Pathogenesis of Pf Bacteriophages in Pseudomonas Cystic Fibrosis lung Infections

项目摘要 铜绿假单胞菌是一种主要的人类致病菌，其毒力取决于其形成的能力。 生物膜-粘稠的多糖层和细菌，使其对抗生素和免疫产生抵抗力 通行证。PA生物膜在囊性纤维化(CF)中尤其有问题，它们是导致 死亡率。我们发现了Pf噬菌体在慢性生物被膜感染中的新作用。我们最近 据报道，由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 克里斯托。这种水晶结构使CF痰更粘稠，更具粘性。它还能捕获抗生素 比如妥布霉素，可以防止它们接触到体内的细菌。 除了这些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。纯净的 PF噬菌体抑制吞噬作用，阻碍清除细菌感染的能力。在初步工作中 在PA感染的CF患者队列中，我们发现Pf噬菌体与PA肺部感染负担有关， 铜绿假单胞菌感染的慢性化、急性发作期间肺功能下降以及对抗生素的耐药性 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 可能通过附着和上皮纤毛刷功能的损害而影响宿主的粘液纤毛清除。 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更差的临床结果 破坏细菌清除机制，促进细菌对抗生素的耐受性。我们将对此进行测试 有以下目的的实验中的假设： 在目标1中，我们将确定PF噬菌体对呼吸道细胞功能的影响。我们的假设是Pf噬菌体 干扰吞噬作用，损害粘液运输，扰乱睫毛刷子的功能。为了测试这一点， 我们将进行一些体外实验来研究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的抗生素耐受性。我们的假设是 PA生物膜的液晶组织防止了抗生素的扩散，并促进了 耐药菌株。为了测试这一点，我们将评估噬菌体介导的隔离如何影响Pf的生物活性。 随着时间的推移，抗生素和耐药PA菌株的出现。 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噬菌体滴度与结晶性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和发生 病情恶化的可能性。我们还将调查CF患者中是否存在针对PF的抗体反应 并将它们的存在与临床结果相关联。 这一建议代表了一种大胆而彻底的非常规方法来治疗PA生物被膜感染和CF 病理生物学。如果成功，这项工作将确定一个新的治疗靶点在CF和其他环境中 生物被膜感染会导致疾病。

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.