Abstract
PBP2x is a primary determinant of beta-lactams resistance in Streptococcus pneumoniae. Altered PBP2x with multiple mutations have a reduced "affinity" for the antibiotics. An important polymorphism is found in PBP2x sequences from clinical resistant strains. To understand the mechanism of resistance, it is necessary to identify and characterize the relevant substitutions. Many similar PBP2x sequences from resistant isolates have the previously studied T338A mutation, adjacent to the active site Ser337. We report here the structural and functional analysis of the M339F substitution that is found in a subset of these sequences, originating from highly resistant strains. The M339F mutation causes a 4-10-fold reduction of the reaction rate with beta-lactams, depending on the molecular context. In addition, release of the inactivated antibiotic from the active site is up to 3-fold faster as a result from the M339F mutation. These effects measured in vitro are correlated with the level of beta-lactam resistance in vivo conferred by several PBP2x variants. Thus, a single amino acid difference between similar PBP2x from clinical isolates can strongly modulate the degree of beta-lactam resistance. The crystal structure of the double mutant T338A/M339F solved to a resolution of 2.4 A shows a distortion of the active site and a reorientation of the hydroxyl group of the active site Ser337, which can explain the kinetic effects of the mutations.
Highlights
Streptococcus pneumoniae is one of the major human pathogens of the upper respiratory tract
5204-PBP2x possesses the M339F mutation in the catalytic motif close to the active site Ser337. 5204-PBP2x has 80 amino acid changes compared with R6-PBP2x, of which 41 are located within the transpeptidase domain. 4790PBP2x has 75 residue substitutions relative to R6-PBP2x, including 39 changes in the transpeptidase domain
Antibiotic resistance has spread in recent years and the current medical practice, in case of severe infection, is to treat with a combination of wide spectrum antibiotics, whereas the pathogen is being identified and its susceptibility to various drugs established by classical microbiology techniques
Summary
Expression Plasmids—S. pneumoniae strains 4790 and 5204 were used as a source of chromosomal DNA for PCR amplification of the pbp2x and pbp1a genes. The pGEX-4790-pbp2x* plasmid was mutated with the following primers and their reverse complements, which introduce the A338T and M339F, respectively, and the respective diagnostic restriction sites ScaI and DraI: 5Ј-CTATGAACCAGGAAGTACTATGAAGGTTATGACGTTAGCTTCTTC-3Ј, 5Ј-CTATGAACCAGGATCAGCCTTTAAAGTTATGACGTTAGCTTCTTC-3Ј. The MIC of the transformed cells for penicillin G and cefotaxime was determined by the agar dilution method. The structure of PBP2x from S. pneumoniae R6 at 2.4 Å (24) (Protein Data Bank accession code 1QME) was used as a search model with residues Thr338 and Met339 changed to glycines. Determination of the Efficiency of Acylation—The k2/K parameter was measured by following the decrease of the intrinsic fluorescence of the protein, at various concentrations of a large excess of antibiotic, using an SFM-400 stopped-flow apparatus (Biol-Logic) (13, 29). The rate constant k3 was determined by nonlinear least-square fitting to the first-order equation [EI*]t ϭ [EI*]0exp(Ϫk3t) using the Kaleidagraph software
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