Identification and Characterization of the Genes Involved in Glycosylation Pathways of Mycobacterial Glycopeptidolipid Biosynthesis

Abstract

Glycopeptidolipids (GPLs) are major components present on the outer layers of the cell walls of several nontuberculous mycobacteria. GPLs are antigenic molecules and have variant oligosaccharides in mycobacteria such as Mycobacterium avium. In this study, we identified four genes (gtf1, gtf2, gtf3, and gtf4) in the genome of Mycobacterium smegmatis. These genes were independently inactivated by homologous recombination in M. smegmatis, and the structures of GPLs from each gene disruptant were analyzed. Thin-layer chromatography, gas chromatography-mass spectrometry, and matrix-assisted laser desorption ionization-time-of-flight mass spectrometry analyses revealed that the mutants ⌬gtf1 and ⌬gtf2 accumulated the fatty acyl-tetrapeptide core having O-methyl-rhamnose and 6-deoxy-talose as sugar residues, respectively. The mutant ⌬gtf4 possessed the same GPLs as the wild type, whereas the mutant ⌬gtf3 lacked two minor GPLs, consisting of 3-O-methylrhamnose attached to O-methyl-rhamnose of the fatty acyl-tetrapeptide core. These results indicate that the gtf1 and gtf2 genes are responsible for the early glycosylation steps of GPL biosynthesis and the gtf3 gene is involved in transferring a rhamnose residue not to 6-deoxy-talose but to an O-methyl-rhamnose residue. Moreover, a complementation experiment showed that M. avium gtfA and gtfB, which are deduced glycosyltransferase genes of GPL biosynthesis, restore complete GPL production in the mutants ⌬gtf1 and ⌬gtf2, respectively. Our findings propose that both M. smegmatis and M. avium have the common glycosylation pathway in the early steps of GPL biosynthesis but differ at the later stages.The mycobacterial cell envelope has a unique structure that contains a complex of covalently linked peptidoglycan, arabinogalactan, and mycolic acids (7, 11). The outer layer of the cell envelope is composed of several types of glycolipids that affect the surface properties of mycobacterial cells (7, 11). Glycopeptidolipids (GPLs) are a major class of glycolipid present on the outer layer of several species of nontuberculous mycobacteria, such as Mycobacterium avium complex, M. scrofulaceum, M. chelonae, M. fortuitum, and M. smegmatis (31). GPLs have a common fatty acyl-tetrapeptide core consisting of tetrapeptide amino alcohol (D-Phe-D-allo-Thr-D-Ala-L-alaninol) and amide-linked long-chain fatty acid (C 26-34 ). The fatty acyl-tetrapeptide core is glycosylated with 6-deoxytalose (6-d-Tal) and variable O-methyl-rhamnose (O-Me-Rha) residues, termed non-serovar-specific GPLs (nsGPLs), which are also the main products of M. smegmatis GPLs (1, 4, 10). The GPLs of M. avium have a more complicated structure, in which an additional Rha residue is added to 6-d-Tal of nsGPLs to be extended with various haptenic oligosaccharides, which are important surface antigens, resulting in serovar-specific GPLs (ssGPLs) (1, 4, 31).There are some evidences that GPLs may be responsible for pathogenicity. It has been shown that the some of the ssGPLs are immunosuppressive and are able to induce a variet...

Structural Characterization of a Specific Glycopeptidolipid Containing a Novel N -Acyl-Deoxy Sugar from Mycobacterium intracellulare Serotype 7 and Genetic Analysis of Its Glycosylation Pathway

“…Krzywinska and Schorey isolated and sequenced a 27.5-kb DNA fragment responsible for the carbohydrate portion of serotype 4 GPL from M. avium strain A5 (24). Recently, enzymatic characterization of the glycosyltransferase and methyltransferase in M. smegmatis, which can synthesize only nonpolar GPLs, has been reported (29,30).…”

Defects in glycopeptidolipid biosynthesis confer phage I3 resistance in Mycobacterium smegmatis

“…GPLs are a group of complex lipids derived from an acylated non-ribosomal tetrapeptide core and carbohydrate moieties, and they are abundant in the outer envelope of M. smegmatis and other mycobacteria (Billman-Jacobe, 2004;Schorey & Sweet, 2008). M. smegmatis produces six subclasses of GPLs, each of which has a common fatty acyl tetrapeptide (FATP) core consisting of a tetrapeptide amino alcohol (D-Phe-D-alloThr-D-Ala-L-alaninol) linked to a C 26 -C 34 fatty acyl chain via an amide bond (Miyamoto et al, 2006; Fig. 3a).…”

“…GPL-1, -2, -3 and -4 contain a rhamnose (with varying levels of Omethylation) and di-acetylated 6-deoxytalose as sugar residues. GPL-5 and -6 contain an additional O-methyl rhamnose attached to the original rhamnose residue (Miyamoto et al, 2006).…”

“…The sequence of assembly of sugar residues in GPLs was identified by Miyamoto et al (2006) by specifically deleting gtf1, gtf2 or gtf3, which are three genes encoding glycosyltransferases in M. smegmatis mc 2 155. Gtf1 is involved in the transfer of deoxytalose to the FATP core, and consequently the Dgtf1 mutant lacked all classes of GPLs, and instead accumulated the intermediates FATP-3,4-di-O-methyl rhamnose and FATP-2,3,4-tri-O-methyl rhamnose (FATP-di-O-Me-Rha and FATP-tri-O-methylrhamnose; Fig.…”

Identification and Characterization of Two Novel Methyltransferase Genes That Determine the Serotype 12-Specific Structure of Glycopeptidolipids of Mycobacterium intracellulare

“…Modification of the GPL structure might play an important role not only in antigenicity but also in host immune responses and bacterial physiology (18). Recently, chemical synthesis of various haptenic OSEs was demonstrated, and the genes encoding glycosylation pathway enzymes for the biosynthesis of GPLs were identified and characterized (8,12,19,21). However, genes responsible for serotype-specific glycosylation have yet to be analyzed for most of the serotypes.…”