Role of GAG-Binding Proteins in Cryptosporidium Infection

项目总结 隐孢子虫(Crypto)在世界范围内引起腹泻疾病，在 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，在这些地区并没有始终如一有效的治疗隐孢子虫病的方法。 弱势群体。我们的长期目标是了解参与调节的隐形蛋白的作用 宿主细胞的附着和入侵，以开发针对隐孢子虫病的新干预措施。我们有 CLEC是一种独特的隐含蛋白，具有三种参与蛋白的特性 依恋和入侵。CLEC是一种糖胺多聚糖(GAG)结合蛋白，具有2)粘蛋白样蛋白和3)C- 键入凝集素结构域。本应用的目的是阐明CLEC在Crypto感染中的作用。CLEC是 一种在微小隐孢子虫、人隐孢子虫和小隐孢子虫中保守的胞外1型膜蛋白是 在体外感染期间表达，定位于根尖表面和致密颗粒的侵袭期，结合 对细胞表面的GAG，特别是肠上皮细胞上的硫酸乙酰肝素蛋白多糖(HSPGs)和 阻断C.细小病毒通过与这些缝隙的相互作用附着和侵入这些细胞。这些发现， 强烈提示CLEC通过介导附着和侵袭在隐形病毒感染中发挥作用，并提高 它可能成为干预的目标。然而，直到最近，还不可能确定 利用分子遗传学研究隐形蛋白的作用。我们的中心假设是CLEC调节Crypto 肠上皮细胞体外黏附和侵袭及与细胞结合促进体内感染 表面硫酸乙酰肝素蛋白多糖。在这个应用中，我们建议在体外和在 使用CRISPR/Cas9介导的基因靶向方法的活体，最近由我们的合作者Dr。 鲍里斯·斯特里潘。具体目标是1)确定微小隐孢子虫CLEC(CpClec)在依附和 用基因打靶方法体外侵袭人肠上皮细胞和2)确定 CpClec在隐孢子虫病动物模型体内感染中的作用。在完成这些目标时，我们 CpClec在体外黏附、侵袭和体内感染中的作用 分子遗传学和互补方法。未来的研究将致力于确定 CLEC的特定结构域，并鉴定和阐明其他GAG结合的隐含蛋白的作用 基因打靶方法。长期目标是确定CpClec和/或其他恶作剧结合 蛋白质可以作为药物或疫苗开发的靶点。

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.