This article details the purification of outer membrane vesicles (OMVs) from Legionella pneumophila, a pathogenic Gram-negative bacterium. The process involves multiple centrifugation and filtration steps to ensure the removal of bacterial contaminants, allowing for the study of host-pathogen interactions.
Begin with a culture of Legionella pneumophila, a pathogenic Gram-negative bacterium that releases outer membrane vesicles.
These OMVs carry lipopolysaccharides and membrane proteins that contribute to bacterial virulence.
Centrifuge the culture to pellet intact bacteria.
Collect the supernatant containing OMVs and centrifuge again to pellet residual bacteria.
Filter the supernatant through a membrane to remove the remaining bacteria while retaining OMVs.
Perform a second filtration using a fresh membrane to remove bacterial debris.
Collect the bacteria-free filtrate and ultracentrifuge it to pellet the OMVs.
Discard the supernatant, resuspend the pellet in buffer, and ultracentrifuge again to remove loosely associated proteins and lipopolysaccharides.
Remove the supernatant and resuspend the purified OMVs in buffer.
Streak an aliquot of the OMVs onto blood agar and BCYE agar plates and incubate.
The lack of colony formation confirms the absence of bacterial contamination.
Store the OMVs at a low temperature for host-pathogen interaction studies.
Add the remaining liquid culture to 90 milliliters of fresh YEB medium and incubate the culture on a rotating shaker until it reaches an OD600 of 3.0 to 3.5. Centrifuge the liquid culture at 4,000 g for 20 minutes to pellet the bacteria. Then transfer the supernatant to fresh centrifuge tubes.
Discard the bacterial pellets and centrifuge the samples again before repeating the centrifugation step once more. Next, filter-sterilize the remaining supernatant twice.
Then transfer the bacteria-free supernatant to ultracentrifuge tubes and spin the samples at 100,000 g in 4 degrees Celsius for 3 hours. Decant the supernatant and discard it. Then use sterile PBS to resuspend the OMV pellets and pool them.
Repeat the ultracentrifugation step to remove contaminating proteins and free LPS. Now, discard the supernatant and dry the ultracentrifuge tube with a sterile cotton swab. Resuspend the OMV pellet with 500 microliters of sterile PBS.
Then, streak 20 microliters each onto blood agar and BCYE agar plates to exclude bacterial contamination of the prepared vesicle. Incubate the blood agar plate overnight and the BCYE agar plate for 3 days.
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