Isolation and Characterization of Extracellular Vesicles Produced by Iron-limited Mycobacteria

This method permits culturing Mycobacterium tuberculosis under strict low iron conditions and purifying membrane vesicles from the low iron culture filtrate. Because iron limitation is known to stimulate membrane vesicle production, this protocol yields high abundance of pure membrane vesicles that can be utilized for further biochemical or functional assays. When we demonstrate the protocol using the Mycobacterium smegmatis, the nonvirulent strain, this protocol can be followed in BSL-2 facilities using Mycobacterium tuberculosis, the virulent strain.

To begin, prepare one liter of minimal medium by dissolving five grams of monopotassium phosphate, five grams of L-asparagine, 20 milliliters of glycerol, and two grams of dextrose in 900 milliliters of deionized water in a plastic container. Adjust the pH to 6.8 with five normal sodium hydroxide and adjust the volume to one liter with deionized water. Then add 50 grams of metal chelating resin and gently agitate using a magnetic stir bar for 24 hours at four degrees Celsius.

The next day, stop the stirring and let the resin sediment for about 30 minutes. Sterilize and remove the remaining resin from the solution by filtration through a 22 micron filter unit with a plastic receiver. Now, supplement minimal medium with 0.5 milligrams per liter of zinc chloride, 40 milligrams per liter of magnesium sulfate, and 0.1 milligrams per liter of manganese sulfate.

Inoculate a single colony of MTB in two milliliters of mycobacterial broth medium supplemented with ADN enrichment. Incubate with agitation at 37 degrees Celsius. When there is visible growth, extract two milliliters of the culture and measure the OD 540 on a spectrophotometer.

Stop the incubation when the OD 540 reaches 0.8 which indicates the late logarithmic phase. Spread 200 microliters of the late logarithmic culture onto the mycobacterial agar plates supplemented with 0.2 glycerol, 0.5%Tween 80, and ADN. Inoculate at least five plates.

Incubate the plates at 37 degrees Celsius. And after one week, the bacterial growth is visible as a confluent layer. Then wet a sterile cotton swab in a low iron minimal medium.

Use this swab to collect bacteria from the agar plates. Inoculate the bacteria in 100 milliliters of low iron minimal media in a tube. Collect sufficient bacteria to prepare a suspension with an OD 540 of one.

Dilute the suspension 10 times to one liter with low iron minimal medium and divide it into two sterile plastic bottles. Next, transfer two milliliters of the culture to a five milliliter culture tube. Add 10 microliters of 10%volume by volume tyloxapol.

Incubate the cultures at 37 degrees Celsius for 14 days. Transfer the culture to five 225 milliliter conical centrifuge tubes and centrifuge at 2, 850 times g for seven minutes at 20 degrees Celsius. Collect the culture supernatant with a 50 milliliter pipette and filter sterilize it through a 0.22 micron filter.

To isolate MEVs, transfer the culture filtrate into a stirred cell ultrafiltration system placed at four degrees Celsius and filter the concentrate at a pressure less than 50 PSI through a 100 kilodalton cutoff membrane. Then centrifuge the concentrated culture filtrate at 15, 000 times g for 15 minutes at four degrees Celsius and collect the supernatant in polycarbonate ultracentrifugation tubes. Centrifuge the supernatant at 100, 000 times g for two hours at four degrees Celsius.

After that, remove the supernatant and resuspend the membranous pellets in a total of one milliliter of sterile PBS by gentle pipetting. Mix 0.5 milliliters of the pellet suspension with 1.5 milliliters of 60%iodixanol solution at the bottom of a polypropylene thin walled ultracentrifuge tube. Overlay this MVE iodixanol 45%suspension with one milliliter of 40%35%30%25%and 20%iodixanol solutions and one milliliter of PBS at the top.

Centrifuge at 100, 000 times g for 18 hours at four degrees Celsius. Next, use a one milliliter Hamilton syringe to collect the one milliliter density gradient fractions starting from the top. Dilute each collected fraction to 20 milliliters with PBS and centrifuge at 100, 000 times g for two hours at four degrees Celsius.

Remove the supernatant and resuspend in 0.5 milliliters of PBS. Store the tubes at four degrees Celsius. To perform membrane lipid analysis, combine 10 microliters of each gradient fraction with the fluorescent membrane probe TMA-DPH at a final concentration of one microgram per milliliter in 50 microliters of PBS in each well of a 96-well black plate.

Incubate the plates at 33 degrees Celsius for 20 minutes. Measure the fluorescence at 360 nanometers for excitation and 430 nanometers for emission. In this protocol, MEVs were purified by differential sedimentation in a density gradient.

Under the conditions described, MEVs were separated mostly in gradient fraction three which corresponds to 25%iodixanol. Vesicle-associated proteins were concentrated in fraction three shown by dot blot analysis. Negative staining also confirmed the presence of MEVs in fraction three and nanoparticle analysis showed the size of MEV was around 100 nanometers.

Protein and lipid concentration normalized to colony forming units showed an approximately eight-fold increase of MEV yield in low iron relative to high iron conditions. Dot blot analysis showed a more intense signal from MEV-associated markers in density gradient fractions from iron limited MTB culture than from iron sufficient MTB cultures. Follow the instructions for the preparation of the minimal medium to assure iron limited conditions and prevent contamination of the membrane vesicles with lipoprotein aggregates.

The mycobacterial membrane vesicles purified in this manner can be used for biochemical characterization and functional analysis.