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

与自由生活的浮游细胞相比，生活在生物膜中的细菌对抗生素治疗的敏感性较低。 抗生素耐受性增加的一个机制是生物膜含有生理上的 异质的细胞亚群，包括耐抗生素的休眠细菌。当细菌 进入休眠状态后，它们会经历各种生理变化，从而降低能源成本 过程，但保护复苏所需大分子的完整性。其中包括 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，可以阻止翻译并保护核糖体免受降解 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 在条件有利的情况下从休眠状态中复苏，导致持续感染。核糖体 进入冬眠和离开冬眠是动态的过程，生物膜亚群具有 核糖体处于不同的状态，取决于当地的环境条件。在研究中 在这里，我们将表征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，允许基于实时荧光的核糖体成像 铜绿假单胞菌生物膜细胞动力学研究这个系统使我们能够对生物膜细菌进行成像 和冬眠的核糖体，并根据核糖体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， 调节冬眠因子的加载。这些研究的目标是：(I)产生分子 为核糖体处于活性状态的细菌提供最佳荧光报告信号的构建 冬眠状态。(Ii)使用荧光报告系统来识别和分类活动和休眠 细菌在生物膜形成过程中的变化，并比较它们的抗生素敏感性谱，以及 (3)确定导致休眠生物膜对抗生素耐受性的其他休眠因素 细胞。由于许多常用的抗生素都是针对细菌翻译的，因此这些 研究是确定分子靶点，当被破坏时，增强生物膜对核糖体的敏感性- 以抗生素为目标。靶向核糖体冬眠因子或其分子伴侣可能 提供一种方法来抑制休眠细菌的活性，从而增加生物膜对 抗生素。

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.