Final Report Summary - TRANSLOCATION (MOLECULAR BASIS OF ANTIBIOTIC TRANSLOCATION)
Scientific coordinator: Mathias Winterhalter, Professor of Biophysics, Jacob University Bremen,
D-28759 Bremen; m.winterhalter@jacobs-university.de
Phone +49 421 200 3248 FAX +49 421 200 3249
Web-page www.itn-translocation.eu
Drug-resistant pathogens, in particular Gram-negative bacteria, are becoming increasingly pervasive. The Outer Membrane in these bacteria represents a physical bottleneck for any antibiotics to reach a potential target. The aim here is to investigate the molecular and cellular mechanisms at the basis of the influx and efflux processes.
A particular strength of this ITN is the integration of the young fellows into a private-public partnership project. Within the New Drugs For bad Bugs platform, a larger European Network within the Innovative Medicine Initiative (IMI) wherein five major pharmaceutical companies defined a number of current bottlenecks in the antibiotic development. In close collaboration with 27 organizations (SME, large companies, universities and research centers) the Translocation consortium is searching for solutions to increase antibiotic concentration inside bacteria. Within this ITN project we integrate twelve young scientists into the larger target driven project and train young scientists with a different scientific background to go beyond the classical faculty border. Bridging nanotechnology, physics, chemistry, computer modelling, pharmacology and microbiology together will facilitate the transfer of expertise acquired within the network in both academia and industry.
The first research project is on permeation of small molecules across membrane channels. For example 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. The Newcastle group obtained a high resolution crystal structure which allowed the theoretical group at JUB-UK to perform all-atom modeling to understand the transport process: 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.
A further project is to find general pattern of molecules able to translocate through closely related porins. For example we apply all-atom MD modeling to OmpF and OmpC to elucidate the effect of hydrated water on the electric field inside the channel. The inner electric field interact with the dipole of the antibiotic molecules and force the penetrating molecule into a particular orientation. Depending on the structure of the drug this inner field might hinder the drug to penetrate the channel constriction zone.
Prior performing MD modelling high resolution structure are necessary. The main aim of the project at Newcastle is to study and understand the antibiotic permeation in the OmpF/C-like outer membrane proteins OmpU and OmpT of Vibrio cholera. The group at St. Andrews is devoted to determine the structure of MOMP and Omp50 porins from the pathogen Campylobacter jejuni and Omp35, Omp36 and Omp37 porins from the pathogen Enterobacter aerogenes. To do so, all the proteins have been expressed in a suitable system, a purification protocol have been optimized and the purified proteins have been crystallized.
A further task is devoted to efflux pumps. We have selected 6 RND multidrug efflux transporter genes from S. typhimurium and C. jejuni for proof-of-principle of a streamlined, time-saving and economical approach to identify stable protein candidates to enter the larger production and purification. Twelve constructs are synthesized in E. coli as a GFP fusion protein using the versatile FX-cloning procedure. Expression yield and stability within different detergents are directly visualized in crude E. coli detergent extracts. From the initial 6 cloned genes, we obtained 3 pure and stable RND efflux transporters, of which two produced crystals in the initial crystallization screen. These results are very promising towards the elucidation of RND efflux transporter structure, which will yield valuable data for the design of new antibiotics/inhibitors in the fight against multiple drug resistance.
The high resolution structure serves as input for all-atom modelling on the efflux pumps of the resistance-nodulation-division (RND) family, the major transporter superfamily responsible for the appearance of multi drug resistance. We are performing a comparative study of transporters of the RND family showing different substrate specificity, i.e. AcrD and AcrB of E. coli and MexY and MexB of P. aeruginosa. For instance, AcrD and MexY transport aminoglycosides but AcrB and MexB do not. The results of this comparative investigation will offer insights into the microscopic details of the functioning of the efflux systems and, when mapped onto the chemical structures of the compounds considered in the present study, will possibly help the design of molecules able to escape and/or to inhibit the efflux systems.
