Multispecies activity screening of microcin J25 mutants yields antimicrobials with increased specificity toward pathogenic Salmonella species relative to human commensal Escherichia coli

Abstract

Modern large-scale agricultural practices that incorporate high density farming with subtherapeutic antibiotic dosing are considered a major contributor to the rise of antibiotic-resistant bacterial infections of humans with species of Salmonella being a leading agriculture-based bacterial infection. Microcin J25, a potent and highly stable antimicrobial peptide active against Enterobacteriaceae, is a candidate antimicrobial against multiple Salmonella species. Emerging evidence supports the hypothesis that the composition of the microbiota of the gastrointestinal tract prevents a variety of diseases by preventing infectious agents from proliferating. Reducing clearance of off-target bacteria may decrease susceptibility to secondary infection. Of the Enterobacteriaceae susceptible to microcin J25, Escherichia coli are the most abundant within the human gut. To explore the modulation of specificity, a collection of 207 mutants encompassing 12 positions in both the ring and loop of microcin J25 was built and tested for activity against Salmonella and E. coli strains. As has been found previously, mutational tolerance of ring residues was lower than loop residues, with 22% and 51% of mutations, respectively, retaining activity toward at least one target within the target organism test panel. The multitarget screening elucidated increased mutational tolerance at position G2, G3, and G14 than previously identified in panels composed of single targets. Multiple mutations conferred differential response between the different targets. Examination of specificity differences between mutants found that 30% showed significant improvements to specificity toward any of the targets. Generation and testing of a combinatorial library designed from the point-mutant study revealed that microcin J25 reduces off-target activity toward commensal human-derived E. coli isolates by 81% relative to Salmonella enterica serovar Enteritidis. 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.

Comprehensive Mutational Analysis of the Lasso Peptide Klebsidin

“…By co-encapsulating bacteria producing novel lanthipeptides with sensor bacterial strains in hydrogel compartments, Schmitt et al screened thousands of variants to identify novel lanthipeptides with antimicrobial activity. These examples, in addition to others, ,− suggest that RiPPs can be engineered for new or improved biological activity.…”

Engineering Selectively Targeting Antimicrobial Peptides

“…The use of genetically encoded peptide libraries in screening has yielded unique, synthetic AMPs. Ritter et al (48) designed a library of 960 microcin J25 mutants to screen for mutants with specific antimicrobial activity against pathogenic Salmonella species. The library was cloned into an E. coli production strain and screened for activity against a panel of bacteria (two pathogenic Salmonella species and two commensal E. coli strains) using an agar diffusion assay.…”

“…The library was cloned into an E. coli production strain and screened for activity against a panel of bacteria (two pathogenic Salmonella species and two commensal E. coli strains) using an agar diffusion assay. Ritter et al (48) discovered a mutant with a single amino acid mutation (I13T) that significantly reduced activity toward commensal E. coli strains relative to the Salmonella species. Tucker et al (49) developed a peptide screening methodology, called Surface Localized Antimicrobial Display, for generating novel, active AMPs targeted toward E. coli.…”

A Platform for Deep Sequence–Activity Mapping and Engineering Antimicrobial Peptides

“…5,6 Many AMPs are ribosomally synthesized, which permits efficient engineering of potency, specificity, and stability via recombinant DNA technology and innovative genotype-phenotype linkage strategies. 7,8 Therefore, rapid responsiveness is enabled to evolve AMPs as infectious microbes emerge.…”

“…Along with improved antibiotic stewardship, innovative antimicrobial therapies and platforms for their efficient discovery are sorely needed. Antimicrobial peptides and proteins (AMPs) are a compelling alternative to traditional antibiotics. , AMPs kill bacteria via various processes and could be administered in combination to potentially reduce resistance. , Many AMPs are ribosomally synthesized, which permits efficient engineering of potency, specificity, and stability via recombinant DNA technology and innovative genotype-phenotype linkage strategies. , Therefore, rapid responsiveness is enabled to evolve AMPs as infectious microbes emerge.…”