Harsha Bajaj

• The outer membrane of bacteria forms an effective barrier; hence hydrophilic antibiotics are known to use porin pathway to reach the target inside the bacterium. With the overuse of antibiotics in clinics, bacteria have evolved to become resistant and one of the ways to do so is to reduce the intake of antibiotics. Increasing antibiotic resistance is recognized as a major public health threat. Antimicrobial drug discovery faces unique challenges, primarily to produce compounds which have sufficient permeation across outer membrane of bacteria. In this regard it becomes indispensable to measure the translocation rate and understand the molecular mechanism of antibiotic transport through porin channel. In this thesis we investigate two aspects: one to study antibiotic transport across clinically relevant porins and understand their contribution in antibiotic resistance; Second, to elucidate the translocation mechanism of the relevant antibiotics across porins from Gram-negative and Gram-positive bacteria. In the first part, we studied antibiotic translocation across, major porin from pathogen Providencia stuartii and porin mutants from Escherichia coli clinical strain. A multidisciplinary approach, including single channel electrophysiology, liposome permeation assay, microbiological assays and molecular dynamic simulations, provided an understanding of permeability from macro scale to atomic scale. In the second part of the thesis, we mainly investigate the interaction of β-lactam and fluoroquinolone class of antibiotics with porins from E. coli and Nocardia farcinica at a single molecule level. An assay based on fluorescence detection in vesicles in microfluidic chambers is employed to obtain translocation rate of fluoroquinolone antibiotic across porins in a quantitative manner. Modulating parameters like pH significantly changes the kinetics of norfloxacin through OmpF in electrophysiology. Voltage dependent transport of antibiotic norfloxacin through single OmpF is reported and we discuss different contributions involved in the translocation of antibiotic through channel. We conclude that altered permeability across porins contributes to reduced susceptibility. By varying different parameters, like effect of point mutations in antibiotic translocation through channels or varying the charge state of antibiotic, we decipher the rate limiting interactions which might be useful for rational drug designing.