Role of Cryptosporidium Mucins in Host-parasite interactions

描述(申请人提供)：隐孢子虫是一种普遍存在的水传播病原体，可在世界范围内引起腹泻疾病。这种病原体具有重要的医学意义，因为它能够在免疫受损的宿主，特别是艾滋病患者中引起危及生命的腹泻疾病。到目前为止，还没有持续有效的治疗隐孢子虫感染的方法，也没有预防感染的疫苗。研究表明，隐孢子虫与其他顶端复合体不同，它依赖粘蛋白样糖蛋白附着和入侵肠道上皮细胞。这些粘蛋白抗原也是保护性细胞免疫反应的目标。尽管粘蛋白抗原对隐孢子虫的生物学很重要，但很少有人对其进行研究。隐孢子虫基因组计划已经确定了31个开放阅读框，编码假定的粘蛋白抗原。这项申请中提出的研究将研究位于2号染色体(CpMuc1-7)上的7个粘蛋白，并确定它们在宿主-寄生虫相互作用中所起的作用。目的#1：研究CpMuc1-7基因座7个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体形成。我们将使用实时定量RT-PCR来定量CpMucs在细胞内发育过程中的表达，确定蛋白质的表达和定位，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体。目的#2：确定CpMuc1-7糖蛋白是否在细胞免疫反应的侵袭和/或刺激中发挥作用。我们将确定抗CpMuc抗体和重组蛋白是否抑制微小弧菌对肠道上皮细胞的入侵，并确定CpMucs是否是小鼠感染期间发展的细胞免疫反应的激活剂。在这些研究中，我们还将探索糖基化在宿主细胞侵袭和免疫激活中的作用。最后，使用非毒力弓形虫株作为抗原传递系统，我们将确定针对其中一种CpMucs的免疫反应是否提供了对隐孢子虫病的保护。这些研究将加深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的作用的理解，并极大地提高我们设计有效的治疗方法来对抗世界各地这一严重的传染病威胁的能力。

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.