(569e) Saturation Mutagenesis Panning Libraries Enable Activity and Specificity Modulation of Microcin J25

Modern large-scale agricultural practices which incorporate high density farming with sub-therapeutic antibiotic dosing are a major contributor to the rise of antibiotic resistant bacterial infections. A leading foodborne pathogen, Salmonella, is present on as much as 20% of all packaged chicken products with 15% of those isolates found to be resistant to 4 or more antibiotics by the Centers for Disease Control. Alternatives to antibiotics, such as antimicrobial peptides (AMP) offer higher levels of specificity and activity towards targets of interest compared to traditional antibiotics. To this end, Microcin J25, a 21 amino acid AMP with a highly stable threaded lasso structure has been studied with single-site saturation mutagenesis to evaluate mutations contributing to the specificity and activity against multiple Salmonella and Escherichia coli strains.

A collection of 218 mutants (91% phenotypic coverage of the 240 possible variants) encompassing 12 positions in both the ring and loop of J25 was built and tested for activity versus Salmonella and Escherichia coli strains in a growth halo assay. Mutational tolerance of ring residues was lower than loop residues, with 24% and 50% of mutations retaining nonzero activity towards at least one target within the target organism test panel, respectively. Multiple mutations conferred differential response between the different targets. For example substitution of position 13 isoleucine with tyrosine retained wild-type activity level against E. coli JJ1887 (a urinary tract infector) while reducing activity against E. coli O157:H7 by 50%. In contrast, substitution with leucine improved activity by 10 and 80% against both E. coli, respectively, while reducing activity against Salmonella enteritidis and Salmonella Tennessee by 40%. In addition, substitution of position 3 alanine for cysteine displayed zero activity against both E. coli strains while retaining activity against both Salmonella. In total an improved specificity response was recorded for 78 of the 218 mutants (p = 0.01). A second generation library simultaneously mutating 6 positions between 2 to 5 phenotypic options was generated to assess the impact of multiple mutations on the specificity of J25. Of these multi-position mutants 53 of 1800 sampled retained some level of activity against the pathogen Salmonella enteritidis. The activity of these mutants will further be determined against a panel of commensal E. coli isolated from fecal and urine samples of healthcare patients, as well as pathogenic strains of enterobacteriaceae. This information will identify J25 variants with improved specificity for particular pathogenic targets.

These in vitro specificity improvements are likely to improve in vivo treatment efficacy by reducing clearance of commensal bacteria in the gastrointestinal tract of hosts. This both reduces selection of resistance within the microflora and reduces the likelihood of secondary infection from opportunistic pathogens, both problems with broad-spectrum antibiotic treatment. In addition, all variants generated were produced through the native microcin J25 synthesis pathway and could be adapted for use in previously described engineered probiotics utilizing J25 (Forkus et. al., 2017).