New era of lipid-targeting peptide antibiotics

The work presented in this thesis describes a detailed investigation to understand the mechanism of lipid targeting antibiotics using an array of methods including ssNMR, advanced microscopy techniques, computational analysis, and biophysical and biochemical assays. Together, these methods enable a holistic description of the supramolecular mechanism of lipid-targeting antibiotics such as teixobactin or clovibactin. Chapter 1 introduces the current scenario antimicrobial resistance (AMR) and highlights the studies conducted on teixobactin to date. This chapter introduces the basics of ssNMR and other complimentary techniques. It also lays the foundation for the methodologies used in the following chapters to gain insights into the mechanism of lipid-targeting antibiotics, teixobactin and clovibactin. Chapter 2 is adapted from a study published in Nature Communications in 2020. Here we describe the mechanism of action of synthetic analogues of teixobactin, namely [R4L10] and [L10]. With the help of ssNMR studies, we could solve the structure of the complex formed between [R4L10]-teixobactin and its target, lipid II directly in membranes. Furthermore, we investigated the impact of the membrane composition on the mode of action of [R4L10]- teixobactin and demonstrate that the bacterial cell membrane affects the binding of the drug. Chapter 3 is adapted from a study published in Nature in 2022 where we describe a new dual mode of action for natural teixobactin. In this chapter, we determine the dual mechanism of teixobactin from an atomic to supramolecular scale using ssNMR, HS-AFM, and confocal microscopy. We present an atomically resolved ssNMR structure of the complex and elaborate on its evolution into fibrils and eventually micrometre-sized clusters which ultimately kill the bacteria. Based on our results we show that teixobactin ventures away from the classic 1-drug to 1-target ideology and uses a new type of antimicrobial action. Chapter 4 is adapted from a publication that is currently under review. This chapter emphasizes the discovery of a new antibiotic called clovibactin from the bacterial “dark matter.” We use our established methodology to study and elucidate the novel mechanism of clovibactin and showcase its multi-targeting action. Conclusively in Chapter 5, we showcase some recently acquired and unpublished data on the effect of anionic lipids on teixobactin’s mechanism of action and propose future studies which can be conducted to expand on the role of anionic membranes on the activity of lipid-targeting antibiotics. We also discuss how the efficient methodology developed can be useful in studying lipid-targeting antibiotics at different length scales under varying membrane compositions to provide a complete overview of their mechanism and help in designing better drugs to tackle the problem of AMR.