The Mycobacterium tuberculosis protein LdtMt2 is a nonclassical transpeptidase required for virulence and resistance to amoxicillin

Abstract

The peptidoglycan layer is a vital component of the bacterial cell wall. The existing paradigm describes the peptidoglycan network as a static structure that is cross-linked predominantly by 4-->3 transpeptide linkages. However, the nonclassical 3-->3 linkages predominate the transpeptide networking of the peptidoglycan layer of nonreplicating Mycobacterium tuberculosis. The molecular basis of these linkages and their role in the physiology of the peptidoglycan layer, virulence and susceptibility of M. tuberculosis to drugs remain undefined. Here we identify MT2594 as an L,D-transpeptidase that generates 3-->3 linkages in M. tuberculosis. We show that the loss of this protein leads to altered colony morphology, loss of virulence and increased susceptibility to amoxicillin-clavulanate during the chronic phase of infection. This suggests that 3-->3 cross-linking is vital to the physiology of the peptidoglycan layer. Although a functional homolog exists, expression of ldtMt2 is dominant throughout the growth phases of M. tuberculosis. 4-->3 transpeptide linkages are targeted by one of the most widely used classes of antibacterial drugs in human clinical use today, beta-lactams. Recently, meropenem-clavulanate was shown to be effective against drug-resistant M. tuberculosis. Our study suggests that a combination of L,D-transpeptidase and beta-lactamase inhibitors could effectively target persisting bacilli during the chronic phase of tuberculosis.

Figure 1

Morphology and growth in vitro. (a) Morphologies of wild-type M. tuberculosis (WT), Ldt_Mt2 mutant (MUT) and the complemented strain (COMP) on solid media after 21 days of growth at 37 °C. (scale = 1 cm).(b) Growth of wild-type M. tuberculosis (WT), Ldt_Mt2 mutant (MUT) and the complemented strain (COMP) in Middlebrook 7H9 liquid medium at 37 °C. The decrease in optical density and CFU at the final time point for WT and COMP is due to clumped cultures.

Figure 2

Characterization of Ldt_Mt2 from M. tuberculosis. (a) Domain composition of L,D-transpeptidases from E. faecium (Ldt_fm) and M. tuberculosis (Ldt_Mt2). Residues 250–377 of Ldt_Mt2 share homology with the catalytic domain of Ldt_fm (Domain II, 338–466). (b) Purification of a soluble fragment of Ldt_Mt2 produced in E. coli. (c) Structure and inferred fragmentation pattern of the peptidoglycan dimer formed in vitro by Ldt_Mt2. The ion at m/z 974.51 was generated by losses of the two GlcNAc-MurNAc residues following fragmentations of the ether links connecting the lactoyl group to the C–3 position of MurNAc. Loss of each sugar decreased the m/z by 203. Cleavage of additional peptide bonds from the ion at m/z 974.51 gave ions at 703.36, 532.28, 433.22 and 272.15 as indicated in the inset. (d) Transcription profile of Ldt_Mt2 (MT2594) and Ldt_Mt1 (MT0125). RNA isolated from wild-type Mtb cultures at growth phases T1 (A_600nm=0.2), T2 (A_600nm=0.5), T3 (A_600nm=0.8), T4 (A_600nm=0.9), T5 (A_600nm=01.9), T6 (A_600nm=3.0), T7 (2 days post A_600nm =3.0) and T8 (3 days post clumping).

Figure 3

Assessment of growth, virulence and susceptibility to amoxicillin in vivo. (a) Bacterial burden in the lungs of mice infected with wild-type M. tuberculosis (WT), Ldt_Mt2 mutant (MUT) or the complemented (COMP) strain are shown. (b) Virulence of each strain was assessed by determining time-to-death following infection of mice, 12 per group, with the three strains. (c) Mice were infected with either WT or the MUT strain. Following two weeks of infection, mice were treated daily with either no drug placebo (d), or 25 mg kg⁻¹ isoniazid (e), or 200 mg kg⁻¹ amoxicillin and 50 mg kg⁻¹ clavulanate (f). Bacterial burden was determined by enumerating CFU from the lungs of mice.

Figure 4

Proposed model for physiology of the peptidoglycan layer in M. tuberculosis. (a) classical model of peptidoglycan crosslinking containing 4→3 inter peptide bonds. (b) model based on recent data: the peptidoglycan is cross-linked with classical 4→3 and non-classical 3→3 inter peptide bonds. Both 4→3 (orange) and 3→3 (green) transpeptidases are involved in maintenance and remodeling of the peptidoglycan layer in the proposed model.