Human Airway Colonization by Mycoplasma pneumoniae

描述(申请人提供)：肺炎支原体是社区获得性呼吸道疾病的主要原因，占美国每年肺炎和住院病例的20%，是年龄较大的儿童和年轻人肺炎的主要原因。感染通常是慢性的，可能导致或加剧包括哮喘和COPD在内的改变生活的疾病，但组织趋向性和呼吸道持久性的分子基础尚不清楚。支原体粘附素蛋白P1直接参与受体结合和滑行运动。唾液酸糖蛋白和硫脂等糖脂已被鉴定为受体，但肺炎支原体细胞与唾液糖蛋白结合时滑动，与硫脂结合时静止。我们的长期目标是了解肺炎支原体和呼吸道上皮之间的动态相互作用如何影响传播、组织趋向性、宿主内传播、免疫逃避和持久性。更好地了解潜在的分子识别事件及其如何影响感染结果将增强我们开发新策略以限制发病率和影响的能力。我们的中心假设是肺炎支原体的黏附和滑动运动受到呼吸道多聚糖密度和结构的影响，进而影响定植模式和宿主内传播。令人惊讶的是，人们对呼吸道多糖的结构和分布知之甚少，尽管它们对各种呼吸道病原体具有根本的重要性。目的1将在潜在肺炎支原体受体的背景下描述人类呼吸道糖糖的特征，解决在人类呼吸道模型和人类呼吸道组织中在哪里和在什么水平发现哪些多糖。我们将利用最先进的碳水化合物结构分析来定义在气液界面培养中生长的正常人类支气管上皮(NHBE)细胞的糖类，我们用它来模拟支原体的定植。我们推测，在这个模型中观察到的不同的空间和时间定植模式是由葡聚糖环境造成的。NHBE细胞糖类分析的结果将与来自上呼吸道和下呼吸道的人体组织样本的全面硫酸盐化糖脂分析进行比较。目的2将确定肺炎支原体结合的特异性和对受体群体的作用力和滑动表型，解决哪些潜在的受体影响结合和滑动活性。我们将筛选一个全面的糖链阵列，以确定结合特异性，通过原子力显微镜确定结合强度，并评估不同相对浓度下单独和组合在唾液酸糖蛋白和硫酸糖脂受体部分上的黏附和滑动。目的3将描述粘附素P1蛋白的一个亚群，该亚群由其特有的单抗标记模式定义，评估该亚群丰度的变化如何与黏附和滑动功能相关，并将电子冷冻断层扫描观察到的P1动态与末端细胞器表面的结构变化相关联。

DESCRIPTION (provided by applicant): Mycoplasma pneumoniae is a major cause of community-acquired respiratory disease, accounting for >20% of all pneumonia and >100,000 hospitalizations annually in the U.S., and is the leading cause of pneumonia in older children and young adults. Infections are often chronic and can precipitate or exacerbate life-altering conditions including asthma and COPD, but the molecular basis for tissue tropism and persistence in the airways is not known. Mycoplasma adhesin protein P1 functions directly in receptor binding and gliding motility. Sialoglycoproteins and sulfated glycolipids such as sulfatide have been identified as receptors, but M. pneumoniae cells glide when bound to sialoglycoproteins and are static when bound to sulfatide. Our long- term goal is to understand how the dynamic interplay between M. pneumoniae and airway epithelium impacts transmission, tissue tropism, within-host spread, immune evasion, and persistence. A greater understanding of the underlying molecular recognition events and how they impact infection outcome will enhance our ability to develop new strategies to limit incidence and impact. Our central hypothesis is that M. pneumoniae adherence and gliding motility are influenced by the density and structure of airway glycans, which in turn impacts colonization pattern and within-host spread. Surprisingly little is known about the structure and distribution of airway glycans, despite their fundamental importance for diverse respiratory pathogens. Aim 1 will characterize the human airway glycome in the context of potential M. pneumoniae receptors, addressing which glycans are found where and at what levels in a human airway model and in human airway tissues. We will utilize state-of- the-art carbohydrate structural analysis to define the glycome of normal human bronchial epithelial (NHBE) cells grown in air-liquid interface culture, with which we model mycoplasma colonization. We speculate that glycan environment contributes to the distinct spatial and temporal colonization patterns observed in this model. Results from NHBE cell glycome analysis will be compared with comprehensive sulfated glycolipid analysis of human tissue samples from upper and lower airways. Aim 2 will define M. pneumoniae binding specificity and force and gliding phenotype on receptor populations, addressing which potential receptors impact binding and gliding activity. We will screen a comprehensive glycan array to determine binding specificity, determine binding strength by atomic force microscopy, and evaluate adherence and gliding on sialoglycoprotein and sulfated glycolipid receptor moieties individually and in combination at different relative concentrations. Aim 3 will characterize a subpopulation of the adhesin P1 protein that is defined by its characteristic monoclonal antibody labeling pattern, assess how changes in the abundance of this P1 subpopulation correlate with adherence and gliding function, and correlate P1 dynamics with structural changes on the terminal organelle surface, as observed by electron cryotomography.