CHARACTERIZATION OF NOVEL ANTIMICROBIAL LASSO PEPTIDES AND DISSECTING THEIR MECHANISM OF ACTION

Abstract

In drug discovery and design, natural products have long served as a rich source of bioactive compounds that have inspired scientific innovation. Through elucidation of the mechanisms that bacteria and other microorganisms use to defend themselves in competitive niches, the twentieth century saw a revolution in antibiotic discovery and allowed us to treat previously lethal infections, leading to a dramatic increase in human life expectancy. However, as bacteria develop resistance to even last-resort antibiotics, new antibiotic discovery is critical to maintaining the quality and longevity of life that has been achieved. In particular, Gram-negative bacteria are notoriously difficult targets for antibiotic development, as their outer membrane serves as a largely impenetrable barrier for many Gram-positive-acting antibiotics. Lasso peptides are a growing class of natural products, a subset of which have been identified as potent antimicrobials, including those with activity against Gram-negative pathogens. My thesis work has focused on identifying, expressing, and characterizing new antimicrobial lasso peptides and elucidating their mechanisms of action. In addition to enhancing our understanding of the ecological roles of lasso peptides, this research aims to advance their potential as novel antibiotics. In Chapters 2 and 3, I discuss the discovery of the achromonodins and cloacaenodin, respectively. After production and isolation of the lasso peptides through heterologous expression in E. coli, the lasso peptides were shown to have potent, selective antimicrobial activity against pathogenic bacterial strains, including drug-resistant clinical isolates. The NMR structures of the lasso peptides were solved and demonstrated a shared structural architecture of Gram-negative-targeting lasso peptides. In Chapter 4, I use genetic techniques to elucidate the full transport pathway of cloacaenodin into susceptible bacteria, offering insight into its focused bioactivity. This led to the discovery and characterization of a new TonB-dependent transporter, which we name CloU, found predominantly in Enterobacter species. In Chapter 5, I present the discovery of pseudomonodin, a lasso peptide from a plant isolate of Pseudomonas. Pseudomonodin shows potent activity against other plant-associated Pseudomonas species, likely influencing the composition of the plant microbiome.