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Evolution of increased ß-lactam resistance in an engineered Metallo-ß-lactamase
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
(English)Manuscript (preprint) (Other academic)
Abstract [en]

The extensive use and misuse of antibiotics during the last 60 years has led to the evolution and global spread of a variety of resistance mechanisms. Of high medical importance are ß-lactamases, a group of enzymes that can hydrolyze the ß-lactam ring present in all ß-lactam antibiotics. Metallo-ß-lactamases (MBLs) are particularly problematic due to their ability to hydrolyze virtually all classes of ß-lactam antibiotics. A novel MBL (evMBL9) with low-level resistance against ß-lactam antibiotics was designed and employed as the ancestral MBL during an experiment to examine how an enzyme evolved towards increased resistance. We designed and synthesized a mutant library in which the substrate binding profile was varied by randomizing six amino acid residues. Mutants with increased resistance against seven different ß-lactam antibiotics (penicillin G, ampicillin, cefalotin, cefaclor, cefuroxime, cefoperazone and cefotaxime) were isolated and characterized. For the majority of mutants, in spite of their significantly increased resistance, both mRNA and protein levels were reduced (up to >20 fold) relative to those of parental evMBL9, indicating that the catalytic activities of these mutant MBLs were highly increased. Multivariate analysis showed that the majority of mutant enzymes became generalists, conferring increased resistance against most of the examined ß-lactams. The increased resistance and decreased protein level suggest that the improved hydrolysis in these novel MBLs is associated with decreased protein stability.

Keyword [en]
Metallo-beta-lactamase, directed protein evolution
National Category
Other Biological Topics
Identifiers
URN: urn:nbn:se:uu:diva-172785OAI: oai:DiVA.org:uu-172785DiVA: diva2:515765
Available from: 2012-04-15 Created: 2012-04-15 Last updated: 2012-08-01
In thesis
1. Dynamics and Mechanisms of Adaptive Evolution in Bacteria
Open this publication in new window or tab >>Dynamics and Mechanisms of Adaptive Evolution in Bacteria
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Determining the properties of mutations is fundamental to understanding the mechanisms of adaptive evolution. The major goal of this thesis is to investigate the mechanisms of bacterial adaptation to new environments using experimental evolution. Different types of mutations were under investigations with a particular focus on genome rearrangements. Adaptive evolution experiments were focused on the development of bacterial resistance to antibiotics.

In paper I, we performed stochastic simulations to examine the role of gene amplification in promoting the establishment of new gene functions. The results show that gene amplification can contribute to creation of new gene functions in nature. In paper II, the evolution of β-lactam resistance was studied by evolving S. typhimurium carrying a β-lactamase gene towards increased resistance against cephalosporins. Our results suggest that gene amplification is likely to provide an immediate solution at the early stage of adaptive evolution and subsequently facilitate further stable adaptation. In paper III, we isolated spontaneous deletion mutants with increased competitive fitness, which indicated that genome reduction could be driven by selection. To test this hypothesis, independent lineages of wild type S. typhimurium were serially passaged for 1000 generations and we observed fixation of deletions that significantly increased bacterial fitness when reconstructed in wild type genetic background. In paper IV, we developed a new strategy combining 454 pyrosequencing technology and a ‘split mapping’ computational method to identify unique junction sequences formed by spontaneous genome rearrangements. A high steady-state frequency of rearrangements in unselected bacterial populations was suggested from our results. In paper V, the rates, mechanisms and fitness effects of colistin resistance in S. typhimurium were determined. The high mutation rate and low fitness costs suggest that colistin resistance could develop in clinical settings. In paper VI, a novel Metallo-β-lactamase (MBL) with low resistance against β-lactam antibiotics was employed as the ancestral protein in a directed evolution experiment to examine how an enzyme evolves towards increased resistance. For most isolated mutants, in spite of their significantly increased resistance, both mRNA and protein levels were decreased as compared with the parental protein, suggesting that the catalytic activity had increased.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2012. 64 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 770
Keyword
adaptive evolution, mutation, genome rearrangements, antibiotic resistance, gene amplification, genome reduction, directed evolution
National Category
Microbiology
Research subject
Biology with specialization in Microbiology
Identifiers
urn:nbn:se:uu:diva-172786 (URN)978-91-554-8354-8 (ISBN)
Public defence
2012-06-05, C10:305, BMC, Husargatan 3, Uppsala, 09:00 (English)
Opponent
Supervisors
Available from: 2012-05-14 Created: 2012-04-15 Last updated: 2012-08-01Bibliographically approved

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