Biochemical characterization of Streptococcus pneumoniae penicillin-binding protein 2b and its implication in beta-lactam resistance.
Extensive use of beta-lactam antibiotics has led to the selection of pathogenic streptococci resistant to beta-lactams due to modifications of the penicillin-binding proteins (PBPs). PBP2b from Streptococcus pneumoniae is a monofunctional (class B) high-molecular-weight PBP catalyzing the transpeptidation between adjacent stem peptides of peptidoglycan. The transpeptidase domain of PBP2b isolated from seven clinical resistant (CR) strains contains 7 to 44 amino acid changes over the sequence of PBP2b from the R6 beta-lactam-sensitive strain. We show that the extracellular soluble domains of recombinant PBP2b proteins (PBP2b*) originating from these CR strains have an in vitro affinity for penicillin G that is reduced by up to 99% from that of the R6 strain. The Thr446Ala mutation is always observed in CR strains and is close to the key conserved motif (S(443)SN). The Thr446Ala mutation in R6 PBP2b* displays a 60% reduction in penicillin G affinity in vitro compared to that for the wild-type protein. A recombinant R6 strain expressing the R6 PBP2b Thr446Ala mutation is twofold less sensitive to piperacillin than the parental S. pneumoniae strain. Analysis of the Thr446Ala mutation in the context of the PBP2b CR sequences revealed that its influence depends upon the presence of other unidentified mutations.
Pagliero
Estelle
E
Laboratoire d'Ingénierie des Macromolécules, Institut de Biologie Structurale Jean-Pierre Ebel (CEA-CNRS UMR 5075-UJF), 38027 Grenoble Cedex 1, France.
Chesnel
Laurent
L
Hopkins
Julie
J
Croizé
Jacques
J
Dideberg
Otto
O
Vernet
Thierry
T
Di Guilmi
Anne Marie
AM
D000595:Amino Acid Sequence; D019881:Aminoacyltransferases; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D006602:Hexosyltransferases / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D002267:Muramoylpentapeptide Carboxypeptidase / Q000235:genetics / Q000378:metabolism*; D009154:Mutation / Q000235:genetics; D010400:Penicillin G / Q000494:pharmacology; D046915:Penicillin-Binding Proteins; D010406:Penicillins / Q000494:pharmacology; D010458:Peptidyl Transferases / Q000235:genetics / Q000378:metabolism*; D011008:Pneumococcal Infections / Q000382:microbiology; D012703:Serotyping; D013296:Streptococcus pneumoniae / Q000235:genetics / Q000378:metabolism*; D018440:beta-Lactam Resistance / Q000235:genetics
D001426:Bacterial Proteins; D002352:Carrier Proteins; D004269:DNA, Bacterial; D046915:Penicillin-Binding Proteins; D010406:Penicillins; D019881:Aminoacyltransferases; C478701:penicillin-binding protein 2b, Streptococcus; D010458:Peptidyl Transferases; D006602:Hexosyltransferases; D002267:Muramoylpentapeptide Carboxypeptidase; D010400:Penicillin G