Project

Discipline

Membrane proteins; Multidrug efflux pumps; ABC transporter; Pathogenic bacteria; Antibiotic resistance; Enterococcus faecalis; Single particle cryo-EM; Structure-based biochemistry; Electron paramagnetic resonance spectroscopy; Deep mutational scanning

Lead
Molekulare Einblicke in Antibiotikapumpen pathogener BakterienWerden Bakterien mittels Antibiotika bekämpft, reagieren diese durch eine erhöhte Produktion von Transportproteinen, welche Antibiotika aus der Bakterienzelle herauspumpen. Durch das gezielte Anschalten solcher Antibiokapumpen erlangt das Bakterium Antibiotikaresistenz und es gewinnt Zeit, um weitere Resistenzen zu erlangen. Dieses Projekt untersucht die molekularen Grundlagen einer Antibiokapumpe aus dem pathogenen Bakterium Enterococcus faecalis mit dem Ziel, den Transportmechanismus besser zu verstehen.
Lay summary
Inhalt und Ziel des Forschungsprojekts Wir untersuchen eine Antibiokapumpe aus dem pathogenen Bakterium Enterococcus faecalis. Dabei handelt es sich um ein Transporterprotein mit dem Namen EfrCD, welches zu den sogenannten ABC Exportern gehört. ABC Exporter werden durch die Spaltung von ATP angetrieben, um Transportsubstrate (in unserem Falle Antibiotika) gegen einen Konzentrationsgradienten zu pumpen. Im Rahmen des Projektes werden wir die dreidimensionale Struktur von EfrCD mittels Elektronenmikroskopie lösen. Mithilfe einer neuen Methode namens „Deep mutational scanning“ werden wir zudem eine Mutantenbibliothek von EfrCD anlegen, welche in einem effizienten Verfahren auf Erhöhung resp. Erniedrigung der Pumpaktivität getestet wird. Die daraus gewonnenen Erkenntisse werden schliesslich mittels biochemischer Analysen von Einzel- oder Kombinationsmutanten weiter verfeinert und detailliert ausgeleuchtet. Das übergeordnete Ziel ist es, die molekularen Mechanismen von Antibiotikapumpen besser zu verstehen.   Wissenschaftlicher und Gesellschaftlicher Kontext des Forschungsprojektes Antibiotikaresistenz ist ein drängendes Problem unserer Zeit und bedroht die Errungenschaften der modernen Medizin. Dieses Grundlagenforschungsprojekt wird wichtige neue Erkenntnisse zum molekularen Mechanismus von Antibiotikapumpen liefern. Ein wichtiges Merkmal von Antibiotikapumpen ist deren Eigenschaft, chemisch sehr unterschiedliche Antibiotika aus der Zelle herauszupumpen. Dieses Projekt möchte insbesondere Licht auf die noch ungeklärte Frage werfen, wie verschiedene Antibiotika durch ein und dieselbe Antibiotikapumpe erkannt und transportiert werden.

Direct link to Lay Summary

Last update: 01.10.2019

Natural persons

Number	Title	Start	Funding scheme

Overexpression of multidrug efflux pumps is a common phenotypic trait of pathogenic bacteria to gain resistance against antibiotics. Despite of rapid progress in the structural elucidation of drug efflux pumps, the molecular determinants of multidrug recognition and transport are only partially understood.In recent years, my lab has identified a set of heterodimeric ABC exporters stemming from the opportunistic pathogen Enterococcus faecalis, which confer resistance against a variety of drugs. These transporters harness the energy of ATP binding and hydrolysis at a pair of nucleotide binding domains (NBDs) to efficiently pump noxious compounds out of the bacterial cell via the transmembrane domains (TMDs). Here I propose to study one of these transporters - called EfrCD - at the functional and structural level by a unique combination of experimental techniques. EfrCD exhibits a particularly strong drug efflux activity when expressed in the model organism Lactococcus lactis and it can be purified as monodisperse protein for structural and biochemical analyses. This makes EfrCD a uniquely suitable model protein to investigate the molecular basis of multidrug efflux.We will apply the method of deep mutational scanning (DMS) to characterize the physicochemical properties of amino acid side chains located in the NBDs and in the drug permeation pathway. In DMS, a library of single mutations is generated, which allows to investigate the entire amino acid space in a defined set of amino acid positions. We have already created and validated a DMS library for EfrCD comprising a total of 81 amino acid positions (14 in the NBDs and 67 in the TMDs). The library is expressed in L. lactis and the cells are subjected to a competitive growth experiment in the presence of drugs. In this manner, mutants that confer higher resistance than the wildtype protein are enriched whereas mutants with decreased drug efflux activity are depleted. The relative abundance of mutants is analyzed by next generation sequencing of the DMS library prior and after the selection experiment. A preliminary dataset for a single drug revealed that around 10 % of the substitutions result in transporter variants exhibiting increased drug efflux activity, whereas around 60 % showed decreased activity. The mechanistic underpinning of selected gain and loss of function mutants will be thoroughly studied at the functional level in vivo as well as at the biochemical level with reconstituted transporter in vitro. To investigate the biophysical basis of substrate promiscuity, DMS selections will be performed in the presence of different drugs. Finally, the DMS analysis will be extended to four heterodimeric ABC exporters to identify similarities and differences among closely related drug efflux pumps. In order to distinguish DMS mutants that directly affect drug recognition from those which alter drug efflux activity allosterically, we aim to solve high resolution structures of EfrCD in complex with different drugs. Preliminary cryo-EM analyzes of nanodisc-reconstituted EfrCD suggest that this aim is feasible. Double electron electron resonance (DEER) will be used to study conformational changes and equilibrium shifts of EfrCD caused by drug binding or the introduction of mutations identified by DMS. To investigate the transport cycle of this transporter directly in native membranes, we will generate natural and synthetic nanobodies against EfrCD and use them as spin-labeled affinity reagents. To directly study drug-transporter interactions, spin-labeled drugs will be synthetized.In summary, our integrative experimental approach will shed new light on promiscuous substrate recognition and transport of multidrug efflux pumps and will allow us to identify molecular hallmarks required for drug efflux activity.