Molecular basis of vancomycin dependence in VanA-type Staphylococcus aureus VRSA-9

Abstract

The vancomycin-resistant Staphylococcus aureus VRSA-9 clinical isolate was partially dependent on glycopeptide for growth. The responsible vanA operon had the same organization as that of Tn1546 and was located on a plasmid. The chromosomal D-Ala:D-Ala ligase (ddl) gene had two point mutations that led to Q260K and A283E substitutions, resulting in a 200-fold decrease in enzymatic activity compared to that of the wild-type strain VRSA-6. To gain insight into the mechanism of enzyme impairment, we determined the crystal structure of VRSA-9 Ddl and showed that the A283E mutation induces new ion pair/hydrogen bond interactions, leading to an asymmetric rearrangement of side chains in the dimer interface. The Q260K substitution is located in an exposed external loop and did not induce any significant conformational change. The VRSA-9 strain was susceptible to oxacillin due to synthesis of pentadepsipeptide precursors ending in D-alanyl-D-lactate which are not substrates for the β-lactam-resistant penicillin binding protein PBP2'. Comparison with the partially vancomycin-dependent VRSA-7, whose Ddl is 5-fold less efficient than that of VRSA-9, indicated that the levels of vancomycin dependence and susceptibility to β-lactams correlate with the degree of Ddl impairment. Ddl drug targeting could therefore be an effective strategy against vancomycin-resistant S. aureus.

FIG. 1.

Glycopeptide dependence of VRSA-9 as tested by disk diffusion. VA, vancomycin; TEC, teicoplanin.

FIG. 2.

Sequence alignment of VRSA-9, VRSA-6, and VRSA-7 Ddls. Dots indicate identical amino acids; active-site residues are highlighted in dark gray; underlined residues are conserved in all d-Ala:d-X ligases; amino acid substitutions in VRSA-7 and VRSA-9 Ddls are indicated in bold. Numbering above the sequences indicates amino acids interacting with ligands (1, d-Ala₁; 2, d-Ala₂; 3, ATP; 4, Mg²⁺).

FIG. 3.

Comparison of the full time courses of VRSA-9 Ddl reactions at various concentrations of d-Ala (40 to 250 mM) with and without preincubation with ATP. (A) The reaction was initiated by the addition of the enzyme. (B) The reaction was initiated by the addition of d-Ala to the Ddl preincubated for 5 min with 10 mM ATP. Curves 1 to 6 are for 40, 80, 120, 160, 200, and 250 mM d-Ala, respectively.

FIG. 4.

Overall structure of the VRSA-9 Ddl dimer (monomer A is shown in light blue and monomer B is in light brown). The positions of mutated Lys260 and Glu283 and the nucleotide binding sites are shown. Residue Lys260 is located in an exposed external loop in the C-terminal domain. Residue Glu283 is located near the dimer interface. The overall structure is similar to that of VRSA-6 Ddl (19).

FIG. 5.

Detailed view of the dimer interface. (1) VRSA-9 Ddl. (2) VRSA-6 Ddl. Residues in monomer A are shown in green and those in monomer B in yellow. Hydrogen-bonding interactions that differ between the two monomers are shown as dashed lines.

FIG. 6.

(A) Superimposed structures of VRSA-9 Ddl (light blue) and VRSA-6 Ddl (light brown) showing the shift of the central domain. (B) Detailed view of the nucleotide binding site and of the serine-serine loop. The phosphoryl phosphinate substrate analog (PHY) was modeled using the structure of LmDdl2 (PDB code 1EHI).