Collectins and Innate Defense against Inhaled Pathogens

描述(申请人提供)：该项目的中心主题是了解两种肺集合素，表面活性蛋白A和D(SP-A，SP-D)对吸入性病原体的天然宿主防御机制。这一认识不仅在流行的呼吸道感染方面很重要，而且在与严重的全球生物危机有关的问题上也很重要--出现具有抗生素耐药性的生物体；更致命的呼吸道病毒的进化和迅速传播；以及可能在生物恐怖主义行为中使用吸入性病原体。有效的肺宿主防御需要及早识别微生物。在生物体沉积到呼吸道后，有一个关键的机会清除病原体；延迟的反应有利于感染。肺集合素在许多革兰氏阴性和革兰氏阳性细菌及其内毒素、分枝杆菌、病原性真菌和专性细胞内病原体(包括甲型流感病毒和其他潜在致死性病毒)的天然防御中发挥着一线作用。肺集合素的天然防御特性依赖于它们的快速识别，在某些情况下，基于高度保守的微生物表面成分的模式识别，如LAV上的N-连接高甘露聚糖和革兰氏阴性脂多糖(内毒素)，对吸入微生物的识别。其他研究表明，肺集合素缺乏的动物对微生物挑战的敏感度显著增加。在小鼠过敏和感染模型中对重组截短SP-A和SP-D的研究提供了这样一种可能性，即这些蛋白的雾化吸入形式可能在控制人类呼吸道感染、炎症和过敏方面具有治疗潜力。在这个计划项目中，一组高度协作和专注的项目使用互补的方法，包括X射线结晶学、突变、光谱学、使用动物模型的体外和体内分析，专门针对集合素和调节其活性的蛋白质(如KGF)对LAV和革兰氏阴性细菌的先天反应，以获得机制上的了解并协助设计潜在的基于集合素的治疗方法，增加抗微生物活性。 项目1：振动光谱学：集合素与生理配体的相互作用(Mendelsohn，R) 项目1描述(申请人提供)：该项目的长期目标是阐明肺中的集合素在宿主防御中对空气传播病原体的第一个挑战的分子基础。表面活性物质特有的收集素SP-A和SP-D通过与脂多糖(LPS)的相互作用，部分地负责这一活动。结合本实验室开发的新型振动光谱方法的技术专长，我们提出了两个特定的目标来表征这两种蛋白质与生理内毒素衍生物的相互作用。有了项目中其他研究人员的专业知识，我们将能够获得这些蛋白质的大量遗传变异，使我们能够精确定位各种生物相关物理准备中的主要相互作用部位。 提出了两个具体目标。首先，由于这两种集合素都识别碳水化合物和其他极性配体位置，我们将使用从单层成分中提取分子结构和取向信息的独特振动光谱实验(红外反射吸收光谱-IRRAS)来确定那些对在空气/水界面的朗缪尔膜(即单分子膜)中与内毒素及其变体结合至关重要的集合素结构因子。这些膜模拟细菌革兰氏阴性外膜单层的极区。这一目标的整合假设是，在分子结构水平上，集合素与脂多糖之间的相互作用取决于特定的蛋白质结构元素，并强调集合素-碳水化合物识别结构域的特定区域。拉曼微晶学是对IRRAS的补充，并提供了关于最初识别事件中涉及的蛋白质侧链的非常具体的结构信息。在第二个目标中，我们将用IR实验来补充蛋白质结构变化的研究，这些实验跟踪脂多糖衍生物的酰基链和极性区域的结构变化。我们将在朗缪尔膜不是唯一合理的实验范式的条件下监测集合素/脂多糖的相互作用。用于获取结构信息的脂质和脂质/蛋白质复合体的物理状态包括囊泡、单层膜、定向支撑的多层膜和胶束。

DESCRIPTION (provided by applicant): The central theme of the Program Project is to understand the innate host defense mechanisms against inhaled pathogens by the two pulmonary collectins, surfactant proteins A and D (SP-A, SP-D). This understanding is important not only in the context of prevalent respiratory infections but also those associated with serious global biothreats - the emergence of antibiotic-resistant organisms; evolution and rapid spread of more lethal respiratory viruses; and potential use of inhaled pathogens in acts of bioterrorism. Effective pulmonary host defense requires early recognition of microorganisms. Following deposition of an organism into the respiratory tract, there is a critical window of opportunity for pathogen clearance; delayed response favors infection. The pulmonary collectins play a front-line role in the innate defense against many Gram-negative and positive bacteria and their endotoxins, mycobacteria, pathogenic fungi, and obligate intracellular pathogens including influenza A (lAV) and other potentially lethal viruses. The innate defense properties of pulmonary collectins depend upon their rapid recognition and, in some cases, neutralization of inhaled microorganisms based on pattern recognition of highly-conserved microbial surface components such as N-linked high-mannose glycans on lAV and Gram-negative lipolysaccharide (endotoxin). Other studies show that animals deficient in lung collectins show significantly increased susceptibility to microbiological challenges. Studies of recombinant truncated SP-A and SP-D in murine models of allergy and infection have offered the possibility that aerosolized forms of these proteins delivered by inhalation may have therapeutic potential in controlling respiratory infection, inflammation, and allergy in humans. In this program project, a highly collaborative and focused group of projects using complementary approaches, including x-ray crystallography, mutagenesis, spectroscopy, in vitro and in vivo analyses using animal models, aim specifically at innate responses of collectins, and proteins such as KGF that modulate their activity, to lAV and Gram-negative bacteria both to gain mechanistic understanding and assist design of potential collectin-based therapeutics with increased antimicrobial activities. PROJECT 1: Vibrational Spectroscopy: Collectin Interactions with Physiological Ligands (Mendelsohn, R) PROJECT 1 DESCRIPTION (provided by applicant): The long term objective of this project is to elucidate the molecular basis by which collectins in the lung provide the first challenge to airborne pathogens in host defense. The surfactant specific collectins SP-A and SP-D, through their interactions with lipopolysaccharides (LPS), are responsible in part for this activity. With the technological expertise in novel vibrational spectroscopic approaches developed in this lab, we propose two specific aims to characterize the interaction of both proteins with physiological LPS derivatives. With the expertise of the other investigators in the program project, we will have access to a large number of genetic variants of these proteins, permitting us to pinpoint the primary interaction sites in a variety of biologically relevant physical preparations. Two specific aims are proposed. First, since both collectins recognize carbohydrate and other polar ligand sites, we will determine those collectin structural factors important for binding to LPS and its variants in Langmuir films (i.e. monolayers) at the air/water interface using a unique vibrational spectroscopy experiment (Infrared Reflection Absorption Spectroscopy- IRRAS) that extracts molecular structural and orientational information from the monolayer constituents. These films mimic polar regions of bacterial Gram negative outer membrane monolayers. The integrating hypothesis for this Aim is that at the level of molecular structure, the interaction between collectins and LPS depends on particular elements of protein structure and emphasizes specific regions of the collectin-carbohydrate recognition domain. Raman micro crystallography complements IRRAS and provides very specific structural information about protein side chains involved in the initial recognition event. In the second Aim, we will complement studies of protein structural changes with IR experiments that track structural changes both in the acyl chains and in the polar regions of the LPS derivatives. We will monitor collectin/LPS interaction under conditions where Langmuir films are not the only reasonable experimental paradigm. Physical states of lipid and lipid/protein complexes from which structural information will be acquired include vesicles, monolayers, supported oriented multibilayers, and micelles.