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:10207216
- 负责人:
- 金额:$ 14.65万
- 依托单位:
- 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 2024-03-31
- 项目状态:已结题
- 来源:
- 关键词:AddressAdherens JunctionAdhesionsAmino AcidsBacteremiaBacteriaBiological AssayBiological ModelsBloodBlood CirculationCD47 geneCell Adhesion MoleculesCell PolarityCell-Cell AdhesionCellsChemotactic FactorsChimera organismClostridium perfringens theta-toxinComputer softwareConfocal MicroscopyDevelopmentDiseaseE-CadherinEffector CellEpithelialEpithelial CellsExcisionFamilyFinancial HardshipFluorescent Antibody TechniqueGoalsHealthImageImage AnalysisImmune responseIn VitroInfectionIntegration Host FactorsIntercellular JunctionsLeadLeukocytesLungLung infectionsMeasuresMediatingMicrobiological TechniquesMigration AssayMolecular BiologyOutcomePathogenesisPatient-Focused OutcomesPatientsPeptide HydrolasesPneumococcal PneumoniaPneumoniaProcessProteinsReactive Oxygen SpeciesResearchRespiratory Tract InfectionsRoleSignal PathwayStainsStatistical Data InterpretationStreptococcus pneumoniaeStreptococcus pneumoniae plY proteinSystemTight JunctionsToxinVirulence FactorsWorkafadincell motilitycombatdesignexperimental studyin vivoinhibitor/antagonistjunctional adhesion moleculemicrobialmicrobial hostmigrationmonolayermortalitynectinneutrophilnovel therapeuticsoccludinpreventquantitative imagingrecruitrespiratory 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.
项目摘要
肺炎链球菌每年在美国引起约900,000例肺炎球菌性肺炎病例
死亡率为5-7%,使这种疾病成为重大的健康和经济负担。肺炎链球菌感染可以
传播到血液(菌血症)并导致严重的患者结局。拟议研究的目标
是阐明微生物(AIM 1)和宿主(AIM 2)因素,使该细菌从肺部转移到
血液,这种能力对于许多呼吸道病原体引起传播感染至关重要。一个重要的
肺炎链球菌感染期间的毒力因子是肺炎(ply),一种形成毒素的孔,它已经
与菌血症的发展有关。肺炎链球菌感染也具有过多的特征
免疫反应主要是由称为多形核细胞(PMN)的白细胞介导的,可能引起
宿主损害并导致致命感染。我们的总体假设是PLY和PMN迁移破坏了肺
上皮,促进从肺部到血液的细菌转移。
我们将通过使用体外thransepithialial迁移测定法研究这一假设,这使我们能够评估
细菌如何跨肺上皮过渡,类似于从肺部传播细菌
体内血液。这种多功能的系统模型各种微环境,易于维护,并集成
与其他分子生物学和微生物学技术无缝。在AIM 1中,我们将确定层如何破坏
肺上皮的细胞间结,以及如何促进肺炎链球菌过渡
独立于PMN。为了评估细胞间连接蛋白的层介导的去除,我们将感染极化
肺上皮单层具有脂肪质(WT)或缺乏层的同生细菌菌株,染色细胞间菌株
与荧光抗体的连接蛋白,通过共聚焦显微镜对单层图像单层图像,并使用图像J和
棱镜软件分别执行定量图像和统计分析。同时,我们将量化S。
遍布肺上皮单层的肺炎转运,以连接细胞间连接的层介导的干扰
在没有PMN的情况下细菌迁移的变化。
在AIM 2中,我们将确定PMN如何破坏肺上皮的细胞间连接以及这种扰动如何
从肺部促进肺炎链球菌过境。评估PMN介导的细胞间连接的去除
蛋白质,我们将在存在或不存在的情况下感染具有wt链球菌的极化肺上皮单层
PMNS,具有荧光抗体的染色细胞间连接蛋白,通过共焦对单层图像单层图像
显微镜,并使用图像J和Prism软件执行定量图像和统计分析,
分别。与这些实验并联,我们将测量肺炎链球菌在肺上皮
单层将PMN介导的单层破坏与细菌迁移的变化联系起来。共同
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,这是肺炎链球菌发病机理和其他肺部感染的基本过程。
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
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Walter Isaiah Adams其他文献
Walter Isaiah Adams的其他文献
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{{ truncateString('Walter Isaiah Adams', 18)}}的其他基金
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:
10596529 - 财政年份:2021
- 资助金额:
$ 14.65万 - 项目类别:
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 - 财政年份:2021
- 资助金额:
$ 14.65万 - 项目类别:
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