Enriching mycobacterial EVs helps delineate their contents, decipher their factions, and dates designing ways to construct and generate recombinant EVs that mainly exploit vaccine candidates. Six series generate from different locations of the bacterial surface. They do not have conserved markers.
Hence, any affinity-based approaches can enrich only a subset of the EVs. In contrast, our method captures all generated mycobacteria EVs. Demonstrating the procedure will be Mohd Ilyas, a PHD student from my laboratory.
To begin, add one milliliter of glycerol stock of wild-type or recombinant Mycobacterium smegmatis to a 50-milliliter centrifuge tube containing 10 milliliters of pre-warmed 7H9 broth. Close the lid and swirl the tube before incubating the culture overnight at 37 degrees Celsius in between 200 to 220 RPM. On the next day, when the optical density at 600 nanometers reaches approximately one, centrifuge the culture at 3, 200 G for 10 minutes at room temperature and discard the supernatant into a liquid discard container.
Add one milliliter of pre-warmed Sautons media and gently resuspend to obtain a uniform suspension. Make up the volume to 10 milliliters using Sautons media. Centrifuge again and discard the supernatant.
After washing, resuspend the bacterial cells. in 20 milliliters of pre-warmed Sautons media and measure the optical density at 600 nanometers. Inoculate the required volume of bacterial suspension into a one to two liter Erlenmeyer flask containing 330 milliliters of Sautons media and incubate at 37 degrees Celsius in 200 RPM until the optical density reaches 0.3.
On the next day, wash the cells once as previously demonstrated and resuspend the pellet in 330 milliliters of Sautons containing 1/10 Tween 80. Distribute 50 milliliters of the cell suspension into six one-liter flasks, each containing 280 milliliters of pre-warmed Sautons containing 1/10 Tween 80. Incubate the cultures at 37 degrees Celsius in 200 RPM until the optical density reaches two to 2.5.
Add two liters of mid-exponential stage cultures to six 400-milliliter bottles and centrifuge at 8, 000 G for 20 minutes at four degrees Celsius. Collect the supernatant and pre-chilled autoclaved flasks or beakers and store an aliquot of the pellet for analytical procedures. The pellet is colored if the EVs are recombinant for mCherry and brown if they are wild-type or recombinant for non-fluorescent proteins.
Filter the culture supernatant through a 0.45-micron disposal filter, followed by filtration through a 0.22-micron filter to remove all traces of bacteria. Next, pre-wash the 30-kilodalton membrane concentrators with 15 milliliters of double-distilled water at 4, 000 G for 20 minutes at four degrees Celsius. Then wash with 15 milliliters of pre-filtered cold Sautons media using the same conditions to remove all traces of chemicals.
After washing, add 15 milliliters of filtered cultured supernatant into a concentrator and concentrate at 4, 000 G for 20 minutes at four degrees Celsius. Transfer the concentrate to a cold 40-milliliter Oak Ridge centrifuge tube or a 50-milliliter fresh Falcon centrifuge tube. Once the entire two liters of culture filtrate is concentrated, transfer the concentrate into a double distilled, washed, and pre-chilled 50-milliliter polypropylene centrifuge tube and subjected to a two-step centrifugation.
First at 4, 000 G, and then at 15, 000 G.Transfer the centrifuged concentrate into a pre-chilled 38.5-milliliter ultracentrifuge tube and spin at 100, 000 G for four hours at four degrees Celsius. Collect the supernatant in a pre-chilled 50 milliliter fresh Falcon centrifuge tube. invert the ultracentrifuge tube on a fresh, lint-free absorbent paper to remove traces of the supernatant and resuspend the pellet in 600 microliters of HEPES buffer.
Layer the resuspended pellet at the bottom of a 13-milliliter pre-chilled UltraClear polypropylene ultracentrifuge tube and gently mix with four milliliters of inert 60%density gradient iodixanol solution. Then overlay with one milliliter each of 40%30%20%and 10%of iodixanol. Fill the tube by adding four milliliters of 6%iodixanol at the top.
After carefully weighing the tube in a glass beaker or a stand, gently transfer it into the swinging buckets and then to the swinging buckets rotor for ultracentrifugation at 141, 000 G for 16 hours at four degrees Celsius. After ultracentrifugation, carefully remove the tube and collect a one milliliter fraction into autoclaved microcentrifuge tubes. TEM analysis showed a mycobacterial EVs are circular and nano-tracking analysis confirmed different sized EVs with diameters ranging between 20 and 250 nanometers.
When the N-terminal end of mCherry was translationally fused to the C-terminal end of CFP-29, a portion of enriched mEVs of Msm turned pink, indicating cf-29's ability to carry a foreign protein of interest. CFP-29 was then used to evaluate EsxA delivery into Msm EVs. Mtb's EsxA was observed in Msm EVs in low quantities.
CFP-29 EsxA 3X FLAG was more stable and accumulated at higher levels in mEVs. Enriching the EVs from mycobacteria grown under physiological different conditions provides insights into their altered contents and modified functions that dictate sustained infectivity, pathogenesis, and pathogen spread.
