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Understanding multidrug efflux at a molecular level

English title Understanding multidrug efflux at a molecular level
Applicant Seeger Markus
Number 144823
Funding scheme SNSF Professorships
Research institution Institut für Medizinische Mikrobiologie Universität Zürich
Institution of higher education University of Zurich - ZH
Main discipline Biochemistry
Start/End 01.06.2013 - 31.05.2017
Approved amount 1'607'756.00
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All Disciplines (8)

Discipline
Biochemistry
Biophysics
Molecular Biology
Experimental Microbiology
Cellular Biology, Cytology
Medical Microbiology
Pharmacology, Pharmacy
Structural Research

Keywords (13)

mycobacterial lipids; X-ray crystallography; Multidrug efflux pumps; protein reconstitution; ABC transporter; virulence factors; Mycobacterium smegmatis; secondary-active transporters; Structure-based biochemistry; Membrane proteins; Mycobacterium tuberculosis; pathogenic bacteria; Lactococcus lactis

Lay Summary (German)

Lead
Molekulare Einblicke in Antibiotika-TransportproteineWerden Bakterien mittels Antibiotika bekämpft, reagieren diese durch eine erhöhte Produktion von Transportproteinen, welche die Antibiotika aus der Bakterienzelle herauspumpen. Dadurch erlangt das Bakterium Antibiotikaresistenz und es gewinnt Zeit um weitere Resistenzen zu erlangen. Wir untersuchen solche Antibiotikapumpen aus dem Verursacher von Tuberkulose, dem Bakterium Mycobacterium tuberculosis (Mtu) und anderen Mikroorganismen.
Lay summary

Inhalt und Ziel des Forschungsprojekts

In unserer Arbeit untersuchen wir die Bedeutung von Antibiotikapumpen im Zusammenhang von Antibiotikaresistenz und Virulenz von pathogenen Bakterien, insbesondere von Mtu. Mittels Röntgenkristallographie und einer Reihe von biochemischen und mikrobiologischen Methoden wollen wir auf molekularer Ebene verstehen, wie genau Antibiotika von diesen Transportproteinen erkannt und über die Membran transportiert werden. Dazu werden wir diese Antibiotikapumpen i) rekombinant in verschiedenen Bakterien in ausreichender Menge produzieren, ii) die Proteine reinigen und biochemisch charakterisieren, iii) die gereinigten Proteine, falls möglich, kristallisieren zwecks Strukturbestimmung mittels Röntgenkristallographie, iv) Antibiotikatransportversuche mit rekombinant-überproduzierten Transportern unternehmen und v) die Gene für diese Transporter in Mtu deletieren und die entsprechenden Mutanten auf Antibiotikaresistenz und Virulenz testen.

 

Wissenschaftlicher und Gesellschaftlicher Kontext des Forschungsprojektes

In den letzten Jahren wurden einige Mtu-Transportproteine als Antibiotikapumpen beschrieben. Trotzdem ist deren Beitrag zur Resistenzbildung als auch deren biochemische und molekulare Funktionsweise nur ansatzweise bekannt. Mit unserer Arbeit möchten wir einen Beitrag zum molekularen Verständnis von Antibiotikapumpen in Mtu leisten. Tuberkulose bleibt eine vor allem in bevölkerungsreichen Staaten Afrikas und Südostasiens weit verbreitete Krankheit, welche zu 1.4 Millionen Todesfällen jährlich führt. Gerade wegen der immer häufiger auftauchenden Antibiotikaresistenzen zieht eine Tuberkuloseerkrankung sehr oft schwierige und langwierige medikamentöse Behandlungen nach sich.

Direct link to Lay Summary Last update: 19.04.2013

Responsible applicant and co-applicants

Employees

Publications

Publication
Split tasks of asymmetric nucleotide‐binding sites in the heterodimeric ABC exporter EfrCD
Hürlimann Lea M., Hohl Michael, Seeger Markus A. (2017), Split tasks of asymmetric nucleotide‐binding sites in the heterodimeric ABC exporter EfrCD, in The FEBS Journal, 284(11), 1672-1687.
Exploring conformational equilibria of a heterodimeric ABC transporter
Timachi M Hadi, Hutter Cedric AJ, Hohl Michael, Assafa Tufa, Böhm Simon, Mittal Anshumali, Seeger Markus A, Bordignon Enrica (2017), Exploring conformational equilibria of a heterodimeric ABC transporter, in eLife, 6, 1-2.
The heterodimeric ABC transporter EfrCD mediates multidrug efflux in Enterococcus faecalis.
Hürlimann Lea M, Corradi Valentina, Hohl Michael, Bloemberg Guido V, Tieleman D Peter, Seeger Markus A (2016), The heterodimeric ABC transporter EfrCD mediates multidrug efflux in Enterococcus faecalis., in Antimicrobial agents and chemotherapy, 60(9), 5400-5411.
A Transporter Motor Taken Apart: Flexibility in the Nucleotide Binding Domains of a Heterodimeric ABC Exporter.
Bukowska Magdalena A, Hohl Michael, Geertsma Eric R, Hürlimann Lea M, Grütter Markus G, Seeger Markus A (2015), A Transporter Motor Taken Apart: Flexibility in the Nucleotide Binding Domains of a Heterodimeric ABC Exporter., in Biochemistry, 54(19), 3086-99.
Bicistronic mRNAs to enhance membrane protein overexpression.
Marino Jacopo, Hohl Michael, Seeger Markus A, Zerbe Oliver, Geertsma Eric R (2015), Bicistronic mRNAs to enhance membrane protein overexpression., in Journal of molecular biology, 427(4), 943-54.
Coupling of remote alternating-access transport mechanisms for protons and substrates in the multidrug efflux pump AcrB.
Eicher Thomas, Seeger Markus A, Anselmi Claudio, Zhou Wenchang, Brandstätter Lorenz, Verrey François, Diederichs Kay, Faraldo-Gómez José D, Pos Klaas M (2014), Coupling of remote alternating-access transport mechanisms for protons and substrates in the multidrug efflux pump AcrB., in eLife, 3, 0.
Structural basis for allosteric cross-talk between the asymmetric nucleotide binding sites of a heterodimeric ABC exporter.
Hohl Michael, Hürlimann Lea M, Böhm Simon, Schöppe Jendrik, Grütter Markus G, Bordignon Enrica, Seeger Markus A (2014), Structural basis for allosteric cross-talk between the asymmetric nucleotide binding sites of a heterodimeric ABC exporter., in Proceedings of the National Academy of Sciences of the United States of America, 111(30), 11025-30.