At Fraunhofer IME the main target is TolC, outer membrane protein of the efflux system AcrAB-TolC present in E. coli. Addressing TolC, the goal is to identify small molecules that can modulate the transport through the channel, interfering with the functionality of the complex. Using a structure-based characterization of the protein, we selected the periplasmic site of TolC as a target for virtual screening campaigns and compound libraries for database setup. The outcome will be analyzed and the selected hits will be tested in cell-free based in vitro assays to confirm and characterize compounds activity. In a collaboration between the European Screening Port/ IME Fraunhofer institute and both ESRs at Jacobs University we are working on a joint project with electrophysiology, all-atom MD simulation and docking studies on TolC.
The Biozentrum group in Basel set up a highly sensitive and accurate Selective Reaction Monitoring technique using heavy isotope labeled peptides to obtain in vivo absolute quantification of the outer membrane proteome of Salmonella i.e. the copy number per cell. This targeted proteomics technique enables the envelope characterization of different subsets of Salmonella mutants in various environments and will be used as a guide to optimize in vitro conditions that mimic the in vivo Salmonella envelope properties.
We designed a novel gene knockout platform, which does not require any antibiotic resistance marker, to be able to do gene deletions in Gram negative multidrug resistant clinical isolates of the ESKAPE pathogens. We applied this system and validated the transcriptional regulator AdeR, suggested to be essential in the tigecycline resistance pathway of Acinetobacter baumannii, as a putative drug target. We confirmed the important role of AdeR in conferring tigecycline resistance by upregulation of the efflux pump AdeABC. However, we demonstrated that targeting AdeR is not sufficient to switch-off tigecycline resistance in clinical isolates to rejuvenate the activity of this antibiotic. Our work clearly devalidated AdeR as a potential drug target. We currently use the developed knockout technology to validate several transcriptional regulators involved in diverse antibiotic resistance pathways as putative drug targets.
The unique comination of microbiology, structural biology and all-atom modelling helped us to understand the permeability of small molecules across the Gram-negative bacteria. Our twelve fellows worked at the interface of disciplines and performed secondments in other research labs as well as in SMEs.
D-28759 Bremen; m.winterhalter@jacobs-university.de
Phone +49 421 200 3248 FAX +49 421 200 3249
Web-page www.itn-translocation.eu
Drug-resistant pathogens, in particular Gram-negative bacteria, are becoming increasingly pervasive. The Outer Membrane in these bacteria represents a physical bottleneck for any antibiotics to reach a potential target. The aim here is to investigate the molecular and cellular mechanisms at the basis of the influx and efflux processes.
A particular strength of this ITN is the integration of the young fellows into a private-public partnership project. Within the New Drugs For bad Bugs platform, a larger European Network within the Innovative Medicine Initiative (IMI) wherein five major pharmaceutical companies defined a number of current bottlenecks in the antibiotic development. In close collaboration with 27 organizations (SME, large companies, universities and research centers) the Translocation consortium is searching for solutions to increase antibiotic concentration inside bacteria. Within this ITN project we integrate twelve young scientists into the larger target driven project and train young scientists with a different scientific background to go beyond the classical faculty border. Bridging nanotechnology, physics, chemistry, computer modelling, pharmacology and microbiology together will facilitate the transfer of expertise acquired within the network in both academia and industry.
The first research project is on permeation of small molecules across membrane channels. For example 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. The Newcastle group obtained a high resolution crystal structure which allowed the theoretical group at JUB-UK to perform all-atom modeling to understand the transport process: 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.
A further project is to find general pattern of molecules able to translocate through closely related porins. For example we apply all-atom MD modeling to OmpF and OmpC to elucidate the effect of hydrated water on the electric field inside the channel. The inner electric field interact with the dipole of the antibiotic molecules and force the penetrating molecule into a particular orientation. Depending on the structure of the drug this inner field might hinder the drug to penetrate the channel constriction zone.
