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 β-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 Ca²⁺ and Mg²⁺. 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

Effect of divalent cations on antibacterial activity.

<p>Mid-log phase bacteria (10⁶ CFU/mL) were incubated with peptides at their LC in the absence or presence of the indicated concentrations of CaCl₂ and MgCl₂. The data are mean values of three independent experiments and the error bars represent standard deviation of the measurements. Standard deviation values ranged between 0.2-2.7.</p

Effect of NaCl on antibacterial activity.

<p>Mid-log phase bacteria (10⁶ CFU/mL) were incubated with peptides at their LC in the absence and presence of the indicated concentrations of NaCl. The data are mean values of three independent experiments and the error bars represent standard deviation of the measurements. Standard deviation values ranged between 0.3-1.5.</p

Confocal microscope images of <i>E. coli</i> in the presence of CF-labeled HC-1(R), [D]HC-1(R) and [D]HC-3(R).

<p>Bacterial cells (1 x 10⁷) were treated with CF labeled peptide and FM4-64 and were incubated for different time points. Panel A represents an early time point where the peptide interacts with the bacterial membrane. Panel B represents a late time point wherein the peptide is found completely diffused inside the cell. The bar represents 5 µm.</p