Structural determinants in a glucose-containing lipopolysaccharide from Mycobacterium tuberculosis critical for inducing a subset of protective T cells

Abstract

Mycobacteria synthesize intracellular, 6-O-methylglucose–containing lipopolysaccharides (mGLPs) proposed to modulate bacterial fatty acid metabolism. Recently, it has been shown that Mycobacterium tuberculosis mGLP specifically induces a specific subset of protective γ9δ2 T cells. Mild base treatment, which removes all the base-labile groups, reduces the specific activity of mGLP required for induction of these T cells, suggesting that acylation of the saccharide moieties is required for γ9δ2 T-cell activation. On the basis of this premise, we used analytical LC/MS and NMR methods to identify and locate the acyl functions on the mGLP saccharides. We found that mGLP is heterogeneous with respect to acyl functions and contains acetyl, isobutyryl, succinyl, and octanoyl groups and that all acylations in mGLP, except for succinyl and octanoyl residues, reside on the glucosyl residues immediately following the terminal 3-O-methylglucose. Our analyses also indicated that the octanoyl residue resides at position 2 of an internal glucose toward the reducing end. LC/MS analysis of the residual product obtained by digesting the mGLP with pancreatic α-amylase revealed that the product is an oligosaccharide terminated by α-(1→4)–linked 6-O-methyl-d-glucosyl residues. This oligosaccharide retained none of the acyl groups, except for the octanoyl group, and was unable to induce protective γ9δ2 T cells. This observation confirmed that mGLP induces γ9δ2 T cells and indicated that the acylated glucosyl residues at the nonreducing terminus of mGLP are required for this activity.

Lipids of Clinically Significant Mycobacteria

“…Various acylation combinations of PMPSs involve acetate, propionate, isobutyrate, octanoate, and succinate (Keller and Ballou 1968;Gray and Ballou 1972;Smith and Ballou 1973;Narumi et al 1973;Jackson and Brennan 2009). Acyl function heterogeneity of the PMPS of M. tuberculosis was confirmed (De et al 2018), so only a representative structure is shown in Fig. 27.…”

“…PMPSs have been described in a variety of mycobacteria and Nocardia (Lee 1966;Smith and Ballou 1973;Tuffal et al 1995Tuffal et al , 1998aJackson and Brennan 2009). These important minor lipophilic PMPS molecules (0.01% of biomass; De et al 2018) are possibly involved in the regulation of fatty and mycolic acid synthesis. The PMPSs and the related 3-O-methyl-mannose-containing polysaccharides from M. smegmatis (MMPs; Gray and Ballou 1971;Maitra and Ballou 1977) are likely fatty-acyl carriers, facilitating processing of long, insoluble fatty-acyl CoAs .…”

Urine lipoarabinomannan in HIV uninfected, smear negative, symptomatic TB patients: effective sample pretreatment for a sensitive immunoassay and mass spectrometry

“…Gas chromatography/mass spectrometry analysis for TBSA and d-ara in urine LAM. The method developed has been described in detail 15,16 . Typically, urine samples (1 mL) were subjected to hydropho- www.nature.com/scientificreports/ bic interaction chromatography (HIC) over Octyl Sepharose (OS)-CL 4B.…”

“…There is extensive evidence that LAM is found in serum and urine in measurable quantities, established using both chemo-analytical assays and immunoassays 7,8,[13][14][15][16] ; however, only the Alere LAM test is currently in the market for clinical usage. The clinical performance of this test has had a questionable lack of sensitivity; this could be due to a low abundance of LAM in urine in TB patients who have a low bacterial load, particularly for patients who are HIV uninfected.…”

O-Methylation in Carbohydrates: An NMR and MD Simulation Study with Application to Methylcellulose

“…Methylation in carbohydrates is found in many places in nature such as bacteria, fungi, worms, mollusks, algae, and plants, but it is still rare compared to other modifications . Methylated oligosaccharides are only found in bacteria and plants, where for instance certain plant hemicelluloses (xylans) are rich in 4-O-methylated glucuronic acids, and 6-O-methyl substitutions are present in glucosyl residues from mycobacterial polysaccharides − and in a polysaccharide from the leaves of the plant Catharanthus rosea …”