The discovery of antibiotics from soil and the characterization of darobactin analogs

Abstract

The spread of antibiotic resistance in bacterial pathogens is limiting our therapeutic options to combat infections. The World Health Organization lists antimicrobial resistance as one of the top 10 threats to global health in 2019, and we desperately need a platform to discover new antibiotics. Moreover, there is a critical need for antibiotics active against drug-resistant Gram-negative pathogens. Gram-negative bacteria maintain an additional outer membrane, preventing penetration to the cell by most drugs. This thesis describes the discovery of a new class of antibiotics, named darobactins, which selectively kill Gram-negative bacteria, including multi-drug resistant pathogens. We discovered the first darobactin by screening Photorhabus isolates, which are symbionts of entomopathogenic nematodes. Darobactin kills selectively by targeting BamA. The BAM complex is responsible for the folding and translocation of outer membrane proteins. Darobactin had good efficacy in animal models of infection, and we showed resistant mutations lead to a loss of virulence. Further work revealed the mode of action, which is to mimic the recognition signal of native BamA substrates and subsequently block binding to the lateral gate of BamA. This work confirmed that the lateral gate of BamA is an attractive target for new antibiotics against Gram-negative pathogens. Additionally, this thesis describes the discovery, production, and characterization of naturally occurring darobactin analogs. These analogs were first identified in bacterial genome sequences and contained one or two amino acid substitutions in the heptapeptide core of darobactin. Darobactin B was shown to be a promising candidate, with equal or improved activity against Gram-negative pathogens when compared to darobactin A. Additional optimization of compounds from the darobactin class will likely expand into several therapeutic candidates. Lastly, this thesis describes methods in the discovery of antibiotics produced by environmental bacteria. We developed differential screens for antibiotics active against Gram-negative bacteria but are not active against Gram-positive bacteria. These screens resulted in the rediscovery of known selective antibiotics such as polymyxins, as well as other novel hits. We also employed 16S metagenomic sequencing to prioritize which soil samples to screen, on metrics of bacterial abundance, diversity, and biosynthetic potential. Finally, solid-state fermentation and elicitation with a compound library was used to induce production of a likely novel compound that selectively kills E. coli, produced by Delftia acidovorans. --Author's abstract