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A microtiter plate-based beta-lactam binding assay for inhibitors of high-molecular-mass penicillin-binding proteins.

ABSTRACT: High-molecular-mass penicillin-binding proteins (HMM PBPs) are essential for bacterial cell wall biosynthesis and are the lethal targets of beta-lactam antibiotics. When purified, HMM PBPs give undetectable or weak enzyme activity. This has impeded efforts to develop assays for HMM PBPs and to develop new inhibitors for HMM PBPs as HMM PBP targeted antibacterial agents. However, even when purified, HMM PBPs retain their ability to bind beta-lactams. Here we describe a fluorescently detected microtiter plate-based assay for inhibitor binding to HMM PBPs based on competition with biotin-ampicillin conjugate (BIO-AMP) binding.

SUBMITTER: Stefanova M

PROVIDER: S-EPMC2790019 | biostudies-literature | 2010 Jan

REPOSITORIES: biostudies-literature

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A microtiter plate-based beta-lactam binding assay for inhibitors of high-molecular-mass penicillin-binding proteins.

Stefanova Miglena M Bobba Sudheer S Gutheil William G WG

Analytical biochemistry 20090911 1

High-molecular-mass penicillin-binding proteins (HMM PBPs) are essential for bacterial cell wall biosynthesis and are the lethal targets of beta-lactam antibiotics. When purified, HMM PBPs give undetectable or weak enzyme activity. This has impeded efforts to develop assays for HMM PBPs and to develop new inhibitors for HMM PBPs as HMM PBP targeted antibacterial agents. However, even when purified, HMM PBPs retain their ability to bind beta-lactams. Here we describe a fluorescently detected micr ...[more]

PMID: 19748471

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Project description:The human society faces a serious problem due to the widespread resistance to antibiotics in clinical practice. Most antibiotic biosynthesis gene clusters in actinobacteria contain genes for intrinsic self-resistance to the produced antibiotics, and it has been proposed that the antibiotic resistance genes in pathogenic bacteria originated in antibiotic-producing microorganisms. The model actinobacteria Streptomyces clavuligerus produces the β-lactam antibiotic cephamycin C, a class A β-lactamase, and the β lactamases inhibitor clavulanic acid, all of which are encoded in a gene supercluster; in addition, it synthesizes the β-lactamase inhibitory protein BLIP. The secreted clavulanic acid has a synergistic effect with the cephamycin produced by the same strain in the fight against competing microorganisms in its natural habitat. High levels of resistance to cephamycin/cephalosporin in actinobacteria are due to the presence (in their β-lactam clusters) of genes encoding PBPs which bind penicillins but not cephalosporins. We have revised the previously reported cephamycin C and clavulanic acid gene clusters and, in addition, we have searched for novel β-lactam gene clusters in protein databases. Notably, in S. clavuligerus and Nocardia lactamdurans, the β-lactamases are retained in the cell wall and do not affect the intracellular formation of isopenicillin N/penicillin N. The activity of the β-lactamase in S. clavuligerus may be modulated by the β-lactamase inhibitory protein BLIP at the cell-wall level. Analysis of the β-lactam cluster in actinobacteria suggests that these clusters have been moved by horizontal gene transfer between different actinobacteria and have culminated in S. clavuligerus with the organization of an elaborated set of genes designed for fine tuning of antibiotic resistance and cell wall remodeling for the survival of this Streptomyces species. This article is focused specifically on the enigmatic connection between β-lactam biosynthesis and β-lactam resistance mechanisms in the producer actinobacteria.

Project description:This study aimed to characterize the role of Pseudomonas aeruginosa low-molecular-mass penicillin-binding proteins (LMM PBPs), namely, PBP4 (DacB), PBP5 (DacC), and PBP7 (PbpG), in peptidoglycan composition, ?-lactam resistance, and ampC regulation. For this purpose, we constructed all single and multiple mutants of dacB, dacC, pbpG, and ampC from the wild-type P. aeruginosa PAO1 strain. Peptidoglycan composition was determined by high-performance liquid chromatography (HPLC), ampC expression by reverse transcription-PCR (RT-PCR), PBP patterns by a Bocillin FL-binding test, and antimicrobial susceptibility by MIC testing for a panel of ?-lactams. Microscopy and growth rate analyses revealed no apparent major morphological changes for any of the mutants compared to the wild-type PAO1 strain. Of the single mutants, only dacC mutation led to significantly increased pentapeptide levels, showing that PBP5 is the major dd-carboxypeptidase in P. aeruginosa. Moreover, our results indicate that PBP4 and PBP7 play a significant role as dd-carboxypeptidase only if PBP5 is absent, and their dd-endopeptidase activity is also inferred. As expected, the inactivation of PBP4 led to a significant increase in ampC expression (around 50-fold), but, remarkably, the sequential inactivation of the three LMM PBPs produced a much greater increase (1,000-fold), which correlated with peptidoglycan pentapeptide levels. Finally, the ?-lactam susceptibility profiles of the LMM PBP mutants correlated well with the ampC expression data. However, the inactivation of ampC in these mutants also evidenced a role of LMM PBPs, especially PBP5, in intrinsic ?-lactam resistance. In summary, in addition to assessing the effect of P. aeruginosa LMM PBPs on peptidoglycan structure for the first time, we obtained results that represent a step forward in understanding the impact of these PBPs on ?-lactam resistance, apparently driven by the interplay between their roles in AmpC induction, ?-lactam trapping, and dd-carboxypeptidase/?-lactamase activity.

Dataset Information

A microtiter plate-based beta-lactam binding assay for inhibitors of high-molecular-mass penicillin-binding proteins.

Publications

A microtiter plate-based beta-lactam binding assay for inhibitors of high-molecular-mass penicillin-binding proteins.

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