Aromatic-rich C-terminal region of LCI is a potent antimicrobial peptide in itself Karabi Saikia, Vinay Kumar Belwal, Debika Datta, Nitin Chaudhary * Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Guwahati, 781 039, India article info Article history: Received 9 August 2019 Accepted 5 September 2019 Available online 10 September 2019 Keywords: Antimicrobial Peptide LCI 22-47 Membrane Fluorescence abstract LCI is a 47-residue antimicrobial peptide produced by Bacillus subtilis. The peptide displays potent ac- tivity against plant pathogens, Xanthomonas and Pseudomonas. The peptide takes a compact 3- dimensional structure characterized by a four-stranded b-sheet. The peptide is unusually rich in aro- matic residues; 10 of the 47 residues are aromatic and 8 of them lie in the C-terminal region, LCI 22-47 . Here we report the antimicrobial activity of this C-terminal region against Gram-positive and Gram- negative bacteria. The C-terminal-amidated peptide displays potent activity against E. coli, methicillin and gentamicin-resistant S. aureus, and Xanthomonas oryzae pv. oryzae with lethal concentrations 4 mM. Membrane-binding assays indicate preferential binding to the negatively-charged lipids. The peptide permeabilizes the outer-membrane of E. coli indicating membrane-permeabilization as one of the mechanisms of killing. Interestingly, however, no inner-membrane permeabilization was observed, indicating that the membrane-permeabilization may not be the sole mechanism of action. © 2019 Elsevier Inc. All rights reserved. 1. Introduction Peptides are ubiquitously used by organisms to defend them- selves from pathogens [1 ,2]. Such host-defense peptides, also known as antimicrobial peptides (AMPs) have been identified in all known genera of living organisms. Bacteria are a great source of antimicrobial peptides. Bacteriocins are AMPs that are produced by a large number of bacteria to protect themselves from their closely related strains. Bacteriocins exhibit antimicrobial properties at very low concentrations making them promising candidates to be used as next-generation antibiotics [3]. LCI is an AMP produced by Bacillus subtilis strain A014 [4,5]. The peptide inhibits the plant pathogens, Xanthomonas and Pseudo- monas. The peptide harbors certain interesting structural features viz. an unusually high thermodynamic stability and richness in aromatic residues. The peptide folds into a highly stable structure characterized by a four-stranded b-sheet [5]. Beta-sheet rich AMPs are often stabilized by one or more disulfide linkages. LCI lacks cysteine but is reported to have retained >80% activity even after 20 min of heating at 80  C. Such remarkable stability for a peptide lacking a disulfide linkage is intriguing. The thermodynamic stability of LCI is believed to be conferred by the aromatic stacking interactions, cation-p interactions, and aromatic-backbone amide interactions. The distribution of the cationic and aromatic residues in the sequence is worth noticing; the first 21 residues harbor only two phenylalanine and one lysine residues. The C-terminal 26 residues, on the other hand, harbor three Trp residues, four Tyr residues, one Phe residue, five Lys residues, and one Arg residue. This implies that 80% of the aromatic residues and ~86% of the cationic residues reside in the C-terminal 26-residue stretch. This stretch contributes two b-strands viz. b3 and b4 to the native LCI structure wherein the two strands are linked through a Type-I b- turn. Such peculiar properties prompted us to investigate the antibacterial properties of this 26-residue stretch. We synthesized the C-terminal amidated peptide (KWIFKSKYYDSSKGYWV- GIYEVWDRK-NH 2 ), referred to as LCI 22-47 hereafter, and investi- gated the antibacterial activity against E. coli, gentamicin and methicillin-resistant S. aureus (gentamicin-resistant MRSA), and Xanthomonas oryzae pv. oryzae (Xoo). The peptide killed all the three organisms efficiently with lethal concentrations of 4 mM or less. Studies carried out with model membranes and E. coli mem- branes suggested membrane-permeabilization as one of the mechanisms of killing. * Corresponding author. E-mail address: chaudhary@iitg.ac.in (N. Chaudhary). Contents lists available at ScienceDirect Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc https://doi.org/10.1016/j.bbrc.2019.09.013 0006-291X/© 2019 Elsevier Inc. All rights reserved. Biochemical and Biophysical Research Communications 519 (2019) 372e377