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

• 批准号: 9004600
• 负责人: DEAN R MADDEN
• 金额: $ 45.48万
• 依托单位: DARTMOUTH COLLEGE
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-03-15 至 2019-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9004600
• 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; Pathogenesis; Pathway interactions; Patient-Focused Outcomes; 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; new therapeutic target; novel; novel therapeutic intervention; pathogen; protein transport; research study; response; targeted treatment; trafficking; transcription factor

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，对与慢性和急性肺部疾病相关的苦难产生了重要贡献。在2400万慢性阻塞性肺部疾病的患者中，它与急性加重的50％有关。这也是医院和社区获得性肺炎的发病率和死亡率的主要因素，并且是CF死亡率的主要原因。铜绿假单胞菌毒力的主要元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，对治疗这种病原体的新型治疗方法迫切需要。我们的团队发现了一种新型的宿主 - 病原体相互作用，该相互作用是由环氧化物水解酶（EH）CIF介导的。 CIF由铜绿假单胞菌的临床分离株分泌，代表了其他机会病原体中也发现了一种新的EH酶。当应用于气道上皮细胞时，CIF会抑制CF跨膜电导调节剂（CFTR）的核细胞去泛素化。结果，CIF抑制了CFTR的细胞表面水平，这是有效的粘膜清除所必需的。 CIF还会导致TAP1肽转运蛋白的损失，这是I类MHC抗原表现所必需的。因此，CIF攻击宿主的先天和获得的免疫系统，可能促进气道殖民化，并屏蔽免疫监测的病毒感染。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：二醇信号来扰动必需ABC转运蛋白的细胞内运输。在临床分离株中，CIF表达受环氧化物反应性CIFR阻遏物的调节。为了发展对CIF/CIFR系统的分子理解，我们提出以下目的：（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环氧化物和二醇群体，并确定那些随着CIF活性的响应而变化的环氧化物和二醇。同时，我们将研究CIF EH活性影响CFTR去泛素化和内吞后运输的机制； （2）测试CIF与生理环氧底物相互作用的假设。作为识别CIF内源性环氧靶标的基础，我们将捕获候选底物并确定底物结合裂解的结构概要。使用一对大规模迁移突变体，我们还将对上皮细胞裂解物进行捕获实验，以识别具有高灵敏度的已知或新型生理底物。 （3）为了建立CIF/CIFR调节量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。同时，我们将利用新开发的遗传和生化报告基因测定法来鉴定结合CIFR并调节CIF表达的内源环氧化物，并为针对CIFR的调节剂开发筛选：环保相互作用。综上所述，我们的研究将利用假单胞菌毒力系统和尖端的代谢组学方法来揭示控制关键宿主贩运和病原体感染途径的新型生物信号传导机制。