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Inhibiting an Epoxide Hydrolase Virulence Factor from Pseudomonas aeruginosa Protects CFTR.

ABSTRACT: Opportunistic pathogens exploit diverse strategies to sabotage host defenses. Pseudomonas aeruginosa secretes the CFTR inhibitory factor Cif and thus triggers loss of CFTR, an ion channel required for airway mucociliary defense. However, the mechanism of action of Cif has remained unclear. It catalyzes epoxide hydrolysis, but there is no known role for natural epoxides in CFTR regulation. It was demonstrated that the hydrolase activity of Cif is strictly required for its effects on CFTR. A small-molecule inhibitor that protects this key component of the mucociliary defense system was also uncovered. These results provide a basis for targeting the distinctive virulence chemistry of Cif and suggest an unanticipated role of physiological epoxides in intracellular protein trafficking.

SUBMITTER: Bahl CD

PROVIDER: S-EPMC4556431 | biostudies-literature | 2015 Aug

REPOSITORIES: biostudies-literature

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Inhibiting an Epoxide Hydrolase Virulence Factor from Pseudomonas aeruginosa Protects CFTR.

Bahl Christopher D CD Hvorecny Kelli L KL Bomberger Jennifer M JM Stanton Bruce A BA Hammock Bruce D BD Morisseau Christophe C Madden Dean R DR

Angewandte Chemie (International ed. in English) 20150701 34

Opportunistic pathogens exploit diverse strategies to sabotage host defenses. Pseudomonas aeruginosa secretes the CFTR inhibitory factor Cif and thus triggers loss of CFTR, an ion channel required for airway mucociliary defense. However, the mechanism of action of Cif has remained unclear. It catalyzes epoxide hydrolysis, but there is no known role for natural epoxides in CFTR regulation. It was demonstrated that the hydrolase activity of Cif is strictly required for its effects on CFTR. A small ...[more]

PMID: 26136396

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Project description:Pseudomonas aeruginosa is an opportunistic bacterium in patients with cystic fibrosis and hospital acquired infections. It presents a plethora of virulence factors and antioxidant enzymes that help to subvert the immune system. In this study, we identified the 2-Cys peroxiredoxin, alkyl-hydroperoxide reductase C1 (AhpC1), as a relevant scavenger of oxidants generated during inflammatory oxidative burst and a mechanism of P. aeruginosa (PA14) escaping from killing. Deletion of AhpC1 led to a higher sensitivity to hypochlorous acid (HOCl, IC50 3.2 ± 0.3 versus 19.1 ± 0.2 μM), hydrogen peroxide (IC50 91.2 ± 0.3 versus 496.5 ± 6.4 μM) and the organic peroxide urate hydroperoxide. ΔahpC1 strain was more sensitive to the killing by isolated neutrophils and less virulent in a mice model of infection. All mice intranasally instilled with ΔahpC1 survived as long as they were monitored (15 days), whereas 100% wild-type and ΔahpC1 complemented with ahpC1 gene (ΔahpC1 attB:ahpC1) died within 3 days. A significantly lower number of colonies was detected in the lung and spleen of ΔahpC1-infected mice. Total leucocytes, neutrophils, myeloperoxidase activity, pro-inflammatory cytokines, nitrite production and lipid peroxidation were much lower in lungs or bronchoalveolar liquid of mice infected with ΔahpC1. Purified AhpC neutralized the inflammatory organic peroxide, urate hydroperoxide, at a rate constant of 2.3 ± 0.1 × 106 M-1s-1, and only the ΔahpC1 strain was sensitive to this oxidant. Incubation of neutrophils with uric acid, the urate hydroperoxide precursor, impaired neutrophil killing of wild-type but improved the killing of ΔahpC1. Hyperuricemic mice presented higher levels of serum cytokines and succumbed much faster to PA14 infection when compared to normouricemic mice. In summary, ΔahpC1 PA14 presented a lower virulence, which was attributed to a poorer ability to neutralize the oxidants generated by inflammatory oxidative burst, leading to a more efficient killing by the host. The enzyme is particularly relevant in detoxifying the newly reported inflammatory organic peroxide, urate hydroperoxide.

Project description:Osteoarthritis (OA) is a degenerative joint disease that causes pain and bone deterioration driven by an increase in prostaglandins (PGs) and inflammatory cytokines. Current treatments focus on inhibiting prostaglandin production, a pro-inflammatory lipid metabolite, with NSAID drugs; however, other lipid signaling targets could provide safer and more effective treatment strategies. Epoxides of polyunsaturated fatty acids (PUFAs) are anti-inflammatory lipid mediators that are rapidly metabolized by the soluble epoxide hydrolase (sEH) into corresponding vicinal diols. Interestingly, diol levels are increased in the synovial fluid of humans with OA, warranting further research on the biological role of this lipid pathway in the progression of OA. sEH inhibitors (sEHI) stabilize these biologically active, anti-inflammatory lipid epoxides, resulting in analgesia in both neuropathic, and inflammatory pain conditions. Most experimental studies testing the analgesic effects of sEH inhibitors have used experimental rodent models, which do not completely represent the complex etiology of painful diseases. Here, we tested the efficacy of sEHI in aged dogs with natural arthritis to provide a better representation of the clinical manifestations of pain. Two sEHI were administered orally, once daily for 5 days to dogs with naturally occurring arthritis to assess efficacy and pharmacokinetics. Blinded technicians recorded the behavior of the arthritic dogs based on pre-determined criteria to assess pain and function. After 5 days, EC1728 significantly reduced pain at a dose of 5 mg/kg compared to vehicle controls. Pharmacokinetic evaluation showed concentrations exceeding the enzyme potency in both plasma and synovial fluid. In vitro data showed that epoxyeicosatrienoic acid (EETs), epoxide metabolites of arachidonic acid, decreased inflammatory cytokines, IL-6 and TNF-?, and reduced cytotoxicity in canine chondrocytes challenged with IL1? to simulate an arthritic environment. These results provide the first example of altering lipid epoxides as a therapeutic target for OA potentially acting by protecting chondrocytes from inflammatory induced cytotoxicity. Considering the challenges and high variability of naturally occurring disease in aged dogs, these data provide initial proof of concept justification that inhibiting the sEH is a non-NSAID, non-opioid, disease altering strategy for treating OA, and warrants further investigation.

Dataset Information

Inhibiting an Epoxide Hydrolase Virulence Factor from Pseudomonas aeruginosa Protects CFTR.

Publications

Inhibiting an Epoxide Hydrolase Virulence Factor from Pseudomonas aeruginosa Protects CFTR.

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