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 Deltagtf1 and Deltagtf2 accumulated the fatty acyl-tetrapeptide core having O-methyl-rhamnose and 6-deoxy-talose as sugar residues, respectively. The mutant Deltagtf4 possessed the same GPLs as the wild type, whereas the mutant Deltagtf3 lacked two minor GPLs, consisting of 3-O-methyl-rhamnose 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 Deltagtf1 and Deltagtf2, 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.

FIG. 1.

Generation of gtf gene disruptants. (A to D) Schematic diagram of each gtf region on the chromosome of the wild-type M. smegmatis mc²155 strain (Wt) and its gene disruptants Δgtf1, Δgtf2, Δgtf3, and Δgtf4. The shaded boxes indicate the regions included in recombinant phage for gene disruption. The black arrows represent the coding region of each gtf gene. The gray boxes represent the hygromycin resistance cassette (hyg). The primers used for PCR analysis are indicated by small arrows. (E) PCR analyses of the wild type and each disruptant using the primers indicated above.

FIG. 2.

TLC analyses of crude GPL extracts from the M. smegmatis mc²155 strain (Wt) and its gene disruptants. The total lipid fraction after mild alkaline hydrolysis was spotted on plates and developed in CHCl₃-CH₃OH (9:1 [vol/vol]). GPLs were visualized by spraying with 10% H₂SO₄ and charring. Each total lipid fraction was extracted from an equal weight of harvested cells.

FIG. 3.

GC/MS analyses of alditol acetates of sugars released from crude GPLs. GPLs were extracted from M. smegmatis strains: (A) mc²155 strain, (B) Δgtf1, and (C) Δgtf2. Alditol acetate derivatives were prepared from the total lipid fraction after mild alkaline hydrolysis, which was extracted from an equal weight of harvested cells.

FIG. 4.

Biochemical characterization of GPL-5 (a), GPL-6 (b), GPL-3 (c), and GPL-4 (d). (A) GC/MS analysis of alditol acetates of sugars released from each purified GPL. (B) MALDI-TOF/MS analysis of total molecular mass of each purified GPLs. (C) MALDI-TOF/MS analysis of total molecular mass of purified GPL-5 (a) and GPL-6 (b), which were subjected to β-elimination.

FIG. 4.

Biochemical characterization of GPL-5 (a), GPL-6 (b), GPL-3 (c), and GPL-4 (d). (A) GC/MS analysis of alditol acetates of sugars released from each purified GPL. (B) MALDI-TOF/MS analysis of total molecular mass of each purified GPLs. (C) MALDI-TOF/MS analysis of total molecular mass of purified GPL-5 (a) and GPL-6 (b), which were subjected to β-elimination.

FIG. 5.

GC/MS analysis of alditol acetates of sugars released from perdeuteriomethylated GPL-5. (A) GC profile. (B) Mass spectrum and fragment ion assignment corresponding to 3-O-Me-Rha. (C) Mass spectrum of fragment ion assignment corresponding to 3,4-di-O-Me-Rha.

FIG. 6.

Proposed structures of GPL-5 and GPL-6. Figure in parentheses shows the structure of GPL-1, GPL-2, GPL-3, and GPL-4, which were characterized in previous studies (10, 16, 25).

FIG. 7.

TLC analyses of crude GPL extracts from the M. smegmatis mc²155 strain (Wt) transformed with gtf expression vectors. Total lipid fraction after mild alkaline hydrolysis was spotted on plates and developed in CHCl₃-CH₃OH (9:1 [vol/vol]). GPLs were visualized by spraying with 10% H₂SO₄ and charring. Each total lipid fraction was extracted from an equal weight of harvested cells.

FIG. 8.

TLC analyses of crude GPL extracts from the M. smegmatis mc²155 strain (Wt) and its gene disruptants transformed with M. avium gtfA and gtfB. Total lipid fraction after mild alkaline hydrolysis was spotted on plates and developed in CHCl₃-CH₃OH (9:1 [vol/vol]). GPLs were visualized by spraying with 10% H₂SO₄ and charring. Each total lipid fraction was extracted from an equal weight of harvested cells.

FIG. 9.

Proposed biosynthetic pathways for GPLs of M. smegmatis and M. avium. FATP core, fatty acyl-tetrapeptide core.