Microbial and Host Factors that Promote Epithelial Disruption and S. pneumoniae Transit out of the Lung

项目摘要 在美国，肺炎链球菌每年导致约90万例肺炎球菌肺炎，其中 死亡率为5%-7%，使这种疾病成为主要的健康和经济负担。肺炎链球菌肺部感染可 扩散到血液中(菌血症)，并导致严重的患者结局。拟议研究的目标是 是阐明微生物(目标1)和宿主(目标2)的因素，使这种细菌能够从肺转移到 血液是许多呼吸道病原体导致播散性感染的关键能力。一个重要的 肺炎链球菌感染过程中的毒力因子是肺炎链球菌溶血素(PLY)，这是一种形成毛孔的毒素，已被 与菌血症的发展有关。肺炎链球菌感染的特征也是过度的 主要由称为多形核细胞(PMN)的白细胞介导的免疫反应可导致 寄主受损并导致致命性感染。我们的总体假设是PLY和PMN的迁移破坏了肺 上皮细胞，促进细菌从肺部进入血液。 我们将通过使用体外跨上皮迁移试验来研究这一假说，这使我们能够评估 细菌如何穿过肺上皮，类似于细菌从肺部传播到 活体内的血流。该多功能系统可对不同的微环境进行建模，易于维护和集成 与其他分子生物学和微生物学技术无缝结合。在目标1中，我们将确定铺层如何破坏 肺上皮细胞间连接及其如何促进肺炎链球菌向肺外传播 独立于PMN。为了评估PLY介导的细胞间连接蛋白的去除，我们将感染极化 PLY熟练(WT)或PLY缺乏的同基因细菌肺上皮单层，细胞间染色 连接蛋白与荧光抗体，通过共聚焦显微镜成像单层，并使用Image J和 PRISM软件分别进行定量图像和统计分析。同时，我们将量化S。 肺炎衣原体穿过肺上皮单层连接PLY介导的细胞间连接中断 在没有PMN的情况下细菌迁移的变化。 在目标2中，我们将确定PMN如何破坏肺上皮细胞间的连接，以及这种干扰是如何发生的。 促进肺炎链球菌从肺部传播出去。评价中性粒细胞介导的细胞间连接的清除 蛋白，我们将在有或没有肺炎链球菌的情况下感染极化的肺上皮细胞单层。 中性粒细胞，用荧光抗体标记细胞间连接蛋白，通过共聚焦成像单层 显微镜，并使用Image J和Prism软件进行定量图像和统计分析。 分别进行了分析。在这些实验的同时，我们将测量肺炎链球菌在肺上皮细胞中的传播。 将PMN介导的单层破坏与细菌迁移的变化联系起来的单层。总而言之， 这些实验将解释微生物和宿主因素如何破坏肺上皮，导致细菌 播散是肺炎链球菌和其他肺部感染的一个基本过程。

Project Summary Streptococcus pneumoniae causes ~900,000 cases of pneumococcal pneumonia annually in the US, with a mortality rate of 5-7%, making this disease a major health and financial burden. S. pneumoniae lung infections can spread to the bloodstream (bacteremia) and lead to severe patient outcomes. The goal of the proposed research is to elucidate the microbial (Aim 1) and host (Aim 2) factors that enable this bacterium to transit from the lung to the blood, an ability that is critical for many respiratory pathogens to cause disseminated infection. An important virulence factor during S. pneumoniae infection is pneumolysin (PLY), a pore forming toxin, which has been implicated in the development of bacteremia. S. pneumoniae infections are also characterized by an excessive immune response mediated primarily by white blood cells called polymorphonuclear cells (PMNs) that can cause host damage and result in lethal infection. Our overall hypothesis is that PLY and PMN migration disrupt the lung epithelium, promoting bacterial transit from the lung into the bloodstream. We will investigate this hypothesis by using an in vitro transepithelial migration assay, which allows us to assess how bacteria transit across the lung epithelium, analogous to bacterial dissemination from the lungs into the bloodstream in vivo. This versatile system models diverse microenvironments, is easy to maintain, and integrates seamlessly with other molecular biology and microbiology techniques. In Aim 1 we will determine how PLY disrupts intercellular junctions of the lung epithelium and how that promotes S. pneumoniae transit out of the lungs independent of PMNs. To assess PLY-mediated removal of intercellular junction proteins, we will infect polarized lung epithelial monolayers with PLY-proficient (WT) or PLY-deficient isogenic bacterial strains, stain intercellular junction proteins with fluorescent antibodies, image the monolayers by confocal microscopy, and use Image J and Prism software to perform quantitative image and statistical analysis, respectively. In parallel, we will quantify S. pneumoniae transit across lung epithelial monolayers to connect PLY-mediated disruptions of intercellular junctions to changes in bacterial migration in the absence of PMNs. In Aim 2 we will identify how PMNs disrupt intercellular junctions of the lung epithelium and how this perturbation promotes S. pneumoniae transit out of the lungs. To evaluate PMN-mediated removal of intercellular junction proteins, we will infect polarized lung epithelial monolayers with WT S. pneumoniae in the presence or absence of PMNs, stain intercellular junction proteins with fluorescent antibodies, image the monolayers by confocal microscopy, and use Image J and Prism software to perform quantitative image and statistical analysis, respectively. In parallel with these experiments, we will measure S. pneumoniae transit across the lung epithelial monolayers to connect PMN-mediated monolayer disruptions with changes in bacterial migration. Collectively, these experiments will explain how microbial and host factors disrupt the lung epithelium, leading to bacterial dissemination, a fundamental process in S. pneumoniae pathogenesis and other lung infections.