白（NcMIC1和NcMIC4）形成复合物。总之，新型微线体蛋白NcMIC6的鉴定和表征可能有助于理解该蛋白在寄生虫运动和宿主细胞侵入过程中的功能。
