Multiple changes of penicillin-binding proteins in penicillin-resistant clinical isolates of Streptococcus pneumoniae

Hakenbeck, Regine; Tarpay, Martha; Tomasz, Alexander

doi:10.1128/aac.17.3.364

Cited by 214 publications

(95 citation statements)

References 28 publications

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“…Both groups of pneumococci expressing the abnormal peptidoglycan compositions were penicillin-resistant isolates, and the peptidoglycan changes may reflect alterations in the properties of peptidoglycan transpeptidases (PBPs) of these bacteria (9,18,29). While there is general agreement concerning the involvement of PBPs in terminal states of peptidoglycan assembly, PBP mutants of E. coli showed no compositional alteration in their peptidoglycan (16a).…”

Section: Discussionmentioning

confidence: 98%

Naturally occurring peptidoglycan variants of Streptococcus pneumoniae

Severin

Tomasz

1996

J Bacteriol

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Analysis by high-performance liquid chromatography of the stem peptide composition of cell walls purified from a large number of pneumococcal strains indicates that these bacteria produce a highly conserved species-specific peptidoglycan independent of serotype, isolation date, and geographic origin. Characteristic features of this highly reproducible peptide pattern are the dominance of linear stem peptides with a monomeric tripeptide, a tri-tetra linear dimer, and two indirectly cross-linked tri-tetra dimers being the most abundant components. Screening of strains with the high-performance liquid chromatography technique has identified two naturally occurring peptidoglycan variants in which the species-specific stem peptide composition was replaced by two drastically different and distinct stem peptide patterns, each unique to the particular clone of pneumococci producing it. Both isolates were multidrug resistant, including resistance to penicillin. In one of these clones-defined by multilocus enzyme analysis and pulsed-field gel electrophoresis of the chromosomal DNAs-the linear stem peptides were replaced by branched peptides that most frequently carried an alanyl-alanine substituent on the amino group of the diamino acid residue. In the second clone, the predominant stem peptide species replacing the linear stem peptides carried a seryl-alanine substituent. The abnormal peptidoglycans may be related to the altered substrate preference of transpeptidases (penicillinbinding proteins) in the pneumococcal variants.Peptidoglycans surround the bacterial cell with an uninterrupted network of covalently interlinked muropeptides which is essential for bacterial survival in most environments. While all eubacterial peptidoglycans are constructed on the basis of similar structural principles, the amino acid composition and cross-linking mode of peptidoglycans show sufficient speciesto-species variation to be used for taxonomic purposes (25,26). In the system of Schleifer and Kandler (25), the nature of the cross-linking peptide bond, the amino acid residues connected, and the chemical nature of the diamino acid component of muropeptides were used as the basis of taxonomic classification. The complexity of peptidoglycan structure has only been fully recognized after the recent introduction of the high-resolution analytical technique of high-performance liquid chromatography (HPLC) (17), which demonstrated the unexpectedly large number of distinct muropeptides present in the peptidoglycans of both gram-negative (1, 14, 23, 28) and gram-positive (4, 16) species. To what extent and with what degree of precision the unique composition of these complex supramolecular structures is reproduced in each cell division are not known. Testing this was one of the main purposes of the studies described here. We found that pneumococcal strains of various serotypes, different isolation dates, and geographic origins produced a species-specific peptidoglycan characterized by highly conserved molar ratios of 18 different muropeptides, re...

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Section: Discussionmentioning

confidence: 98%

Naturally occurring peptidoglycan variants of Streptococcus pneumoniae

Severin

Tomasz

1996

J Bacteriol

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

Laible

Hakenbeck

1991

J Bacteriol

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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...

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“…The development of penicillin-resistant pneumococci has occurred as a result of more than 40 years of penicillin therapy by the evolution of penicillin-resistant forms of each of the five high molecular weight penicillin-binding proteins (PBPs) (lA, 1B, 2A, 2X, and 2B) (6)(7)(8)(9). The origin(s) of penicillin-resistant pneumococci is unclear.…”

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Horizontal transfer of penicillin-binding protein genes in penicillin-resistant clinical isolates of Streptococcus pneumoniae.

Dowson

Hutchison

Brannigan

et al. 1989

Proc. Natl. Acad. Sci. U.S.A.

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256

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Resistance to penicillin in clinical isolates of Streptococcus pneumoniae has occurred by the development of altered penicillin-binding proteins (PBPs) that have greatly decreased affinity for the antibiotic. We have investigated the origins of penicillin-resistant strains by comparing the sequences of the transpeptidase domain of PBP2B from 6 penicillin-sensitive and 14 penicillin-resistant strains. In addition we have sequenced part of the amylomaltase gene from 2 of the sensitive and 6 of the resistant strains. The sequences of the amylomaltase gene of all of the strains and of the PBP2B gene of the penicillin-sensitive strains show that S. pneumoniae is genetically very uniform. In contrast the PBP2B genes of the penicillin-resistant strains show -14% sequence divergence from those of the penicillin-sensitive strains and the

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Multiple changes of penicillin-binding proteins in penicillin-resistant clinical isolates of Streptococcus pneumoniae

Cited by 214 publications

References 28 publications

Naturally occurring peptidoglycan variants of Streptococcus pneumoniae

Naturally occurring peptidoglycan variants of Streptococcus pneumoniae

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

Horizontal transfer of penicillin-binding protein genes in penicillin-resistant clinical isolates of Streptococcus pneumoniae.

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