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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI091699-02
Application #
8440732
Study Section
Bacterial Pathogenesis Study Section (BACP)
Program Officer
Taylor, Christopher E,
Project Start
2012-03-15
Project End
2017-02-28
Budget Start
2013-03-01
Budget End
2014-02-28
Support Year
2
Fiscal Year
2013
Total Cost
$428,949
Indirect Cost
$139,771
Name
Dartmouth College
Department
Biochemistry
Type
Schools of Medicine
DUNS #
041027822
City
Hanover
State
NH
Country
United States
Zip Code
03755
Bahl, Christopher D; Hvorecny, Kelli L; Bridges, Andrew A et al. (2014) Signature motifs identify an Acinetobacter Cif virulence factor with epoxide hydrolase activity. J Biol Chem 289:7460-9
Ballok, Alicia E; Filkins, Laura M; Bomberger, Jennifer M et al. (2014) Epoxide-mediated differential packaging of Cif and other virulence factors into outer membrane vesicles. J Bacteriol 196:3633-42
Gifford, Alex H; Alexandru, Diana M; Li, Zhigang et al. (2014) Iron supplementation does not worsen respiratory health or alter the sputum microbiome in cystic fibrosis. J Cyst Fibros 13:311-8
Amacher, Jeanine F; Cushing, Patrick R; Bahl, Christopher D et al. (2013) Stereochemical determinants of C-terminal specificity in PDZ peptide-binding domains: a novel contribution of the carboxylate-binding loop. J Biol Chem 288:5114-26
Ballok, Alicia E; O'Toole, George A (2013) Pouring salt on a wound: Pseudomonas aeruginosa virulence factors alter Na+ and Cl- flux in the lung. J Bacteriol 195:4013-9