The peptidoglycan of stationary-phase Mycobacterium tuberculosis predominantly contains cross-links generated by L,D-transpeptidation

Abstract

Our understanding of the mechanisms used by Mycobacterium tuberculosis to persist in a "dormant" state is essential to the development of therapies effective in sterilizing tissues. Gene expression profiling in model systems has revealed a complex adaptive response thought to endow M. tuberculosis with the capacity to survive several months of combinatorial antibiotic treatment. We show here that this adaptive response may involve remodeling of the peptidoglycan network by substitution of 4-->3 cross-links generated by the D,D-transpeptidase activity of penicillin-binding proteins by 3-->3 cross-links generated by a transpeptidase of L,D specificity. A candidate gene, previously shown to be upregulated upon nutrient starvation, was found to encode an L,D-transpeptidase active in the formation of 3-->3 cross-links. The enzyme, Ldt(Mt1), was inactivated by carbapenems, a class of beta-lactam antibiotics that are poorly hydrolyzed by the M. tuberculosis beta-lactamases. Ldt(Mt1) and carbapenems may therefore represent a target and a drug family relevant to the eradication of persistent M. tuberculosis.

FIG. 1.

Schematic representation of peptidoglycan synthesis. The nucleotide UDP-MurNAc-pentapeptide is formed in the cytoplasm by sequential addition of l-Ala, d-Glu, meso-diaminopimelic acid (mDap), and the dipeptide d-Ala-d-Ala. The membrane steps of peptidoglycan synthesis involve the transfer of the phospho-MurNAc-pentapeptide moiety of the nucleotide to the lipid carrier (undecaprenyl-phosphate) and the addition of the second sugar (GlcNAc). The complete precursor linked to the lipid carrier (lipid II) is translocated through the membrane and polymerized by glycosyltransferases (formation of glycan strands) and by the d,d-transpeptidase activity of PBPs (formation of the cross-links). These enzymes cleave the d-Ala⁴-d-Ala⁵ bond of a pentapeptide donor and link the carbonyl of d-Ala⁴ to the side chain amine of mDap at the third position of an acceptor stem (4→3 cross-links). β-Lactam antibiotics are structural analogues of the d-Ala⁴-d-Ala⁵ extremity of the precursors and act as suicide substrates of the d,d-transpeptidases. l,d-Transpeptidases cleave the mDap³-d-Ala⁴ bond of a tetrapeptide donor and link the carbonyl of mDap³ to the acceptor stem (3→3 cross-links).

FIG. 2.

Structure of the peptidoglycan of M. tuberculosis. (A) rp-HPLC profile of peptidoglycan fragments obtained by digestion of the peptidoglycan of strain H37Rv with muramidases and treatment with ammonium hydroxide. mAU, absorbance unit × 10³ at 210 nm. (B) Structure of monomers. d-iGln, d-iso-glutamine; d-iGlu, d-iso-glutamic acid; d-Lac, d-lactate; GlcNAc-MurNGlyc^anh, N-acetylglucosamine-anhydro-N-glycolylmuramic acid; mDap, meso-diaminopimelic acid; mDap_NH2, mDap with an amidated ɛ carboxyl group. (C) Peptidoglycan composition. The relative abundance (%) was calculated by integration of the absorbance. “Mass” refers to observed monoisotopic mass. NA, not applicable; ND, not determined. E, d-iGlu. (D) Sequencing of a dimer generated by l,d-transpeptidation. Tandem mass spectrometry was performed on the [M+H]⁺ ion at m/z 974.6 (peak 10). Boxes indicate ions generated by cleavage at single peptide bonds. (E) Sequencing of a dimer generated by d,d-transpeptidation (peak 13; [M+H]⁺ ion at m/z 1,045.4).

FIG. 3.

Characterization of Ldt_Mt1 from M. tuberculosis. (A) Domain composition of l,d-transpeptidases from E. faecium (Ldt_fm) and M. tuberculosis (Ldt_Mt1). Residues 121 to 251 of Ldt_Mt1 are related (29% identity) to the catalytic domain of Ldt_fm (domain II, positions 338 to 466). Hatched boxes represent hydrophobic regions that could act as a membrane anchor for Ldt_fm and as a signal peptide for Ldt_Mt1. (B) Purification of a soluble fragment of Ldt_Mt1 produced in E. coli. (C) Analysis of a dimer formed in vitro by Ldt_Mt1. Fragmentation was performed on the [M+H]⁺ ion at m/z 974.5. Boxes indicate ions generated by cleavage at single peptide bonds. (D) Structure of the dimer and inferred fragmentation pattern.

FIG. 4.

Formation of adducts between Ldt_Mt1 and β-lactams. (A) Ldt_Mt1 (12.5 μM) was incubated without antibiotic (left) or with 125 μM imipenem (right). Proteins and adducts were detected by electrospray mass spectrometry. Peaks at m/z 848.75 and 879.03 correspond to the [M+29H]²⁹⁺ and [M+28H]²⁸⁺ ions of the native protein, respectively (deduced average mass of 24,584.4). Peaks at m/z 859.08 and 889.65 correspond to the [M+29H]²⁹⁺ and [M+28H]²⁸⁺ ions of the Ldt_Mt1-imipenem adduct (deduced average mass of 24,883.5). (B) Formation of adducts between Ldt_Mt1 and various β-lactams. NA, not applicable; ND, not detected.