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

• 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
• 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

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.

项目摘要 铜绿假单胞菌（PA）是一种主要的人类病原体，其毒力是基于其形成的能力的 生物膜 - 多糖和细菌的粘层，赋予对抗生素和免疫的耐药性 清除。 PA生物膜在囊性纤维化（CF）中尤其有问题，其中它们是造成的主要原因 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 报道PA由PA生产的PF噬菌体将痰液中存在于液体中的宿主和微生物聚合物 水晶。这种结晶结构使CF痰液更具粘性和更具粘合剂。它还捕获抗生素 像毒素一样，阻止它们到达生活中的细菌。 除了对生物膜形成和功能的影响，PF噬菌体还直接干扰宿主免疫。纯化 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 PA感染CF患者的队列，我们​​发现PF噬菌体与PA肺部感染负担之间的关联， PA感染的慢性性，加重过程中肺功能下降以及对抗生素的抗生素抗性 伪抗生素。鉴于CF痰液中PF的高丰度，我们还假设PF液晶 可能会通过附着和上皮睫毛刷功能的附着和损害影响宿主的粘膜纤毛清除率。 鉴于这些令人兴奋的初步数据，我们假设PF噬菌体会导致CF的临床结果较差 破坏细菌清除机制并促进对抗生素的细菌耐受性。我们将测试这个 实验中的假设以下目的： 在AIM 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，会损害粘液转运并破坏睫毛刷的功能。为了测试这一点， 我们将进行许多体外实验，以研究PF病原体的机制。 在AIM 2中，我们将阐明PF噬菌体生产如何驱动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的扩散，并促进 抗性菌株。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 随着时间的流逝，抗生素和抗生素耐药性PA的出现。 在AIM 3中，我们将表征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有关。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和发生 恶化。我们还将在CF患者中调查是否存在针对PF的抗体反应 并将其存在与临床结果相关联。 该提案代表了PA生物膜感染和CF的一种大胆且根本非常规的方法 病理生物学。如果成功，这项工作将确定在CF和其他情况下的新型治疗靶标 生物膜感染引起疾病。