The Structural Determinants of Innate Immune Modulation by Francisella LPS

描述(申请人提供)：革兰氏阴性细菌经常导致人类感染。在这些细菌的外膜中发现了一种丰富的糖脂，脂多糖(LPS)，形成了它们结构的组成部分，并在感染过程中调节与免疫系统的多种相互作用。近年来，许多革兰氏阴性病原菌在适应宿主的过程中改变了其内毒素的结构，以优化免疫逃避。这项建议寻求应用新的实验方法来更好地理解方济各氏菌在细胞内生长过程中类脂A结构修饰的机制和后果。这将是对内毒素的第一次详细描述 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内毒素不同，弗朗西斯杆菌内毒素是非炎症性的。它既不刺激也不拮抗内毒素感应宿主机制，我们之前已经证明它无法与重要的内毒素感应分子相互作用，这些分子通常促进有效地消除革兰氏阴性细菌。这些独特的特征可能来自于其不寻常的脂类A和核心多糖结构。弗朗西斯氏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A组成，缺乏核心和O抗原多糖。弗氏杆菌脂多糖的惰性的确切结构决定因素以及存在如此多疏水游离脂A的功能后果尚不完全清楚。大量疏水性内毒素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类吞噬细胞内用[14C]醋酸酯标记复制的方济氏菌的方法。该标记被结合到脂肪酸中，提供了一种灵敏的、定量的方法来详细分析弗朗西塞氏菌的结构。越来越多的证据表明，在方济各氏菌入侵和传播时所作的适应中，其脂类A/脂多糖的结构组成发生了变化。我们开发的基于特定结构特征的标记、分离和定量脂类A/内毒素分子的方法将使我们能够详细分析方济氏菌对天然免疫的调节。根据我们的初步数据，我们假设Francisella在适应哺乳动物细胞胞浆的过程中改变了它的类脂A的结构，这些变化导致了一种具有独特性质的类脂A，有助于逃避天然免疫。为了验证这一假说，我们提出了以下具体目标：1.确定图拉氏丝孢子菌类脂A异质性的决定因素2.确定在受感染的宿主细胞中选择性积累的类脂A物种的形成是如何被调节的3.确定在细胞内感染过程中发生的类脂A变化的功能后果

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