(65a) Mutagenesis of the Lasso Antibiotic Peptide Microcin J25: Discovery of Variants with Increased Potency and New Insights Into the Structure-Activity Relationship

Infections caused by drug-resistant Gram-negative bacteria pose a rising threat as some strains become ever more resistant to virtually all existing antibiotics. Microcin J25 (MccJ25) is a ribosomally-synthesized antibacterial peptide isolated from Escherichia coli that exhibits potent mechanisms of action against pathogenic strains of Salmonella, E. coli, and Shigella. MccJ25 has an unusual threaded lasso structure in which the C-terminal “tail” of the peptide is fed through a macrocyclic “ring” formed by the N-terminal residues. Production of MccJ25 in E. coli is dependent on a four-gene cluster encoding the structural gene mcjA, two maturation enzymes mcjB and mcjC, and an immunity factor, mcjD, in the form of an MccJ25 export pump. Here we have developed a system for orthogonal control of the expression of the structural gene and the export pump thus permitting independent control of MccJ25 production and export/immunity in E. coli. We used this system to screen saturation mutagenesis libraries targeted to the ring and tail portions of MccJ25. While multiple amino acid substitutions in the tail portion of the peptide were well-tolerated, mutagenesis of the ring portion of the peptide was detrimental to the antimicrobial function of MccJ25. We demonstrated that substitutions in the ring portion of the peptide likely disrupt the transport of these variants into the cytoplasm of susceptible strains. Additionally, we found several MccJ25 variants from the tail library with up to nearly 5-fold improvement in potency toward the strains E. coli and Salmonella enterica serovar Newport.