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

IbpA 在维持铜绿假单胞菌生物膜持续细胞活力中的作用

• 批准号: 8223135
• 负责人: MICHAEL J FRANKLIN
• 金额: $ 17.81万
• 依托单位: MONTANA STATE UNIVERSITY - BOZEMAN
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-02-15 至 2015-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8223135
• 关键词: 5' Untranslated Regions; Aging; Antibiotic Resistance; Antibiotic Therapy; Antibiotics; Artificial Implants; Bacteria; Bacterial Infections; Binding; Biological Assay; Cell Aging; Cell Fraction; Cells; Chimeric Proteins; Chronic; Development; Enzymes; Escherichia coli; Goals; Green Fluorescent Proteins; Individual; Infection; Laser Microscopy; Laser Scanning Confocal Microscopy; Longitudinal Studies; Lung; Mediating; Messenger RNA; Microbial Biofilms; Molecular; Molecular Chaperones; Molecular Target; Oxidants; Oxidative Stress; Patients; Pattern; Peptide Hydrolases; Process; Proteins; Pseudomonas aeruginosa; Reporter Genes; Research; Resistance; Resuscitation; Role; Sigma Factor; Stress; Surface; Time; Tissues; aged; biological adaptation to stress; cell age; cystic fibrosis patients; implantable device; killings; laser capture microdissection; mRNA Stability; mutant; pressure; protein aggregate; protein expression; protein misfolding; public health relevance; transcriptomics

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.

描述（由申请人提供）：铜绿假单胞菌在囊性纤维化（CF）患者的肺组织上形成生物膜和慢性感染。对个别患者的纵向研究表明，随着时间的推移，铜绿假单胞菌菌株通常会出现克隆，这表明未被抗生素杀死的细菌（持久细胞）会在治疗后重新填充生物膜。持久细胞处于代谢休眠状态，对大多数抗生素不太敏感。然而，它们还受到其他环境压力的影响，包括宿主防御细胞的氧化压力和细胞老化。这些压力会导致蛋白质错误折叠、失活和聚集。使用激光捕获显微切割 (LCM) 和转录组学，我们确定 ibpA 是铜绿假单胞菌生物膜休眠细胞部分中最丰富的 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和蛋白酶以进行蛋白质重折叠或降解。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。在这项研究中，我们将：（i）表征铜绿假单胞菌生物膜中 ibpA 的空间和时间表达模式。使用绿色荧光蛋白 (GFP) 融合和延时共焦扫描激光显微镜 (CSLM)，我们将描述转录和转录后过程在 ibpA 表达中的作用。 GFP 融合体将用于确定铜绿假单胞菌 ibpA 是否在所有细胞或老化生物膜细胞的子集中表达，以及 IbpA 蛋白是否分隔错误折叠的蛋白。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。使用 ibpA 和 rpoH 缺失突变体，我们将确定 IbpA 在使暴露于应激（包括衰老、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重新激活中的作用。最终，我们将确定 IbpA 是否可以作为分子靶标与其他抗生素治疗相结合，以消除引起慢性铜绿假单胞菌肺部感染的持续细胞亚群。 ） 公共卫生相关性：与表面（包括肺组织或人工植入装置）相关的细菌感染通常对抗生素治疗具有耐药性。耐药性可能是由休眠细胞亚群介导的，该亚群在治疗后重新填充感染。这项研究的目的是表征铜绿假单胞菌（一种引起肺组织感染的细菌）休眠细胞亚群的分子活性。我们将确定应激反应蛋白的作用，该蛋白使细菌能够在长时间的休眠中存活并恢复成肺部生物膜。

项目成果

A Pseudomonas aeruginosa EF-hand protein, EfhP (PA4107), modulates stress responses and virulence at high calcium concentration.

铜绿假单胞菌 EF 手蛋白 EfhP (PA4107) 可调节高钙浓度下的应激反应和毒力。

• DOI: 10.1371/journal.pone.0098985
• 发表时间: 2014
• 期刊: PloS one
• 影响因子: 3.7
• 作者: Sarkisova,SvetlanaA;Lotlikar,ShalakaR;Guragain,Manita;Kubat,Ryan;Cloud,John;Franklin,MichaelJ;Patrauchan,MariannaA
• 通讯作者: Patrauchan,MariannaA