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Molecular basis of antibiotic translocation

Final Activity Report Summary - TRANSLOCATION (Molecular basis of antibiotic translocation)

Gram-negative bacteria are responsible for a large proportion of antibiotic resistant bacterial diseases. These bacteria have a complex cell envelope that comprises an outer membrane and an inner membrane that delimit the periplasm. The outer membrane contains various protein channels, called porins, which are involved in the influx of various compounds, including several classes of antibiotics. Bacterial adaptation to reduce influx through porins is an increasing problem worldwide that contributes, together with efflux systems, to the emergence and dissemination of antibiotic resistance. Within this project we investigated the molecular basis of membrane impermeability as a bacterial resistance mechanism. As a novel approach we applied high resolution electrophysiology to quantify the influx through bacterial channels at a single molecular level. Metadynamic all-atom computer modelling allowed to simulate the pathway of an antibiotic penetrating the membrane channel. Combining both allows the identification of the rate limiting interaction of the antibiotic with the channel.

Within this project we;
- Improved the time resolution of our electrophysiological set-up.
- Combining high resolution electrophysiology and all atom molecular modelling allowed us now to identify the rate limiting interaction of the drug with the channel.

- Together with our colleagues in clinical microbiology and our partner from a pharmaceutical company we characterized the transport of several ß-lactam, cephalosporin and fluoroquinolone antibiotics through E. coli and Campylobacter porins. We further isolated and characterised two porins from Providencia stuartii.

The Initial Training Network 'Molecular basis of antibiotic translocation' served to train scientists with different scientific background to go beyond the classical borders of the individual disciplines. During four year experts in nanotechnology, physics, chemistry, computer modelling, pharmacology, and microbiology worked together. In addition to the directly financed 460 man month of researcher also other group members and guests participated in this project. Moreover to integrate this network in the larger community, we initiated a COST action (BM0701 ATHENS in 2008) and to render this network more sustainable we prepared a European Master in Biophysics on Molecular Transport across Bacterial Walls. Furthermore, we annually organized and continue to organise research workshops on the topic of this proposal for about 100 participants.

To conclude the main results are:
- We elucidated the rate limiting interaction of ampicillin during the passage across OmpF from E.coli. This approach can be generalised for many other transport processes through channels (antibiotics, nutrition, substrates, peptides, proteins etc).
- We developed a novel set-up with very low background capacitance (<2pF) allowing to record very small channels with high precision.
- We provided training in electrophysiology for a large number of interested researcher outside our network.
- We provided a platform for novel techniques in electrophysiology by offering an annual workshop for more than 100 participants. Special attention was paid to integrate biologists, chemist, physicists, engineers and researcher from industry.