Role of GAG-Binding Proteins in Cryptosporidium Infection

项目摘要 隐孢子虫（Crypto）在世界范围内引起炭疽病，这可能危及生命， 免疫功能低下的宿主，如未经治疗的艾滋病毒/艾滋病患者和营养不良的儿童， 受限制的国家。然而，对于这些隐孢子虫病没有一致有效的治疗方法， 弱势群体。我们的长期目标是了解密码蛋白在介导 宿主细胞附着和侵入，以便开发新的隐孢子虫病干预措施。我们有 鉴定和表征Clec，一种独特的密码蛋白，具有三种蛋白质特征， 依恋与入侵Clec是1）糖胺聚糖（GAG）结合蛋白，具有2）粘蛋白样和3）C- 型凝集素结构域。本申请的目的是阐明Clec在Crypto感染中的作用。克莱克是 胞外1型膜蛋白，其在C. parvum、小隐翅虫C. hominis和C.穆里斯群岛 在体外感染过程中表达，定位于侵袭阶段的顶端表面和致密颗粒，结合 - 细胞表面GAG，特别是肠上皮细胞上的硫酸乙酰肝素蛋白聚糖（HSPG）， C区。细小病毒通过与这些GAG的相互作用附着和侵入这些细胞。这些发现， 这强烈表明Clec通过介导附着和侵袭，并提高细胞的粘附能力，在隐孢子虫感染中发挥作用。 它可能成为干预的目标。然而，直到最近， 用分子遗传学研究密码蛋白的作用。我们的中心假设是Clec介导了Crypto 在体外粘附和侵入肠上皮细胞，并通过与细胞结合促进体内感染 表面硫酸乙酰肝素蛋白聚糖。在本申请中，我们建议在体外和体内测试这一假设。 体内使用CRISPR/Cas9介导的基因靶向方法，最近由我们的合作者，Dr. 鲍里斯·斯特里彭具体目的是：1）确定C。parvum Clec（CpClec）附着于 使用基因靶向方法体外检测人肠上皮细胞的侵袭; 2）确定 CpClec在使用隐孢子虫病动物模型的体内感染中的作用。在完成这些目标后，我们 预期已经建立了CpClec在体外附着和侵袭以及体内感染中的作用， 分子遗传学和互补方法。未来的研究将致力于确定 Clec的特异性结构域，并使用 基因靶向方法。长期目标是确定CpClec和/或其他GAG结合蛋白是否能够抑制肿瘤的生长。 蛋白质可以作为药物或疫苗开发的目标。

PROJECT SUMMARY Cryptosporidium (Crypto) causes diarrheal disease worldwide, which can be life-threatening in immunocompromised hosts such as untreated HIV/AIDS patients and malnourished children in resource- constrained countries. However, there is no consistently effective treatment for cryptosporidiosis in these vulnerable populations. Our long-term goal is to understand the role of Crypto proteins involved in mediating host cell attachment and invasion in order to develop novel interventions for cryptosporidiosis. We have identified and characterized Clec, a unique Crypto protein with three characteristics of proteins involved in attachment and invasion. Clec is 1) a glycosaminoglycan (GAG)-binding protein with 2) mucin-like and 3) C- type lectin domains. The objective of this application is to elucidate the role of Clec in Crypto infection. Clec is an extracellular type 1 membrane protein, which is conserved in C. parvum, C. hominis and C. muris, is expressed during infection in vitro, localizes to the apical surface and dense granules of invasive stages, binds to cell surface GAGs, specifically heparan sulfate proteoglycans (HSPGs) on intestinal epithelial cells and blocks C. parvum attachment to and invasion of these cells via interactions with these GAGs. These findings, strongly suggest that Clec plays a role in Crypto infection by mediating attachment and invasion and raise the possibility that it may serve as a target for intervention. However, until recently, it was not possible to determine the role of Crypto proteins using molecular genetics. Our central hypothesis is that Clec mediates Crypto attachment to and invasion of intestinal epithelial cells in vitro and promotes infection in vivo by binding to cell surface heparan sulfate proteoglycans. In this application, we propose to test this hypothesis in vitro and in vivo using CRISPR/Cas9 mediated gene-targeting approaches recently pioneered by our collaborator, Dr. Boris Striepen. The Specific Aims are 1) to determine the role of C. parvum Clec (CpClec) in attachment to and invasion of human intestinal epithelial cells in vitro using gene-targeting approaches and 2) To determine the role of CpClec in infection in vivo using animal models of cryptosporidiosis. At the completion of these aims we expect to have established the role of CpClec in attachment and invasion in vitro and in infection in vivo using molecular genetics and complementary approaches. Future studies will be directed at determining the role of specific domains of Clec, and identifying and elucidating the roles of other GAG-binding Crypto proteins using gene-targeting approaches. The long-term goal is to determine whether CpClec and/or other GAG-binding proteins can serve as targets for drug or vaccine development.