Engineering of a linear inactive analog of human β-defensin 4 to generate peptides with potent antimicrobial activity

Abstract

Human β-defensins (HBDs) are cationic antimicrobial peptides constrained by three disulfide bridges. They have diverse range of functions in the innate immune response. It is of interest to investigate whether linear analogs of defensins can be generated, which possess antimicrobial activity. In this study, we have designed linear peptides with potent antimicrobial activity from an inactive peptide spanning the N-terminus of HBD4. Our results show that l-arginine to d-arginine substitution imparts considerable antimicrobial activity against both bacteria and Candida albicans. Increase in hydrophobicity by fatty acylation of the peptides with myristic acid further enhances their potency. In the presence of high concentrations of salt, antimicrobial activity of the myristoylated peptide with l-arginine is attenuated relatively to a lesser extent as compared with the linear active peptide with d-arginine. Substitution of cysteine with the hydrophobic helix-promoting amino acid α-aminoisobutyric acid favors candidacidal activity but not antibacterial activity. The mechanism of killing by d-arginine substituted unacylated analog involves transient interaction with the bacterial membrane followed by translocation into the cytoplasm without membrane permeabilization. Accumulation of peptides in the cytoplasm can affect various cellular processes that lead to cell death. However, the peptide causes membrane permeabilization in case of C. albicans. Myristoylation results in greater interaction of the peptide chain with the microbial cell surface and causes membrane permeabilization. Results described in the study demonstrate that it is possible to generate highly active linear analogs of defensins by selective introduction of d-amino acids and fatty acids, which could be attractive candidates for development as therapeutic agents.

Antimicrobial activity of human α-defensin 5 and its linear analogs: N-terminal fatty acylation results in enhanced antimicrobial activity of the linear analogs

“…Although fatty acylation improved antibacterial activity, myristoylated peptides showed potent hemolytic activity unlike the myristoylated analogs of HBD4 [54]. However lauryl peptides were not hemolytic.…”

“…walls [66]. It appears that binding and destabilization of outer membrane of Gram-negative bacteria is an essential step for the activity of HD5 and analogs [54]. Efficient interaction with bacterial surfaces enhances the antibacterial activity of defensin derived peptides.…”

“…The effect of altering the orientation of the side chains of arginine residues by substituting them (except the C-terminal arginine) with D-isomer (D5r) on the antimicrobial activity was investigated. It has been observed that attachment of fatty acids to linear cationic antimicrobial peptides can modulate their antimicrobial activity [50][51][52][53][54]. We therefore examined the effect of covalently attaching fatty acids at the N-terminus of D5R and D5r, which though rich in cationic amino acids do not have sequence similarity or rich in hydrophobic amino acids as compared to cecropins and magainins [55].…”

Variations in the interaction of human defensins with Escherichia coli: Possible implications in bacterial killing

“…In fact, Alves et al ., have reported a correlation between extent of surface charge neutralizing ability and minimum inhibitory concentrations for amphipathic CAMPs [50]. We have previously shown that effective interaction of human defensin analogs with bacterial surfaces enhances antibacterial potency [39, 51, 52]. With a view to understand, how human defensins vary in their ability to interact with bacterial surfaces, we compared the extent of electrostatic interactions between the E .…”

Perspectives for clinical use of engineered human host defense antimicrobial peptides

“…2013; Wood et al. 2013; Mathew and Nagaraj 2015a,b; Olli, Nagaraj and Motukupally 2015; Sharma, Mathew and Nagaraj 2015; Tripathi et al. 2015).…”