Biochemical characterization of Streptococcus pneumoniae penicillin-binding protein 2b and its implication in β-lactam resistance

The high-molecular-weight (high-Mr) penicillin-binding proteins (PBPs) carry out the extracellular steps of peptidoglycan synthesis. These enzymes catalyze the glycosyltransferase reaction leading to the polymerization of the glycan chains and the transpeptidation (TP) activity establishing a covalent bond between two juxtaposed peptides (3, 15, 32). Streptococcus pneumoniae class A high-Mr PBPs (PBP1a, PBP1b, and PBP2a) are bifunctional enzymes bearing both activities, whereas class B high-Mr PBPs (PBP2b and PBP2x) have so far only been associated with the TP activity (15). Due to their structural similarity to the natural substrates, the d-Ala-d-Ala stem peptides, β-lactam molecules inhibit the TP reaction leading to bacterial cell lysis (35). S. pneumoniae is a major human pathogen and the causative agent of ear infections in children, as well as meningitis and pneumonia. Extensive use of β-lactam antibiotics over the last 4 decades has led to the selection of pathogenic streptococci resistant to their action. The resistance to β-lactams in S. pneumoniae results from a decreased affinity of PBPs for the antibiotics. This phenotype results from genetic reshuffling. Mosaic genes encoding PBPs with lower affinity for β-lactams have been generated by homologous recombination events with other streptococcal species (18). The mosaicity of the pneumococcal pbp2b gene has been well established in penicillin clinical resistant (CR) isolates as resulting from recombination events between S. pneumoniae and Streptococcus mitis (8-10). DNA sequence analysis has produced evidence that, under clinical and laboratory conditions, mutants of the pbp2x and pbp2b genes are the first to be selected upon treatment with cefotaxime and piperacillin, respectively, indicating that PBP2x and PBP2b are primary resistance determinants for these antibiotics (22, 29). To characterize the biochemical properties of PBP2b, we have expressed the periplasmic domain of PBP2b from the S. pneumoniae R6 sensitive strain (PBP2b*R6) and from seven CR isolates (PBP2b*CR). These latter proteins show a marked reduction in affinity for penicillin G and, when recombined in the R6 strain, significantly increase the MIC of piperacillin for the host strain. The preponderance of the Thr446Ala mutation in clinical strains, as well as its juxtaposition to the key motif (S443SN) of the PBP TP function, led us to investigate the role of this mutation in more detail. The Thr446Ala mutation lowers the affinity for β-lactams and may thus play a role in the development of antibiotic resistance.