The Central Kink Region of Fowlicidin-2, an α-Helical Host Defense Peptide, Is Critically Involved in Bacterial Killing and Endotoxin Neutralization

Abstract

Fowlicidins are a group of newly identified chicken cathelicidin host defense peptides. We have shown that the putatively mature fowlicidin-2 of 31 amino acid residues possesses potent antibacterial and lipopolysaccharide (LPS)- neutralizing activities, but with a noticeable toxicity to mammalian cells. As a first step in exploring the structure-activity relationships of fowlicidin-2, in this study we determined its tertiary structure by nuclear magnetic resonance spectroscopy. Unlike the majority of cathelicidins, which are composed of a predominant α-helix with a short hinge sequence near the center, fowlicidin-2 consists of 2 well-defined α-helical segments (residues 6–12 and 23–27) connected by a long extensive kink (residues 13–20) induced by proline. To further investigate the functional significance of each of these structural components, several N- and C-terminal deletion analogs of fowlicidin-2 were synthesized and analyzed for their antibacterial, cytotoxic and LPS-neutralizing activities. Our results indicated that neither the N- nor C-terminal α-helix alone is sufficient to confer any function. Rather, fowlicidin-2(1–18) and fowlicidin-2(15–31), 2 α-helical segments with inclusion of the central cationic kink region, retained substantial capacities to kill bacteria and neutralize the LPS-induced proinflammatory response, relative to the parent peptide. More desirably, these 2 peptide analogs showed substantially reduced toxicity to human erythrocytes and epithelial cells, indicative of improved potential as antibacterial and antisepsis agents. To our knowledge, fowlicidin-2 is the first α-helical cathelicidin, with the central kink region shown to be critically important in killing bacteria and neutralizing LPS.

Chicken cathelicidin-2-derived peptides with enhanced immunomodulatory and antibacterial activities against biological warfare agents

“…As reported earlier by Xiao et al [8] and confirmed by us, M a n u s c r i p t 12 CATH-2 is resistant to salt, but C1-15 almost completely lost its antibacterial activity in the presence of 100 mM NaCl. However, we could restore the antibacterial activity in the presence of salt by Phe to Trp substitutions.…”

In vitro antibacterial and hemolytic activities of crotamine, a small basic myotoxin from rattlesnake Crotalus durissus

“…14,15 Briefly, bacteria at the mid-logarithmic phase were washed twice in 10 mM sodium phosphate buffer, pH 7.4, diluted to 0.03 A 600 nm (equivalent to 2.5-5Â10 7 CFU ml À1 ) in the same buffer containing 1% trypticase soy broth. In a 96-well tissue culture plate, 80 ml bacteria was then mixed with native or reduced crotamine to a final concentration of 50 mg ml À1 , followed by addition of 1.5 mM of a chromogenic substrate, o-nitrophenyl-b-D-galactopyranoside (Sigma, St Louis, MO, USA), to a total volume of 100 ml per well.…”

The Central Hinge Link Truncation of the Antimicrobial Peptide Fowlicidin-3 Enhances Its Cell Selectivity without Antibacterial Activity Loss

“…The antibacterial activities of Fow-3(1-15), Fow-3(1-19), and Fow-3(20-27) were attenuated or even nearly lost, and Fow-3(1-15-20-27) displayed antibacterial activity similar to that of Fow-3. Fow-3 (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19) showed obvious cytotoxicity compared with other analogs, which demonstrated that the central hinge link (-AGIN-) was crucial to the cytotoxicity of the peptide. Fow-3 (1-15-20-27) showed good tolerance to salt ions and a considerably higher level of cell selectivity than Fow-3.…”

“…Many studies were carried out to do this by altering certain physicochemical and structural properties, such as charge, amphipathicity, hydrophobicity, degree of structuring (␣-helix or ␤-sheet), D-isomerization, and cyclization (9,10,11,12). In addition, truncation of natural AMPs has been shown to be a simple and effective approach for the design and/or optimization of AMPs (12,13,14).…”