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Deciphering bacterial membrane transport at the molecular level - drugs, iron and lipids

English title Deciphering bacterial membrane transport at the molecular level - drugs, iron and lipids
Applicant Seeger Markus
Number 170625
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 01.06.2017 - 31.05.2019
Approved amount 799'941.00
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All Disciplines (8)

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

Keywords (15)

Membrane proteins; Multidrug efflux pumps; ABC transporter; MFS transporter; Pathogenic bacteria; Antibiotic resistance; Mycobacterium tuberculosis; Enterococcus faecalis; Drug efflux; Virulence factors; Mycobacterial lipids; Siderophore transport; Iron sequestration; X-ray crystallography; Structure-based biochemistry

Lay Summary (German)

Lead
Molekulare Einblicke in Transportproteine aus pathogenen BakterienWerden 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. Zudem dienen Transportproteine der Aufnahme von wichtigen Nährstoffen, wie zum Beispiel Eisen und sind für den Aufbau der Zellmembran unerlässlich. Wir untersuchen Transportproteine, die für den Membrantransport von Antibiotika und Virulenzfaktoren aus pathogenen Bakterien verantwortlich sind. Ein spezielles Augenmerk richten wir auf das Bakterium Mycobacterium tuberculosis, dem Erreger der Tuberkulose.
Lay summary

Inhalt und Ziel des Forschungsprojekts

In unserer Arbeit untersuchen wir die Bedeutung von Transportprozessen im Zusammenhang von Antibiotikaresistenz und Virulenz von pathogenen Bakterien, insbesondere von Mycobacterium tuberculosis. Mittels Röntgenkristallographie und einer Reihe von biochemischen und mikrobiologischen Methoden wollen wir auf molekularer Ebene verstehen, wie genau Antibiotika und andere chemische Verbindungen von diesen Transportproteinen erkannt und über die Membran transportiert werden. Dazu werden i) Transportproteine rekombinant in verschiedenen Bakterien in ausreichender Menge produziert, ii) die Proteine gereinigt und biochemisch charakterisiert, iii) die gereinigten Proteine kristallisiert für die Strukturbestimmung mittels Röntgenkristallographie, iv) Transportexperimente in vivo und in vitro durchgeführt und schliesslich v) Gene für diese Transporter in den entsprechenden pathogenen Bakterien deletiert und die entsprechenden Mutanten auf Antibiotikaresistenz und Virulenz getestet.

 

Wissenschaftlicher und Gesellschaftlicher Kontext des Forschungsprojektes

Die 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. Zudem sind Antibiotika-resistente Bakterien weltweit auf dem Vormarsch und stellen eine grosse Bedrohung der Weltgesundheit dar. Mit unserer Arbeit werden wir einen Beitrag zum molekularen Verständnis von Transportprozessen in pathogenen Bakterien leisten. Insbesondere haben wir uns zum Ziel gesetzt, die Bedeutung von Antibiotikapumpen im Zusammenhang von Antibiotikaresistenzen zu verstehen.

Direct link to Lay Summary Last update: 31.05.2017

Responsible applicant and co-applicants

Employees

Publications

Publication
The extracellular gate shapes the energy profile of an ABC exporter
Hutter Cedric A. J., Timachi M. Hadi, Hürlimann Lea M., Zimmermann Iwan, Egloff Pascal, Göddeke Hendrik, Kucher Svetlana, Štefanić Saša, Karttunen Mikko, Schäfer Lars V., Bordignon Enrica, Seeger Markus A. (2019), The extracellular gate shapes the energy profile of an ABC exporter, in Nature Communications, 10(1), 2260-2260.
Engineered peptide barcodes for in-depth analyses of binding protein libraries
Egloff Pascal, Zimmermann Iwan, Arnold Fabian M., Hutter Cedric A. J., Morger Damien, Opitz Lennart, Poveda Lucy, Keserue Hans-Anton, Panse Christian, Roschitzki Bernd, Seeger Markus A. (2019), Engineered peptide barcodes for in-depth analyses of binding protein libraries, in Nature Methods, 16(5), 421-428.
Identification of conformation-selective nanobodies against the membrane protein insertase BamA by an integrated structural biology approach
Seeger Markus A., Hiller Sebastian, Kaur Hundeep, Hartmann Jean-Baptiste, Jakob Roman P., Zahn Michael, Zimmermann Iwan, Maier Timm (2019), Identification of conformation-selective nanobodies against the membrane protein insertase BamA by an integrated structural biology approach, in Journal of Biomolecular NMR, 1-2.
Increased drug permeability of a stiffened mycobacterial outer membrane in cells lacking MFS transporter Rv1410 and lipoprotein LprG
Hohl Michael, Remm Sille, Eskandarian Haig A., Dal Molin Michael, Arnold Fabian M., Hürlimann Lea M., Krügel Andri, Fantner Georg E., Sander Peter, Seeger Markus A. (2019), Increased drug permeability of a stiffened mycobacterial outer membrane in cells lacking MFS transporter Rv1410 and lipoprotein LprG, in Molecular Microbiology, 111(5), 1263-1282.
Structural basis for pH-dependent retrieval of ER proteins from the Golgi by the KDEL receptor
Bräuer Philipp, Parker Joanne L., Gerondopoulos Andreas, Zimmermann Iwan, Seeger Markus A., Barr Francis A., Newstead Simon (2019), Structural basis for pH-dependent retrieval of ER proteins from the Golgi by the KDEL receptor, in Science, 363(6431), 1103-1107.

