Figure 5

Structural analysis of the GPL content in Δtle.A, TLC analysis representative from three independent experiments of native apolar (left) and polar (right) lipid extracts. Lipids were separated using chloroform/methanol/water (90:10:1, v/v/v) followed by spraying with orcinol and charring. GPL-1b and GPL-2b previously described (35) from Δtle display a lower R_f than WT GPL-2a and GPL-3, which are restored in Δtle::C. TDM: trehalose dimycolate; DAT: Di-O-acyl trehalose. B, TLC analysis of native (NaOH−) and saponified (NaOH+) polar fractions. Lipids were separated using chloroform/methanol/water (90:10:1, v/v/v) followed by spraying with orcinol and charring. The R_f of GPL-2a and GPL-3 are identical in S and Δtle::C but not in Δtle. C, MALDI-TOF positive MS spectra of native polar lipid fraction (black, NaOH−) from the S and Δtle::C strains show ions for GPL-2a at m/z 1257.9/1285.9 and for GPL-3 at m/z 1403.9/1432.0. MS spectra from saponified polar lipid fraction (red, NaOH+) show a 84 u.m.a decrease, to 1173.4/1201.4 and 1319.3/1347.4, corresponding to deacetylated dGPL-2a and dGPL-3, as previously described (34). Conversely, ions for GPL-1b at m/z 1013.8/1041.8 and from GPL-2b at 1187.9 were intense in native and saponified polar extracts from Δtle. D, relative proportion of each monosaccharides was determined in the GPL-enriched saponified polar lipid fraction. 6-d-Tal could only be detected in S and Δtle::C.