Let-7b in Extracellular Vesicles Secreted by Bronchial Epithelial Cells Increases the Antibiotic Sensitivity of Pseudomonas

支气管上皮细胞分泌的胞外囊泡中的 Let-7b 增加假单胞菌的抗生素敏感性

• 批准号: 10319005
• 负责人: Bruce A. Stanton
• 金额: $ 53.94万
• 依托单位: DARTMOUTH COLLEGE
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-12-15 至 2024-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10319005
• 关键词: Acute; Acute Respiratory Distress Syndrome; Adult; Aftercare; American; Anti-Inflammatory Agents; Antibiotic Resistance; Antibiotics; Bacterial Infections; Bacterial Pneumonia; Biological Assay; Bronchiectasis; Cause of Death; Chronic; Chronic Obstructive Pulmonary Disease; Clinical; Clinical Trials; Coculture Techniques; Code; Cystic Fibrosis; Data; Development; Diabetes Mellitus; Disease Management; Epithelial Cells; Event; Family; Film; Fluoroquinolones; Gene Expression; Gene Proteins; Genes; Goals; Growth; Human; Immune response; Individual; Inflammation; Inflammatory; Lead; Liquid substance; Lung; Lung infections; Mass Spectrum Analysis; Messenger RNA; MicroRNAs; Microbe; Microbial Biofilms; Modeling; Monobactams; Nucleotides; Obesity; Partner in relationship; Patient-Focused Outcomes; Patients; Persons; Pharmaceutical Preparations; Pneumonia; Predisposition; Pseudomonas; Pseudomonas aeruginosa; Pseudomonas aeruginosa infection; Pulmonary Cystic Fibrosis; RNA analysis; Research; Techniques; Testing; Western Blotting; Work; beta-Lactamase; beta-Lactams; bioinformatics tool; bronchial epithelium; cystic fibrosis infection; cystic fibrosis patients; efflux pump; extracellular vesicles; improved; inflammatory milieu; member; new therapeutic target; novel; novel strategies; opportunistic pathogen; pathogen; resistant strain; transcriptome sequencing; uptake

The research described in this proposal will test the hypothesis that the secretion of let-7b—a 22-nucleotide micro-RNA (miRNA)—by human bronchial epithelial cells (HBEC) in extracellular vesicles increases the antibiotic sensitivity of and reduces biofilm formation by Pseudomonas aeruginosa, and is thereby an important mechanism of host-pathogen interactions in the lungs. P. aeruginosa is an opportunistic pathogen that infects the lungs of individuals with chronic obstructive pulmonary disease (COPD), cystic fibrosis (CF), and pneumonia. P. aeruginosa contributes to 5–10% of the acute-exacerbation events that can occur with COPD, which afflicts 24 million Americans and is the 3rd-leading cause of death in the U.S. Let-7b, like all miRNAs, suppresses gene expression, is highly conserved across species, and regulates the innate host immune responses to pathogens. In preliminary studies, this research team made the unique observations that: (1) extracellular vesicles secreted by primary HBEC deliver let-7b into P. aeruginosa; and (2) that let-7b increases the ability of front-line antibiotics to kill P. aeruginosa and reduce biofilm formation by targeting genes coding for an RND efflux pump, genes essential for biofilm formation, and genes coding for β-lactamases. The team also made the novel observation that let-7b is dramatically reduced in extracellular vesicles isolated from the bronchoalveolar fluid (BALF) of CF patients, compared to BALF from healthy subjects, leading them to conclude that let-7b secretion in extracellular vesicles is defective in CF. Aside from the clinical implications, these preliminary data are exciting because they are the first direct demonstration that a eukaryotic miRNA can regulate prokaryotic function. Building on these findings, the team propose three specific aims: (1) Test the hypothesis that let-7b increases P. aeruginosa sensitivity to fluoroquinolone antibiotics by reducing the RND efflux pump, MexGHI-OpmD; (2) Test the hypothesis that let-7b inhibits the formation of P. aeruginosa biofilms by targeting genes essential for biofilm formation; and (3) Test the hypothesis that let-7b increases P. aeruginosa sensitivity to β-lactam antibiotics by targeting β-lactamases. Work in this project will utilize: (a) RNA-seq and mass spectrometry to identify genes and proteins downregulated by let-7b in P. aeruginosa; (b) GRIL-seq to directly identify let-7b mRNA targets in P. aeruginosa; and (c) a co-culture model developed by the team to study P. aeruginosa biofilms growing on HBEC. Combined, this work will elucidate the mechanisms whereby let-7b, which is anti-inflammatory, enhances the ability of antibiotics to kill P. aeruginosa and reduce biofilm formation. Since several members of the let-7 family are currently in clinical trials, and because chronic lung infections of P. aeruginosa in COPD and CF are associated with a hyper-inflammatory state, the long-term goal of this work is to develop an approach utilizing let-7b in combination with antibiotics to reduce P. aeruginosa lung infections—and the accompanying hyper-inflammatory state—in patients with CF and COPD.

