Novel mechanism of resistance to glycopeptide antibiotics in Enterococcus faecium

Abstract

Glycopeptides and beta-lactams are the major antibiotics available for the treatment of infections due to Gram-positive bacteria. Emergence of cross-resistance to these drugs by a single mechanism has been considered as unlikely because they inhibit peptidoglycan polymerization by different mechanisms. The glycopeptides bind to the peptidyl-D-Ala(4)-D-Ala(5) extremity of peptidoglycan precursors and block by steric hindrance the essential glycosyltransferase and D,D-transpeptidase activities of the penicillin-binding proteins (PBPs). The beta-lactams are structural analogues of D-Ala(4)-D-Ala(5) and act as suicide substrates of the D,D-transpeptidase module of the PBPs. Here we have shown that bypass of the PBPs by the recently described beta-lactam-insensitive L,D-transpeptidase from Enterococcus faecium (Ldt(fm)) can lead to high level resistance to glycopeptides and beta-lactams. Cross-resistance was selected by glycopeptides alone or serially by beta-lactams and glycopeptides. In the corresponding mutants, UDP-MurNAc-pentapeptide was extensively converted to UDP-MurNAc-tetrapeptide following hydrolysis of D-Ala(5), thereby providing the substrate of Ldt(fm). Complete elimination of D-Ala(5), a residue essential for glycopeptide binding, was possible because Ldt(fm) uses the energy of the L-Lys(3)-D-Ala(4) peptide bond for cross-link formation in contrast to PBPs, which use the energy of the D-Ala(4)-D-Ala(5) bond. This novel mechanism of glycopeptide resistance was unrelated to the previously identified replacement of D-Ala(5) by D-Ser or D-lactate.

Fig. 1. Peptidoglycan assembly in Enterococcus faecium

(A) In wild-type strains, β-lactams act as suicide substrates and inactivate the D,D-transpeptidase module of penicillin-binding proteins (PBPs). Glycopeptides bind to the peptidyl-D-Ala⁴-D-Ala⁵ extremity of peptidoglycan precursors and block transglycosylation by steric hindrance. Binding of the latter drugs to lipid intermediate II at the outer surface of the membrane (as represented) is thought to sequester the lipid carrier. Binding of glycopeptides to pentapeptide stems also inhibits the D,D-transpeptidase activity of the PBPs. (B) Activation of the L,D-transpeptidation pathway in mutants of E. faecium results from production of cytoplasmic precursors containing a tetrapeptide stem. The resulting precursors do not interact with glycopeptides and are cross-linked by the β-lactam-insensitive L,D-transpeptidase (Ldt_fm). D-iAsx; D-iso-aspartyl or D-iso-asparaginyl residue; D-iGln, D-iso-glutaminyl; D-iGlu, D-iso-glutamyl; GlcNAc, N-acetylglucosamine; MurNAc, N-acetylmuramic acid; P, phosphate..

Fig. 2. MICs of vancomycin, teicoplanin, and ampicillin for derivatives of E. faecium D344S

(A) Mutant M1, M2, M3, M4, and M512 were obtained by five consecutive selections steps on increasing concentration of ampicillin (18). Four additional selection steps with glycopeptides were required to obtain high-level cross-resistance to vancomycin, teicoplanin, and ampicillin. (B) Mutants G1 to G9 were obtained by serial selection with glycopeptides.

Fig. 3. MS/MS analysis of peak at m/z 1,079.35 corresponding to UDP-MurNAc-tetrapeptide

Cytoplasmic nucleotide precursors of mutant M9 were purified, separated by rp-HPLC, and identified by mass spectrometry and tandem mass spectrometry. Fragmentation of the peak at m/z 1,079.35 gave ions at m/z 675.27 and 490.19 corresponding to the MurNAc-tetrapeptide and lactoyl-tetrapeptide moieties of the molecule. Peak at m/z 401.21 matched the predicted value for loss of the D-alanine residue at the C-terminal position of lactoyl-tetrapeptide (m/z 490.19). Loss of additional L-Lys and D-Glu gave ion at m/z 273.14 and 144.05, respectively. The peaks at m/z 347.19, 258.12, 218.14, and 129.09 matched the expected mass of D-iGlu-L-Lys-D-Ala, D-iGlu-L-Lys, L-Lys-D-Ala, and L-Lys, respectively.

Fig. 4. Structure of the peptidoglycan of mutant M9

(A) rp-HPLC profile of lactoyl-peptides. Peptidoglycan was digested with muramidases and treated with ammonium hydroxide to cleave the ether link internal to MurNAc. This treatment also converts D-iso-asparaginyl into D-iso-aspartatyl (D-iAsp) residues. mAU, absorbance unit × 10³ at 210 nm. (B) Schematic representation of dimers generated by L,D-transpeptidation. All multimers of M9 contained L-Lys³→D-iAsp-L-Lys³ cross-links. Variations occurred in the free side chain (presence or absence of D-iAsp) and at the 4^th position of the acceptor stem peptide (presence of Gly, D-Ala or absence of a residue). The diversity of monomers resulted from variations at the same positions. (C) Peptidoglycan composition. The relative abundance (%) of the material in the peaks was calculated by integration of the absorbance at 210 nm. The structure of lactoyl-peptides was deduced from the mass and confirmed by tandem mass spectrometry for all monomers and dimers. Mass, observed monoisotopic mass.

Fig. 5. Sequencing of a dimer of M9 (peak 11) by tandem mass spectrometry

Fragmentation of the ion at m/z 1,118.5 (A) and inferred structure (B). Boxes indicate ions generated by cleavage at single peptide bounds. D-Lac, D-lactoyl.