Disruption of the SucT acyltransferase in Mycobacterium smegmatis abrogates succinylation of cell envelope polysaccharides

Abstract

Similar to other prokaryotes, mycobacteria decorate their major cell envelope glycans with minor covalent substituents whose biological significance remains largely unknown. We report on the discovery of a mycobacterial enzyme, named here SucT, that adds succinyl groups to the arabinan domains of both arabinogalactan (AG) and lipoarabinomannan (LAM). Disruption of the SucT-encoding gene in Mycobacterium smegmatis abolished AG and LAM succinylation and altered the hydrophobicity and rigidity of the cell envelope of the bacilli without significantly altering AG and LAM biosynthesis. The changes in the cell surface properties of the mutant were consistent with earlier reports of transposon mutants of the closely related species Mycobacterium marinum and Mycobacterium avium harboring insertions in the orthologous gene whose ability to microaggregate and form biofilms were altered. Our findings point to an important role of SucT-mediated AG and LAM succinylation in modulating the cell surface properties of mycobacteria.

Keywords: Mycobacterium smegmatis; arabinogalactan; cell surface; lipoarabinomannan; mycobacteria; polysaccharide; succinylation; tuberculosis.

Figure 1.

Detail of the chemical modifications affecting the internal arabinan domains of AG and LAM in M. tuberculosis. Note that the succinylation of the mycolylated chains in arabinogalactan has been reported to be diminished or absent (15).

Figure 2.

Electrophoretic mobility of lipoglycans from WT M. smegmatis mc²155, the sucT mutant, and the complemented sucT mutant. Lipoglycans extracted from WT mc²155, mc²ΔsucT, and mc²ΔsucT/pMVGH1-sucT (ΔsucT comp) were run on a 10–20% Tricine gel, followed by periodic acid–silver staining. PIMs, phosphatidylinositol mannosides.

Figure 3.

Succinate content of AG and LAM prepared from WT M. smegmatis mc²155, the ΔsucT mutant, and the complemented mutant strain. A, the amounts of succinates and arabinose residues in the same LAM and mAGP samples prepared from the WT, mutant, and complemented mutant (ΔsucT compl) strains were quantified as described in the Supporting Methods. Results are expressed as average ± S.D. succinate/arabinose molar ratios from three technical replicates. B, endogenous endoarabinanase digestion of mAGP from the WT, mutant, and complemented mutant strains. Analysis of the products of the reaction by QTOF LC/MS in the negative ion mode revealed characteristic [M-2H]⁻² ions corresponding to oligoarabinosides with succinyl groups in the WT and complemented mutant that are not found in the sucT mutant mAGP.

Figure 4.

NMR analysis of LAM prepared from the WT, mutant, and complemented mutant strains. Shown are 1D ¹H (A, C, and E) and 2D ¹H-¹³C (B, D, and F) HMQC NMR spectra. Arrows point to the signals typifying succinate detection and localization (see text for details).

Figure 5.

Alterations in the cell surface properties of the M. smegmatis sucT mutant. A, colony morphology of WT mc²155, mc²ΔsucT, and mc²ΔsucT/pMVGH1-sucT (ΔsucT comp) after 3 days of incubation on 7H11-OADC agar at 37 °C. B, Congo red binding on a TS agar plate and in TS liquid medium (graph). Shown on the graph are the average ± S.D. absorbances of acetone extracts measured for three biological replicates. C, analysis of the cell surface rigidity of the WT, mutant, and complemented mutant strains by correlated optical fluorescence and AFM. Two-sided rank sum test demonstrates a statistically significant difference in stiffness between the sucT mutant and both the WT (p = 0.0214) and the sucT complemented mutant (p = 0.0011).