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Research Methodology and Approach

Here is a detailed description of the methodology we used in our Work Packages.

cropped-cropped-marie_curie-imi-page.jpgWork package WP1: Quantification of antibiotic influx (performed by Jacobs University, Nanion Technologies, University of Cagliari, Newcastle University, University of Saint Andrews). The influx of hydrophilic molecules across the outer membrane is largely controlled by porins, which are water-filled open channels spanning the outer membrane that allow the passive penetration of hydrophilic molecules. For example, high resolution structures for porins from E. coli indicate a conserved 16-strand trimeric beta-barrel structure. Each monomer contains a channel restricted at its midpoint by a long loop bent inside the pore. This ‘eyelet’ region, which governs the channel size and ion selectivity, has positively charged amino acid residues on one side of the lumen facing negatively charged residues on the other. This creates a strong electrostatic field that influences translocation through the porin and has a major influence on the influx of antibiotics. Mutations in this area alter the levels of susceptibility to antibiotics. ß-lactams and fluoroquinolones are the most prominent antibiotic classes in our current antibacterial arsenal and known to enter through porins. Concerning other antibiotic classes, e.g. aminoglycosides and macrolides, the diffusion process across the outer membrane remains to be quantified.
This work package is based on our ongoing collaborations and here we will mainly characterize antibiotic permeation across porins localized in the outer membrane of E. coli, Salmonella, Vibrio cholerae and Enterobacteria. High resolution electrophysiology allows now to quantify the flux through single porins. However, this technique is very time consuming requiring well trained collaborators to characterize about one compound per day. To overcome the labour intensive needs of electrophysiology in general, automatic patch clamping techniques have been recently developed by several companies including our partner Nanion Technologies. This technique is routinely applied in drug research for channel blockers and here it will be implemented by Nanion Technologies and by Jacobs University to allow now to screen the activity of larger compound libraries on reconstituted systems. On early stage researcher will perform all-atom computer simulations to identify the drug pathway through various porin channels and in particular to quantify the rate limiting interaction with the channel. Two researchers will work on determining novel high-resolution structures of porins. One of them will be also engaged in crystallization of RamA needed in WP3.

The outcome of this biophysical approach is molecular information on the influx pathways through porin channels with identification of the rate limiting residues involved in drug–channel interaction. Furthermore the aim is to obtain a commercially available robust chip to quantify translocation through channels in a High-Throughput format.

cropped-antibio.jpgWork Package 2. Understanding the active process: efflux (performed by University of Cagliari, Jacobs University, Goethe University). Efflux pumps are an important component of MDR in clinical isolates as they are often over-expressed under antibiotic therapy. The poly specificity of bacterial efflux transporters, especially those belonging to resistance-nodulation division (RND) family, contribute to the acquisition of additional mechanisms of resistance, such as mutation of antibiotic targets or production of enzymes that degrade antibiotics, and also reinforces the effects of these acquired mechanisms. To combat MDR “superbugs”, it is necessary to define the molecular bases of the efflux mechanisms. The RND efflux systems of Gram-negative bacteria are comprised of an inner membrane transporter (IMT), an outer membrane channel (OMC), and a membrane fusion protein (MFP) located in the periplasm. Despite the importance of RND efflux systems, the mechanism of drug expulsion by these transporters is still poorly understood and in part due to the persistent lack of structural data. Only for E. coli and P. aeruginosa high-resolution structures of all three components are available but the interplay between the three units including dynamical features is only slowly unravelled. Among the key elements necessary to understand the mechanism of efflux are the structure, the stoichiometry and the stability of the functional complex, the extrusion pathway(s) of antibiotics, the dynamics and the amount of structural changes coupled to drug efflux, the role of the solvent, the role of cooperativity.
The project aims at providing insights into the above question. It will benefit from the comparisons among efflux systems of different bacteria as well as from the high spatial and time resolution of the interdisciplinary techniques available in this Work Package 2 and at Aix Université Marseille and Fraunhofer IME ScreeningPort concerning inhibitors and in general with Work Package 3 concerning biochemical and genetic data. The experience/protocols developed by WP2 for structural and dynamical features characterizing the functioning of RND efflux systems in E. coli and P. aeruginosa will be a very valuable starting point for the project that will extend the strategies to other RND systems of E. aerogenes, K. pneumoniae, Salmonella sp., and Campylobacter sp. The structures of the pumps from several organisms solved by the other units within the WP will constitute a solid base for comparison and functional prioritization analysis. Emphasis will be put on the co-crystallization of IMT (RND) components from different organisms with drug substrates and inhibitors (from Aix Marseille Université in Work Package 3). Furthermore, the MFP and OMC subunits will be analysed via X-ray crystallography and computational studies. Within this Work Package computational state-of-the-art approaches might furnish details on affinity sites, inventory of the key interactions, role of the solvents offering a valid base to rationalize the structural and biochemical data acquired by the other units.

The outcome of the Work Package 2 is the knowledge on the substrate recognition and function of efflux pumps, especially focusing on the assembly of the entire systems and on the interaction with substrates.

rnd_bigWork package 3: Development of an in vitro assay that mimic envelope permeability, expression and regulation of bacterial membrane proteins (performed by Fraunhofer IME ScreeningPort, University of Basel, Aix Marseille Université, BioVersys).
Current whole-cell assays for screening antimicrobials rely on standardized, well-accepted in vitro conditions. Although useful, such conditions may not fully reproduce relevant conditions that pathogens encounter in infected host tissues. Gram-negative bacteria such as Pseudomonas readily adapt to different conditions by comprehensively remodelling their cell envelope properties such as differential expression of one or more of the some 30 porins, induction of one or more of their ~20 efflux pumps, or modifications to the lipopolysaccharide. This envelope remodelling can substantially affect envelope penetration of antimicrobials. As a consequence, antimicrobials with promising activity under standard in vitro conditions might fail under relevant in vivo conditions because of insufficient penetration or increased expulsion/degradation in that environment.

The first outcome of Work Package 3 is how and how fast bacterial pathogens adapt their membrane permeability to host conditions and to treatment with certain antibiotics in vivo and in vitro. This knowledge allows clinicians to achieve a maximum efficiency by adopting dosages/combinations depending on the drug transporters expressed. A second outcome is the identification of the pharmacophoric groups involved in drug permeation allowing the design of new antibacterial molecules (inhibitors) or the development of diagnosis tests for the detection of the membrane-based resistance in MDR clinical isolates.