The Galactosamine Residue in Mycobacterial Arabinogalactan Is α‑Linked

Previous studies have demonstrated that cell wall arabinogalactan from mycobacteria possesses a single galactosamine (GalN) residue. This moiety, which is one of the rare natural occurrences of galactosamine lacking an acetyl group on the nitrogen, has been identified as a pendant substituent attached to a highly branched arabinofuranose residue in the arabinan core. However, the stereochemistry by which the GalN residue is linked to the polysaccharide remains unknown. We report here the synthesis of two tetrasaccharides, <b>1</b> and <b>2</b>, consisting of GalN attached through either an α- or β-linkage to a trisaccharide fragment of mycobacterial arabinan. These molecules represent the first synthetic GalN-containing oligosaccharides, and the preparation of both targets was achieved from a single donor species by modulation of the reaction solvent. Comparison of the NMR spectra of <b>1</b> and <b>2</b> with those obtained from a sample derived from the natural glycan revealed that the GalN residue in the polysaccharide is attached via an α-linkage

Correction: LprG-Mediated Surface Expression of Lipoarabinomannan Is Essential for Virulence of <i>Mycobacterium tuberculosis</i>

<p>Correction: LprG-Mediated Surface Expression of Lipoarabinomannan Is Essential for Virulence of <i>Mycobacterium tuberculosis</i></p

The glycosyl composition of the delipidated cells, capsules and culture filtrates of the H37Rv and <i>lprG</i> mutant (Δ<i>lprG</i>) strains.

<p>The glycosyl composition of the delipidated cells, capsules and culture filtrates of the H37Rv and <i>lprG</i> mutant (Δ<i>lprG</i>) strains.</p

The <i>lprG</i> mutant is impaired for macrophages entry.

<p>(A) Resting BMMΦ from C57BL/6 mice were infected with wild-type (H37Rv), <i>lprG</i> mutant (Δ<i>lprG</i>), and Δ<i>lprG</i> complemented with <i>lprG</i>-Rv1410c (::<i>lprG</i>) at MOI of 1∶10 (Input). At 4 h post-infection cells were washed and intracellular bacteria (Output) were enumerated on agar. Bar graph shows average CFU counts ±SEM of three independent experiments performed in triplicate wells. (B–D) RAW 264.7 macrophages treated with and without 4 mg/ml mannan or 0.5 µg/ml anti-MMR pAb were infected with FITC-labeled bacteria at MOI of 1∶100 for 2 h. Cells were washed, mounted and visualized using a fluorescent microscope. Graphs show percentage of infected cells with no treatment (B), treated with mannan (C) and treated with anti-MMR pAb (D). Bars show average percentage ±SEM of two independent experiments performed in triplicates. Δ<i>lprG</i> and ::<i>lprG</i> were compared to H37Rv. *P<0.05.</p

The <i>lprG</i> mutant has normal LAM content in the cell envelope.

<p>(A) SDS/PAGE analysis of phosphatidylinositol mannosides (PIMs), lipomannan (LM) and LAM prepared from wild-type (H37Rv), <i>lprG</i> mutant (Δ<i>lprG</i>), and Δ<i>lprG</i> complemented with <i>lprG</i>-Rv1410c (::<i>lprG</i>). 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 Δ<i>lprG</i>. 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 Δ<i>lprG</i> 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. (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.</p

Surface staining reveals reduced LAM on the <i>lprG</i> mutant cells.

<p>Indirect fluorescent antibody staining of wild-type (H37Rv), <i>lprG</i> mutant (Δ<i>lprG</i>), and Δ<i>lprG</i> complemented with <i>lprG</i>-Rv1410c (::<i>lprG</i>) with anti-LAM antibodies. Bacilli cultured in shaking broth cultures were washed, fixed in 4% paraformaldehyde, and then suspended in 5% bovine serum albumin for 1 h. Bacilli were incubated with primary antibodies (1° Ab) rabbit anti-LAM pAb (α-LAM) and mouse anti-LAM mAb (CS-35), washed, and then incubated with secondary antibodies (2° Ab), goat anti-rabbit IgG Fab2-Alexa fluor and goat anti-mouse IgG IgG-Dylight, respectively. Bacilli were mounted with DAPI-containing medium and visualized using a fluorescent microscope. (A) Confocal images showing positive staining bacteria with α-LAM (arrow). (B and C) Bar graphs show average percent of bacteria staining positive with α-LAM (B) and CS-35 (C) ±SD of three independent experiments. Δ<i>lprG</i> and ::<i>lprG</i> were compared to H37Rv. **P<0.01; ***P<0.001.</p