Pathogenesis of Pf Bacteriophages in Pseudomonas Cystic Fibrosis lung Infections

Pf 噬菌体在假单胞菌囊性纤维化肺部感染中的发病机制

基本信息

批准号：
10463594
负责人：
Carlos Milla
金额：
$ 41.95万
依托单位：
STANFORD UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-08-01 至 2024-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10463594
关键词：
Adhesives Adult Affect Aminoglycosides Antibiotic Resistance Antibiotics Antibodies Antibody Response Architecture Aztreonam Bacteria Bacterial Infections Bacteriophages Cell physiology Characteristics Charge Chronic Ciliary epithelium Clinical Crystallization Cystic Fibrosis Cystic Fibrosis Transmembrane Conductance Regulator Cystic Fibrosis sputum Data Development Diffusion Disease Disease Progression Effectiveness Elastases Electrostatics Entropy Epithelial Extracellular Matrix Immune response Immunity Impairment In Vitro Infection Inflammatory Inhalation Ion Transport Ions Light Liquid substance Longitudinal Studies Lung Lung Transplantation Lung infections Mediating Meropenem Microbial Biofilms Movement Mucociliary Clearance Mucous body substance Outcome Pathogenesis Pathogenicity Patients Phagocytes Phagocytosis Polymers Polysaccharides Production Pseudomonas Pseudomonas aeruginosa Pseudomonas aeruginosa infection Pulmonary Cystic Fibrosis Reporting Role Sampling Sputum Suggestion Surface Testing Therapeutic Intervention Time Tobramycin Translations Virulence Virulence Factors Virus Work antibiotic tolerance base chronic infection cohort crystallinity cystic fibrosis patients cytokine experimental study extracellular human pathogen immune clearance in vitro Model infection burden liquid crystal microbial mortality neutrophil new therapeutic target novel pathogenic bacteria pediatric patients prevent pulmonary function resistance mutation resistant strain response treatment strategy

项目摘要

Project Summary Pseudomonas aeruginosa (Pa) is a major human pathogen whose virulence is predicated on its ability to form biofilms - slimy layers of polysaccharides and bacteria that confer resistance to antibiotics and immune clearance. Pa biofilms are particularly problematic in Cystic Fibrosis (CF), where they are a major cause of mortality. We have uncovered novel roles for Pf bacteriophages in chronic biofilm infections. We recently reported that Pf phages produced by Pa organize host and microbial polymers present in sputum into a liquid crystal. This crystalline architecture makes CF sputum more viscous and more adhesive. It also traps antibiotics like tobramycin and prevents them from reaching the bacteria living within. Along with these effects on biofilm formation and function, Pf phages directly interfere with host immunity. Purified Pf phage inhibits phagocytosis and hampers the ability to clear bacterial infections. In preliminary work with a cohort of Pa infected CF patients, we found associations between Pf phage and Pa lung infection burden, chronicity of Pa infection, declines in pulmonary function during exacerbation, and antibiotic resistance to anti- Pseudomonal antibiotics. Given the high abundance of Pf in CF sputum we also postulate that Pf liquid crystals may affect host mucociliary clearance by attachment and impairment of the epithelial ciliary brush function. In light of these exciting preliminary data, we hypothesize that Pf phage cause worse clinical outcomes in CF by disrupting bacterial clearance mechanisms and promoting bacterial tolerance to antibiotics. We will test this hypothesis in experiments with the following aims: In Aim 1 we will determine how Pf phage impacts cell function in the airways. Our hypothesis is that Pf phage interferes with phagocytosis, impairs mucus transport and disrupts the function of the ciliary brush. To test this, we will conduct a number of in vitro experiments to investigate the mechanisms of Pf pathogenecity. In Aim 2 we will elucidate how Pf phage production drives Pa antibiotic tolerance. Our hypothesis is that the liquid crystalline organization of Pa biofilms prevents diffusion of antibiotics and promotes the emergence of resistant strains. To test this, we will assess how Pf phage-mediated sequestration impacts the bioactivity of antibiotics and the emergence of antibiotic resistant Pa isolates over time. In Aim 3 we will characterize how Pf phage impacts clinical outcomes in CF patients. Our hypothesis is that higher Pf phage titers are associated with crystalline sputum and worse clinical outcomes. To test this, we will perform longitudinal studies to determine how Pa colonization influences pulmonary function and the occurrence of exacerbations. We will also investigate in CF patients for the presence of antibody responses directed at Pf and correlate their presence with clinical outcomes. This proposal represents a bold and radically unconventional approach to Pa biofilm infections and CF pathobiology. If successful, this work will identify a novel therapeutic target in CF and other settings where Pa biofilm infections cause disease.

项目概要铜绿假单胞菌 (Pa) 是一种主要的人类病原体，其毒力取决于其形成细菌的能力。生物膜 - 多糖和细菌的粘稠层，赋予抗生素和免疫抗性清除。 Pa 生物膜在囊性纤维化 (CF) 中尤其成问题，它们是导致囊性纤维化 (CF) 的主要原因死亡。我们发现了 Pf 噬菌体在慢性生物膜感染中的新作用。我们最近据报道，Pa 产生的 Pf 噬菌体将痰中存在的宿主和微生物聚合物组织成液体水晶。这种结晶结构使 CF 痰液更加粘稠且更具粘附性。它还捕获抗生素就像妥布霉素一样，可以防止它们接触到体内的细菌。除了对生物膜形成和功能的影响外，Pf 噬菌体还直接干扰宿主免疫。纯化的 Pf 噬菌体抑制吞噬作用并阻碍清除细菌感染的能力。在初步工作中与 Pa 感染 CF 患者队列，我们发现 Pf 噬菌体与 Pa 肺部感染负担之间存在关联， Pa 感染的慢性性、恶化期间肺功能的下降以及抗生素耐药性假单胞菌抗生素。鉴于 CF 痰中 Pf 的高丰度，我们还假设 Pf 液晶可能通过附着和损害上皮纤毛刷功能来影响宿主粘膜纤毛清除。根据这些令人兴奋的初步数据，我们假设 Pf 噬菌体通过以下方式导致 CF 的临床结果更差破坏细菌清除机制并促进细菌对抗生素的耐受性。我们将测试这个实验中的假设具有以下目标：在目标 1 中，我们将确定 Pf 噬菌体如何影响气道中的细胞功能。我们的假设是 Pf 噬菌体干扰吞噬作用，损害粘液运输并破坏睫状刷的功能。为了测试这一点，我们将进行大量的体外实验来研究Pf的致病机制。在目标 2 中，我们将阐明 Pf 噬菌体的产生如何驱动 Pa 抗生素耐受性。我们的假设是 Pa 生物膜的液晶组织可防止抗生素扩散并促进抗生素的出现耐药菌株。为了测试这一点，我们将评估 Pf 噬菌体介导的隔离如何影响随着时间的推移，抗生素和抗生素耐药 Pa 分离株的出现。在目标 3 中，我们将描述 Pf 噬菌体如何影响 CF 患者的临床结果。我们的假设是较高的 Pf 噬菌体滴度与结晶痰和较差的临床结果相关。为了测试这一点，我们将进行纵向研究以确定 Pa 定植如何影响肺功能及其发生的恶化。我们还将调查 CF 患者是否存在针对 Pf 的抗体反应并将它们的存在与临床结果相关联。该提案代表了一种针对 Pa 生物膜感染和 CF 的大胆且完全非常规的方法病理学。如果成功，这项工作将确定 CF 和其他需要 Pa 治疗的新治疗靶点生物膜感染引起疾病。