IDENTIFICATION OF A STRUCTURAL DETERMINANT FOR RESISTANCE TO BETA-LACTAM ANTIBIOTICS IN GRAM-POSITIVE BACTERIA

Streptococcus pneumoniae is the main causal agent of pathologies that are increasingly resistant to antibiotic treatment, Clinical resistanc e of S, pneumoniae to beta-lactam antibiotics is linked to multiple mu tations of high molecular mass penicillin-binding proteins (H-PBPs), e ssential enzymes involved in the final steps of bacterial cell wall sy nthesis. H-PBPs from resistant bacteria have a reduced affinity for be ta-lactam and a decreased hydrolytic activity on substrate analogues. In S, pneumoniae, the gene coding for one of these H-PBPs, PBP2x, is l ocated in the cell division cluster (DCW). We present here structural evidence linking multiple beta-Lactam resistance to amino acid substit utions in PBP2x within a buried cavity near the catalytic site that co ntains a structural water molecule, Site-directed mutation of amino ac ids in contact with this mater molecule in the ''sensitive'' form of P BP2x produces mutants similar, in terms of beta-lactam affinity and su bstrate hydrolysis, to altered PBP2x produced in resistant clinical is olates, A reverse mutation in a PBP2x variant from a clinically import ant resistant clone increases the acylation efficiency for beta-lactam s and substrate analogues. Furthermore, amino acid residues in contact with the structural water molecule are conserved in the equivalent H- PBPs of pathogenic Gram-positive cocci. We suggest that, probably via a local structural modification, the partial or complete loss of this water molecule reduces the acylation efficiency of PBP2x substrates to a point at which cell wall synthesis still occurs, but the sensitivit y to therapeutic concentrations of beta-lactam antibiotics is lost.