Abstract

   The rapid dissemination of antibiotic resistance accelerates the desire
   for new antibacterial agents. Here, a class of antimicrobial peptides
   (AMPs) is designed by modifying the structural parameters of a natural
   chickpea‐derived AMP–Leg2, termed “functionalized chickpea‐derived Leg2
   antimicrobial peptides” (FCLAPs). Among the FCLAPs, KTA and KTR show
   superior antibacterial efficacy against the foodborne pathogen
   Escherichia coli (E. coli) O157:H7 (with MICs in the range of
   2.5–4.7 µmol L^−1) and demonstrate satisfactory feasibility in
   alleviating E. coli O157:H7‐induced intestinal infection. Additionally,
   the low cytotoxicity along with insusceptibility to antimicrobial
   resistance increases the potential of FCLAPs as appealing
   antimicrobials. Combining the multi‐omics profiling andpeptide‐membrane
   interaction assays, a unique dual‐targeting mode of action is
   characterized. To specify the antibacterial mechanism, microscopical
   observations, membrane‐related physicochemical properties studies, and
   mass spectrometry assays are further performed. Data indicate that KTA
   and KTR induce membrane damage by initially targeting the
   lipopolysaccharide (LPS), thus promoting the peptides to traverse the
   outer membrane. Subsequently, the peptides intercalate into the
   peptidoglycan (PGN) layer, blocking its synthesis, and causing a
   collapse of membrane structure. These findings altogether imply the
   great potential of KTA and KTR as promising antibacterial candidates in
   combating the growing threat of E. coli O157:H7.

   Keywords: antimicrobial peptides, dual‐targeting mechanism of action,
   Escherichia coli O157:H7, foodborne pathogen intervention,
   membrane‐mediated antimicrobial mechanism
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   Functionalized chickpea‐derived Leg2 antimicrobial peptides (FCLAPs)
   show superior antibacterial activity, and they combat Escherichia coli
   O157:H7 through a dual‐targeting mechanism. First, FCLAPs induce
   membrane damage by initially targeting the lipopolysaccharide, thus
   promoting the peptides to traverse the outer membrane. Subsequently,
   the peptides intercalate into the peptidoglycan layer, blocking its
   synthesis, and causing a collapse of membrane structure.

   graphic file with name ADVS-10-2205301-g010.jpg

1. Introduction

   The ever‐growing prevalence of foodborne infections caused by
   Gram‐negative microbes, especially E. coli O157:H7, Salmonella, and
   Pseudomonas aeruginosa, has been recognized as a critical healthcare
   issue that necessitates the development of new antimicrobial agents.^[
   [44]^1 ^] However, the pipeline for the discovery of novel
   antimicrobials that target Gram‐negative bacteria remains empty.^[
   [45]^2 ^] This problem is partly attributed to their intrinsic
   resistance to most antibiotics that are currently available in clinical
   applications.^[ [46]^3 ^] Another reason is that Gram‐negative bacteria
   evolved a unique outer membrane (OM) that serves as a highly
   impermeable barrier to protect themselves from harmful compounds.^[
   [47]^4 ^] Although recent efforts have been devoted to antimicrobial
   development, most of the new compounds functioned similarly in their
   mechanisms to those of traditional antibiotics.^[ [48]^5 ^] These
   worrisome limitations portend the introduction of drugs to clinical
   applications might be a long and arduous process. Under these
   circumstances, developing ideal promising antibiotic alternatives that
   selectively kill Gram‐negative bacteria is imperative to address the
   urgent medical need.

   Antimicrobial peptides (AMPs) have received extensive attention during
   the past three decades as a new generation of antibiotics.^[ [49]^6 ^]
   Different from most conventional antibiotics that act on specific
   intracellular targets, AMPs combat bacteria primarily through
   electrostatic interactions and physically destroying the microbial
   lipid bilayers.^[ [50]^7 ^] The nature of the membrane‐active mechanism
   renders bacteria with minimal probability to evolve resistance to AMPs,
   primarily due to the need for a range of genetic mutations to alter the
   whole components of the bacterial cell membrane.^[ [51]^8 ^] At
   present, more than 3000 AMPs have been documented in the antimicrobial
   peptide database, however, only seven of them have been approved by the
   U.S. Food and Drug Administration for clinical application.^[ [52]^9 ^]
   The reduced efficacy in clinically relevant environments, high toxicity
   toward mammalian cells, and proteolytic instability of natural AMPs
   were the major challenges that slowed down their clinical
   implementation.^[ [53]^10 ^] and application in the food industry.^[
   [54]^11 ^] Therefore, new potent AMPs with improved in vivo
   antimicrobial performance and reduced cytotoxicity are stringently
   needed to overcome the limitations of natural AMPs.^[ [55]^12 ^] In
   this regard, researchers have been focused on modifying the AMPs
   isolated from natural sources for stronger properties.^[ [56]^13 ^]

   Great efforts have been made to explore the relationships between the
   physicochemical parameters of AMPs (i.e., net charge, hydrophobicity,
   amphiphilicity, and structural propensity) and the antibacterial
   efficacy which provide references for rational modification and