PBP4 activity and its overexpression are necessary for PBP4-mediated high-level β-lactam resistance

Abstract

Background: PBP4 is typically considered unimportant for conferring high-level β-lactam resistance in Staphylococcus aureus. Mutations in PBP4 have been associated with β-lactam non-susceptibility among natural strains of S. aureus. We have previously shown that PBP4 can mediate high-level β-lactam resistance in laboratory-generated strains passaged in β-lactam antibiotics. Mutations in the pbp4 promoter that up-regulate its expression and missense mutations that surround PBP4's active site were detected in high frequencies among passaged strains, suggesting PBP4 plays a key role in resistance. How these mutations participate in PBP4's ability to provide high-level β-lactam resistance is unknown.

Objectives: To determine whether enzymatic activity of PBP4 is required for high-level β-lactam resistance and to investigate how the pbp4-associated mutations provide β-lactam resistance.

Methods: The catalytic activity of PBP4 was disabled through introduction of a serine to alanine point mutation in its active site (Ser-75→Ala) in a representative and well-studied passaged strain, CRB. pbp4 promoter and missense mutations detected in CRB were reconstituted in a WT strain individually and in combination. β-Lactam resistance of the resultant strains was evaluated by population analysis. Bacterial peptidoglycan composition of the pbp4 mutants was evaluated with and without antibiotic treatment using LC.

Results: PBP4 inactivation imparted complete β-lactam susceptibility of CRB. Reconstitution of PBP4 missense mutations alone did not impart β-lactam resistance, but did so in synergism with pbp4 promoter mutation. A similar synergistic interaction of pbp4 mutations was observed in enhanced peptidoglycan cross-linking upon antibiotic treatment.

Conclusions: PBP4's activity and overexpression both contribute to high-level β-lactam resistance.

Figure 1.

Activity of PBP4 is important to confer β-lactam resistance in strain CRB. Population analysis in nafcillin was carried out with highly resistant CRB (filled circles), its Δpbp4 deletion mutant (open circles) and its PBP4 (S-75→A) substitution mutant (open squares). Two-way ANOVA of the data revealed a significant difference (P < 0.0001) between CRB and the other strains.

Figure 2.

PBP4 missense mutations provide β-lactam resistance in synergism with its promoter mutation. Population analysis in nafcillin was carried out with COLnex (filled circles), COLnex pbp4^{(E-183→A and F-241→R)} (open triangles), COLnex Ppbp4^(CRB) (filled diamonds), COLnex Ppbp4^(CRB)pbp4^{(E-183→A and F-241→R)} (open diamonds) and COLnex Ppbp4^(CRB)pbp4^{(E-183→A and F-241→R) (S-75→A)} (filled squares). Two-way ANOVA of the data revealed a significant difference (P < 0.0001) between COLnex Ppbp4^(CRB), COLnex Ppbp4^(CRB)pbp4^{(E-183→A and F-241→R)} and the other strains.