gms | German Medical Science

10. Kongress für Infektionskrankheiten und Tropenmedizin (KIT 2010)

Deutsche Gesellschaft für Infektiologie,
Deutsche AIDS-Gesellschaft,
Deutsche Gesellschaft für Tropenmedizin und Internationale Gesundheit,
Paul-Ehrlich-Gesellschaft für Chemotherapie

23.06. - 26.06.2010, Köln

Gene-specific random mutagenesis of AcrB from Escherichia coli in vitro permits isolation of mutants with altered resistance spectrum and/or altered efficiency of known efflux pump inhibitors

Genspezifische in vitro-Random-Mutagenese von Escherichia coli-AcrB erlaubt die Generierung von Mutanten mit verändertem Resistenzspektrum und/oder veränderter Wirksamkeit von Effluxpumpen-Inhibitoren

Meeting Abstract

  • S. Schuster - Universitätsklinikum Freiburg, Zentrum Infektiologie und Reisemedizin, Freiburg i.Br., Germany
  • C. Biegert - Universitätsklinikum Freiburg, Zentrum Infektiologie und Reisemedizin, Freiburg i.Br., Germany
  • A. Sakaras - Universitätsklinikum Freiburg, Zentrum Infektiologie und Reisemedizin, Freiburg i.Br., Germany
  • J. Bohnert - Universitätsklinikum Freiburg, Zentrum Infektiologie und Reisemedizin, Freiburg i.Br., Germany
  • W.V. Kern - Universitätsklinikum Freiburg, Zentrum Infektiologie und Reisemedizin, Freiburg i.Br., Germany

10. Kongress für Infektionskrankheiten und Tropenmedizin (KIT 2010). Köln, 23.-26.06.2010. Düsseldorf: German Medical Science GMS Publishing House; 2010. DocP12

DOI: 10.3205/10kit068, URN: urn:nbn:de:0183-10kit0689

Published: June 2, 2010

© 2010 Schuster et al.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en). You are free: to Share – to copy, distribute and transmit the work, provided the original author and source are credited.


Outline

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Introduction: AcrB is the periplasmic space spanning pump component of the tripartite AcrAB-TolC complex, the major RND (resistance nodulation cell division) efflux pump in E.coli. RND efflux pumps are widespread in gram-negative bacteria and typically provide resistance to a wide variety of antimicrobial agents. Knowledge about the target of efflux pump inhibitors (EPIs) reported to be active against AcrAB-TolC (e.g. NMP and PAβN) and to restore partially antibiotic susceptibility is limited. Directed evolution methods may be helpful in better define EPI targets and EPI/substrate interaction specificities for AcrB.

Methods: We initially replaced the whole acrB gene from an AcrAB over expressing E.coli strain (3AG100) by an rpsLneo cassette using the Red/ET homologous recombination method. In a second step we exchanged the rpsLneo cassette by error prone PCR products from acrB generated with MutazymII. After confirmation of successful replacement, screening for alteration in drug susceptibility was done by microdilution assays. For altered EPI efficiency we selected on appropriate drug concentrations in combination with NMP or PAβN, respectively. For further investigations of selected mutants we used accumulation and efflux assays with and without EPIs. Mutations were detected by nt sequencing.

Results: So far, we have generated a library of 581 mutants. For confirming the efficiency of the mutation method we tested 82 mutants for changes in drug susceptibility. 34 of them (42%) showed an at least 4 fold alteration in resistance level for one or more agent tested. Beside silent mutations we detected between one and four efficient mutations in acrB of those mutants. Partially “EPI-resistant” clones resulted from co-selection of the whole mutant pool with different antibiotics in combination with NMP or PAβN. A total of 90 clones were isolated and 97% of them showed an at least 4-fold decreased MIC reducing capacity of PAβN or NMP for one or more drug tested. AcrB from five of those mutants were sequenced and again, between one and four mutations were identified.

Conclusion: In vitro random mutagenesis is known to be a powerful strategy for investigating structure function relationship of enzymes. Here we showed its potency for detecting binding sites of substrates and EPIs of a bacterial efflux pump. Beside crystallographic studies this method could provide further valuable informtions concerning structural requirements for efficient drug design.