Antibiotic Action Beyond Simple Target Binding: Exploring Supramolecular Mechanisms of Membrane-Active Antibiotics

Abstract

Antibiotics are cornerstone drugs of modern medicine. However, their effectiveness is declining rapidly due to the emergence and spread of antimicrobial resistance. It is therefore critical that new antibiotics are developed that evade current resistance mechanisms and are additionally challenging for bacteria to become resistant to. Few candidate antibiotics have been discovered over the recent decades, and therefore we must make the most out of what we have. Unfortunately, the further development of key antimicrobial candidates is severely hampered by the lack of knowledge of the finer atomic-level details of how they work. Investigating these details is particularly troublesome for antibiotics that target the bacterial membrane (the fatty layer that separates the in- and outside of the bacterium) because membranes are poorly amendable to the most used analytical techniques that can provide atomic resolution information. In this thesis, we apply so-called solid-state nuclear magnetic resonance spectroscopy (ssNMR spectroscopy) towards solving this problem. We develop general methodology to produce isotopically enriched phospholipids (the major component of bacterial membranes) and demonstrate their use by investigating the interaction between antibiotic Cinnamycin and the phospholipid PE. Next, we investigate the mode of action of plectasin, a fungal antibiotic that binds to Lipid II, a crucial intermediate in the synthesis of the bacterial cell wall. By integrating the structural information from ssNMR with biochemical assays and other analytical techniques, we uncover how plectasin binds to Lipid II in membranes and that upon binding the complex further self-assembles on the membrane, forming a network, stabilized by calcium, that tightly sequesters Lipid II, making it inaccessible for the bacterium.