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

这项提案中描述的研究将检验这样一种假设，即let-7b-a 22核苷酸的分泌 微小RNA(MiRNA)-由人支气管上皮细胞(HBEC)在细胞外小泡中增加 铜绿假单胞菌对抗生素的敏感性和减少生物膜的形成，因此是一个重要的 肺部宿主-病原体相互作用的机制。铜绿假单胞菌是一种条件致病菌，可感染 慢性阻塞性肺疾病(COPD)、囊性纤维化(CF)和 肺炎。在COPD可能发生的急性加重事件中，铜绿假单胞菌占5%-10%， 它折磨着2400万美国人，是美国第三大死亡原因。let-7b和所有miRNAs一样， 抑制基因表达，在物种间高度保守，并调节天然宿主免疫 对病原体的反应。在初步研究中，该研究小组进行了独特的观察：(1) 原代HBEC分泌的胞外小泡将let-7b送入铜绿假单胞菌；(2)let-7b增加 一线抗生素通过靶向基因编码杀死铜绿假单胞菌和减少生物被膜形成的能力 对于RND外排泵，生物膜形成所必需的基因，以及编码β-内酰胺酶的基因。该队 也做了新的观察到，let-7b在分离的细胞外小泡中显著减少 CF患者的BALF与健康人的BALF进行比较，导致他们 结论：CF细胞外小泡中let-7b的分泌存在缺陷。除了临床意义之外， 这些初步数据令人兴奋，因为它们是第一次直接证明真核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-7b通过降低RND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被膜形成的假说 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. 铜绿假单胞菌对β-内酰胺类抗生素的靶向β-内酰胺酶敏感性。该项目的工作将利用：(A) RNA-SEQ和质谱仪鉴定铜绿假单胞菌中LET-7b下调的基因和蛋白质；(B) GRIL-SEQ用于直接确定铜绿假单胞菌中let-7b的mRNA靶点；以及(C)由 研究生长在HBEC上的铜绿假单胞菌生物膜的团队。结合起来，这项工作将阐明 抗炎的let-7b增强抗生素杀灭铜绿假单胞菌能力的机制 并减少生物膜的形成。由于LET-7家族的几个成员目前正在进行临床试验，以及 因为慢性阻塞性肺疾病和慢性阻塞性肺疾病患者的慢性铜绿假单胞菌感染与高炎症相关 这项工作的长期目标是开发一种结合使用let-7b和抗生素的方法来 减少CF患者的铜绿假单胞菌肺部感染及伴随的高炎症状态 和慢性阻塞性肺病。

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.