title: Enterocin A mutants identified by saturation mutagenesis enhance potency towards vancomycin-resistant Enterococci.
abstract: Vancomycin-resistant Enterococci infections are a significant clinical problem. One proposed solution is to use probiotics, such as lactic acid bacteria, to produce antimicrobial peptides at the site of infection. Enterocin A, a class 2a bacteriocin, exhibits inhibitory activity against E. faecium and E. faecalis, which account for 86% of vancomycin-resistant Enterococci infections. In this study, we aimed to engineer enterocin A mutants with enhanced potency within a lactic acid bacterial production system. Peptide mutants resulting from saturation mutagenesis at sites A24 and T27 were efficiently screened in a 96-well plate assay for inhibition of pathogen growth. Several mutants exhibit increased potency relative to wild-type enterocin A in both liquid- and solid-medium growth assays. In particular, A24P and T27G exhibit enhanced inhibition of multiple strains of E. faecium and E. faecalis, including clinically isolated vancomycin-resistant strains. A24P and T27G enhance killing of E. faecium 8 by 13 ± 3- and 18 ± 4-fold, respectively. The engineered enterocin A/lactic acid bacteria systems offer significant potential to combat antibiotic-resistant infections.
authors.affiliation: Department of Chemical Engineering and Materials Science, University of Minnesota-Twin Cities, Minneapolis, Minnesota, 55455.
authors.forename: Maria K
authors.initials: MK
authors.lastname: McClintock
authors.affiliation: Department of Chemical Engineering and Materials Science, University of Minnesota-Twin Cities, Minneapolis, Minnesota, 55455.
authors.forename: Yiannis N
authors.initials: YN
authors.lastname: Kaznessis
authors.affiliation: Department of Chemical Engineering and Materials Science, University of Minnesota-Twin Cities, Minneapolis, Minnesota, 55455. hackel@umn.edu.
authors.forename: Benjamin J
authors.initials: BJ
authors.lastname: Hackel
mesh_terms: D001430:Bacteriocins / Q000235:genetics* / Q000378:metabolism*; D013294:Lactococcus lactis / Q000235:genetics / Q000378:metabolism*; D008826:Microbial Sensitivity Tests; D050296:Microbial Viability / Q000187:drug effects; D016296:Mutagenesis; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D065507:Vancomycin-Resistant Enterococci / Q000187:drug effects*
chemical_list: D001430:Bacteriocins; D050505:Mutant Proteins; C107799:enterocin A
keywords: antimicrobial peptide; enterocin A; lactic acid bacteria; peptide engineering; saturation mutagenesis