In the case of multimethyl-branched FA found in PDIM, SGL,
PGL, or PPT of the M. tuberculosis complex (Fig. 1), the stereochemistry of the carbon bearing the methyl branches at positions
2, 4, 6, or more, but always at even carbons, is different in mycocerosates (found in PDIM and PGL of M. tuberculosis, M. leprae,
M. kansasii, M. gastri) and in phthioceranates (found in SGL of
M. tuberculosis and compounds structurally related to PDIM and
PGL in M. marinum and M. ulcerans), reflecting differences in
enzymatic systems used for their biosynthesis [44, 45]. In the
levorotatory-branched fatty acids or mycocerosic (M in Fig. 2),
the chiral centers have D configuration. Therefore, the C32 mycocerosic acid is the 2D, 4D, 6D, 8D-tetramethyl octacosanoic acid
esterifying the phthiocerol in PDIM and PGL (Fig. 1). The other
family of branched FA esterifying trehalose in SGL has similar
structure (phthioceranic acids, also labeled M in Fig. 2) but the
chiral centers bearing the methyl branches have L configurations,
conferring a positive value to their molecular rotation. More
recently, the absolute stereochemistry of the carbon bearing the
hydroxyl in the hydroxy-phthioceranic acid constitutive of diacylated sulfoglycolipids (Ac 2 SGL) has been demonstrated to be
R [46]. It is worth mentioning here other important FAs found
in virulent strains of M. tuberculosis, the so-called phthienoic acids
(P in Fig. 2); they are easily characterized by their high dextrorotation value [α] D + 18
. The main C 27 homologue of this series is
2,4L,6L-trimethyl 2-trans tetracosanoic acid [45] (Fig. 1) esterifying trehalose and yielding PPT [47].
Chiral molecules are characterized by their property to rotate
the plane of polarized light. The determination of specific rotation
[α] D is realized on purified lipid solubilized in chloroform with a
polarimeter apparatus at a defined temperature of 20
C.
The present chapter describes the current methods used in
isolating, fractionating, purifying, and analyzing the major types
of biologically active mycobacterial lipids and LAM. We describe
methods used for global and rapid analyses of mycobacterial
extractable lipids and their constituents, cell-wall-linked MA, and
LAM, which require simple chromatographic techniques such as
TLC, GC-MS, and SDS-PAGE. Then, we present methods that
allow the purification of the most commonly isolated biologically
active lipids. The most commonly used techniques for structural
elucidation of mycobacterial lipids are also briefly described.
2 Materials
2.1 Extraction of Free
Lipids
1. A biosafety cabinet in the appropriate laboratory (see Note 1).
2. 50 mL screw-capped glass round-bottom flasks (250 mL).
3. Glass funnels.
4. Glass Pasteur pipettes.
116
Marie-Antoinette Lane ´ elle et al.
PGL, or PPT of the M. tuberculosis complex (Fig. 1), the stereochemistry of the carbon bearing the methyl branches at positions
2, 4, 6, or more, but always at even carbons, is different in mycocerosates (found in PDIM and PGL of M. tuberculosis, M. leprae,
M. kansasii, M. gastri) and in phthioceranates (found in SGL of
M. tuberculosis and compounds structurally related to PDIM and
PGL in M. marinum and M. ulcerans), reflecting differences in
enzymatic systems used for their biosynthesis [44, 45]. In the
levorotatory-branched fatty acids or mycocerosic (M in Fig. 2),
the chiral centers have D configuration. Therefore, the C32 mycocerosic acid is the 2D, 4D, 6D, 8D-tetramethyl octacosanoic acid
esterifying the phthiocerol in PDIM and PGL (Fig. 1). The other
family of branched FA esterifying trehalose in SGL has similar
structure (phthioceranic acids, also labeled M in Fig. 2) but the
chiral centers bearing the methyl branches have L configurations,
conferring a positive value to their molecular rotation. More
recently, the absolute stereochemistry of the carbon bearing the
hydroxyl in the hydroxy-phthioceranic acid constitutive of diacylated sulfoglycolipids (Ac 2 SGL) has been demonstrated to be
R [46]. It is worth mentioning here other important FAs found
in virulent strains of M. tuberculosis, the so-called phthienoic acids
(P in Fig. 2); they are easily characterized by their high dextrorotation value [α] D + 18
. The main C 27 homologue of this series is
2,4L,6L-trimethyl 2-trans tetracosanoic acid [45] (Fig. 1) esterifying trehalose and yielding PPT [47].
Chiral molecules are characterized by their property to rotate
the plane of polarized light. The determination of specific rotation
[α] D is realized on purified lipid solubilized in chloroform with a
polarimeter apparatus at a defined temperature of 20
C.
The present chapter describes the current methods used in
isolating, fractionating, purifying, and analyzing the major types
of biologically active mycobacterial lipids and LAM. We describe
methods used for global and rapid analyses of mycobacterial
extractable lipids and their constituents, cell-wall-linked MA, and
LAM, which require simple chromatographic techniques such as
TLC, GC-MS, and SDS-PAGE. Then, we present methods that
allow the purification of the most commonly isolated biologically
active lipids. The most commonly used techniques for structural
elucidation of mycobacterial lipids are also briefly described.
2 Materials
2.1 Extraction of Free
Lipids
1. A biosafety cabinet in the appropriate laboratory (see Note 1).
2. 50 mL screw-capped glass round-bottom flasks (250 mL).
3. Glass funnels.
4. Glass Pasteur pipettes.
116
Marie-Antoinette Lane ´ elle et al.