Mechanism of Cif Virulence: A Bacterial Strategy to Subvert Host-Cell Defenses

Cif 毒力机制：破坏宿主细胞防御的细菌策略

基本信息

批准号：
8811911
负责人：
DEAN R MADDEN
金额：
$ 45.71万
依托单位：
DARTMOUTH COLLEGE
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-03-15 至 2016-02-29
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8811911
关键词：
ATP-Binding Cassette Transporters Active Sites Acute Address Affect Analytical Chemistry Antibiotic Resistance Antigen Presentation Bacteria Bacterial Infections Binding Biochemical Biological Biological Assay Cell surface Cells Cellular biology Cessation of life Chronic Chronic Obstructive Airway Disease Cleaved cell Clinical Collaborations Combined Modality Therapy Community Hospitals Cystic Fibrosis Cystic Fibrosis Transmembrane Conductance Regulator DNA DNA Binding Defense Mechanisms Deubiquitination Development Elements Enzymes Epithelial Cells Epoxide hydrolase Epoxy Compounds Exhibits Family Gene Expression Genetic Glycols Hospital Mortality Host Defense Hydrolysis Immune response Immune system Immunologic Surveillance In Vitro Incidence Individual Infection Inflammation Laboratories Lung Lung diseases MHC Class I Genes Mass Spectrum Analysis Mediating Microbe Molecular Morbidity - disease rate Mucociliary Clearance Outcome Pathogenesis Pathway interactions Patients Peptides Physiological Play Pneumonia Population Predisposition Process Protein Secretion Pseudomonas Pseudomonas aeruginosa Recording of previous events Recruitment Activity Refractory Regulation Regulon Reporter Research Research Design Role Signal Transduction Structure System TAP1 gene Techniques Testing Validation Vasodilation Viral Viral Antigens Virulence Virulence Factors Virus Diseases Work adaptive immunity airway epithelium antimicrobial base clinically relevant cystic fibrosis serum factor in vivo inhibitor/antagonist insight member metabolomics microbial mortality mucoid mutant novel novel therapeutic intervention pathogen protein transport research study response targeted treatment therapeutic target trafficking transcription factor

项目摘要

DESCRIPTION (provided by applicant): P. aeruginosa is an opportunistic pathogen that contributes significantly to the suffering associated with chronic and acute lung disease. Among the 24 million patients with chronic obstructive pulmonary disease, it is associated with >50% of acute exacerbations. It is also a major factor in the incidence and mortality of hospital- and community-acquired pneumonias and is the predominant cause of CF mortality. A major element of P. aeruginosa virulence is its exceptional antibiotic resistance and its synergistic interactions with viral infections. Thus, there is a critical need for novel therapeutic approaches to treat this pathogen. Our team has found a novel host-pathogen interaction, mediated by the epoxide hydrolase (EH) Cif. Cif is secreted by clinical isolates of P. aeruginosa and represents a new family of EH enzymes also found in other opportunistic pathogens. When applied to airway epithelial cells, Cif inhibits post-endocytic deubiquitination of the CF transmembrane conductance regulator (CFTR). As a result, Cif suppresses cell-surface levels of CFTR, which is required for effective mucociliary clearance. Cif also causes loss of the TAP1 peptide transporter, which is required for class I MHC antigen presentation. Thus, Cif attacks both the innate and acquired immune systems of the host, likely facilitating airway colonization and shielding coincident viral infections from immune surveillance. Our studies reveal that Cif-mediated inhibition of CFTR requires a functional active site, and that Cif likely exploits an endogenous epoxide:diol signal to perturb the intracellular trafficking of essential ABC transporters. In clinical isolates, Cif expression is regulated by the epoxide-responsive CifR repressor. To develop a molecular understanding of the Cif/CifR system, we propose the following aims: (1) To identify the impact of Cif EH activity on airway epithelial cells. We will characterize the epoxide and diol populations in epithelial cells and identify those that change in response to Cif activity. In parallel, we will investigate the mechanism(s) by which Cif EH activity affects CFTR deubiquitination and post-endocytic trafficking; (2) To test the hypothesis that Cif interacts with a physiological epoxide substrate. As a basis for identifying endogenous epoxide targets of Cif, we will trap candidate substrates and determine the structural outlines of the substrate-binding cleft. Using a pair of mass-shifted mutants, we will also perform trapping experiments on epithelial-cell lysates to identify known or novel physiological substrates with high sensitivity; (3) To establish the clinical relevance of the Cif/CifR regulon in airway colonization, we will assess Cif expression levels in early and late clinical isolates of P. aeruginosa. In parallel, we will utilize newly developed genetic and biochemical reporter assays to identify endogenous epoxides that bind CifR and regulate Cif expression, and to develop screens for modulators targeting the CifR:epoxide interaction. Taken together, our studies will exploit a Pseudomonas virulence system and cutting-edge metabolomics approaches to uncover novel biological signaling mechanisms that control key host-trafficking and pathogen-virulence pathways.