Prior performing MD modelling high resolution structure are necessary. The main aim of the project at Newcastle is to study and understand the antibiotic permeation in the OmpF/C-like outer membrane proteins OmpU and OmpT of Vibrio cholera. The group at St. Andrews is devoted to determine the structure of MOMP and Omp50 porins from the pathogen Campylobacter jejuni and Omp35, Omp36 and Omp37 porins from the pathogen Enterobacter aerogenes. To do so, all the proteins have been expressed in a suitable system, a purification protocol have been optimized and the purified proteins have been crystallized.
A further task is devoted to efflux pumps. We have selected 6 RND multidrug efflux transporter genes from S. typhimurium and C. jejuni for proof-of-principle of a streamlined, time-saving and economical approach to identify stable protein candidates to enter the larger production and purification. Twelve constructs are synthesized in E. coli as a GFP fusion protein using the versatile FX-cloning procedure. Expression yield and stability within different detergents are directly visualized in crude E. coli detergent extracts. From the initial 6 cloned genes, we obtained 3 pure and stable RND efflux transporters, of which two produced crystals in the initial crystallization screen. These results are very promising towards the elucidation of RND efflux transporter structure, which will yield valuable data for the design of new antibiotics/inhibitors in the fight against multiple drug resistance.
The high resolution structure serves as input for all-atom modelling on the efflux pumps of the resistance-nodulation-division (RND) family, the major transporter superfamily responsible for the appearance of multi drug resistance. We are performing a comparative study of transporters of the RND family showing different substrate specificity, i.e. AcrD and AcrB of E. coli and MexY and MexB of P. aeruginosa. For instance, AcrD and MexY transport aminoglycosides but AcrB and MexB do not. The results of this comparative investigation will offer insights into the microscopic details of the functioning of the efflux systems and, when mapped onto the chemical structures of the compounds considered in the present study, will possibly help the design of molecules able to escape and/or to inhibit the efflux systems.
At Fraunhofer IME the main target is TolC, outer membrane protein of the efflux system AcrAB-TolC present in E. coli. Addressing TolC, the goal is to identify small molecules that can modulate the transport through the channel, interfering with the functionality of the complex. Using a structure-based characterization of the protein, we selected the periplasmic site of TolC as a target for virtual screening campaigns and compound libraries for database setup. The outcome will be analyzed and the selected hits will be tested in cell-free based in vitro assays to confirm and characterize compounds activity. In a collaboration between the European Screening Port/ IME Fraunhofer institute and both ESRs at Jacobs University we are working on a joint project with electrophysiology, all-atom MD simulation and docking studies on TolC.
The Biozentrum group in Basel set up a highly sensitive and accurate Selective Reaction Monitoring technique using heavy isotope labeled peptides to obtain in vivo absolute quantification of the outer membrane proteome of Salmonella i.e. the copy number per cell. This targeted proteomics technique enables the envelope characterization of different subsets of Salmonella mutants in various environments and will be used as a guide to optimize in vitro conditions that mimic the in vivo Salmonella envelope properties.
We designed a novel gene knockout platform, which does not require any antibiotic resistance marker, to be able to do gene deletions in Gram negative multidrug resistant clinical isolates of the ESKAPE pathogens. We applied this system and validated the transcriptional regulator AdeR, suggested to be essential in the tigecycline resistance pathway of Acinetobacter baumannii, as a putative drug target. We confirmed the important role of AdeR in conferring tigecycline resistance by upregulation of the efflux pump AdeABC. However, we demonstrated that targeting AdeR is not sufficient to switch-off tigecycline resistance in clinical isolates to rejuvenate the activity of this antibiotic. Our work clearly devalidated AdeR as a potential drug target. We currently use the developed knockout technology to validate several transcriptional regulators involved in diverse antibiotic resistance pathways as putative drug targets.
The unique comination of microbiology, structural biology and all-atom modelling helped us to understand the permeability of small molecules across the Gram-negative bacteria. Our twelve fellows worked at the interface of disciplines and performed secondments in other research labs as well as in SMEs.