Collaboration

Group / person Country
Types of collaboration
Prof. Hubert Hilbi, Institute of Medical Microbiology, University of Zurich Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
- Research Infrastructure
Prof. Ohad Medalia, Department of Biochemistry, University of Zurich Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
- Research Infrastructure
Prof. Lars Schäfer, Ruhr University, Bochum Germany (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
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
- Exchange of personnel
Prof. Enrica Bordignon, Ruhr University Bochum Germany (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
- Research Infrastructure
- Exchange of personnel
Prof. Jonas Barandun, Umeå University Sweden (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Research Infrastructure
- Exchange of personnel
Prof. Peter Sander, Institute of Medical Microbiology, University of Zurich Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
- Research Infrastructure
- Exchange of personnel

Scientific events

Active participation

Title Type of contribution Title of article or contribution Date Place Persons involved
Gorden Research Conference on Tuberculosis Drug Discovery and Development Talk given at a conference Siderophore import by an unusual mycobacterial ABC exporter 24.06.2019 Castelldefels, Spain Seeger Markus;
Mechanisms of Membrane Transport - Gordon Research Conference Poster Conformational trapping of a heterodimeric ABC exporter by synthetic nanobodies 25.06.2017 Boston, United States of America Seeger Markus;


Communication with the public

Communication Title Media Place Year
New media (web, blogs, podcasts, news feeds etc.) Transportproteine vom Transport abhalten Presseinformation Ruhr Universität Bochum International 2019

Awards

Title Year
ERC consolidator grant 2017

Associated projects

Number Title Start Funding scheme
183382 A state-of-the-art detector for cryo-electron microscopy 01.06.2019 R'EQUIP
144823 Understanding multidrug efflux at a molecular level 01.06.2013 SNSF Professorships
126338 Targeting multidrug efflux proteins: Studying transport processes across the membrane using designed ankyrin repeat proteins and antibody fragments 01.10.2009 Ambizione
144520 Structure-function studies of heterodimeric ABC transporters 01.10.2012 Ambizione
188817 Molecular mechanisms of bacterial multidrug efflux 01.10.2019 Project funding (Div. I-III)

Abstract

Uptake and extrusion of molecules across the lipid bilayer are vital cellular processes mediated by transmembrane proteins. Despite their physiological importance, membrane transporters are poorly characterized at the biochemical and structural level.ATP binding cassette (ABC) exporters and major facilitator superfamily (MFS) transporters belong to two large transporter superfamilies and pump substrates against their concentration gradient by harnessing the energy of ATP hydrolysis and the proton motive force, respectively. ABC exporters are closely related at the sequence level and share the same structural fold. However, their substrate specificities and physiological functions are remarkably diverse. Because only few ABC exporter structures exist, molecular determinants of substrate specificity remain largely elusive. In pathogenic bacteria, ABC exporters pump antibiotics and other noxious substances out of the cell and thereby mediate multidrug resistance. The molecular mechanism by which drug efflux pumps are capable of extruding a myriad of different compounds remains ill-defined. Transport processes play also a vital role in the uptake of essential nutrients such as iron and the export of lipids from the cytoplasm. M. tuberculosis depends on its iron siderophores mycobactin and carboxymycobactin to replicate in macrophages. Mycobactin-mediated iron uptake requires the heterodimeric ABC exporter IrtAB, but the actual substrate recognized by this transporter remains to be elucidated. Mycobacteria synthesize glycosylated lipids called lipoarabinomannans (LAMs), which need to be incorporated into the complex mycobacterial cell wall in a process that is crucial for virulence of M. tuberculosis. The MFS transporter Rv1410 and the liproprotein LprG are responsible for LAM incorporation by a mechanism that remains elusive.By combining protein biochemistry, X-ray crystallography, molecular microbiology and bacterial genetics we will obtain novel insights into:1) The molecular mechanism of heterodimeric ABC exporters by solving the structure of TM287/288 in its outward-facing and/or outward-occluded state and by studying the functional role of its extracellular gate. 2) Molecular hallmarks of multidrug ABC exporters by investigating seven closely related heterodimeric ABC exporters of E. faecalis of which only a subset is capable of multidrug efflux.3) The functional role of the mycobacterial ABC exporter IrtAB and its attached siderophore interaction domain in the transport and reduction of iron bound to mycobactin and carboxymycobactin.4) The molecular interplay between the MFS transporter Rv1410 and the liproprotein LprG to display LAMs at the cellular surface of Mycobacteria.In summary, our work will shed light on the molecular mechanisms of transporters responsible for drug efflux, iron uptake and lipid transport in pathogenic bacteria.
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