描述（由申请人提供）：铜绿假单胞菌是一种机会性病原体，它对慢性和急性肺病相关的痛苦有显着的影响。在 2400 万慢性阻塞性肺病患者中，超过 50% 的急性加重与该病相关。它也是医院和社区获得性肺炎发病率和死亡率的主要因素，也是CF死亡的主要原因。铜绿假单胞菌毒力的一个主要因素是其特殊的抗生素耐药性及其与病毒感染的协同相互作用。因此，迫切需要新的治疗方法来治疗这种病原体。我们的团队发现了一种新的宿主-病原体相互作用，由环氧化物水解酶 (EH) Cif 介导。 Cif 由铜绿假单胞菌临床分离株分泌，代表了在其他机会性病原体中也发现的新 EH 酶家族。当应用于气道上皮细胞时，Cif 会抑制 CF 跨膜电导调节因子 (CFTR) 的内吞后去泛素化。因此，Cif 抑制细胞表面 CFTR 水平，而 CFTR 是有效粘膜纤毛清除所必需的。 Cif 还会导致 I 类 MHC 抗原呈递所需的 TAP1 肽转运蛋白丢失。因此，Cif 攻击宿主的先天性和后天性免疫系统，可能促进气道定植并保护同时发生的病毒感染免受免疫监视。我们的研究表明，Cif 介导的 CFTR 抑制需要功能性活性位点，并且 Cif 可能利用内源性环氧化物：二醇信号来干扰重要 ABC 转运蛋白的细胞内运输。在临床分离株中，Cif 表达受到环氧化物反应性 CifR 阻遏蛋白的调节。为了发展对 Cif/CifR 系统的分子理解，我们提出以下目标：（1）确定 Cif EH 活性对气道上皮细胞的影响。我们将表征上皮细胞中的环氧化物和二醇群体，并识别那些响应 Cif 活性而变化的环氧化物和二醇群体。与此同时，我们将研究 Cif EH 活性影响 CFTR 去泛素化和内吞后转运的机制； (2) 检验 Cif 与生理环氧化物底物相互作用的假设。作为识别 Cif 内源环氧化物靶标的基础，我们将捕获候选底物并确定底物结合裂口的结构轮廓。使用一对质量转移突变体，我们还将对上皮细胞裂解物进行捕获实验，以高灵敏度识别已知或新颖的生理底物； (3) 为了确定 Cif/CifR 调节子在气道定植中的临床相关性，我们将评估铜绿假单胞菌早期和晚期临床分离株中的 Cif 表达水平。与此同时，我们将利用新开发的遗传和生化报告分析来鉴定结合 CifR 并调节 Cif 表达的内源环氧化物，并开发针对 CifR:环氧化物相互作用的调节剂的筛选。总而言之，我们的研究将利用假单胞菌毒力系统和尖端代谢组学方法来揭示控制关键宿主贩运和病原体毒力途径的新型生物信号机制。