title: 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 host-relevant 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.
authors.affiliation: Department of Microbiology and Immunology, Geisel School of Medicine at Dartmouth, Hanover, New Hampshire, USA.
authors.forename: Alicia E
authors.initials: AE
authors.lastname: Ballok
authors.affiliation: Department of Microbiology and Immunology, Geisel School of Medicine at Dartmouth, Hanover, New Hampshire, USA.
authors.forename: Laura M
authors.initials: LM
authors.lastname: Filkins
authors.affiliation: Department of Microbiology and Immunology, Geisel School of Medicine at Dartmouth, Hanover, New Hampshire, USA.
authors.forename: Jennifer M
authors.initials: JM
authors.lastname: Bomberger
authors.affiliation: Department of Microbiology and Immunology, Geisel School of Medicine at Dartmouth, Hanover, New Hampshire, USA.
authors.forename: Bruce A
authors.initials: BA
authors.lastname: Stanton
authors.affiliation: Department of Microbiology and Immunology, Geisel School of Medicine at Dartmouth, Hanover, New Hampshire, USA georgeo@dartmouth.edu.
authors.forename: George A
authors.initials: GA
authors.lastname: O'Toole
mesh_terms: D000469:Alkaline Phosphatase; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D004852:Epoxy Compounds / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology*; D010740:Phospholipases / Q000378:metabolism; D021381:Protein Transport; D011550:Pseudomonas aeruginosa / Q000235:genetics / Q000378:metabolism*; D013312:Stress, Physiological; D037521:Virulence Factors / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; C547935:Cif protein, Pseudomonas aeruginosa; D004852:Epoxy Compounds; C547605:OprF protein, Pseudomonas aeruginosa; D037521:Virulence Factors; D010740:Phospholipases; D000469:Alkaline Phosphatase
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