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Five independent combinations of mutations can result in low-affinity penicillin-binding protein 2x of Streptococcus
1Max-Planck Institut für Molekulare Genetik, Berlin, Germany.
Abstract:
Penicillin-binding protein 2x (PBP 2x) of Streptococcus pneumoniae is one of the high-molecular-weight PBPs involved in the development of intrinsic beta-lactam resistance. Point mutations in the PBP 2x genes (pbpX) have now been characterized in five independent spontaneous laboratory mutants in order to identify protein regions which are important for interaction with beta-lactam antibiotics. All mutant genes contained two to four mutations resulting in amino acid substitutions within the penicillin-binding domain of PBP 2x, and none of the mutants carried an identical set of mutations. For one particular mutant, C606, carrying four mutations in pbpX, the mutations at positions 601 and 597 conferred first- and second-level resistance when introduced into the susceptible parent strain S. pneumoniae R6. However, the other two mutations, at amino acid positions 289 and 422, which were originally selected at the fifth and sixth isolation steps, did not contribute at all to resistance in similar experiments. This suggests that they are phenotypically expressed only in combination with mutations in other genes. Three PBP 2x regions were mutated in from two to all four mutants carrying a low-affinity PBP 2x. However, in a fifth mutant containing a PBP 2x with apparent zero affinity for beta-lactams, the three mutations in pbpX mapped at entirely different positions. This demonstrates that different mutational pathways exist for remodeling this PBP during resistance development.
Insights
Mutations in penicillin-binding protein 2x (PBP 2x) genes of Streptococcus pneumoniae reveal diverse pathways for beta-lactam resistance. Different mutation combinations in PBP 2x can lead to varying levels of antibiotic resistance.
Area of Science:
- Microbiology
- Molecular Biology
- Genetics
Background:
- Penicillin-binding protein 2x (PBP 2x) in Streptococcus pneumoniae is crucial for intrinsic beta-lactam resistance.
- Understanding mutations in the PBP 2x gene (pbpX) is key to deciphering antibiotic resistance mechanisms.
Purpose of the Study:
- To identify specific protein regions of PBP 2x involved in beta-lactam antibiotic interaction.
- To investigate the role of point mutations in the development of beta-lactam resistance in S. pneumoniae.
Main Methods:
- Characterization of point mutations in the pbpX gene of five independent spontaneous laboratory mutants.
- Introduction of specific mutations into the susceptible S. pneumoniae R6 strain to assess their contribution to resistance.
- Analysis of mutation locations within the penicillin-binding domain of PBP 2x.
Main Results:
- All five mutants exhibited two to four mutations in pbpX, leading to amino acid substitutions in the penicillin-binding domain.
- Specific mutations (e.g., at positions 601 and 597) conferred significant beta-lactam resistance, while others (e.g., at 289 and 422) showed no effect alone, suggesting epistatic interactions.
- Three distinct regions of PBP 2x were commonly mutated in low-affinity variants, but a fifth mutant with near-zero affinity showed mutations in different locations, indicating multiple resistance pathways.
Conclusions:
- Different mutational pathways exist for PBP 2x remodeling during the development of antibiotic resistance.
- The study highlights the complex genetic basis of beta-lactam resistance in S. pneumoniae, involving both direct resistance-conferring mutations and potentially silent mutations that act in combination.