N Glycolylation of the nucleotide precursors of peptidoglycan biosynthesis of Mycobacterium spp. is altered by drug treatment

Abstract

The peptidoglycan of Mycobacterium spp. reportedly has some unique features, including the occurrence of N-glycolylmuramic rather than N-acetylmuramic acid. However, very little is known of the actual biosynthesis of mycobacterial peptidoglycan, including the extent and origin of N glycolylation. In the present work, we have isolated and analyzed muramic acid residues located in peptidoglycan and UDP-linked precursors of peptidoglycan from Mycobacterium tuberculosis and Mycobacterium smegmatis. The muramic acid residues isolated from the mature peptidoglycan of both species were shown to be a mixture of the N-acetyl and N-glycolyl derivatives, not solely the N-glycolylated product as generally reported. The isolated UDP-linked N-acylmuramyl-pentapeptide precursor molecules also contain a mixture of N-acetyl and N-glycolyl muramyl residues in apparent contrast to previous observations in which the precursors isolated after treatment with d-cycloserine consisted entirely of N-glycolyl muropeptides. However, nucleotide-linked peptidoglycan precursors isolated from M. tuberculosis treated with d-cycloserine contained only N-glycolylmuramyl-tripeptide precursors, whereas those from similarly treated M. smegmatis consisted of a mixture of N-glycolylated and N-acetylated residues. The full pentapeptide intermediate, isolated following vancomycin treatment of M. smegmatis, consisted of the N-glycolyl derivative only, whereas the corresponding M. tuberculosis intermediate was a mixture of both the N-glycolyl and N-acetyl products. Thus, treatment with vancomycin and d-cylcoserine not only caused an accumulation of nucleotide-linked intermediate compounds but also altered their glycolylation status, possibly by altering the normal equilibrium maintained by de novo biosynthesis and peptidoglycan recycling.

FIG. 1.

MS analysis of the N-acylmuramic acid residues of mature peptidoglycan isolated from M. tuberculosis. The trimethylsilane derivatives of the N-acylmuramic acids were analyzed by GC-MS as described in Materials and Methods. (A) Total ion chromatogram of the GC run. (B) Mass spectrum of the peak at 11.37 min. The structure of the TMS derivative of N-acetyl muramic acid and the diagnostic fragment (m/z 187.0) used as positive identification are inset. (C) Mass spectrum of the peak at 13.25 min. The structure of the TMS derivative of N-glycolylmuramic acid and the diagnostic fragment with the structure of the diagnostic fragment (m/z 275.1) used as positive identification are inset.

FIG. 2.

Elution patterns from Sephadex G-25 columns of the TCA-soluble material from M. smegmatis cell lysates obtained from treated and untreated cells (10 g [wet weight]). The elution was monitored at 262 nm. (A) TCA-soluble material obtained from untreated cells. (B) TCA-soluble material from the cells treated with d-cycloserine. (C) TCA-soluble material from vancomycin-treated cells. The arrows indicate the elution position of nucleotide-linked peptidoglycan precursors.

FIG. 3.

Positive ion mass spectrum of the UDP-N-acylmuramyl-l-Ala-d-Glu-DAP-d-Ala-d-Ala isolated from untreated M. tuberculosis. The m/z 808.2 ion represents MurNAc-l-Ala-d-Glu-DAP-d-Ala-d-Ala, and m/z 830.3 was the corresponding monosodium adduct. m/z 824.2 was identified as MurNGlyc-l-Ala-d-Glu-DAP-d-Ala-d-Ala. Ions corresponding to the mono- and disodium adducts (m/z 846.3 and m/z 868.2, respectively) were also observed.

FIG. 4.

Tandem mass spectrum of the molecular ion m/z 808.2. The structures of daughter ions labeled with m/z values in panel A are indicated in panel B, which shows the deduced fragmentation pattern of MurNAc-l-Ala-d-Glu-DAP-d-Ala-d-Ala. The ion with an m/z value of 790.1 represents the molecular ion after the neutral loss of H₂O. The structures of unlabeled ions were not solved.

FIG. 5.

Positive ion mass spectrum of the nucleotide-linked intermediates isolated from M. tuberculosis H37Rv treated with d-cycloserine. The mass spectrum is dominated by the ion with an m/z value of 682.2, which represents MurNGlyc-l-Ala-d-Glu-DAP, and the corresponding monosodium adduct (m/z of 704.2).