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Project Objectives and Results

 

figura-silvia-1Multidrug resistant (MDR) bacteria are ubiquitous in both hospitals and the larger community. Moreover, MDR is not restricted to bacteria but also relevant for drugs against parasite and in cancer chemotherapy. The dissemination of MDR Escherichia coli in US, the recent NDM-1 mechanism involved in the carbapenem resistance reported in various enterobacterial infections in Europe, and the resurrection of tuberculosis are examples demonstrating the risk associated with evolved drug resistance. Unfortunately many pharmaceutical companies abandoned this field and no truly novel active antibacterial compounds are currently in clinical trials. Obviously new antibacterial molecules and novel strategies to combat MDR bacteria are needed.
Several experimental findings suggest that among other factors porins and efflux pumps are involved in MDR. The current bottleneck is in addition to toxicity is the low effective permeability of the drugs. In Enterobacteriaceae such as E. coli, E. aerogenes, K. pneumonia and Salmonella enterica several genes are involved in the control of membrane permeability and consequently in the emergence of resistant isolates and in the dissemination of a multi-drug resistant phenotype.

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For example, enterobacterial MDR strains exhibit a reduction of functional outer membrane porins by decreasing or even by a complete shutdown of their biosynthesis, or they express altered porins combined with a high expression of efflux systems. The combined action of these mechanisms during an infection confers a significant decrease in bacterial susceptibility to antibiotherapy. MDR Pseudomonas aeruginosa and Acinetobacter employ similar mechanisms of antibiotic resistance. Infections with these MDR strains have been increasing severely in hospitals. Obviously, antibiotic resistance depends on a multitude of parameters; here we restrict ourselves on the membrane permeability only. To this end we focus on the modulation of the in- and efflux of ß-lactam and fluoroquinolone antibiotics across the cell wall of bacteria belonging to the family of Enterobacteriaceae as well as Pseudomonas aeruginosa, Salmonella, Vibrio cholerae or Acinetobacter.
Here we apply new technologies to quantify rate limiting steps involved in antibiotic penetration and active pumping out. Our whole cell approach identifies bottlenecks of existing antibiotics at a cellular level. Our findings might contribute to novel antibiotic therapy. As a novel approach, we will quantify the flux of antibiotics across porin channels and active transporters and within a systems biology approach we will connect the individual flux with the global genetic regulation of membrane permeability. The following technological developments render this proposal timely: the increasing number of available high resolution structures and new algorithms combined with powerful supercomputers allows molecular modeling to simulate the kinetics of the translocation pathway with atomistic precision and to identify rate limiting interactions of drugs with particular amino acids inside the channel or transporter.

 

fabio-3High resolution electrophysiology makes it possible to follow the passage of individual antibiotic molecules across a single porin channel and to understand the molecular adaptation used by bacterial cells faced with the environmental stresses. In addition, new rational chemical syntheses based on determination of pharmacophoric molecular groups have been reported allowing the production of molecules exhibiting appropriate functionalities to alter the activity of efflux pumps or to facilitate the drug penetration. Moreover, novel highly sensitive approaches for protein identification and absolute quantification enable systematic analysis of membrane proteomes as a basis for systems biology approaches for quantitative comprehensive modeling of transport across the cell envelope. We expect that a combination of these novel techniques allows us to determine rate limiting steps in antibiotic action.