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

描述（由申请方提供）：铜绿假单胞菌是一种机会致病菌，可显著导致慢性和急性肺部疾病相关的痛苦。在2400万慢性阻塞性肺疾病患者中，它与>50%的急性加重相关。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，是CF死亡率的主要原因。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由铜绿假单胞菌的临床分离株分泌，代表了在其他机会致病菌中也发现的EH酶的新家族。当应用于气道上皮细胞时，Cif抑制CF跨膜传导调节因子（CFTR）的内吞后去泛素化。因此，Cif抑制CFTR的细胞表面水平，这是有效的粘膜纤毛清除所必需的。Cif还导致TAP 1肽转运蛋白的丢失，这是I类MHC抗原呈递所必需的。因此，Cif攻击宿主的先天性和获得性免疫系统，可能促进气道定殖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介导的抑制CFTR需要一个功能活性位点，Cif可能利用内源性环氧化物：二醇信号干扰细胞内运输的基本ABC转运蛋白。在临床分离株中，Cif表达受环氧化物响应性CifR阻遏物调节。为了从分子水平上理解Cif/CifR系统，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，并确定那些响应Cif活性的变化。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）验证Cif与生理性环氧化物底物相互作用的假设。作为确定Cif的内源性环氧化物靶点的基础，我们将捕获候选底物并确定底物结合裂缝的结构轮廓。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，以高灵敏度鉴定已知或新的生理底物;（3）为了建立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的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物化学报告分析，以确定内源性环氧化物结合CifR和调节Cif表达，并开发针对CifR的调节剂筛选：环氧化物相互作用。总之，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毒力途径的新型生物信号传导机制。 公共卫生相关性：铜绿假单胞菌是一种感染气道的细菌，直接导致患者痛苦和死亡，特别是在已经生病或住院的脆弱个体中。我们试图了解帮助假单胞菌破坏宿主防御的机制，使其能够建立感染并促进随后的病毒感染。这项工作将为开发针对这种重要微生物感染的新疗法奠定基础。

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. PUBLIC HEALTH RELEVANCE: Pseudomonas aeruginosa is a bacterium that infects the airways and contributes directly to patient suffering and death, especially in vulnerable individuals who are already ill or hospitalized. We seek to understand mechanisms that help Pseudomonas undermine host defenses, allowing it to establish infections and facilitate subsequent viral infections. This work will form the basis for development of novel therapies targeting infections by this important microbe.