(A) SDS/PAGE analysis of phosphatidylinositol mannosides (PIMs), lipomannan (LM) and LAM prepared from wild-type (H37Rv), lprG mutant (ΔlprG), and ΔlprG complemented with lprG-Rv1410c (::lprG). LM and LAM extracted from equal amounts of bacterial cells were separated on a 10–20% Tricine gel and visualized by periodic acid/Schiff reagent staining. (B) Thin-layer chromatograms of total lipids extracted from H37Rv and ΔlprG. The same amounts of total lipids extract from bacilli grown in GAS medium were loaded for each strain. Thin-layer chromatogram plates were run in the solvent system CHCl₃/CH₃OH/H₂O (65:25:4, by vol.) and revealed with α-naphthol. SL, sulfolipid; TMM, trehalose monomycolates; DAT, diacyltrehaloses; PE, phosphatidylethanolamine; CL, cardiolipin; PIM₂, phosphatidylinositol dimannoside; PI, phosphatidylinositol; PIM₆, phosphatidylinositol hexamannosides. (C) SDS/PAGE immunoblot for LAM analysis in H37Rv and ΔlprG cellular extracts. Extracts normalized to protein concentration were separated on a 15% SDS/PAGE gel and transferred to PVDF membrane. The blot was blocked, and then stained with anti-LAM pAb (α-LAM) followed by goat anti-rabbit IgG-HRP secondary antibody. The blot was washed and imaged after adding 30% 3,3’-diaminobenzidine tetrahydrochloride solution plus 0.0005% H₂O₂. LAM Std, purified H37Rv LAM standard. Lanes shown formed part of a larger gel. (D) Spot immunoblot for analysis of capsular α-glucan. Capsular content extracted from equal numbers of bacteria were spotted on PVDF membrane and stained with goat anti-phosphatidylinositol-glycans pAb followed by donkey anti-goat IgG-HRP secondary antibody. The membrane was developed and imaged as described in C. Dilutions of extract spotted on membrane are shown. Data is representative of two independent experiments.

https://doi.org/10.1371/journal.ppat.1005336.g001