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Alessia successfully defended her PhD at Jacobs University (Bremen, Germany) on July the 14th, 2017!
Here is a summary.
Novel approaches to identify small molecules modulating E.coli TolC protein function
The urge of new strategies to overcome the world wide health problem of antibiotic resistance induced researchers as well governmental and private institution to come together to increase the medical and scientific knowledge on this topic.
This PhD project is part of the ITN Translocation, whose aim was to investigate the molecular and cellular mechanism at the basis of influx and efflux processes in gram-negative bacteria. The work behind the scientific advances reached during my 3-year-journey has been accomplished mainly between the Fraunhofer IME ScreeningPort in Hamburg and the Jacobs University Bremen, enriched by external collaborations with the University of Frankfurt and the Helmholtz Center for Infection Research.
Through an interdisciplinary approach, from in silico studies to biophysical characterization in vitro, small molecules hits to be developed as efflux pump inhibitors (EPIs) were identified. In particular, these compounds were shown to bind TolC, part of the major efflux systems in E.coli, in docking studies targeting an acidic pocket present in the periplasmic tip of the channel; to selectively bind TolCWT against a recombinant version, where key residues in the target site are mutated, in a biophysical setup allowing the determination of binding constant; to modulate ion current in reconstituted TolCWT in single-channel electrophysiology measurements.
Overall, the findings reported in this PhD thesis increase the knowledge of the biophysical characteristics of TolC through the use of cutting-edge methods and technologies, in particular in the field of membrane channels, and allowed the identification of promising compounds hits to support the development of EPIs to be employed as adjuvants in antimicrobial therapies.
Satya successfully defended her PhD at Jacobs University on May 17th, 2017!
“Structure-function relation and transport across Gram-negative outer membrane channels investigated by Electrophysiology”.
The outer membrane (OM) of Gram-negative bacteria contains channels involved in small molecule uptake and information exchange. Based on the energy used for the molecular transport, these channels are classified as passive diffusion channels and active transporters. For selective uptake of molecules throughtranslocation, channels display various structural features such as long extracellular loops, residue constellations near their constriction region and various periplasmic N-terminus extensions.
This thesis focusses on two main concepts: the structure-function relationship of OM channels and the translocation of molecules through these channels. For the investigated passive diffusion channels and active transporters, the role of N-termini in their structure-function relationship was studied using electrophysiology. Furthermore,translocation was probed for both charged and uncharged molecules.
The existence and role of electro-osmosis in substratetranslocation is the most exciting result, along with the gating behaviour of the N-terminus of the uncharged cyclodextrin specific channel CymA from Klebsiella oxytoca. Moreover, the electro-osmosis phenomenon is not bound to this specific channel.
For the putative channel DcaP from Acinetobacter baumannii, its existence as a trimeric channel and exclusive anion selectivity was revealed. It possesses a long N-terminus and its role in the uptake of charged dicarboxylic acids was established.
For the SusCD protein complex involved in the glycan metabolism of human gut bacteria, the role of N-terminus as a plug, occluding the SusC transporter was established. The structure-function relation between two interacting proteins was also elucidated and coined as pedal-bin mechanism.
For the efflux protein OM component TolC, its channel opening was explained at extreme applied voltages.
Overall, the results of my doctoral study encompass conclusions on the role of N-termini, the presence and role of electro-osmosis, the uptake of charged dicarboxylic acids, a novel mechanism for inter-protein interactions, and even on channel opening mechanisms.
On June 16th, 2017 Vincent Trebosc successfully defended his thesis at Bioversys!
Antimicrobial resistance is a serious threat for public health worldwide. The risk to enter a post-antibiotic era has been raised due to the emergence of pathogens resistant to most of available antibiotics. Beside research on novel antibiotics, innovative approaches may provide another solution to combat infections. One of the innovative approach resides in adjuvant therapies that have the capability to revert antibiotic resistances. This approach has been successfully applied during decades with β-lactams inhibitors but never expanded to other antibiotics. My work aimed to investigate the adjuvant therapy approach in Acinetobacter baumannii and Mycobacterium tuberculosis, which are two pathogens of great importance with constantly increasing drug resistance rates.
The development of a genome engineering method allowed us to characterize the different mechanisms employed by A. baumannii to resist the antibacterial action of tigecycline and colistin antibiotics during patient treatment. Overall, we showed that the mechanisms employed to resist the action of a specific antibiotic may be diverse due to the treatment history of the individual clinical strains. This impairs the development of adjuvant drug to overcome tigecycline and colistin resistances in A. baumannii and it implies a careful evaluation of drug target relevance before to develop inhibitors that are specific of one resistance pathway. Nevertheless, our data highlight phosphoethanolamine transferase enzymes as attractive targets to restore polymyxin sensitivity in A. baumannii.
Furthermore, on M. tuberculosis, we successfully demonstrated that adjuvant approaches have the potential to overcome antibiotic resistance. By designing a synthetic mammalian gene regulation system, we assisted the
development of EthR2 inhibitory compounds that boost the newly discovered M. tuberculosis ethionamide bioactivation pathway. These compounds not only revert ethionamide resistance in MDR M. tuberculosis, but also boost the ethionamide efficacy in drug susceptible strains, rendering ethionamide into a more potent antibiotic. These adjuvant compounds are very promising chemical entities and they are currently in pre-clinical development.
The research performed in this thesis demonstrated that special attention should be paid to drug target evaluation. The non-essential nature of these drug targets promotes a higher diversity and heterogeneity and careful target validation is required before starting a drug development program. However, the successful development of ethionamide boosting compounds that also switch off bacterial resistance traits demonstrates the potential of novel therapeutic adjuvant approaches.
This project has received funding from the European Union’s Seventh Framework Programme for research, technological development and demonstration under grant agreement no 607694.