Pratik Singh

• Bacterial cell envelope acts as a first line of defense against various antibacterial compounds. Antibacterial compounds such as antibiotics and antimicrobial peptides need to penetrate the outer membrane barrier of the cell envelope to reach their target site. Hydrophilic antibiotics such as penicillins and carbapenems are known to utilize water-filled protein channels in the outer-membrane to diffuse inside the cell periplasm. Down-regulation in expression of channel proteins and mutations in important amino acid residues of the proteins channels are often induced to limit the permeation of penetrating antibiotics. This thesis highlights an interdisciplinary approach to comprehend the mechanisms of antibiotics translocation through the bacterial cell envelope. In vitro single channel electrophysiology experiments, in vivo biological assays and molecular dynamics simulation studies are combined together to obtain an atomistic and molecular detail of antibiotic permeation through the outer membrane protein channels. Antibiotics interaction with the outer-membrane proteins channels from Gram-negative bacteria such as Escherichia coli and Enterobacter aerogenes and Gram-positive bacteria Nocardia farcinica have been characterized in detail. Here, we present two fascinating aspects that could alter the interaction of antibiotic with the protein channels. First, the biophysical characteristics of membrane proteins play a significant role in determining the translocation of antibiotics. For example, we have shown how the negatively charged N. farcinica porin allow the permeation of positively charged antibiotics; however neutralizing the negatively charged residues of the pore showed a depleted interaction of positively charged antibiotics. Second, the external conditions can also drastically change the kinetics of antibiotic interaction with the channel protein. For instance, we have shown that the presence of magnesium ion in the solution can modulate the kinetics of enrofloxacin interaction with the E. coli OmpF porin. Similarly, changing the external electrolyte solution from potassium chloride to bulky ionic liquid solution can drastically slow down the kinetics of antibiotic with the channel protein. Overall, we have explored various aspects involved in understanding the permeation of hydrophilic antibiotics through the outer-membrane protein channels.