Collaboration

Group / person Country
Types of collaboration
Prof. Gabriele Meloni, The University of Texas at Dallas United States of America (North America)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
- Research Infrastructure
Prof. Peter Sander, Institute of Medical Microbiology, University of Zurich Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Research Infrastructure
Prof. Peter Tieleman, Dept. of Biological Sciences, University of Calgary Canada (North America)
- in-depth/constructive exchanges on approaches, methods or results
- Research Infrastructure
Prof. Hubert Hilbi Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
- Research Infrastructure
- Exchange of personnel
Prof. Eric Geertsma, Institute of Biochemistry, Goethe-University Frankfurt Germany (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
- Research Infrastructure
- Exchange of personnel
Prof. Enrica Bordignon, Fakultät für Chemie und Biochemie, Ruhr Unversity Bochum Germany (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
- Research Infrastructure
- Exchange of personnel

Associated projects

Number Title Start Funding scheme
188817 Molecular mechanisms of bacterial multidrug efflux 01.10.2019 Project funding (Div. I-III)
126338 Targeting multidrug efflux proteins: Studying transport processes across the membrane using designed ankyrin repeat proteins and antibody fragments 01.10.2009 Ambizione
170625 Deciphering bacterial membrane transport at the molecular level - drugs, iron and lipids 01.06.2017 SNSF Professorships
150766 UV-Fluorescence microscopy based incubation and imaging platform for protein and nucleic acid crystals 01.12.2013 R'EQUIP
144520 Structure-function studies of heterodimeric ABC transporters 01.10.2012 Ambizione

Abstract

Membrane transporters play a central role in every living cell. ATP-binding cassette (ABC) transporters for example are found in all phyla of life and use the energy from binding and hydrolysis of ATP to shuttle a wide variety of substrates across the membrane. Many ABC exporters actively extrude drugs and thereby contribute to the development of multidrug resistance in cancers and pathogenic microorganisms which causes severe problems in the treatment of bacterial infections and chemotherapy. Recently, we solved the crystal structure of the heterodimeric ABC exporter TM287/288 from Thermotoga maritima at a resolution of 2.9 Å. Biochemical analyses revealed that TM287/288 exhibits ATPase activity both in detergent-solution and reconstituted in membranes and is capable of extruding a variety of drugs including the anticancer agent daunomycin. Currently, TM287/288 and close bacterial homologues serve us as ideal model systems to investigate the multidrug transport mechanism and substrate recognition of ABC exporters at a molecular level. Crystal structures provide highly accurate snapshots of proteins under study, but are themselves not sufficient to understand the transport mechanism. To overcome this limitation, we use electron paramagnetic resonance (EPR) spectroscopy and single molecule FRET to study the conformational cycling of the membrane transporters both in detergent solution as well as in lipid membranes. Engineered disulfide cross-links and targeted mutations are introduced to trap ABC transporters in defined conformational states. By combining X-ray crystallography and biochemical methods, we aim to establish structure-function relationships which will ultimately allow for the detailed description of the transport cycle of heterodimeric ABC transporters. Importantly, half of the approximately 40 human ABC transporters are heterodimers like TM287/288. Therefore, our studies also contribute to the molecular understanding of mammalian ABC transporters of high clinical relevance.Tuberculosis is one of the world’s most pressing health problems, and the spread of multidrug resistant Mycobacterium tuberculosis strains makes it increasingly difficult to treat infections with the available arsenal of antibiotics. Drug resistance mediated by efflux pumps of M. tuberculosis has received increasing attention in the past decade and a few transport systems have been examined, mainly using microbiological methods. However, biochemical and structural information about these drug efflux pumps is lacking. Building on methods that I have established to study the structure and function of TM287/288, I aim to purify and characterize membrane transporters of M. tuberculosis that are involved in drug efflux and the export of virulence factors. The purified efflux pumps will be characterized at the biochemical and structural level using substrate binding assays and X-ray crystallography and functionally reconstituted in proteoliposomes to perform transport assays in vitro. Drug transport will also be studied in vivo by expressing the efflux proteins in Lactococcus lactis and Mycobacterium smegmatis. Targeted transporter gene deletions in M. tuberculosis will be characterized with respect to susceptibility to antitubercular drugs and in virulence assays using macrophage growth assays and a mouse model for tuberculosis. In summary, my research aims to answer the question how exactly transporters are capable of shuttling drugs and virulence factors across the membrane of pathogenic bacteria such as M. tuberculosis.
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