Role of pathogen-derived capping carbohydrates in altering immunity

描述(申请人提供)：病原体的外表面大量表达结构多样的碳水化合物。该项目利用来自利什曼原虫和分枝杆菌的两种表面碳水化合物的帽糖，分别是脂磷酸多糖(LPG)和脂阿拉伯甘露聚糖(LAM)。这两条来自强毒物种的碳水化合物链延迟了吞噬小体的成熟，并促进了局部或系统的耐受性免疫反应，支持慢性感染的持续。独特的帽糖与毒力分枝杆菌有关；目前尚不清楚LPG/LAM的哪些特定部分会导致免疫变化。困难的碳水化合物化学排除了专门测试LAM/LPG最外部部分的作用的能力。在这项提案中，通过化学家尼古拉·波尔博士提供的突破性技术，这个问题被克服了。涂有LAM和LPG帽糖的珠子已经生产出来。这一提议挑战了两个中心假设：A)帽糖是LAM和LPG的免疫抑制部分；B)四种结构不同的病原体衍生的帽糖在体外和体内的反应将在病原体的处理和破坏的改变方面有所不同。本建议中概述的实验旨在1)确认合成的CAPS糖具有正确的构象和生物活性2)确定LAM或LPG甘露糖帽糖衣小球与对照吞噬小球相比在巨噬细胞中的过程，3)测试微珠处理的巨噬细胞杀死内生病原体的比较能力，4)表征LAM或LPG盖帽糖衣小球在体内的免疫反应。我们预测，包裹有帽糖的珠子来自致病性分枝杆菌或利什曼原虫。会阻碍吞噬小体成熟和巨噬细胞病原体的清除。此外，我们预测这些糖帽将抑制体内的珠子清除和Th1细胞因子反应的促进。由于病原体表面表达的帽子糖部分决定了细菌和寄生虫的致病菌株，这些研究将确定碳水化合物分子帽子结构的变化如何导致免疫结果改变的差异。公共卫生报道：了解帽糖在改变宿主免疫反应中的特定作用，可以立即导致在病原体组装方面的重要发现，这些发现应该成为药物发现的目标。这些病原体表面表达的帽子糖已被证明可以预测致病和非致病细菌和寄生虫菌株之间的差异，因此拟议的研究将提供一个模型系统，以确定这些分子在确定毒力中的作用，以及帽子结构的变化如何导致免疫反应的结果改变差异。鉴于碳水化合物在指导抗病原体免疫反应中的重要作用，有必要确定病原菌来源的帽糖在改变体内和体外免疫反应中的特定作用。

DESCRIPTION (provided by applicant): Structurally diverse carbohydrates are abundantly expressed on outer surfaces of pathogens. This project utilizes cap sugars of two surface carbohydrates, Lipophosphoglycans (LPG) and lipoarabinomannans (LAM), from Leishmania and Mycobacterium respectively. These two carbohydrate chains from virulent species delay phagosome maturation and promote a localized or systemic tolerogenic immune response that supports continuation of chronic infection. Unique cap sugars are associated with virulent Mycobacterial spp.; it is not known which specific portions of LPG/LAM cause immune alterations. Difficult carbohydrate chemistry precluded the ability to specifically test the role of this most-external portion of LAM/LPG. In this proposal, through breakthrough technology provided by chemist Dr. Nicola Pohl, this problem was overcome. Beads coated with LAM and LPG cap sugars have been produced. This proposal challenges two central hypotheses A) cap sugars are an immune- inhibitory portion of LAM and LPG and B) the in vitro and in vivo responses to four structurally different pathogen-derived cap sugars will differ in alteration of pathogen processing and destruction. Experiments outlined in this proposal are designed to 1) Confirm that synthetically-produced caps sugars have correct conformation and biologic activity 2) Determine how LAM or LPG mannose cap sugar-coated beads process in macrophages as compared to control phagocytosed beads, 3) Test the compared ability of bead-treated macrophages to kill internalized pathogens, 4) Characterize the in vivo immune response to LAM or LPG cap sugar-coated beads. We predict that beads coated with cap sugars derived from virulent Mycobacterium or Leishmania sp. will deter phagolysosome maturation and macrophage pathogen removal. In addition we predict that these cap sugars will inhibit in vivo bead clearance and promotion of a Th1 cytokine response. As pathogen-surface-expressed cap sugars partially determine pathogenic strains of bacteria and parasites, these studies will identify how changes in carbohydrate molecule cap structure lead to outcome-altering differences in immunity. PUBLIC HEALTH RELEVENCE: Understanding the specific role of cap sugars, the external surface coat for many pathogens, in altering host immune responses can lead to immediate and important discoveries in pathogen assemby that should be targeted for drug discovery. These pathogen surface- expressed cap sugars have been shown to predict differences between pathogenic and non- pathogenic strains of bacteria and parasites, therefore the proposed studies will provide a model system to identify the role of these molecules in determining virulence and how changes in cap structure leads to outcome-altering differences in immune responses. Due to the important function of carbohydrates in directing anti-pathogen immune responses, it is necessary to determine the specific role of pathogen-derived cap sugars in altering the in vivo and in vitro immune response.