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Jacobs University Project – Satya Prathyusha Bhamidimarri

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Antibiotic Translocation

Fellow: Satya Prathyusha Bhamidimarri

Supervisors: Mathias Winterhalter, Ulrich Kleinekathöfer (Jacobs University Bremen)

Co-supervisor: Jean-Marie Pagès (Aix Marseille Université), Niels Fertig (Nanion), Bert van den Berg (Newcastle University)

bhamidimarri_picture-2015The CymA porin from Klebsiella oxytoca is responsible for passive cyclodextrin uptake. Passive uptake of such large molecules by a porin channel is very much surprising and we subsequently investigated transport properties at single molecular level. Based on the high resolution crystal structure obtained in Newcastle the theory group at JUB-UK performed all-atom modelling to obtain a complete picture: Cyclodextrin binds to the extracellular side of CymA with its plane parallel to the membrane, tilts vertically and glides through the channel. At the periplasmic side there is a flexible loop which acts as a gate preventing free flow of molecules but allows the transport of cyclodextrin through the channel.

In addition to a general understanding CymA might be used as a potent new type of biosensor. Cyclodextrin may be bound covalently at the entrance which allows in developing a specific cyclodextrin based sensor for small molecules. Overall this will provide a sound understanding on molecular transport across channels.

Using electrophysiology and complementary techniques, we characterize the influx of antibiotics or efflux pump blockers. Here we could finish a first investigation ahead of time. For Campylobacter we are finalizing a ms. whereas for Providencia we have a ms. just accepted and one under review. We identified a further bacterial channel with the unusual property to allow passive diffusion of large carbohydrates like alpha-cyclodextrin. The objective during the first period was to elucidate the underlying mechanism of transport of such a bulky molecule through the nanopore and to exploit this to further understand the biophysics behind this which will aid in understand the transport of molecules across a porin in general.

satya-1In Gram negative bacteria the outer membrane acts as a selective uptake barrier. It contains protein channels (porins) which provide an entry pathway for hydrophilic molecules like small nutrient molecules and β-lactam antibiotics. Understanding uptake of these molecules via porins is vital to comprehend the transport mechanism across the cell membrane. Electrophysiology forms a promising approach to study the permeation of molecules across outer membrane and thereby understanding molecular interactions with the channel. However effects due to external applied voltage on the molecular permeation in porins remain unclear in this technique. Voltage effects are even prominent on neutral molecules which are related to the presence of electro osmosis flow. Here we present biophysical characterization of CymA from K.oxytoca which has the ability to take up cyclodextrin (neutral molecules). Detailed single channel analysis revealed inherent asymmetric gating characteristics of the channel. And also voltage dependent interaction of neutral molecule (cyclodextrin) with CymA presumed due to Electro osmosis.
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To further elucidate these effects, substrate interaction studies are performed under various external conditions. To obtain an atomistic view we complement our studies with all-atom molecular dynamics simulation to study the flow of water and also its directionality in the channel in the absence of substrate and in the presence as well. For the first time we have explicitly shown the existence of such an effect in the outer membrane uptake channel-