Role of IbpA in maintaining viability of P. aeruginosa biofilm persister cells

描述（由申请人提供）：铜绿假单胞菌在囊性纤维化（CF）患者的肺组织中形成生物膜和慢性感染。个体患者的纵向研究表明，铜绿假单胞菌菌株往往随着时间的推移而克隆，这表明未被抗生素杀死的细菌（持久性细胞）在治疗后重新填充生物膜。持久性细胞处于代谢休眠状态，对大多数抗生素不太敏感。然而，它们也受到其他环境压力的影响，包括宿主防御细胞的氧化应激和细胞老化。这些压力导致蛋白质错误折叠、失活和聚集。利用激光捕获显微解剖（LCM）和转录组学技术，我们发现在铜绿假单胞菌生物膜的休眠细胞片段中，ibpA是最丰富的mRNA。在大肠杆菌中，IbpA/B负责结合错误折叠的蛋白质聚集体，并将其传递给其他伴侣蛋白和蛋白酶，以进行蛋白质的再折叠或降解。本研究的目的是确定IbpA在维持铜绿假单胞菌生物膜中休眠持续细胞活力中的作用。在本研究中，我们将：(1)表征铜绿假单胞菌生物膜中ibpA的时空表达模式。利用绿色荧光蛋白（GFP）融合和延时共聚焦扫描激光显微镜（CSLM），我们将表征ibpA表达中转录和转录后过程的作用。GFP融合将用于确定铜绿假单胞菌ibpA是在所有细胞中表达还是在老化的生物膜细胞亚群中表达，以及ibpA蛋白是否将错误折叠的蛋白区隔。我们还将：（ii）表征IbpA的分子活性及其在休眠铜绿假单胞菌生物膜细胞存活中的作用。利用ibpA和rpoH缺失突变体，我们将确定ibpA在暴露于应激（包括老化、氧化剂和抗生素）的休眠细胞复苏中的作用。功能酶测定将用于表征铜绿假单胞菌IbpA在错误折叠蛋白的结合或再激活中的作用。最终，我们将确定IbpA是否可以作为分子靶点与其他抗生素治疗联合使用，以消除引起慢性铜绿假单胞菌肺部感染的持久性细胞亚群。)

DESCRIPTION (provided by applicant): Pseudomonas aeruginosa forms biofilms and chronic infections on pulmonary tissue of patients with cystic fibrosis (CF). Longitudinal studies of individual patients indicate that P. aeruginosa strains are often clonal over time, suggesting that bacteria not killed by antibiotics (persister cells) repopulate the biofilms following treatments. Persister cells are metabolically dormant and less susceptible to most antibiotics. However, they are subject to other environmental stresses including oxidative stress from host defensive cells and cell aging. These stresses cause protein misfolding, inactivation, and aggregation. Using laser capture microdissection (LCM) and transcriptomics, we identified ibpA as the most abundant mRNA in the dormant cell fraction of P. aeruginosa biofilms. In Escherichia coli, IbpA/B is responsible for binding misfolded protein aggregates and delivering them to other chaperones and proteases for protein refolding or degradation. The goal of this research is to determine the role of IbpA in maintaining the viability of dormant persister cells in P. aeruginosa biofilms. In this research we will: (i) characterize the spatial and temporal expression patterns of ibpA in P. aeruginosa biofilms. Using green fluorescent protein (GFP) fusions and time-lapse confocal scanning laser microscopy (CSLM) we will characterize the roles of transcriptional and post-transcriptional processes in ibpA expression. GFP fusions will be used to determine if P. aeruginosa ibpA is expressed in all cells or in subset of aged biofilm cells, and if the IbpA protein compartmentalizes misfolded proteins. We will also: (ii) characterize the molecular activities of IbpA and its role in survival of dormant P. aeruginosa biofilm cells. Using ibpA and rpoH deletion mutants, we will determine the role of IbpA in allowing resuscitation of dormant cells exposed to stresses, including aging, oxidizing agents, and antibiotics. Functional enzyme assays will be used to characterize the role of P. aeruginosa IbpA in binding or reactivation of misfolded proteins. Ultimately, we will determine if IbpA may be used as a molecular target in combination with other antibiotic treatments to eliminate the persister cell subpopulations that cause chronic P. aeruginosa pulmonary infections. ) PUBLIC HEALTH RELEVANCE: Bacterial infections associated with surfaces, including pulmonary tissue or artificial implant devices, are often resistant to antibiotic treatment. Resistance may be mediated by a dormant cell subpopulation that repopulates the infection following treatment. The goals of this research are to characterize molecular activities of the dormant cell subpopulations of Pseudomonas aeruginosa, a bacterium that causes infections on pulmonary tissue. We will determine the role of a stress response protein that allows the bacteria to survive prolonged dormancy and resuscitate into pulmonary biofilms.