Chimeric analogs of human ß-defensin 1 and θ-defensin disrupt pre-established bacterial biofilms.

Antibiofilm activity of several human defensin analogs that have the ability to kill planktonic bacteria, against pre-established biofilms of Escherichia coli MG1655 and Staphylococcus aureus NCTC 8530 were examined. Linear and linear fatty acylated analogs did not show any activity while disulfide constrained analogs disrupted pre-established S. aureus biofilms. Chimeric analogs of human ß-defensin 1 and θ-defensin, hBTD-1 and [d]hBTD-1 were highly active against S. aureus biofilms. Among the analogs tested, only the d-enantiomer [d]hBTD-1 showed activity against E. coli biofilm. Our study provides insights into the structural requirements for the eradication of pre-established biofilms in defensin analogs.

A hybrid cationic peptide composed of human ß-defensin-1 and humanized θ-defensin sequences exhibits salt-resistant antimicrobial activity.

We have designed a hybrid peptide by combining sequences of human ß-defensin-1 (HBD-1) and θ-defensin, in an attempt to generate a molecule that combines the diversity in structure and biological activity of two different peptides to yield a promising therapeutic candidate. HBD-1 was chosen as it is a natural defensin of humans that is constitutively expressed, but its antibacterial activity is considerably impaired by elevated ionic strength. θ-Defensins are expressed in human bone marrow as a pseudogene and are homologous to rhesus monkey circular minidefensins. Retrocyclins are synthetic human θ-defensins. The cyclic nature of the θ-defensin peptides makes them salt resistant, nonhemolytic, and virtually noncytotoxic in vitro. However, a nonhuman circular molecule developed for clinical use would be less viable than a linear molecule. In this study, we have fused the C-terminal region of HBD-1 to the nonapeptide sequence of a synthetic retrocyclin. Cyclization was achieved by joining the terminal ends of the hybrid peptide by a disulfide bridge. The hybrid peptide with or without the disulfide bridge exhibited enhanced antimicrobial activity against both Gram-negative and Gram-positive bacteria as well as against fungi, including clinical bacterial isolates from eye infections. The peptide retained activity in the presence of NaCl and serum and was nonhemolytic in vitro. Thus, the hybrid peptide generated holds potential as a new class of antibiotics.

Effect of selectively introducing arginine and D-amino acids on the antimicrobial activity and salt sensitivity in analogs of human beta-defensins.

We have examined the antimicrobial activity of C-terminal analogs of human ß-defensins HBD-1 and-3 wherein lysines have been selectively replaced by L- and D-arginines and L-isoleucine substituted with its D-enantiomer. The analogs exhibited antibacterial and antifungal activities. Physiological concentration of NaCl did not attenuate the activity of the peptides against Gram-negative bacteria considerably, while some attenuation of activity was observed against S. aureus. Variable attenuation of activity was observed in the presence of Ca²âº and Mg²âº. Introduction of D-amino acids abrogated the need for a disulfide bridge for exhibiting activity. Confocal images of carboxyfluorescein (CF) labeled peptides indicated initial localization on the membrane and subsequent translocation into the cell. Analogs corresponding to cationic rich segments of human defensins substituted with L- and D-arginine, could be attractive candidates for development as future therapeutic drugs.

Cloning, characterization and antifungal activity of defensin Tfgd1 from Trigonella foenum-graecum L.

Defensins are small cysteine rich peptides with a molecular mass of 5-10 kDa and some of them exhibit potent antifungal activity. We have cloned the coding region of a cDNA of 225 bp cysteine rich defensin, named as Tfgd1, from the legume Trigonella foenum-graecum. The amino acid sequence deduced from the coding region comprised 74 amino acids, of which the N-terminal 27 amino acids constituted the signal peptide and the mature peptide comprised 47 amino acids. The protein is characterized by the presence of eight cysteine resisdues, conserved in the various plant defensins forming four disulphide bridges, which stabilize the mature peptide. The recombinant protein expressed in E coli exhibited antifungal activity against the broad host range fungus, Rhizoctonia solani and the peanut leaf spot fungus, Phaeoisariopsis personata.