An Investigation of the Stability and Activity of a Lasso Peptide from Members of the Enterobacter cloacae Complex

Abstract

Lasso peptides are a subset of ribosomally synthesized and post-translationally modified peptides (RiPPs) that are natively encoded by diverse phyla of bacteria. Their structure is composed of an N-terminal ring, usually made up of 7 to 9 amino acids, followed by a C-terminal segment that is fed through the ring, hence the name lasso peptide. The threaded nature of lasso peptides is stabilized by steric interactions from large side chains on the C-terminal tail, locking the threaded tail in place within the ring. Lasso peptides are important to study due to their wide variety of biological functions, such as antimicrobial activity. Previous researchers in the Link Lab identified and expressed a lasso peptide that is encoded by species of Enterobacterales which exhibits antimicrobial activity against various other strains. While most lassoes studied to date have been resistant to unthreading, this lasso peptide displays some unthreading behavior even at room temperature in water. Understanding the unthreading behavior is important because once the lasso peptide undergoes unthreading, it is irreversible, and only the threaded form is bioactive. This thesis research aims to investigate the role of specific amino acids within the peptide on its unthreading behavior and antimicrobial activity through mutagenesis. The serine-24 to glycine (S24G) mutation led to an entirely unthreaded lasso peptide, which was unexpected since serine is not expected to serve as a steric lock due to its small size. A mutagenesis study was carried out to determine whether other substitutions at the C-terminus would affect the stability of the lasso peptide. Mutations at the C-terminus were tolerated, but the other mutations, such as G1A and E9D, were not tolerated. Most variants were expressed except for the G1A and E9D mutants. The antimicrobial activity of the Y10A mutant was tested, and the results indicated that there was a drastic loss of its bioactivity.