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 resistance of S. pneumoniae to beta-lactam antibiotics is linked to multiple mutations of high molecular mass penicillin-binding proteins (H-PBPs), essential enzymes involved in the final steps of bacterial cell wall synthesis. H-PBPs from resistant bacteria have a reduced affinity for beta-lactam and a decreased hydrolytic activity on substrate analogues. In S. pneumoniae, the gene coding for one of these H-PBPs, PBP2x, is located in the cell division cluster (DCW). We present here structural evidence linking multiple beta-lactam resistance to amino acid substitutions in PBP2x within a buried cavity near the catalytic site that contains a structural water molecule. Site-directed mutation of amino acids in contact with this water molecule in the "sensitive" form of PBP2x produces mutants similar, in terms of beta-lactam affinity and substrate hydrolysis, to altered PBP2x produced in resistant clinical isolates. A reverse mutation in a PBP2x variant from a clinically important resistant clone increases the acylation efficiency for beta-lactams 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 sensitivity to therapeutic concentrations of beta-lactam antibiotics is lost.
Mouz
N
N
Laboratoire d'Ingénierie des Macromolécules, Institut de Biologie Structurale Jean-Pierre Ebel (Commissariat à l'Energie Atomique-Centre National de la Recherche Scientifique), 41, avenue des Martyrs, F-38027 Grenoble Cedex 1, France.
Gordon
E
E
Di Guilmi
A M
AM
Petit
I
I
Pétillot
Y
Y
Dupont
Y
Y
Hakenbeck
R
R
Vernet
T
T
Dideberg
O
O
D019943:Amino Acid Substitution; D001426:Bacterial Proteins; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics*; D006602:Hexosyltransferases; D002267:Muramoylpentapeptide Carboxypeptidase / Q000737:chemistry* / Q000235:genetics*; D016297:Mutagenesis, Site-Directed; D046915:Penicillin-Binding Proteins; D010458:Peptidyl Transferases; D013296:Streptococcus pneumoniae / Q000737:chemistry* / Q000235:genetics*; D018440:beta-Lactam Resistance / Q000235:genetics*
D001426:Bacterial Proteins; D002352:Carrier Proteins; D046915:Penicillin-Binding Proteins; D010458:Peptidyl Transferases; D006602:Hexosyltransferases; D002267:Muramoylpentapeptide Carboxypeptidase