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Role of Ribosome Hibernation in the Tolerance of P. aeruginosa Biofilms to Antibiotics

核糖体冬眠在铜绿假单胞菌生物膜对抗生素耐受性中的作用

基本信息

批准号：
10218370
负责人：
MICHAEL J FRANKLIN
金额：
$ 21.6万
依托单位：
MONTANA STATE UNIVERSITY - BOZEMAN
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-04-01 至 2023-03-31
项目状态：
已结题

项目摘要

Bacteria living in biofilms are less susceptible to antibiotic treatments than free-living planktonic cells. One mechanism for this increased antibiotic tolerance is that biofilms contain physiologically heterogeneous subpopulations of cells, including antibiotic-tolerant dormant bacteria. When bacteria enter a dormant state, they undergo a variety of physiological changes that reduce energy-expensive processes, yet protect the integrity of macromolecules required for resuscitation. Among these physiological changes is inactivation and preservation of ribosomes. The ribosome accessory proteins (RMF and HPF) are hibernation factors that block translation and protect ribosomes from degradation when the cells are dormant. By protecting ribosomes, ribosome hibernation factors enable the cells to resuscitate from dormancy when conditions are favorable, resulting in persistent infections. Ribosome entry into and exit from hibernation are dynamic processes, and biofilm subpopulations have cells with ribosomes in differing states, depending on the local environmental conditions. In the research proposed here, we will characterize the role of ribosome hibernation in the antibiotic tolerance of biofilm bacteria, by characterizing protein-protein interactions between ribosomal proteins and ribosome accessory factors. In preliminary work, we developed a bimolecular fluorescence complementation (BiFC) system that allows real-time fluorescence-based imaging of ribosome dynamics in Pseudomonas aeruginosa biofilm cells. This system allows us to image biofilm bacteria with hibernating ribosomes, and to sort the cells based on their ribosome state. The BiFC system also allows us to identify additional ribosome hibernation factors, such as molecular chaperones, that mediate the loading of hibernation factors. The goals of these studies are to: (i) generate molecular constructs that provide optimal fluorescent reporter signals for bacteria with ribosomes in active and hibernating states. (ii) Use the fluorescent reporter systems to identify and sort active from dormant bacteria over the course of biofilm development, and compare their antibiotic sensitivity profiles, and (iii) identify additional dormancy factors that contribute to the antibiotic tolerance of dormant biofilm cells. Since many commonly used antibiotics target bacterial translation, the ultimate goal of these studies is to identify molecular targets that, when disrupted, enhance biofilm sensitivity to ribosome- targeting antibiotics. Targeting ribosome hibernation factors, or their molecular chaperones, may provide a means to inhibit the viability of dormant bacteria, thereby increasing biofilm sensitivity to antibiotics.

生活在生物膜中的细菌对抗生素治疗的敏感性低于自由生活的细胞。这种增加的抗生素耐受性的一种机制是生物膜含有生理上的细胞的异质亚群，包括耐缺氧的休眠细菌。当细菌进入休眠状态，他们经历了各种生理变化，减少能源昂贵过程，但保护复苏所需的大分子的完整性。其中生理变化是核糖体的失活和保存。核糖体辅助蛋白 (RMF和HPF）是冬眠因子，其阻断翻译并保护核糖体免于降解当细胞处于休眠状态时通过保护核糖体，核糖体冬眠因子使细胞能够当条件有利时从休眠中复苏，导致持续感染。核糖体进入和离开冬眠是动态过程，并且生物膜亚群具有细胞，核糖体在不同的状态，这取决于当地的环境条件。研究中在这里提出，我们将描述核糖体冬眠在抗生素耐受性中的作用，生物膜细菌，通过表征核糖体蛋白和蛋白质之间的蛋白质-蛋白质相互作用核糖体辅助因子在前期工作中，我们开发了一种双分子荧光互补（BiFC）系统，其允许核糖体的基于荧光的实时成像铜绿假单胞菌生物膜细胞中的动力学。这个系统可以让我们对生物膜细菌成像与冬眠的核糖体结合，并根据核糖体的状态对细胞进行分类。BiFC系统还使我们能够识别额外的核糖体冬眠因子，如分子伴侣，介导冬眠因子的负荷。这些研究的目标是：（i）产生分子构建体，其为具有活性核糖体的细菌提供最佳荧光报告信号，冬眠状态(ii)使用荧光报告系统来识别和分类活跃的休眠细菌在生物膜发展过程中，并比较其抗生素敏感性概况， (iii)鉴定有助于休眠生物膜的抗生素耐受性的其它休眠因子细胞由于许多常用的抗生素靶向细菌翻译，这些抗生素的最终目标是抑制细菌翻译。研究的目的是确定分子靶点，当被破坏时，增强生物膜对核糖体的敏感性，针对抗生素。靶向核糖体冬眠因子或其分子伴侣，提供了一种抑制休眠细菌活力的方法，从而增加了生物膜对抗生素