Role of Cryptosporidium Mucins in Host-parasite interactions

描述（由申请人提供）：隐孢子虫是一种普遍存在的水传播病原体，在世界范围内引起腹泻病。这种病原体具有重要的医学意义，因为它能够在免疫功能低下的宿主，特别是艾滋病患者中引起危及生命的疟疾。迄今为止，对于隐孢子虫感染没有持续有效的治疗方法，也没有预防感染的疫苗。研究表明，隐孢子虫，不像其他apicomplexans，依赖粘蛋白样糖蛋白附着和侵入肠上皮细胞。这些粘蛋白抗原也是保护性细胞免疫应答的靶标。尽管粘蛋白抗原对隐孢子虫生物学的重要性，这些抗原很少被研究。隐孢子虫基因组计划已经鉴定了31个编码推定粘蛋白抗原的开放阅读框架。本申请中提出的研究将研究位于2号染色体上单个位点（CpMuc1 - 7）的7种粘蛋白，并确定它们在宿主-寄生虫相互作用中的作用。目标一：表征CpMuc1 - 7基因座的7种粘蛋白样糖蛋白的表达、定位、翻译后修饰和潜在的多蛋白复合物形成。我们将使用实时RT-PCR在细胞内发育期间定量CpMucs的表达，确定蛋白质表达和定位，阐明天然抗原的糖基化模式，并确定这些抗原是否形成多蛋白复合物。目的#2：确定CpMuc1 - 7糖蛋白是否在侵袭和/或刺激细胞介导的免疫应答中发挥作用。我们将确定抗CpMuc抗体和重组蛋白是否抑制C。本发明的目的是研究CpMucs对肠上皮细胞侵袭的影响，并确定CpMucs是否是小鼠感染期间产生的细胞免疫应答的激活剂。在这些研究中，我们还将探讨糖基化在宿主细胞侵袭和免疫激活中的作用。最后，采用无毒T.弓形虫菌株作为抗原递送系统，我们将确定针对CpMucs之一的免疫应答是否提供对隐孢子虫病的保护。这些研究将增强我们对粘蛋白样糖蛋白在隐孢子虫感染的建立和解决中所起作用的理解，并大大提高我们设计有效治疗方法的能力，以对抗这种严重的全球感染威胁。

DESCRIPTION (provided by applicant): Cryptosporidium is a ubiquitous waterborne pathogen that causes diarrheal disease worldwide. This pathogen is of significant medical importance because of its capacity to cause life-threatening diarrheal disease in immunocompromised hosts, particularly AIDS patients. To date there is no consistently effective treatment for Cryptosporidium infection, and no vaccine to prevent infection. Studies suggest that Cryptosporidium, unlike other apicomplexans, relies on mucin-like glycoproteins to attach to and invade intestinal epithelial cells. These mucin antigens are also targets of protective cellular immune responses. Despite the importance of mucin antigens to Cryptosporidium biology, few of these antigens have been investigated. The Cryptosporidium genome project has identified 31 open reading frames encoding putative mucin antigens. The studies proposed in this application will investigate 7 mucins that reside on a single locus on chromosome 2 (CpMuc1-7) and determine the role they play in host-parasite interactions. Aim #1: To characterize expression, localization, post-translational modifications, and potential multi-protein complex formation of the seven mucin-like glycoproteins of the CpMuc1-7 locus. We will quantify expression of the CpMucs during intracellular development using real-time RT-PCR, determine protein expression and localization, elucidate the glycosylation patterns of the native antigens, and determine if these antigens form multi-protein complexes. Aim #2: To determine if the CpMuc1-7 glycoproteins play a role in invasion and/or stimulation of cell mediated immune responses. We will determine if anti-CpMuc antibodies and recombinant proteins inhibit C. parvum invasion of intestinal epithelial cells, and determine if the CpMucs are activators of cellular immune responses that develop during infection in mice. In these studies, we will also explore the effect of glycosylation in host cell invasion and immune activation. Finally, employing a non-virulent T. gondii strain as an antigen delivery system, we will determine if immune responses targeted against one of the CpMucs provides protection from cryptosporidiosis. These studies will enhance our understanding of the role mucin-like glycoproteins play in the establishment and resolution of Cryptosporidium infections and greatly advance our ability to design effective treatments to combat this serious infectious threat around the world.