Mutation-Based Antibiotic Resistance Mechanism in Methicillin-Resistant Staphylococcus aureus Clinical Isolates

Abstract

β-Lactam antibiotics target penicillin-binding proteins and inhibit the synthesis of peptidoglycan, a crucial step in cell wall biosynthesis. Staphylococcus aureus acquires resistance against β-lactam antibiotics by producing a penicillin-binding protein 2a (PBP2a), encoded by the mecA gene. PBP2a participates in peptidoglycan biosynthesis and exhibits a poor affinity towards β-lactam antibiotics. The current study was performed to determine the diversity and the role of missense mutations of PBP2a in the antibiotic resistance mechanism. The methicillin-resistant Staphylococcus aureus (MRSA) isolates from clinical samples were identified using phenotypic and genotypic techniques. The highest frequency (60%, 18 out of 30) of MRSA was observed in wound specimens. Sequence variation analysis of the mecA gene showed four amino acid substitutions (i.e., E239K, E239R, G246E, and E447K). The E239R mutation was found to be novel. The protein-ligand docking results showed that the E239R mutation in the allosteric site of PBP2a induces conformational changes in the active site and, thus, hinders its interaction with cefoxitin. Therefore, the present report indicates that mutation in the allosteric site of PBP2a provides a more closed active site conformation than wide-type PBP2a and then causes the high-level resistance to cefoxitin.

Keywords: PBP2a; allosteric site; mecA; methicillin-resistant Staphylococcus aureus; mutation.

Figure 1

The detected variations in the mecA gene in the MRSA isolates (MR-13, -19, -4, -33, -26, -29, and -14). (a) Chromatograms showing nucleotide substitutions at different locations. (b) Multiple DNA sequence alignment. (c) Multiple protein sequence alignment. The sign “>” represents a substitution mutation. The left “E>K”, “E>R”, “G>E”, and the right “E>K” refer to E239K, E239R, G246E, and E447K, as shown by the dashed arrows. The left “G>A”, “A>G”, and “G>A”, and the right “G>A” refer to G715A, A716G, G737A, and G1339A. “Ref” represents the reference sequence (GenBank accession no. NC_002745). The MRSA isolates (MR-13, -14, and -33) show amino acid substitution(s) in PBP2a.

Figure 2

The protein-ligand docking of cefoxitin with the wild-type PBP2a and E239R-mutant PBP2a of MRSA. (a) Cefoxitin (shown in capped sticks with carbon in cyan, oxygen in red, nitrogen in blue, and sulfur in yellow) interacts with the Glu-239 (E239, yellow) of the wild-type PBP2a. The docked ligand trajectory shows that cefoxitin is located in close proximity to E239. (b) Cefoxitin interacts with Gln-521 (orange) and Gly-522 (orange) instead of Arg-239 (R239, pink) in the E239R-mutant PBP2a. (c) Peptidoglycan (red, GlcNAc-MurNAc pentapeptide) interacts with E239 and other residues of the wild-type PBP2a. (d) Surface representation of the wild-type PBP2a (green) showing that the ligand (shown in caped sticks) is positioned at the allosteric site (yellow). (e) The ligand (shown in capped sticks) is pointed away from the allosteric site (pink) in the E239R-mutant PBP2a. All of the interactions are shown as black dashed lines. Glu, glutamic acid; Ser, serine; Gln, glutamine; Gly, glycine; Arg, arginine; Thr, threonine; Tyr, tyrosine; Lys, lysine.