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. 2006 Aug;8(9-10):2400-8.
doi: 10.1016/j.micinf.2006.05.001. Epub 2006 Jun 5.

Purification of outer membrane vesicles from Pseudomonas aeruginosa and their activation of an IL-8 response (VSports最新版本)

Susanne J Bauman  1 , Meta J Kuehn
Affiliations

V体育官网 - Purification of outer membrane vesicles from Pseudomonas aeruginosa and their activation of an IL-8 response

"VSports最新版本" Susanne J Bauman et al. Microbes Infect. 2006 Aug.

V体育平台登录 - Abstract

Considerable lung injury results from the inflammatory response to Pseudomonas aeruginosa infections in patients with cystic fibrosis (CF). The P. aeruginosa laboratory strain PAO1, an environmental isolate, and isolates from CF patients were cultured in vitro and outer membrane vesicles from those cultures were quantitated, purified, and characterized. Vesicles were produced throughout the growth phases of the culture and vesicle yield was strain-independent. Strain-dependent differences in the protein composition of vesicles were quantitated and identified VSports手机版. The aminopeptidase PaAP (PA2939) was highly enriched in vesicles from CF isolates. Vesicles from all strains elicited IL-8 secretion by lung epithelial cells. These results suggest that P. aeruginosa colonizing the CF lung may produce vesicles with a particular composition and that the vesicles could contribute to inflammation. .

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Fig. 1
Fig. 1
Quantitative analyses of vesicle production. A. Vesicle yield for strains S470 (S4), CF2 (C), Soil (So), and PAO1 (P) was determined by immunoblotting cell-free culture supernatants and whole culture lysates for outer membrane proteins (anti-OMP) or for OprF alone (anti-OprF) and dividing the amount in the cell-free supernatant with the amount in the culture lysate. Samples were analyzed in duplicate for each experiment. The average and SEM are shown; n = 4 for anti-OMP experiments and n = 2 for anti-OprF experiments. B. The vesicle production of PAO1 during the growth of a culture was monitored by collecting cell-free supernatants at the indicated times and immunoblotting with anti-OMP (diamonds). For each experiment, values were compared to the amount of OMPs detected at the final time point (590 min) which was set to 100%. The average and SEM are shown; n = 3. The OD600 of the culture is shown (open squares).
Fig. 2
Fig. 2
Protein profiles and electron micrographs of density gradient fractions. Ammonium sulfate-precipitated material from culture supernatants was loaded on the bottom of density gradients and centrifuged overnight. Fractions removed sequentially from the top of each gradient were TCA-precipitated and analyzed by Coomassie-stained SDS-PAGE (Fraction 1 = lightest, Fraction 12 = heaviest). Bracketed fractions indicate the pure vesicle-containing fractions that were pooled and further characterized from strains S470 (A), CF2 (B), soil (C), and PAO1 (D). The migration of molecular weight standards are indicated (kDa). Right panels: electron micrographs representative of pooled upper one-third (light) and lowest one-third (heavy) fractions of the vesicle purification gradients for each strain. Samples were negatively stained with uranyl acetate and visualized at 15,000–91,000 × magnification. Arrows indicate pyocins (nail-like structures) and pili or flagella. Bar = 100 nm in all panels.
Fig. 3
Fig. 3
Vesicle protein profiles resemble purified outer membranes and PaAP is enriched in vesicles from CF isolates. Samples (20 μg) of inner membrane (IM), outer membrane (OM), and vesicles (V) from each strain were TCA-precipitated and visualized by Coomassie-stained SDS-PAGE. (+), proteins enriched in OM over vesicles; (*), proteins enriched in vesicles over OM; arrows, proteins that were sequenced from PAO1 vesicles, except PaAP (PA2939), which was sequenced from CF2 vesicles. Molecular weight standards are indicated (kDa). †OprD migrates at a lower molecular weight in PAO1 than it does in other strains, as noted previously [31].
Fig. 4
Fig. 4
Different protein compositions of PAO1 and S470 vesicle proteins revealed by 2-D DIGE. A. Fluorescent image overlay showing 2-D DIGE of PAO1 vesicles (150 μg) labeled with Cy 3 (green) and S470 vesicles (150 μg) labeled with Cy 5 (red). Green spots indicate polypeptides that are more abundant in PAO1 vesicles, red spots indicate a higher abundance in S470 vesicles, and yellow spots indicate polypeptides found in equal abundance in both vesicle preparations. B. Positive changes in abundance (grey bars) indicate proteins that were more abundant in S470 vesicles. Negative changes in abundance (open bars) indicate proteins that were more abundant in PAO1 vesicles. Bar numbers correspond to spot numbers given in C and D. Values are the average of two 2-D DIGE gels, error bars indicate SEM. Grey-scale image of Cy 5 (S470) image (C), and Cy3 (PAO1) image (D). Size and pH axes are indicated on gel images. Arrows in B, C, and D indicate spots that were sequenced and are color coded by protein: Red, PaAP; Green, OprF; Black, OprE; Blue, PA0070. (*), spots that matched with more than one P. aeruginosa protein sequence.
Fig. 5
Fig. 5
Vesicles induce dose-dependent IL-8 production in human airway cells that exceeds LPS response. The concentration of IL-8 was measured by ELISA in supernatants removed from A549 cells (A) or HBE cells (B) incubated without vesicles (CTRL) or with 2.5 μg vesicles (+VES) for 24 h. Triplicate incubations were analyzed by ELISA in duplicate, and SEM is indicated for 2 to 7 separate experiments; *, p < 0.005 compared to control incubations.
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VSports最新版本 - References

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