Five independent combinations of mutations can result in low-affinity penicillin-binding protein 2x of Streptococcus pneumoniae

Abstract

Penicillin-binding protein 2x (PBP 2x) Intrinsic penicillin resistance in pneumococci and various other pathogenic bacteria is mediated by the production of low-penicillin-affinity variants of essential high-molecularweight penicillin-binding proteins (PBPs), the target proteins of ,-lactam antibiotics (11,25,30,34). These PBPs are believed to be essential enzymes functioning during late stages of murein biosynthesis. They interact with ,-lactams by enzymatically forming a covalent penicilloyl (cephalosporoyl) complex which is enzymatically inactive. ,3-Lactams are recognized by PBPs because of their structural similarity to substrate molecules (i.e., muropeptides), and inhibitors as well as substrates interact with the same active-site serine (for a review, see reference 8). Therefore, mutations responsible for low affinity must be carefully positioned within the protein in order to still allow for its actual in vivo function.We have recently isolated a series of spontaneous, independent mutants with stepwise increasing resistance to cefotaxime (17). All higher-level-resistance mutants contained low-affinity variants of PBP 2x and, in some cases, of PBP 2a as well. Reduction in the penicillin binding of PBP 2x at ddiferent selection steps was detected when mutants from different mutant families were compared, indicating that the development of resistance does not follow a strictly predetermined pathway.The gene coding for the 750-amino-acid PBP 2x (pbpX) has been sequenced, and the mutations in one of the mutants (C506) have been mapped (18). We have now determined the sequence of mutations that occurred with increasing cefotaxime resistance in one mutant family and analyzed their contribution to resistance development. Furthermore, mutations in the pbpX genes of another four independent mutants, obtained after five or six selection steps, were characterized.* Corresponding author. MATERIALS AND METHODSBacterial strains, phages, and plasmids. The penicillinsusceptible laboratory strain Streptococcus pneumoniae R6 is an unencapsulated derivative of the Rockefeller University strain R36A (1). The cefotaxime-resistant mutants, all derived from the R6 strain, are independent, spontaneous mutants, as described previously (17). The mutant family C006 is composed of six mutants with increasing levels of resistance, from C106 (first-step isolate) to C606 (sixth-step isolate). Mutants C501, C503, C604, and C505 were obtained after five or six selection steps (indicated by the first digit) in mutant families 1, 3, 4, and 5, respectively (indicated by the last digit).The phages Phagescript SK (Stratagene, San Diego, Calif.) and M13mp8/9 (22) were propagated in Escherichia coli JM103 (21), and derivatives of the plasmids pJDC9 (4) and pR28 (20) were propagated in E. coli DH5. Transformation of S. pneumoniae (32) and of E. coli (3) followed published procedures.Isolation of DNA. Details of the isolation of chromosomal DNA and the preparation of phage and plasmid DNA have been described recently (2, 18).Isolation and sequ...

The CiaRH System ofStreptococcus pneumoniaePrevents Lysis during Stress Induced by Treatment with Cell Wall Inhibitors and by Mutations inpbp2xInvolved in β-Lactam Resistance

“…The second-and third-step mutants of this family, C203 and C303, contained mutations in pbp2x, resulting in the amino acid changes PBP2x-G597D and T550A (27) (Fig. 1).…”

FemABX Peptidyl Transferases: a Link between Branched-Chain Cell Wall Peptide Formation and β-Lactam Resistance in Gram-Positive Cocci

“…Additional gene diversity is created by horizontal exchange of DNA fragments between strains or even with commensal streptococci, resulting in highly mosaic genes. Some very low affinity PBP2x variants from clinical isolates contain more than 100 amino acid substitutions spread over the entire sequence (47,61,83). Mutations in PBP2x and PBP2b are sufficient for midlevel resistance, but to achieve high-level penicillin resistance, all six endogenous PBPs can be modified.…”

A novel resistance mechanism against beta-lactams in Streptococcus pneumoniae involves CpoA, a putative glycosyltransferase

“…Not only were different PBPs affected in cefotaxime-versus piperacillinresistant mutants (17,28), but distinct "non-PBP" genes also contributed to resistance, demonstrating that gram-positive organisms also have an unanticipated variety of defense mechanisms against such drugs. Whatever these mechanisms are in molecular terms, in every mutant lineage inevitably a deficiency in genetic competence became apparent, indicating that the integrity of the cell wall, which is threatened by ␤-lactam treatment, is required for competence and transformability (16).…”