wall arabinogalactan, which in turn is linked to peptidoglycan
[4]. The outer leaflet of the mycomembrane is composed of a
diversity of exotic lipidic molecules, which include ubiquitous
trehalose-containing glycolipids, trehalose mono- and di-mycolate
(TMM and TDM, respectively), and species-specific lipids such as
diesters of phthiocerol (PDIM), phenolic glycolipids (PGL), sulfoglycolipids (SGL), and glycopeptidolipids (GPL) [5–8]. Lipoarabinomannan (LAM) is a lipoglycan distributed between the plasma
membrane and the outermost layers of the mycobacterial cell
envelope.
1.1 Extraction
of Mycobacterial
Lipids and Isolation
of Their Constituents
Lipids are soluble in organic solvents, e.g., chloroform or diethyl
ether, and are easily and routinely extracted from (preferentially)
wet cell pellets, provided that they are not covalently linked to the
cell wall such as MA attached to the arabinogalactan–peptidoglycan
complex. Due to the complexity of mycobacterial extractable “free”
lipids mixture, from the very apolar PDIM to the polar phosphatidylinositol mannosides (PIM), fractionation of the different classes
is generally realized by solvent precipitation, mild alkaline hydrolysis, and combination of various chromatographic techniques.
The MA linked to the cell wall arabinogalactan and the fatty
acids (FA), commonly esterifying alcohols in extractable complex
lipids, are released by alkaline hydrolysis for further structural analyses. Several methods have been proposed for their isolation.
Saponification is the method of choice to break the ester linkage
and to obtain free fatty acids. The method of saponification should
allow a good yield in FA released from all mycobacterial complex
lipids containing ester linkage, not only triacylglycerol (TAG),
phospholipids, trehalose esters, and glycerol monomycolate
(GroMM) but also PDIM; indeed, in this last case, the presence
of multibranched FA hinders the attack of the ester linkage by
alkali. A caution has to be kept in mind for the choice of the method
of saponification for releasing MA since it has been shown that too
mild alkaline conditions cause racemization of the chiral center in
carbon 2. The two diastereoisomers, namely the 2R, 3R (natural
isomer) and the 2S, 3R (artifact isomer), have an opposite value of
their molecular rotations (positive and negative values, respectively)
and show different migrations on TLC (lower and upper spot,
respectively), leading to a multiplication of the spots, 2 for each
class of MA [9, 10]. Acidic conditions for releasing FA have to be
avoided as they lead to the degradation of some structures, e.g., the
aperture of epoxy ring, leading to a complex thin-layer chromatography (TLC) MA methyl ester (MAME) profile [11, 12].
1.2 Structural
Analyses
of Mycobacterial
Lipids
Although the structural elucidation of mycobacterial lipids is sometimes very challenging and time-consuming, the recent progress in
analytical techniques, mainly mass spectrometry (MS), has greatly
facilitated the determination of molecules holistically by reducing
sample consumption.
110
Marie-Antoinette Lane ´ elle et al.
[4]. The outer leaflet of the mycomembrane is composed of a
diversity of exotic lipidic molecules, which include ubiquitous
trehalose-containing glycolipids, trehalose mono- and di-mycolate
(TMM and TDM, respectively), and species-specific lipids such as
diesters of phthiocerol (PDIM), phenolic glycolipids (PGL), sulfoglycolipids (SGL), and glycopeptidolipids (GPL) [5–8]. Lipoarabinomannan (LAM) is a lipoglycan distributed between the plasma
membrane and the outermost layers of the mycobacterial cell
envelope.
1.1 Extraction
of Mycobacterial
Lipids and Isolation
of Their Constituents
Lipids are soluble in organic solvents, e.g., chloroform or diethyl
ether, and are easily and routinely extracted from (preferentially)
wet cell pellets, provided that they are not covalently linked to the
cell wall such as MA attached to the arabinogalactan–peptidoglycan
complex. Due to the complexity of mycobacterial extractable “free”
lipids mixture, from the very apolar PDIM to the polar phosphatidylinositol mannosides (PIM), fractionation of the different classes
is generally realized by solvent precipitation, mild alkaline hydrolysis, and combination of various chromatographic techniques.
The MA linked to the cell wall arabinogalactan and the fatty
acids (FA), commonly esterifying alcohols in extractable complex
lipids, are released by alkaline hydrolysis for further structural analyses. Several methods have been proposed for their isolation.
Saponification is the method of choice to break the ester linkage
and to obtain free fatty acids. The method of saponification should
allow a good yield in FA released from all mycobacterial complex
lipids containing ester linkage, not only triacylglycerol (TAG),
phospholipids, trehalose esters, and glycerol monomycolate
(GroMM) but also PDIM; indeed, in this last case, the presence
of multibranched FA hinders the attack of the ester linkage by
alkali. A caution has to be kept in mind for the choice of the method
of saponification for releasing MA since it has been shown that too
mild alkaline conditions cause racemization of the chiral center in
carbon 2. The two diastereoisomers, namely the 2R, 3R (natural
isomer) and the 2S, 3R (artifact isomer), have an opposite value of
their molecular rotations (positive and negative values, respectively)
and show different migrations on TLC (lower and upper spot,
respectively), leading to a multiplication of the spots, 2 for each
class of MA [9, 10]. Acidic conditions for releasing FA have to be
avoided as they lead to the degradation of some structures, e.g., the
aperture of epoxy ring, leading to a complex thin-layer chromatography (TLC) MA methyl ester (MAME) profile [11, 12].
1.2 Structural
Analyses
of Mycobacterial
Lipids
Although the structural elucidation of mycobacterial lipids is sometimes very challenging and time-consuming, the recent progress in
analytical techniques, mainly mass spectrometry (MS), has greatly
facilitated the determination of molecules holistically by reducing
sample consumption.
110
Marie-Antoinette Lane ´ elle et al.