Alanine 501 Mutations in Penicillin-Binding Protein 2 from Neisseria gonorrhoeae: Structure, Mechanism, and Effects on Cephalosporin Resistance and Biological Fitness

Abstract

Resistance of Neisseria gonorrhoeae to expanded-spectrum cephalosporins such as ceftriaxone and cefixime has increased markedly in the past decade. The primary cephalosporin-resistance determinant is a mutated penA gene, which encodes the essential peptidoglycan transpeptidase, penicillin-binding protein 2 (PBP2). Decreased susceptibility and resistance can be conferred by mosaic penA alleles containing upwards of 60 amino acid changes relative to wild-type PBP2, or by non-mosaic alleles with relatively few mutations, the most important of which occurs at Ala501 located near the active site of PBP2. Recently, fully cefixime- and ceftriaxone-resistant clinical isolates were identified that harbored a mosaic penA allele with an A501P mutation. To examine the potential of mutations at Ala501 to increase resistance to expanded-spectrum cephalosporins, we randomized codon-501 in a mosaic penA allele and transformed N. gonorrhoeae to increased cefixime resistance. Interestingly, only five substitutions of Ala501 (A501V, A501T, A501P, A501R, A501S) were isolated that increased resistance and preserved essential transpeptidase function. To understand their structural implications, these mutations were introduced into the non-mosaic PBP2-6140CT, which contains four C-terminal mutations present in PBP2 from the penicillin-resistant strain FA6140. The crystal structure of PBP2-6140CT-A501T was solved and revealed ordering of a loop near the active site and a new hydrogen bond involving Thr501 that connects the loop and the SxxK conserved active-site motif. The structure suggests that increased rigidity in the active site region is a mechanism for cephalosporin resistance mediated by Ala501 mutations in PBP2.

Recognition of the β-lactam carboxylate triggers acylation of Neisseria gonorrhoeae penicillin-binding protein 2

“…This position constitutes the transition point between ␤3 and the ␤3-␤4 loop. The crystal structure of an A501T mutant has been solved and, similar to G545S, the side chain hydroxyl of Thr-501 forms a new hydrogen bond (with Glu-307 prior to the SXXK motif) that would be expected to reduce flexibility in this region (44). Such similarity in mechanisms at different positions in the protein may be the beginning of a consistent pattern emerging to explain cephalosporin resistance mediated by mosaic variants of PBP2.…”

The sudden emergence of aNeisseria gonorrhoeaestrain with reduced susceptibility to extended-spectrum cephalosporins, Norway

“…The penA gene exhibited significant allelic diversity, with the 13.001 allele being dominant in Clade A. The penA 13.001 allele is non-mosaic but carries an A501V mutation and has been shown to be associated with moderately elevated ESC MICs of ≥ 0.03 µg/ml (32,33) . Across the ST, primarily in Clade A, we also identified repeated acquisition of a porB allele carrying G120K and A121D mutations.…”

In Vivo -Selected Compensatory Mutations Restore the Fitness Cost of Mosaic penA Alleles That Confer Ceftriaxone Resistance in Neisseria gonorrhoeae

“…The ESCs are substrate analogs of the C-terminal acyl-D-Ala-D-Ala peptide chain of peptidoglycan that kill bacteria by covalently inhibiting the PBP transpeptidases that cross-link peptidoglycan. The mosaic variants of PBP2 from H041 and F89 display marked decreases (2,000-to 10,000-fold) in the second-order rate constants (k 2 /K S ) for acylation by ESCs, highlighting the remarkable remodeling of PBP2 to exclude substrate analogs (␤-lactam antibiotics) while retaining sufficient essential transpeptidase activity to support cell viability (21)(22)(23).…”