The Structural Determinants of Innate Immune Modulation by Francisella LPS

弗朗西斯菌 LPS 调节先天免疫的结构决定因素

• 批准号: 8694303
• 负责人: JASON Holt BARKER
• 金额: $ 37.75万
• 依托单位: UNIVERSITY OF IOWA
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-02-15 至 2019-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8694303
• 关键词: Acetates; Affect; Bacteria; Cell Death; Cells; Characteristics; Cytosol; Data; Disease; Environment; Fatty Acids; Francisella; Francisella tularensis; Gene-Modified; Glycolipids; Gram-Negative Bacteria; Growth; Health; Heterogeneity; Human; Immune; Immune system; Infection; Inflammatory; Invaded; Label; Laboratories; Life; Lipid A; Lipopolysaccharides; Mammalian Cell; Membrane; Methods; Microbe; Modification; Natural Immunity; O Antigens; Organism; Phagocytes; Polysaccharides; Population; Production; Property; Structure; Surface; Testing; Tularemia; Wit; base; immunoregulation; interest; novel; pathogen; response

DESCRIPTION (provided by applicant): Gram-negative bacteria frequently cause infections in humans. An abundant glycolipid found in the outer membrane of these bacteria, lipopolysaccharide (LPS), forms an integral part of their structure and modulates multiple interactions with the immune system during infection. It has become clear in recent years that many Gram-negative pathogens alter the structure of their LPS during adaptation to the host, in order to optimize immune evasion. This proposal seeks to apply novel experimental approaches to understand better the mechanisms and consequences of lipid A structural modification by Francisella during intracellular growth. This would represent the first detailed description of LPS adaptation in a cytosolic pathogen. Unlike the LPS of many medically-important Gram-negative microbes, Francisella LPS is non- inflammatory. It neither stimulates nor antagonizes LPS-sensing host machinery, and we have previously documented that it fails to interact with important LPS-sensing molecules that normally promote efficient elimination of Gram-negative bacteria. These unique characteristics likely derive from its unusual lipid A and core polysaccharide structures. The overall composition of Francisella LPS is also highly unusual in that a majority of the LPS molecules consist of free lipid A, lacking both core and O-antigen polysaccharides. The precise structural determinants of the inertness of Francisella LPS and the functional consequences of the presence of so much hydrophobic free lipid A are not fully understood. The presence of a large population of hydrophobic LPS molecules complicates some aspects of structural analysis, and in response to this challenge, we have developed a means to label replicating Francisella with [14C]acetate in both laboratory media and within human phagocytes. The label is incorporated into fatty acids, providing a sensitive, quantitative method to analyze the structure of Francisella LPS in detail. Mounting evidence indicates that among the adaptations made by Francisella as it invades and disseminates are changes in the structural composition of its lipid A/LPS. The methods we have developed to label, isolate, and quantify lipid A/LPS molecules based on specific structural characteristics will permit a detailed analysis of modulation of innate immunity by Francisella. Based on our preliminary data, we hypothesize that Francisella alters the structure of its lipid A during adaptation to the cytosol o mammalian cells, and that these changes result in a lipid A with distinct properties that facilitat evasion of innate immunity. To test this hypothesis, we propose the following specific aims: 1. Characterize the determinants of lipid A heterogeneity of F. tularensis 2. Determine how the formation of lipid A species accumulating selectively in infected host cells is regulated 3. Determine the functional consequences of the lipid A alterations that occur during intracellular infection

描述（由申请人提供）：革兰氏阴性菌经常引起人类感染。在这些细菌的外膜中发现的丰富的糖脂，脂多糖（LPS），形成其结构的组成部分，并在感染期间调节与免疫系统的多种相互作用。近年来已经清楚的是，许多革兰氏阴性病原体在适应宿主期间改变其LPS的结构，以优化免疫逃避。该提案旨在应用新的实验方法，以更好地了解弗朗西斯菌在细胞内生长过程中脂质A结构修饰的机制和后果。这将代表LPS的第一个详细描述 在胞质病原体中的适应。 与许多医学上重要的革兰氏阴性微生物的LPS不同，弗朗西斯菌属LPS是非炎症性的。它既不刺激也不拮抗LPS传感宿主机制，我们以前已经证明，它不能与通常促进有效消除革兰氏阴性菌的重要LPS传感分子相互作用。这些独特的特征可能来自其不寻常的脂质A和核心多糖结构。弗朗西斯菌脂多糖的总体组成也非常不寻常，因为大多数脂多糖分子由游离脂质A组成，缺乏核心多糖和O-抗原多糖。Francisella LPS惰性的精确结构决定因素和如此多的疏水游离脂质A的存在的功能后果尚未完全理解。 大量疏水性LPS分子的存在使结构分析的某些方面变得复杂，为了应对这一挑战，我们开发了一种在实验室介质和人吞噬细胞中用[14 C]乙酸盐标记复制弗朗西斯菌的方法。该标记物被掺入脂肪酸中，提供了一种灵敏的定量方法来详细分析弗朗西斯菌LPS的结构。越来越多的证据表明，弗朗西斯菌在入侵和传播时所做的适应性改变是其脂质A/LPS结构组成的变化。我们已经开发的方法来标记，分离和定量脂质A/LPS分子的基础上的特定结构特征将允许一个详细的分析调制的先天免疫弗朗西斯。 基于我们的初步数据，我们假设弗朗西斯菌在适应哺乳动物细胞的胞质溶胶过程中改变了其脂质A的结构，并且这些变化导致脂质A具有独特的特性，有助于逃避先天免疫。为了验证这一假设，我们提出了以下具体目标：1。描述F. tularensis 2.确定如何形成的脂质A种类的选择性积累在受感染的宿主细胞的调节3。确定在细胞内感染期间发生的脂质A改变的功能后果