Meropenem inhibits D,D-carboxypeptidase activity in Mycobacterium tuberculosis

Abstract

Carbapenems such as meropenem are being investigated for their potential therapeutic utility against highly drug-resistant tuberculosis. These β-lactams target the transpeptidases that introduce interpeptide cross-links into bacterial peptidoglycan thereby controlling rigidity of the bacterial envelope. Treatment of Mycobacterium tuberculosis (Mtb) with the β-lactamase inhibitor clavulanate together with meropenem resulted in rapid, polar, cell lysis releasing cytoplasmic contents. In Mtb it has been previously demonstrated that 3-3 cross-linkages [involving two diaminopimelate (DAP) molecules] predominate over 4-3 cross-linkages (involving one DAP and one D-alanine) in stationary-phase cells. We purified and analysed peptidoglycan from Mtb and found that 3-3 cross-linkages predominate throughout all growth phases and the ratio of 4-3/3-3 linkages does not vary significantly under any growth condition. Meropenem treatment was accompanied by a dramatic accumulation of unlinked pentapeptide stems with no change in the tetrapeptide pools, suggesting that meropenem inhibits both a D,D-carboxypeptidase and an L,D-transpeptidase. We purified a candidate D,D-carboxypeptidase DacB2 and showed that meropenem indeed directly inhibits this enzyme by forming a stable adduct at the enzyme active site. These results suggest that the rapid lysis of meropenem-treated cells is the result of synergistically inhibiting the transpeptidases that introduce 3,3-cross-links while simultaneously limiting the pool of available substrates available for cross-linking.

Fig. 1

Sensitivity of Mtb to various cell-wall damaging agents. (A) Kill curve of Mtb in presence of various agents. (B) Lysis of Mtb upon treatment with these agents measured by quantitating release of cytosolic GFP in cell-free culture supernatant (from a GFP-expressing Mtb/pMSP12 strain). As indicated in the graphs following agents were used either at 1-time (1X) or 10-times (10X) their corresponding MIC: Meropenem-clavulanate (MCA, MIC for meropenem 2 μM), penicillin G-clavulanate (PCA, MIC for penicillin G 2 μM), cephaloridine-clavulanate (CCA, MIC for cephaloridine 1 μM), clavulanate was used at 100 μM throughout, isoniazid (INH, 2 μM) and ethambutol (EMB, 62.5 μM).

Fig. 2

SEM images showing effects of meropenem exposure on Mtb cell shape. (A & B) untreated controls, (C & D) treated with 1X MCA, (E & F) with 10X MCA or (G & H) with 10X INH (Bar=1 μm). The lower magnification pictures shown above (A, C, E, G) were captured at 8,000 X and corresponding higher magnification pictures shown below (B, D, F, I) were captured at 25, 000 X.

Fig. 3

RP-HPLC profile of muropeptides prepared by muramidase digestion and ammonium hydroxide treatment from PG isolated from exponentially growing (OD₆₅₀ = 0.12) Mtb. The absorbance in mAU (absorbance unit × 10³) was recorded at 210 nm.

Fig. 4

Characterization of amidation sites in monomeric and dimeric muropeptides by MS/MS analysis. The expected MS/MS fragmentation pattern is shown in the boxes above each m/z chromatogram. (A) MS/MS profile of [M+H]⁺ ion at m/z 532.2, the observed ions (443.2, 389.2, 272.1, MS/MS/MS of 172.2 showed it corresponded to DAP_NH2: data not shown) matched with the expected ions for Tetra, D-Lac-L-Ala-D-iGln-mDAP_NH2-D-Ala. (B) MS/MS profile of ion m/z 533.2 corresponding to Tetra(E), D-Lac-L-Ala-D-iGlu-mDAP_NH2-D-Ala, MS/MS/MS of 171.9 showed it corresponded to DAP_NH2: data not shown. (C) MS/MS profile ion at m/z 974.4 in peak 8 (Table 1) showing 4-3 cross-linked Tetra-Tri. (D) MS/MS profile ion at m/z 974.4 in peak 10 (Table 1) showing 3-3 cross-linking Tetra-Tri.

Fig. 5

Effect of meropenem/clavulanic acid on PG structure. RP-HPLC profile of muropeptides from PG isolated from Mtb exposed to the following concentrations of meropenem and clavulanic acid: (A) 100 μM clavulanic acid only (B) 0.2 μM meropenem and 100 μM clavulanic acid (C) 0.5 μM meropenem and 100 μM clavulanic acid (D) 2.0 μM meropenem and 100 μM clavulanic acid (E) 5.0 μM meropenem and 100 μM clavulanic acid (F) 50 μM meropenem and 100 μM clavulanic acid.

Fig. 6

D, D-Carboxypeptidase activity assay. The D-ala release assay performed using penta-peptide substrates (Penta-DAP: L-Ala-γ-D-Gln-DAP-D-Ala-D-Ala; Penta-Lys: L-Ala-γ-D-Glu-L-Lys-D-Ala-D-Ala). (A) D-Ala release by DacB1 (B) D-Ala release by DacB2. (C) Inhibition of enzymatic activity of DacB2 by meropenem.