Role of pathogen-derived capping carbohydrates in altering immunity

描述（由申请人提供）：结构多样的碳水化合物在病原体的外表面大量表达。本项目利用两种表面碳水化合物的帽糖，脂磷聚糖（LPG）和脂arabinmanans (LAM)，分别来自利什曼原虫和分枝杆菌。来自有毒物种的这两条碳水化合物链延迟吞噬体成熟，促进局部或全身的耐受性免疫反应，支持慢性感染的持续。独特的帽糖与毒性分枝杆菌有关；目前尚不清楚LPG/LAM的哪个特定部分引起免疫改变。复杂的碳水化合物化学使我们无法专门测试LAM/LPG最外部部分的作用。在这个提案中，通过化学家Nicola Pohl博士提供的突破性技术，这个问题被克服了。包覆有LAM和LPG帽糖的珠子已经生产出来。这一建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)对四种结构不同的病原体衍生帽糖的体外和体内反应在改变病原体的加工和破坏方面存在差异。本提案中概述的实验旨在1)证实合成的帽糖具有正确的构象和生物活性2)确定LAM或LPG甘露糖帽包糖珠与被吞噬的对照珠相比如何在巨噬细胞中加工，3)测试经珠处理的巨噬细胞杀死内化病原体的比较能力4)表征对LAM或LPG帽包糖珠的体内免疫反应。我们预测，包被来自毒性分枝杆菌或利什曼原虫的帽糖的珠将阻止吞噬溶酶体的成熟和巨噬细胞病原体的去除。此外，我们预测这些帽糖将抑制体内头清除和促进Th1细胞因子反应。由于病原体表面表达的帽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结构的变化如何导致免疫结果改变的差异。公共卫生相关性：了解帽糖（许多病原体的外表面外壳）在改变宿主免疫反应中的特定作用，可以立即发现病原体组装的重要发现，这应该是药物发现的目标。这些病原体表面表达的帽糖已被证明可以预测致病性和非致病性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，以确定这些分子在决定毒力方面的作用，以及帽结构的变化如何导致免疫反应结果改变的差异。由于碳水化合物在指导抗病原体免疫反应中的重要作用，有必要确定病原体来源的帽糖在改变体内和体外免疫反应中的具体作用。

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.