Pneumococcal β-Lactam Resistance Due to a Conformational Change in Penicillin-binding Protein 2x

Abstract

Streptococcus pneumoniae is a life-threatening human pathogen that is increasingly resistant to a wide array of drugs. Resistance to β-lactams, the most widely used antibiotics, is correlated with tens of amino acid substitutions in their targets; that is, the penicillin-binding proteins (PBPs), resulting from multiple events of recombination. To discriminate relevant substitutions from those that are incidental to the recombination process, we report the exhaustive characterization of all the mutations in the transpeptidase domain of PBP2x from the highly resistant strain 5204. A semi-automated method combining biochemical and microbiological approaches singled out 6 mutations of 41 (15%) that are essential for high level resistance. The hitherto uncharacterized I371T, R384G, M400T, and N605T together with the previously studied T338M and M339F account for nearly all the loss of affinity of PBP2x for β-lactams. Most interestingly, I371T and R384G cause the conformational change of a loop that borders the entrance of the active site cavity, hampering antibiotic binding. For the first time all the mutations of a PBP relevant to β-lactam resistance have been identified, providing new mechanistic insights. Most notable is the relationship between the decreased susceptibility to β-lactams and the dynamic behavior of a loop. Streptococcus pneumoniae is a life-threatening human pathogen that is increasingly resistant to a wide array of drugs. Resistance to β-lactams, the most widely used antibiotics, is correlated with tens of amino acid substitutions in their targets; that is, the penicillin-binding proteins (PBPs), resulting from multiple events of recombination. To discriminate relevant substitutions from those that are incidental to the recombination process, we report the exhaustive characterization of all the mutations in the transpeptidase domain of PBP2x from the highly resistant strain 5204. A semi-automated method combining biochemical and microbiological approaches singled out 6 mutations of 41 (15%) that are essential for high level resistance. The hitherto uncharacterized I371T, R384G, M400T, and N605T together with the previously studied T338M and M339F account for nearly all the loss of affinity of PBP2x for β-lactams. Most interestingly, I371T and R384G cause the conformational change of a loop that borders the entrance of the active site cavity, hampering antibiotic binding. For the first time all the mutations of a PBP relevant to β-lactam resistance have been identified, providing new mechanistic insights. Most notable is the relationship between the decreased susceptibility to β-lactams and the dynamic behavior of a loop. The discovery of penicillin more than 60 years ago launched the antibiotic era, and β-lactams are still the most widely used drugs to combat bacterial infections today. β-Lactam antibiotics inhibit an essential step in bacterial cell wall synthesis, namely the cross-linking of the peptidoglycan by transpeptidases (TPs). 2The abbreviations used are: TPtranspeptidasePBPpenicillin-binding proteinMICminimal inhibitory concentration. The antibiotics hamper the TP activity by acylating the active site serine of the penicillin-binding proteins (PBPs) enzymes (1Waxman D.J. Strominger J.L. Annu. Rev. Biochem. 1983; 52: 825-869Crossref PubMed Scopus (479) Google Scholar). The active site of PBPs is bordered by three defining conserved motifs, SXXK with the catalytic serine, SXN, and KSG. transpeptidase penicillin-binding protein minimal inhibitory concentration. However, several mechanisms have arisen that greatly threaten the clinical efficacy of β-lactams. Depending on the organisms, the strategies vary and can be combined. β-Lactamases can be expressed that degrade the antibiotics. Altered endogenous PBPs or an additional non-susceptible PBP can be expressed. In Gram-negative bacteria, the outer membrane permeability to the drugs can be decreased and/or the drugs can be actively expelled from the perip