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性病原体，与慢性和急性肺部疾病相关的痛苦有很大的贡献。在2400万慢性阻塞性肺疾病患者中，50%的急性加重与其有关。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，它是由环氧化物水解酶(EH)Cif介导的。CIF是由临床分离的铜绿假单胞菌分泌的，代表着在其他条件致病菌中也发现的一个新的EH酶家族。当应用于呼吸道上皮细胞时，Cif抑制内吞后跨膜电导调节器(CFTR)的去泛素化。因此，Cif抑制细胞表面CFTR的水平，这是有效的粘液纤毛清除所必需的。CIF还会导致TAP1肽转运蛋白的丢失，这是I类MHC抗原呈递所必需的。因此，Cif同时攻击宿主的先天和后天免疫系统，可能促进呼吸道定植，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抑制需要一个功能活性部位，Cif可能利用内源性环氧化物：二醇信号来扰乱必要的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的CIFR阻遏物的调节。为了加深对Cif/CIFR系统的分子理解，我们提出了以下目标：(1)确定Cif EH活性对呼吸道上皮细胞的影响。我们将表征上皮细胞中的环氧化物和二醇群体，并确定那些随着Cif活性的变化而改变的群体。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(S)；(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内源性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解的结构轮廓。使用一对质量移位突变体，我们还将对上皮细胞裂解物进行捕获实验，以确定已知或新的高敏感性生理底物；(3)为了建立Cif/CIFR调节子在呼吸道定植中的临床相关性，我们将评估Cif在铜绿假单胞菌临床分离株早期和晚期的表达水平。同时，我们将利用新开发的遗传和生化报告分析来鉴定与CIFR结合并调节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。综上所述，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.