An unprecedented alteration in mode of action of IsCT resulting its translocation into bacterial cytoplasm and inhibition of macromolecular syntheses.

Jitendra K Tripathi,Manoj Kathuria,Amit Kumar,Jimut K Ghosh,Kalyan Mitra

doi:10.1038/srep09127

Abstract

IsCT, a 13-residue, non-cell-selective antimicrobial peptide is comprised of mostly hydrophobic residues and lesser cationic residues. Assuming that placement of an additional positive charge in the non-polar face of IsCT could reduce its hydrophobic interaction, resulting in its reduction of cytotoxicity, an analog, I9K-IsCT was designed. Two more analogs, namely, E7K-IsCT and E7K,I9K-IsCT, were designed to investigate the impact of positive charges in the polar face as well as polar and non-polar faces at a time. These amino acid substitutions resulted in a significant enhancement of therapeutic potential of IsCT. IsCT and E7K-IsCT seem to target bacterial membrane for their anti-bacterial activity. However, I9K-IsCT and E7K,I9K-IsCT inhibited nucleic acid and protein syntheses in tested E. coli without perturbing its membrane. This was further supported by the observation that NBD-IsCT localized onto bacterial membrane while NBD-labeled I9K-IsCT and E7K,I9K-IsCT translocated into bacterial cytoplasm. Interestingly, IsCT and E7K-IsCT were significantly helical while I9K-IsCT and E7K,I9K-IsCT were mostly unstructured with no helix content in presence of mammalian and bacterial membrane-mimetic lipid vesicles. Altogether, the results identify two novel cell-selective analogs of IsCT with new prototype amino acid sequences that can translocate into bacterial cytoplasm without any helical structure and inhibit macromolecular syntheses.

Highlights

IntroductionThe results identify two novel cell-selective analogs of IsCT with new prototype amino acid sequences that can translocate into bacterial cytoplasm without any helical structure and inhibit macromolecular syntheses
IsCT is one of the shortest natural cytotoxic peptides consisting of only 13 amino acid residues with a-helical secondary structure and antimicrobial property which has been isolated from the scorpion, Opisthacanthus madagascariensis[22]
Functional and biological properties of the native peptide and its analogs were carried out which revealed the design of novel analogs of IsCT that selectively translocated into bacterial cytoplasm and impaired its biomolecular syntheses without causing any damage to the membrane

Summary

Introduction

The results identify two novel cell-selective analogs of IsCT with new prototype amino acid sequences that can translocate into bacterial cytoplasm without any helical structure and inhibit macromolecular syntheses. Substitution of www.nature.com/scientificreports amino acids at ‘a’ and/or ‘d’ positions of leucine zipper like sequences of these antimicrobial peptides with alanine residues drastically reduced their cytotoxicity probably as a result of decrease in peptide-induced permeabilization of mammalian cell membrane[1,2,5,24]. To look into the influence of additional positive charge in the polar face of the molecule, a glutamic acid residue at 7th position was replaced by lysine residue This analog was reported some time ago by others whose antimicrobial and cytotoxic properties were only studied[22]. Functional and biological properties of the native peptide and its analogs were carried out which revealed the design of novel analogs of IsCT that selectively translocated into bacterial cytoplasm and impaired its biomolecular syntheses without causing any damage to the membrane

Methods

Results

Conclusion