Abstract
Marine fish antimicrobial peptide, chrysophsin-1 possesses versatile biological activities but its non-selective nature restricts its therapeutic possibilities. Often small alterations in structural motifs result in significant changes in the properties of concerned proteins/peptides. We have identified GXXXXG motif in chrysophsin-1. Glycine residue(s) of this motif in Chrysophsin-1 was/were replaced with alanine, valine and proline residue(s). Of these, proline-substituted Chrysophsin-1 analogs exhibited significantly reduced cytotoxicity towards mammalian cells. Further, these analogs showed broad-spectrum activity against Gram-positive, Gram-negative bacteria, Methicillin-resistant Staphylococcus aureus strains and fungi and also retained antibacterial activity in presence of physiological salts, serum and at elevated temperatures indicative of their therapeutic potential. These Chrysophsin-1 analogs also inhibited lipopolysaccharide (LPS) induced pro-inflammatory responses in THP-1 cells and in murine primary macrophages. One of these single proline-substituted Chrysophsin-1 analogs inhibited LPS-stimulated pro-inflammatory cytokine production in BALB/c mice and elicited appreciable survival of mice administered with a lethal dose of LPS in a model of severe sepsis. The data for the first time showed the implication of GXXXXG motifs in functional and biological properties of an antimicrobial peptide and could be useful to design novel anti-microbial and anti-endotoxin peptides by employing this motif.
Highlights
The clinical efficacies of most antibiotics have declined drastically as a result of the emergence of dominant resistance in microorganisms against them[1]
Since glycine to proline substitutions do not change the physiochemical properties of the peptide, proline substitutions were preferred for looking into the role of GXXXXG motif in Chrysophsin-1 (Table 2)
The key finding of this study is the identification of GXXXXG motifs in Chrysophsin-1 for the first time and introduction of small amino acid substitution in this motif to yield non-toxic variants of this highly cytotoxic antimicrobial peptides (AMPs) without compromising its antimicrobial and anti-endotoxin activities
Summary
The clinical efficacies of most antibiotics have declined drastically as a result of the emergence of dominant resistance in microorganisms against them[1] It seems that the indiscriminate use of antibiotics put an evolutionary pressure on pathogens to develop strategies for nullifying their effects[2]. The structural motifs present in AMPs play crucial roles in the biological activity of their parent peptides. We identified a combination of two glycine residues with four amino acid residues in between them in Chrysophsin-1 what we call as ‘GXXXXG’ i.e. there is a recurrence of glycine after every four amino acids throughout the sequence of Chrysophsin-1 which was not reported before in this peptide This motif is present in Chrysophsin-2, the second isoform of Chrysophsin which is cytotoxic. Realizing the plausible implication of this motif in the biological activity of Chrysophsin-1, we set to unravel the importance of this motif in cytotoxicity, anti-bacterial and anti-endotoxic activities of this AMP by selectively replacing the glycine residue(s) of these motifs with proline, alanine and valine residue(s)
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