Characterization of the fucosylation pathway in the biosynthesis of glycopeptidolipids from Mycobacterium avium complex

Abstract

The cell envelopes of several species of nontuberculous mycobacteria, including the Mycobacterium avium complex, contain glycopeptidolipids (GPLs) as major glycolipid components. GPLs are highly antigenic surface molecules, and their variant oligosaccharides define each serotype of the M. avium complex. In the oligosaccharide portion of GPLs, the fucose residue is one of the major sugar moieties, but its biosynthesis remains unclear. To elucidate it, we focused on the 5.0-kb chromosomal region of the M. avium complex that includes five genes, two of which showed high levels of similarity to the genes involved in fucose synthesis. For the characterization of this region by deletion and expression analyses, we constructed a recombinant Mycobacterium smegmatis strain that possesses the rtfA gene of the M. avium complex to produce serovar 1 GPL. The results revealed that the 5.0-kb chromosomal region is responsible for the addition of the fucose residue to serovar 1 GPL and that the three genes mdhtA, merA, and gtfD are indispensable for the fucosylation. Functional characterization revealed that the gtfD gene encodes a glycosyltransferase that transfers a fucose residue via 1-->3 linkage to a rhamnose residue of serovar 1 GPL. The other two genes, mdhtA and merA, contributed to the formation of the fucose residue and were predicted to encode the enzymes responsible for the synthesis of fucose from mannose based on their deduced amino acid sequences. These results indicate that the fucosylation pathway in GPL biosynthesis is controlled by a combination of the mdhtA, merA, and gtfD genes. Our findings may contribute to the clarification of the complex glycosylation pathways involved in forming the oligosaccharide portion of GPLs from the M. avium complex, which are structurally distinct.

FIG. 1.

Schematic presentation of the cloning procedure for the 5.0-kb gtfC-gtfD region (A) and its gene-deleted constructs, which were inserted into an expression cassette made up of the hsp60 promoter and the terminator of pMV261 (B). The primers used for PCR amplification of the three fragments are indicated by filled triangles. In pMVΔgtfC and pMVΔgtfD, the genes gtfC and gtfD were completely deleted from the gtfC-gtfD region of pMVgtfCD. In-frame deletions were designed for the construction of the expression cassettes of pMVΔmdhtA, pMVΔmerA, and pMVΔmtfF to prevent the polar effect on each downstream gene. P, PstI; H, HindIII; K, KpnI; E, EcoRI.

FIG. 2.

Functional analyses of the gtfC-gtfD region. (A) TLC analysis of crude GPL extracts from rtfA-int/pMV261 (lane a), rtfA-int/pMVgtfCD (lane b), Wt/pMV261 (lane c), and Wt/pMVgtfCD (lane d). The total lipid fraction after mild alkaline hydrolysis was spotted on plates and was developed in CHCl₃-CH₃OH (9:1 [vol/vol]). (B) GC-MS analyses of alditol acetates of sugars released from crude GPL extracts of rtfA-int/pMV261 (a) and rtfA-int/pMVgtfCD (b). Alditol acetate derivatives were prepared from the total lipid fraction after mild alkaline hydrolysis. Asterisks indicate noncarbohydrates.

FIG. 3.

GC-MS analyses of alditol acetates of sugars released from crude GPLs. GPLs were extracted from rtfA-int/pMVΔgtfC (A), rtfA-int/pMVΔmdhtA (B), rtfA-int/pMVΔmerA (C), rtfA-int/pMVΔmtfF (D), and rtfA-int/pMVΔgtfD (E). Alditol acetate derivatives were prepared from the total lipid fraction after mild alkaline hydrolysis. Asterisks indicate noncarbohydrates.

FIG. 4.

TLC analyses of crude GPL extracts from rtfA-mdhtA-merA-int/pMVgtfD (lane A) and rtfA-mdhtA-merA-int/pMV261 (lane B). The total lipid fraction after mild alkaline hydrolysis was spotted onto plates and developed in CHCl₃-CH₃OH (9:1 [vol/vol]).

FIG. 5.

GC mass spectra and fragment ion assignment of Fuc (A), Rha (B), and 6-d-Tal (C), which are derived from alditol acetates of sugars released from deuteriomethylated GPL-S2. Ac, acetate; D, deuterium.

FIG. 6.

Proposed structure and biosynthetic pathway of fucosylated GPL (GPL-S2).