Analysis of the Lipid Composition of Mycobacteria by Thin Layer Chromatography

Summary

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.

Abstract

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.

Introduction

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....

Protocol

1. Extraction of the total non-covalent-linked lipids from mycobacteria (Figure 1)

1. Scratch 0.2 g of mycobacteria from a solid media and add to a glass tube with a polytetrafluoroethlene (PTFE) liner screw caps. Add a solution consisting of 5 mL of chloroform and 10 mL of methanol (chloroform:methanol, 1:2).
   NOTE: When organic solvents are used, only glass recipient should be used. No plastic containers are allowed. Moreover, PTFE liner screw caps for bottles are needed.
   CAUTION: Chloroform is a potentially toxic and extremely hazardous substance. It must be used in a laminar flow hood wearing appropriate personal....

Results

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.......

Discussion

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 mixture^{23,24<.......}

Materials

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.