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

• 批准号: 10370434
• 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
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-04-01 至 2024-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10370434
• 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; Epithelial; Epithelial Cells; Excision; Family; Financial Hardship; Fluorescent Antibody Technique; Goals; Health; Image; Image Analysis; Immune response; In Vitro; Infection; Integration Host Factors; Intercellular Junctions; Lead; Leukocytes; Lung; Lung infections; Measures; Mediating; Microbiological Techniques; 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; Tight Junctions; Toxin; Virulence Factors; Work; afadin; cell motility; combat; design; experimental study; in vivo; inhibitor; junctional adhesion molecule; microbial; microbial host; migration; monolayer; mortality; nectin; neutrophil; novel therapeutics; occludin; prevent; quantitative imaging; recruit; respiratory pathogen

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.

项目摘要 肺炎链球菌在美国每年引起约90万例肺炎球菌肺炎， 死亡率为5- 7%，使这种疾病成为主要的健康和经济负担。S.肺炎性肺部感染可 扩散到血液中（菌血症）并导致严重的患者后果。拟议研究的目标 目的是阐明微生物（目的1）和宿主（目的2）的因素，使这种细菌从肺转移到 血液是许多呼吸道病原体引起传播性感染的关键能力。一个重要 S.肺炎链球菌感染是肺炎球菌溶血素（pneumolysin，pneumolysin），一种成孔毒素， 与菌血症的发展有关S.肺炎感染的特征还在于 主要由称为多形核白细胞（PMNs）的白色血细胞介导的免疫应答， 宿主损伤并导致致命感染。我们的总体假设是，肺泡上皮细胞和中性粒细胞迁移破坏了肺 上皮细胞，促进细菌从肺进入血流。 我们将通过体外跨上皮迁移试验来研究这一假设， 细菌如何穿过肺上皮，类似于细菌从肺传播到 体内血流这一多功能系统可模拟不同的微环境，易于维护， 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， 肺上皮细胞间连接以及如何促进S.肺炎从肺部排出 独立于PMNs。为了评估PLY介导的细胞间连接蛋白的去除，我们将感染极化的 肺上皮单层与PLY-熟练（WT）或PLY-缺陷的同基因细菌菌株，染色细胞间 连接蛋白与荧光抗体，图像单层共聚焦显微镜，并使用图像J和 Prism软件分别进行定量图像和统计分析。与此同时，我们将量化S。 肺炎杆菌穿越肺上皮细胞单层连接PLY介导的细胞间连接破坏 在没有中性粒细胞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细胞间的连接，以及这种干扰是如何发生的。 促进了S.肺炎从肺中排出。评估PMN介导的细胞间连接的去除 蛋白质，我们将用WT S感染极化的肺上皮单层。肺炎，存在或不存在 PMNs，用荧光抗体染色细胞间连接蛋白，通过共聚焦显微镜成像单层细胞， 显微镜，并使用Image J和Prism软件进行定量图像和统计分析， 分别在这些实验的同时，我们将测量S。肺炎菌穿过肺上皮细胞 单细胞层连接PMN介导的单层破坏与细菌迁移的变化。总的来说， 这些实验将解释微生物和宿主因素是如何破坏肺上皮细胞，导致细菌感染的。 传播是S.肺炎发病机制和其他肺部感染。