Snelling William J, Lin Qishan, Moore John E, Millar B Cherie, Tosini Fabio, Pozio Edoardo, Dooley James S G, Lowery Colm J
Centre for Molecular Biosciences, School of Biomedical Sciences, University of Ulster, Cromore Road, Coleraine, County Londonderry BT52 1SA, Northern Ireland.
Mol Cell Proteomics. 2007 Feb;6(2):346-55. doi: 10.1074/mcp.M600372-MCP200. Epub 2006 Nov 23.
Cryptosporidiosis, caused by coccidian parasites of the genus Cryptosporidium, is a major cause of human gastrointestinal infections and poses a significant health risk especially to immunocompromised patients. Despite intensive efforts for more than 20 years, there is currently no effective drug treatment against these protozoa. This study examined the zoonotic species Cryptosporidium parvum at two important stages of its life cycle: the non-excysted (transmissive) and excysted (infective) forms. To increase our understanding of the molecular basis of sporozoite excystation, LC-MS/MS coupling with a stable isotope N-terminal labeling strategy using iTRAQ reagents was used on soluble fractions of both non-excysted and excysted sporozoites, i.e. sporozoites both inside and outside oocysts were examined. Sporozoites are the infective stage that penetrates small intestinal enterocytes. Also to increase our knowledge of the C. parvum proteome, shotgun sequencing was performed on insoluble fractions from both non-excysted and excysted sporozoites. In total 303 C. parvum proteins were identified, 56 of which, hitherto described as being only hypothetical proteins, are expressed in both excysted and non-excysted sporozoites. Importantly we demonstrated that the expression of 26 proteins increases significantly during excystation. These excystation-induced proteins included ribosomal proteins, metabolic enzymes, and heat shock proteins. Interestingly three Apicomplexa-specific proteins and five Cryptosporidium-specific proteins augmented in excysted invasive sporozoites. These eight proteins represent promising targets for developing vaccines or chemotherapies that could block parasite entry into host cells.
隐孢子虫病由隐孢子虫属的球虫寄生虫引起，是人类胃肠道感染的主要原因，尤其对免疫功能低下的患者构成重大健康风险。尽管经过20多年的深入研究，但目前尚无针对这些原生动物的有效药物治疗方法。本研究在其生命周期的两个重要阶段对人兽共患物种微小隐孢子虫进行了研究：未脱囊（可传播）和脱囊（感染性）形式。为了加深我们对子孢子脱囊分子基础的理解，使用iTRAQ试剂的稳定同位素N端标记策略与LC-MS/MS联用，对未脱囊和脱囊子孢子的可溶性部分进行了分析，即对卵囊内外的子孢子都进行了检测。子孢子是穿透小肠肠上皮细胞的感染阶段。同样，为了增加我们对微小隐孢子虫蛋白质组的了解，对未脱囊和脱囊子孢子的不溶性部分进行了鸟枪法测序。总共鉴定出303种微小隐孢子虫蛋白质，其中56种迄今仅被描述为假设蛋白质，在脱囊和未脱囊子孢子中均有表达。重要的是，我们证明了26种蛋白质的表达在脱囊过程中显著增加。这些脱囊诱导蛋白包括核糖体蛋白、代谢酶和热休克蛋白。有趣的是，三种顶复门特异性蛋白和五种隐孢子虫特异性蛋白在脱囊后的侵袭性子孢子中增加。这八种蛋白质是开发可阻断寄生虫进入宿主细胞的疫苗或化疗药物的有希望的靶点。
