Role of Phenylalanine and Valine10 Residues in the Antimicrobial Activity and Cytotoxicity of Piscidin-1

Abstract

Piscidin-1 (Pis-1) is a linear antibacterial peptide derived from mast cells of aquacultured hybrid striped bass that comprises 22 amino acids with a phenylalanine-rich amino-terminus. Pis-1 exhibits potent antibacterial activity against pathogens but is not selective for distinguishing between bacterial and mammalian cells. To determine the key residues for its antibacterial activity and those for its cytotoxicity, we investigated the role of each Phe residue near the N-terminus as well as the Val10 residue located near the boundary of the hydrophobic and hydrophilic sectors of the helical wheel diagram. Fluorescence dye leakage and tryptophan fluorescence experiments were used to study peptide-lipid interactions, showing comparable depths of insertion of substituted peptides in different membranes. Phe2 was found to be the most deeply inserted phenylalanine in both bacterial- and mammalian-mimic membranes. Each Phe was substituted with Ala or Lys to investigate its functional role. Phe2 plays key roles in the cytotoxicity as well as the antibacterial activities of Pis-1, and Phe6 is essential for the antibacterial activities of Pis-1. We also designed and synthesized a piscidin analog, Pis-V10K, in which Lys was substituted for Val10, resulting in an elevated amphipathic α-helical structure. Pis-V10K showed similar antibacterial activity (average minimum inhibitory concentration (MIC) = 1.6 µM) to Pis-1 (average MIC = 1.5 µM). However, it exhibited much lower cytotoxicity than Pis-1. Lys10-substituted analogs, Pis-F1K/V10K, Pis-F2K/V10K, and Pis-F6K/V10K in which Lys was substituted for Phe retained antibacterial activity toward standard and drug-resistant bacterial strains with novel bacterial cell selectivity. They exert anti-inflammatory activities via inhibition of nitric oxide production, TNF-α secretion, and MIP-1 and MIP-2 production. They may disrupt the binding of LPS to toll-like receptors, eventually suppressing MAPKs-mediated signaling pathways. These peptides may be good candidates for the development of peptide antibiotics with potent antibacterial activity but without cytotoxicity.

The use of the antimicrobial peptide piscidin (PCD)-1 as a novel anti-nociceptive agent

“…1) cells is suppressed by the presence of non-toxic concentrations of PCD-1. The ability of PCD-1 to inhibit upregulation of iNOS is consistent with a previous study that showed that PCD-1 reduced NO production in LPS-stimulated RAW264.7 cells [59]. These observations suggest a possible anti-neuropathic property of PCD-1.…”

“…Thus, based on the previous and present findings, we conclude that PCD-1 may exert its analgesic effects (at least in part) through inhibition of the inflammatory response (up-regulated IL-1b and phospho-mTOR). The ability of PCD-1 to inhibit upregulation of spinal IL-1b in in vivo experiments is consistent with a previous in vitro study that reported that PCD analogs suppressed the mRNA expression of IL-1b, as well as production of other inflammatory cytokines, in LPS-stimulated RAW264.7 cells [59]. In addition, down-regulation of TGF-b1 in astrocytes or neuronal cells of CCI rats was effectively reversed by PCD-1 administration (Figs.…”

Single Amino Acid Substitutions at Specific Positions of the Heptad Repeat Sequence of Piscidin-1 Yielded Novel Analogs That Show Low Cytotoxicity and In Vitro and In Vivo Antiendotoxin Activity

“…Annexin V-Alexa Fluor staining of hRBCs suggested that V12I-piscidin-1, piscidin-1, and the three single valine-substituted piscidin-1 analogs significantly damaged the organization of the hRBC membrane, while the three single alanine-substituted piscidin-1 analogs were appreciably inactive in this regard, suggesting that these peptides have hemolytic activities lower than those of the former group peptides. Previous studies also reported the hemolytic activity of piscidin-1 against human RBCs as well as its cytotoxicity toward a human cell line(s) (5), which are findings consistent with those of the present study (Fig. 2D).…”

“…Piscidin-1 shows significant activity against bacteria, fungi, parasites, and cancer cells (1)(2)(3)(4)(5)(6)(7). It can also neutralize lipopolysaccharide (LPS)-induced proinflammatory responses in macrophage cells (5).…”

Piscidin-1-analogs with double L- and D-lysine residues exhibited different conformations in lipopolysaccharide but comparable anti-endotoxin activities

“…NMR studies suggest that piscidins adopt an amphipathic α–helical conformation upon interaction with model membranes11 or detergent micelles512. Piscidin-1 possesses LPS-neutralizing property1314, and has potent activity against a variety of microbes, comprising of filamentous fungi, yeast, and Gram-positive and negative bacteria and their resistant versions. However, this peptide causes haemolysis of RBCs and is cytotoxic, which may limit its therapeutic applications1516171819.…”