Abstract:

Objective To compare two methods for extracting the outer membrane vesicle (OMV) from Pseudomonas aeruginosa (PA) and analyze it's purity, yield, and the content of exotoxin A (ETA). Methods The OMV of PA was extracted by ultrafiltration concentration and Optiprep density gradient centrifugation. The purity, yield, and content of ETA were detected by negative-stain electron microscopy, BCA method, and Western blotting, respectively. Human lung epithelial cells of A549 were immunized with these two OMVs and the IL-8 was determined by ELISA. Results There were more impurities in OMV extracted by ultrafiltration concentration than OMV extracted by Optiprep density gradient centrifugation. The yield of these two OMVs were 21.3 μg/10¹⁰ bacteria and 48.7 μg/10¹⁰ bacteria respectively. OMV extracted by Optiprep density gradient centrifugation had more ETA and stronger ability to increase the IL-8 level of A549 cell. Conclusion The OMV of PA extracted by Optiprep density gradient centrifugation have higher purity, yield, and content of ETA, and can induce stronger inflammatory response of host cells.

Key words: Pseudomonas aeruginosa, out membrane vesicle, ultrafiltration concentration, density gradient centrifugation, exotoxin A, IL-8