Antibacterial properties of dermaseptin S4 derivatives with in vivo activity

Abstract

Derivatives of the cytotoxic peptide dermaseptin S4 have recently emerged as potential antimicrobial agents. Here, we report on the antibacterial properties of three derivatives with improved toxicity profiles: a 28-residues K4K20-S4 and two shorter versions, K4-S4(1-16) and K4-S4(1-13). The range of MICs of K4K20-S4 against clinical isolates of Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli were, respectively, 1 to 4, 1 to 4, and 1 to 16 microg/ml. MICs of the short derivatives were rather similar or two to fourfold higher. Each of the three peptides was rapidly bactericidal in vitro, reducing the number of viable CFU of either E. coli or S. aureus by 6 log units in 30 min or less. Compared with MSI-78 or PG-1, K4-S4(1-13) was at least as potent against bacteria (assessed at two MIC multiples) but displayed lesser toxicity against human erythrocytes. Serial passage in subinhibitory concentrations led to emergence of resistance to commercial antibiotics but not to the L- or D isomer of either of the dermaseptin derivatives. The short derivatives were further investigated for antibacterial activity in vivo, using a peritonitis model of mice infected with P. aeruginosa. Naive mice in the vehicle control group exhibited 75% mortality, compared to 18 or 36% mortality in mice that received a single intraperitoneal injection (4.5 mg/kg) of K4-S4(1-16) or K4-S4(1-13), respectively. In vivo bactericidal activity was confirmed in neutropenic mice, where intraperitoneal administration of K4-S4(1-16) reduced the number of viable CFU in a dose-dependent manner by >3 log units within 1 h of exposure, and this was sustained for at least 5 h. Overall, the data suggest that dermaseptin S4 derivatives could be useful in treatment of infections, including infections caused by multidrug-resistant bacteria.

FIG. 1.

Bactericidal kinetics and hemolytic activities of K₄-S4(1-13), MSI-78, and PG-1. A peptide concentration of 20 μM [corresponding to 30, 50, and 43 μg/ml, respectively, for K₄-S4(1-13) (asterisks), MSI-78 (squares), and PG-1 (circles)] was used to treat cultures of E. coli (A) and S. aureus (B). After 0, 5, 30, and 60 min of exposure time at 37°C, aliquots were diluted (serial 10-fold dilutions) and plated for CFU counts after overnight incubation at 37°C. The peptide hemolytic activity (C) was assessed against human erythrocytes (1% hematocrit). Hemolysis was determined by measuring absorbance (405 nm) of the supernatants after 4 h of incubation at 37°C in the presence of water (100% hemolysis), PBS (baseline), or serial twofold dilutions of peptides in PBS starting from 200 μM [corresponding to 300, 500, and 430 μg/ml, respectively, for K₄-S4(1-13), MSI-78, and PG-1]. Plotted values correspond to the means ± standard deviations.

FIG. 2.

Evolution of MICs after successive exposures of bacteria to sublytic concentrations of the specified antimicrobial agent. (A and B) Evolution of MICs after 15 and 10 serial passages of P. aeruginosa and S. aureus, respectively. The relative MIC is the normalized ratio of the MIC obtained for a given subculture to the MIC (determined concomitantly) obtained for first-time exposure.

FIG. 3.

In vivo bactericidal activity of K₄-S4(1-16). Neutropenic mice infected i.p. with P. aeruginosa were treated i.p. with PBS alone or with PBS containing the specified peptide doses. Animals (six mice/group) were sacrificed at 0, 1, and 5 h after bacterial infection, and peritoneal fluids were removed as described in Materials and Methods. Viable bacterial counts were determined (in triplicate) by plating diluted peritoneal fluids on Mueller-Hinton agar plates. The results shown correspond to the means ± standard deviations.