Collectins and Innate Defense against Inhaled Pathogens

描述（由申请人提供）：该计划项目的中心主题是了解两种肺聚集素，表面活性蛋白A和D（SP-A，SP-D）对吸入病原体的先天宿主防御机制。这一认识不仅在呼吸道感染流行的情况下很重要，而且在与严重的全球生物威胁有关的情况下也很重要-抗药性生物体的出现;更致命的呼吸道病毒的演变和迅速传播;以及在生物恐怖主义行为中可能使用吸入的病原体。有效的肺部宿主防御需要早期识别微生物。微生物沉积到呼吸道后，有一个关键的机会窗口清除病原体;延迟的反应有利于感染。肺聚集蛋白在针对许多革兰氏阴性和阳性细菌及其内毒素、分枝杆菌、致病性真菌和专性细胞内病原体（包括甲型流感（IAV）和其他潜在致死病毒）的先天防御中发挥一线作用。肺胶原凝集素的先天防御特性取决于它们的快速识别，并且在某些情况下，基于高度保守的微生物表面组分（例如IAV上的N-连接的高甘露糖聚糖和革兰氏阴性脂多糖（内毒素））的模式识别来中和吸入的微生物。其他研究表明，缺乏肺聚集蛋白的动物对微生物挑战的易感性显着增加。在小鼠过敏和感染模型中对重组截短SP-A和SP-D的研究提供了这样的可能性，即通过吸入递送的这些蛋白质的雾化形式在控制人类呼吸道感染、炎症和过敏中可能具有治疗潜力。在这个项目中，一个高度合作和集中的项目组使用互补的方法，包括X射线晶体学，诱变，光谱学，使用动物模型的体外和体内分析，专门针对collectins的先天反应，以及调节其活性的蛋白质，如KGF，以获得机理理解并帮助设计具有增加的抗微生物活性的潜在的基于聚集蛋白的治疗剂。 项目1：振动光谱学：凝集素与生理配体的相互作用（Mendelsohn，R） 项目1描述（由申请人提供）：本项目的长期目标是阐明肺中的聚集素在宿主防御中对空气传播病原体提供首次挑战的分子基础。表面活性剂特异性凝集素SP-A和SP-D通过其与脂多糖（LPS）的相互作用部分地负责该活性。凭借本实验室开发的新型振动光谱方法的技术专长，我们提出了两个具体目标来表征两种蛋白质与生理LPS衍生物的相互作用。凭借该项目其他研究人员的专业知识，我们将获得这些蛋白质的大量遗传变异，使我们能够在各种生物相关的物理制剂中确定主要的相互作用位点。 提出了两个具体目标。首先，由于这两个collectins识别碳水化合物和其他极性配位体的网站，我们将确定这些collectins的结构因素，重要的结合到LPS和它的变体在朗缪尔膜（即单层）在空气/水界面使用一个独特的振动光谱实验（红外反射吸收光谱- IRRAS），提取分子结构和取向信息的单层成分。这些膜模拟细菌革兰氏阴性外膜单层的极性区域。该目标的整合假设是，在分子结构水平上，凝集素和LPS之间的相互作用取决于蛋白质结构的特定元素，并强调凝集素-碳水化合物识别结构域的特定区域。拉曼微晶体学补充了IRRAS，并提供了非常具体的结构信息，蛋白质侧链参与的初始识别事件。在第二个目标中，我们将补充研究蛋白质的结构变化与IR实验，跟踪结构变化的酰基链和极性区域的LPS衍生物。我们将监测收集/LPS相互作用的条件下，朗缪尔膜不是唯一合理的实验范式。脂质和脂质/蛋白质复合物的物理状态，结构信息将被收购包括囊泡，单层，支持定向多层，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.