本提案中描述的研究将检验以下假设：let-7b（一种 22 核苷酸）的分泌 微小RNA（miRNA）——由人支气管上皮细胞（HBEC）在细胞外囊泡中增加 铜绿假单胞菌的抗生素敏感性并减少生物膜的形成，因此是一个重要的 肺部宿主与病原体相互作用的机制 铜绿假单胞菌是一种感染的机会病原体。 患有慢性阻塞性肺病 (COPD)、囊性纤维化 (CF) 和 铜绿假单胞菌导致 5-10% 的 COPD 急性加重事件， 它困扰着 2400 万美国人，是美国第三大死亡原因 Let-7b 与所有 miRNA 一样， 抑制基因表达，在物种间高度保守，并调节宿主先天免疫 在初步研究中，该研究小组做出了独特的观察：（1） 初级 HBEC 分泌的细胞外囊泡将 let-7b 递送至铜绿假单胞菌中；并且 (2) let-7b 增加 一线抗生素通过靶向编码基因杀死铜绿假单胞菌并减少生物膜形成的能力 RND 外排泵、生物膜形成所必需的基因以及 β-内酰胺酶的编码基因 还进行了新的观察，即从分离的细胞外囊泡中let-7b显着减少 CF 患者的支气管肺泡液 (BALF) 与健康受试者的支气管肺泡液 (BALF) 相比，导致他们 结论是除了临床意义外，CF 中细胞外囊泡的 let-7b 分泌存在缺陷。 这些初步数据令人兴奋，因为它们首次直接证明真核生物 miRNA 可以 基于这些发现，该团队提出了三个具体目标：（1）测试 假设 let-7b 通过降低 RND 来增加铜绿假单胞菌对氟喹诺酮类抗生素的敏感性 外排泵，MexGHI-OpmD；（2）检验let-7b抑制铜绿假单胞菌生物膜形成的假设 通过靶向生物膜形成所必需的基因；以及 (3) 检验 let-7b 增加 P 的假设。 通过针对 β-内酰胺酶，铜绿假单胞菌对 β-内酰胺抗生素的敏感性将利用： (a) RNA-seq 和质谱法鉴定铜绿假单胞菌中 let-7b 下调的基因和蛋白质 (b)； GRIL-seq 直接识别铜绿假单胞菌中的 let-7b mRNA 靶标；以及 (c) 由以下公司开发的共培养模型： 该团队研究在 HBEC 上生长的铜绿假单胞菌生物膜，这项工作将阐明这一点。 具有抗炎作用的let-7b 增强抗生素杀死铜绿假单胞菌的能力的机制 并减少生物膜的形成，因为 let-7 家族的几个成员目前正在进行临床试验，并且 因为 COPD 和 CF 中铜绿假单胞菌的慢性肺部感染与过度炎症有关 国家，这项工作的长期目标是开发一种利用let-7b与抗生素相结合的方法 减少 CF 患者的铜绿假单胞菌肺部感染以及伴随的高炎症状态 和慢性阻塞性肺病。