Functional Characterisation of Three O-methyltransferases Involved in the Biosynthesis of Phenolglycolipids in Mycobacterium tuberculosis

Abstract

Phenolic glycolipids are produced by a very limited number of slow-growing mycobacterial species, most of which are pathogen for humans. In Mycobacterium tuberculosis, the etiologic agent of tuberculosis, these molecules play a role in the pathogenicity by modulating the host immune response during infection. The major variant of phenolic glycolipids produced by M. tuberculosis, named PGL-tb, consists of a large lipid core terminated by a glycosylated aromatic nucleus. The carbohydrate part is composed of three sugar residues, two rhamnosyl units and a terminal fucosyl residue, which is per-O-methylated, and seems to be important for pathogenicity. While most of the genes responsible for the synthesis of the lipid core domain and the saccharide appendage of PGL-tb have been characterized, the enzymes involved in the O-methylation of the fucosyl residue of PGL-tb remain unknown. In this study we report the identification and characterization of the methyltransferases required for the O-methylation of the terminal fucosyl residue of PGL-tb. These enzymes are encoded by genes Rv2954c, Rv2955c and Rv2956. Mutants of M. tuberculosis harboring deletion within these genes were constructed. Purification and analysis of the phenolglycolipids produced by these strains, using a combination of mass spectrometry and NMR spectroscopy, revealed that Rv2954c, Rv2955c and Rv2956 encode the methyltransferases that respectively catalysed the O-methylation of the hydroxyl groups located at positions 3, 4 and 2 of the terminal fucosyl residue of PGL-tb. Our data also suggest that methylation at these positions is a sequential process, starting with position 2, followed by positions 4 and 3.

Lipid metabolism and Type VII secretion systems dominate the genome scale virulence profile of Mycobacterium tuberculosis in human dendritic cells

“…Mutants with fitness costs included multi-gene loci associated with specific functional groups such as: the synthesis of lipids e.g. phthiocerol dimycolates (pDIMs) [ 44 ], phenolic glycolipids (PGLs) [ 45 ] and sulfolipids; cholesterol metabolism [ 46 - 48 ] and ESX systems [ 49 ] (Figure 2 c and Figure 3 ). Other functions associated with fitness are more scattered across the genome, but included PPE genes, the genes required for nitrate reduction and genes involved in the metabolism of methionine/cysteine.…”

Effects of Lipid-Lowering Drugs on Vancomycin Susceptibility of Mycobacteria

“…Lipids from M. tuberculosis H37Rv (a 350-ml 7H9 culture without Tween 80 at an OD 600 of 0.5) treated with THL (50 g/ ml) or treated with the same volume of dimethyl sulfoxide (DMSO) as a control were extracted first with CHCl 3 /CH 3 OH (1:2 [vol/vol]) for 24 h at room temperature and then twice with CHCl 3 /CH 3 OH (2:1 [vol/vol]) for 2 days. Crude extracts were washed twice with distilled water and evaporated to dryness according to standard protocols as described previously (34).…”

I3-Ag85 effect on phthiodiolone dimycocerosate synthesis

“…I3-Ag85 inhibitory effect should therefore rely on the inhibition of additional targets, including potentially orthologous Ag85A or B proteins, given that the KO strain is devoid of Ag85C but shows the same MIC to the inhibitor as the WT strain. We therefore analyzed their lipid composition by high-pressure thin-layer chromatography (HPTLC) as previously described [10], comparing midlog-phase growing Mtb CDC1551 WT and KO cultures (with inoculum size 100 fold higher compared to drug susceptibility assays) , treated or untreated 24 h with 44 µg/ml I3-Ag85 [17]. As described by Warrier et al, we observed a slight decrease of TDM and an increase of DAT+TMM in the treated WT strain (Fig.…”