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

描述（由申请人提供）：铜绿假单胞菌是一种机会致病菌，对慢性和急性肺部疾病有重要影响。在2400万慢性阻塞性肺疾病患者中，50%的急性加重与bbb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是由铜绿假单胞菌的临床分离株分泌的，代表了一个新的EH酶家族，也在其他条件致病菌中发现。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，而CFTR是有效清除粘液纤毛所必需的。Cif还会导致TAP1肽转运体的丢失，而TAP1肽转运体是I类MHC抗原呈递所必需的。因此，Cif同时攻击宿主的先天免疫系统和获得性免疫系统，可能促进气道定植，并屏蔽同步病毒感染免受免疫监视。我们的研究表明，Cif介导的CFTR抑制需要一个功能性活性位点，并且Cif可能利用内源性环氧化物：二醇信号来干扰必需ABC转运蛋白的细胞内运输。在临床分离株中，Cif的表达受环氧化物反应性CifR抑制因子的调节。为了进一步了解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。我们将描述上皮细胞中的环氧化物和二醇群体，并确定那些响应Cif活性而变化的群体。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与生理环氧化物底物相互作用的假设。作为鉴定Cif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间隙的结构轮廓。使用一对质量转移突变体，我们还将在上皮细胞裂解物上进行捕获实验，以高灵敏度识别已知或新的生理底物；(3)为了确定Cif/CifR调控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的Cif表达水平。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表达的内源性环氧化物，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的生物信号传导机制。

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.