Epoxide-Mediated Differential Packaging of Cif and Other Virulence Factors into Outer Membrane Vesicles

Abstract

Pseudomonas aeruginosa produces outer membrane vesicles (OMVs) that contain a number of secreted bacterial proteins, including phospholipases, alkaline phosphatase, and the CFTR inhibitory factor (Cif). Previously, Cif, an epoxide hydrolase, was shown to be regulated at the transcriptional level by epoxides, which serve as ligands of the repressor, CifR. Here, we tested whether epoxides have an effect on Cif levels in OMVs. We showed that growth of P. aeruginosa in the presence of specific epoxides but not a hydrolysis product increased Cif packaging into OMVs in a CifR-independent fashion. The outer membrane protein, OprF, was also increased under these conditions, but alkaline phosphatase activity was not significantly altered. Additionally, we demonstrated that OMV shape and density were affected by epoxide treatment, with two distinct vesicle fractions present when cells were treated with epibromohydrin (EBH), a model epoxide. Vesicles isolated from the two density fractions exhibited different protein profiles in Western blotting and silver staining. We have shown that a variety of clinically or hostrelevant treatments, including antibiotics, also alter the proteins packaged in OMVs. Proteomic analysis of purified OMVs followed by an analysis of transposon mutant OMVs yielded mutants with altered vesicle packaging. Finally, epithelial cell cytotoxicity was reduced in the vesicles formed in the presence of EBH, suggesting that this epoxide alters the function of the OMVs. Our data support a model whereby clinically or host-relevant signals mediate differential packaging of virulence factors in OMVs, which results in functional consequences for host-pathogen interactions.

Pseudomonas aeruginosa sabotages the generation of host proresolving lipid mediators

“…We recently showed that P. aeruginosa secretes the CFTR inhibitory factor (Cif), an epoxide hydrolase that triggers the degradation of ABC transporter family members, including CFTR (4-8). Cif transcripts have been observed in sputum from patients with CF, and longitudinal studies of clinical isolates from individual patients confirm that P. aeruginosa maintains Cif expression for up to 15 y (7,9). Nonetheless, Cif's role in CF pathogenesis and the identity of possible host epoxide substrates have remained unclear.…”

The cif Virulence Factor Gene Is Present in Isolates From Patients With Pseudomonas aeruginosa Keratitis

“…These OMVs diffuse through overlying mucus and fuse with membrane lipid rafts, delivering virulence factors into the host cytoplasm. 30 OMVs are proinflammatory and cytotoxic to both the pulmonary and corneal epithelia. 31 The immune response to P. aeruginosa strains in the eye is dominated by dense neutrophil infiltrates forming neutrophil extracellular traps.…”

“…31 The immune response to P. aeruginosa strains in the eye is dominated by dense neutrophil infiltrates forming neutrophil extracellular traps. 30,31 Cytotoxic strains that are associated with higher rates of morbidity, including vision impairment, are able to evade this neutrophil capture mechanism by secreting high amounts of OMVs. 31 This mechanism suggests OMVs provide necessary factors for P. aeruginosa to establish corneal infection.…”

Cyclodextrins reduce the ability of Pseudomonas aeruginosa outer-membrane vesicles to reduce CFTR Cl− secretion

“…aeruginosa and OMV isolation. P. aeruginosa [strain PAO1 and clinical strain SMC1585 (23)] was grown overnight in lysogeny broth (LB) to an optical density of 1, and OMVs were isolated as described elsewhere in detail (3,(5)(6)(7)19). Briefly, overnight cultures of P. aeruginosa were centrifuged for 1 h at 3,500 rpm (4°C) to pellet bacteria.…”

“…P. aeruginosa secretes outer membrane vesicles (OMVs) that fuse with cholesterol-rich lipid rafts in the apical plasma membrane of airway epithelial cells and deliver many virulence factors, including Cif, into the cytoplasm, where it reduces wt-CFTR Cl Ϫ secretion (3,6,7,31,32). Cif enhances the ubiquitination and lysosomal degradation of wt-CFTR, thereby reducing CFTR Cl Ϫ secretion, mucociliary transport by airway epithelial cells, and the ability of mouse lungs to clear P. aeruginosa (18,31).…”