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

促进上皮破坏和肺炎链球菌从肺中转运的微生物和宿主因素

基本信息

批准号：
10596529
负责人：
Walter Isaiah Adams
金额：
$ 14.65万
依托单位：
SAN JOSE STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-04-01 至 2025-03-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10596529
关键词：
Address Adherens Junction Adhesions Amino Acids Bacteremia Bacteria Biological Assay Biological Models Blood Blood Circulation CD47 gene Cell Adhesion Molecules Cell Polarity Cell-Cell Adhesion Cells Chemotactic Factors Chimera organism Clostridium perfringens theta-toxin Computer software Confocal Microscopy Development Disease E-Cadherin Effector Cell Enabling Factors Epithelial Cells Epithelium Excision Family Financial Hardship Fluorescent Antibody Technique Goals Health Image Image Analysis Immune response In Vitro Infection Integration Host Factors Intercellular Junctions Leukocytes Lung Lung infections Measures Mediating Microbiology Migration Assay Molecular Biology Outcome Pathogenesis Patient-Focused Outcomes Patients Peptide Hydrolases Pneumococcal Pneumonia Pneumonia Process Proteins Reactive Oxygen Species Research Respiratory Tract Infections Role Signal Pathway Stains Statistical Data Interpretation Streptococcus pneumoniae Streptococcus pneumoniae plY protein System Techniques Tight Junctions Toxin Virulence Factors Work afadin cell motility combat design experimental study in vivo inhibitor junctional adhesion molecule microbial migration monolayer mortality nectin neutrophil novel therapeutics occludin prevent quantitative imaging recruit respiratory pathogen

项目摘要

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.

项目概要在美国，肺炎链球菌每年引起约 900,000 例肺炎球菌肺炎病例，其中死亡率为5-7%，使这种疾病成为重大的健康和经济负担。肺炎链球菌肺部感染可扩散到血液（菌血症）并导致严重的患者后果。拟议研究的目标目的是阐明使该细菌能够从肺部转移到肺部的微生物（目标 1）和宿主（目标 2）因素。血液，这种能力对于许多呼吸道病原体引起播散性感染至关重要。一个重要的肺炎链球菌感染期间的毒力因子是肺炎链球菌溶血素（PLY），一种成孔毒素，已被与菌血症的发展有关。肺炎链球菌感染的另一个特点是过度主要由称为多形核细胞 (PMN) 的白细胞介导的免疫反应，可导致宿主受损并导致致命的感染。我们的总体假设是 PLY 和 PMN 迁移会破坏肺部上皮细胞，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测定来研究这一假设，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肺部体内的血液。这种多功能系统可以对不同的微环境进行建模，易于维护并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 PLY 如何颠覆肺上皮细胞间连接及其如何促进肺炎链球菌从肺部转运独立于 PMN。为了评估 PLY 介导的细胞间连接蛋白去除，我们将感染极化具有 PLY 丰富 (WT) 或 PLY 缺陷等基因细菌菌株的肺上皮单层，细胞间染色连接蛋白与荧光抗体，通过共焦显微镜对单层进行成像，并使用 Image J 和 Prism 软件分别执行定量图像和统计分析。同时，我们将量化 S。肺炎杆菌穿过肺上皮单层连接 PLY 介导的细胞间连接破坏在没有 PMN 的情况下细菌迁移的变化。在目标 2 中，我们将确定中性粒细胞如何破坏肺上皮的细胞间连接以及这种扰动如何促进肺炎链球菌从肺部排出。评估 PMN 介导的细胞间连接去除蛋白质，我们将在存在或不存在的情况下用 WT 肺炎链球菌感染极化的肺上皮单层 PMN，用荧光抗体染色细胞间连接蛋白，通过共聚焦对单层进行成像显微镜，并使用Image J和Prism软件进行定量图像和统计分析，分别。与这些实验并行，我们将测量肺炎链球菌穿过肺上皮的情况单层细胞将 PMN 介导的单层细胞破坏与细菌迁移的变化联系起来。总的来说，这些实验将解释微生物和宿主因素如何破坏肺上皮，导致细菌传播是肺炎链球菌发病机制和其他肺部感染的基本过程。