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

在这项提案中描述的研究将测试let-7 b-a 22-核苷酸的分泌 人支气管上皮细胞（HBEC）细胞外囊泡中的微小RNA（miRNA）增加了 抗生素敏感性和减少生物膜形成的铜绿假单胞菌，从而是一个重要的 肺中宿主-病原体相互作用的机制。铜绿假单胞菌是一种机会致病菌， 患有慢性阻塞性肺病（COPD）、囊性纤维化（CF）和 肺炎铜绿假单胞菌导致5-10%的COPD急性加重事件， 它折磨着2400万美国人，是美国第三大死亡原因。Let-7 b，像所有的miRNAs一样， 抑制基因表达，在物种间高度保守，并调节先天宿主免疫 对病原体的反应。在初步研究中，该研究小组提出了以下独特的观察结果：（1） 由原代HBEC分泌的细胞外囊泡将let-7 b递送到铜绿假单胞菌中;以及（2）let-7 b增加了 一线抗生素杀死铜绿假单胞菌并通过靶向编码 用于RND外排泵的基因、生物膜形成所必需的基因和编码β-内酰胺酶的基因。球队 也进行了新的观察，let-7 b在从细胞膜分离的细胞外囊泡中显著减少， CF患者的支气管肺泡液（BALF）与健康受试者的BALF相比，导致他们 结论CF细胞外囊泡let-7 b分泌缺陷。除了临床意义之外， 这些初步的数据是令人兴奋的，因为它们是第一个直接证明真核细胞的miRNA可以 调节原核生物的功能。基于这些发现，该团队提出了三个具体目标：（1）测试 假设let-7 b通过降低RND增加铜绿假单胞菌对氟喹诺酮类抗生素敏感性 外排泵，MexGHI-OpmD;（2）检验let-7 b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;和（3）测试let-7 b增加P. 铜绿假单胞菌对β-内酰胺类抗生素的敏感性。本项目的工作将利用： RNA-seq和质谱法鉴定铜绿假单胞菌中let-7 B下调的基因和蛋白质;（B） GRIL-seq直接鉴定铜绿假单胞菌中的let-7 b mRNA靶标;以及（c）由GRIL-seq开发的共培养模型。 研究在HBEC上生长的铜绿假单胞菌生物膜。结合起来，这项工作将阐明 抗炎的let-7 b增强抗生素杀死铜绿假单胞菌的能力的机制 并减少生物膜形成。由于let-7家族的几个成员目前正在进行临床试验， 因为COPD和CF中铜绿假单胞菌的慢性肺部感染与高炎症相关， 这项工作的长期目标是开发一种利用let-7 b与抗生素联合的方法， 减少CF患者的铜绿假单胞菌肺部感染和伴随的高度炎症状态 和COPD。

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.