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A protocol is presented to extract the total lipid content of the cell wall of a wide range of mycobacteria. Moreover, extraction and analytical protocols of the different types of mycolic acids are shown. A thin-layer chromatographic protocol to monitor these mycobacterial compounds is also provided.
Mycobacteria species can differ from one another in the rate of growth, presence of pigmentation, the colony morphology displayed on solid media, as well as other phenotypic characteristics. However, they all have in common the most relevant character of mycobacteria: its unique and highly hydrophobic cell wall. Mycobacteria species contain a membrane-covalent linked complex that includes arabinogalactan, peptidoglycan, and long-chains of mycolic acids with types that differ between mycobacteria species. Additionally, mycobacteria can also produce lipids that are located, non-covalently linked, on their cell surfaces, such as phthiocerol dimycocerosates (PDIM), phenolic glycolipids (PGL), glycopeptidolipids (GPL), acyltrehaloses (AT), or phosphatidil-inositol mannosides (PIM), among others. Some of them are considered virulence factors in pathogenic mycobacteria, or critical antigenic lipids in host-mycobacteria interaction. For these reasons, there is a significant interest in the study of mycobacterial lipids due to their application in several fields, from understanding their role in the pathogenicity of mycobacteria infections, to a possible implication as immunomodulatory agents for the treatment of infectious diseases and other pathologies such as cancer. Here, a simple approach to extract and analyze the total lipid content and the mycolic acid composition of mycobacteria cells grown in a solid medium using mixtures of organic solvents is presented. Once the lipid extracts are obtained, thin-layer chromatography (TLC) is performed to monitor the extracted compounds. The example experiment is performed with four different mycobacteria: the environmental fast-growing Mycolicibacterium brumae and Mycolicibacterium fortuitum, the attenuated slow-growing Mycobacterium bovis bacillus Calmette-Guérin (BCG), and the opportunistic pathogen fast-growing Mycobacterium abscessus, demonstrating that methods shown in the present protocol can be used to a wide range of mycobacteria.
Mycobacterium is a genus that comprises pathogenic and non-pathogenic species, characterized by having a highly hydrophobic and impermeable cell wall formed by their peculiar lipids. Specifically, the mycobacterial cell wall contains mycolic acids, which are α-alkyl and β-hydroxy fatty acids, in which the α-branch is constant in all mycolic acids (except for the length) and the β-chain, called the meromycolate chain, is a long aliphatic chain that may contain different functional chemical groups described along with the literature (α-, α'-, methoxy-, κ-, epoxy-, carboxy-, and ω-1-methoxy- mycolates), therefore produ....
1. Extraction of the total non-covalent-linked lipids from mycobacteria (Figure 1)
With the aim of showing a wide range of lipids present in different mycobacteria species, M. bovis BCG was selected as it is rough and slow-growing mycobacteria. The rough and fast-growing M. fortuitum and M. brumae were added in the procedure and, finally, the smooth morphotype of M. abscessus was also included. These four species permit us to visualize a broad spectrum of mycobacteria-derived lipids such as acyltrehaloses (AT), GPLs, PDIM, PGL, PIM, TDM, and TMM. Moreover, all four s.......
A simple protocol considered as the gold standard method for the extraction of noncovalently linked lipid compounds from the mycobacterial cell wall is presented. Further visualization by one- and two-dimensional TLCs from the extracted lipids of four different mycobacteria is shown.
Two consecutive combined mixtures of chloroform and methanol to recover the lipidic content of mycobacterial cells is the most widely used solvent mixture23,24<.......
The authors have nothing to disclose.
This research was funded by the Spanish Ministry of Science, Innovation and Universities (RTI2018-098777-B-I00), the FEDER Funds, and the Generalitat of Catalunya (2017SGR-229). Sandra Guallar-Garrido is the recipient of a PhD contract (FI) from the Generalitat de Catalunya.
....Name | Company | Catalog Number | Comments |
Acetic Acid | Merck | 100063 | CAUTION. Anhydrous for analysis EMSURE® ACS,ISO,Reag. Ph Eur |
Acetone | Carlo Erba | 400971N | CAUTION. ACETONE RPE-ACS-ISO FOR ANALYS ml 1000 |
Anthrone | Merck | 8014610010 | Anthrone for synthesis. |
Benzene | Carlo Erba | 426113 | CAUTION. Benzene RPE - For analysis - ACS 2.5 l |
Capillary glass tube | Merck | BR708709 | BRAND® disposable BLAUBRAND® micropipettes, intraMark |
Chloroform | Carlo Erba | 412653 | CAUTION. Chloroform RS - For HPLC - Isocratic grade - Stabilized with ethanol 2.5 L |
Dry block heater | J.P. Selecta | 7471200 | |
Dicloromethane | Carlo Erba | 412622 | CAUTION. Dichloromethane RS - For HPLC - Isocratic grade - Stabilized with amylene 2.5 L |
Diethyl ether | Carlo Erba | 412672 | CAUTION. Diethyl ether RS - For HPLC - Isocratic grade - Not stabilized 2.5 L |
Ethyl Acetate | Panreac | 1313181211 | CAUTION. Ethyl acetate (Reag. USP, Ph. Eur.) for analysis, ACS, ISO |
Ethyl Alcohol Absolute | Carlo Erba | 4146072 | CAUTION. Ethanol absolute anhydrous RPE - For analysis - ACS - Reag. Ph.Eur. - Reag. USP 1 L |
Glass funnel | VidraFOC | DURA.2133148 1217/1 | |
Glass tube | VidraFOC | VFOC.45066A-16125 | Glass tube with PTFE recovered cap |
Methanol | Carlo Erba | 412722 | CAUTION. Methanol RS - For HPLC - GOLD - Ultragradient grade 2.5 L |
Molybdatophosphoric acid hydrate | Merck | 51429-74-4 | CAUTION. |
Molybdenum Blue Spray Reagent, 1.3% | Sigma | M1942-100ML | CAUTION. |
n-hexane | Carlo Erba | 446903 | CAUTION. n-Hexane 99% RS - ATRASOL - For traces analysis 2.5 L |
n-nitroso-n-methylurea | Sigma | N4766 | CAUTION |
Orbital shaking platform | DDBiolab | 995018 | NB-205L benchtop shaking incubator |
Petroleum ether (60-80ºC) | Carlo Erba | 427003 | CAUTION. Petroleum ether 60 - 80°C RPE - For analysis 2.5 L |
Sprayer | VidraFOC | 712/1 | |
Sodium sulphate anhydrous | Merck | 238597 | |
Sulfuric acid 95-97% | Merck | 1007311000 | CAUTION. Sulfuric acid 95-97% |
TLC chamber | Merck | Z204226-1EA | Rectangular TLC developing tanks, complete L × H × W 22 cm × 22 cm × 10 cm |
TLC plate | Merck | 1057210001 | TLC SilicaGel 60- 20x20 cm x 25 u |
TLC Plate Heater | CAMAG | 223306 | CAMAG TLC Plat Heater III |
Toluene | Carlo Erba | 488551 | CAUTION. Toluene RPE - For analysis - ISO - ACS - Reag.Ph.Eur. - Reag.USP 1 L |
Vortex | Fisher Scientific | 10132562 | IKA Agitador IKA vórtex 3 |
1-naphthol | Sigma-Aldrich | 102269427 | CAUTION. |
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