Eshita Paul

• Antibiotic resistance poses severe threats on mankind and ruins the greatest of medical advancements. At the economic level, it not only causes loss of productivity but also creates huge burden on healthcare expenses. Regarding antibiotic resistance Gram-negative bacteria especially the ESCAPE pathogens are in the limelight as they are responsible for the highest mortality rates. Their multidrug resistance results from a combination of factors including modification of antibiotic uptake pathway as well as decreased intracellular concentration of drugs. Reduced intracellular drug retention is a result of drug elimination and is mostly driven by RND-family of efflux pumps. AcrAB-TolC is a prototype of this family in E. coli. TolC being the outer membrane component of this efflux machinery is readily available for reconstitution on synthetic lipid bilayers and thus allows substrate interactions to be studied at a single channel level. In the present study, biophysical approach to identify small chemical molecules as TolC inhibitors is described. In order to observe their interaction with TolC at a single molecule level, traditional electrophysiology technique has been used and method’s resolution is extended using principles of protein engineering as well as novel data analysis considering also the ion current fluctuations. At the same time, porin dependent translocation of a therapeutically important class of antibiotics is studied and novel uptake pathways are reported. Although, not directly beneficial to clinical development, outcomes of these studies will help to open new ways for further research and I hope this thesis will be useful despite having many open-ended questions.