Production of Purified Mycobacterial Extracellular Vesicles
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Production and Qualification of Mycobacterial Extracellular Vesicles for Novel Diagnostic Assay Trials
Nicole A Kruh-Garcia
Mycobacterium tuberculosis and other mycobacterial species secrete a diverse array of proteins, protein complexes and extracellular vesicles (MEVs) into the surrounding environment. MEVs are vesicles derived directly from the bacillus, the membrane and internal contents are comprised of mycobacterial lipids, proteins and nucleic acids. The presence of MEVs in biofluids during mycobacterial infection is suggestive of the presence of live bacteria and can be used as a tool for diagnosis and treatment monitoring.
MEVs can be produced in vitro from cultured mycobacteria and can be isolated from the culture filtrate proteins (CFP). Purification of MEVs from CFP entails a multistep workflow including centrifuge filtration and size exclusion chromatography. The yield of MEVs is determined by nanoparticle tracking analysis (NTA). Similarly, NTA is used to determine the size range of the purified MEVs, to ensure consistency between preparations; the average size is around 80-120 nm. Additional quality control of MEVs can be performed by western blot of known MEV components. Using liquid chromatography tandem mass spectrometry, we have characterized the protein content of both in vitro and in vivo purified MEVs. The dominant components consist of lipoprotein antigens including: the 19-kDa lipoprotein (LpqH), LprA and LprG, as well as the superoxide dismutase, SodC. The proteins, as well as the glycolipid, lipoarabinomannan (LAM), can be used to verify the presence of purified MEVs. In contrast, the chaperone GroEL can be used to monitor cell lysis during production, which can contaminate the CFP with intracellular vesicles.
Last updated: December 2019