Killing of Staphylococcus aureus and Salmonella enteritidis and neutralization of lipopolysaccharide by 17-residue bovine lactoferricins: improved activity of Trp/Ala-containing molecules

Abstract

Bovine lactoferricin (LfcinB) has potent antibacterial, antifungal and antiparasitic activities but is also hemolytic. Our objective was to identify LfcinB17-31 derivatives with reduced hemolysis and improved antimicrobial activity via substituting Cys3, Arg4, Gln7, Met10, and Gly14 with more hydrophobic residues. Two peptides, Lfcin4 and Lfcin5, showed higher activity against Staphylococcus aureus and Salmonella enteritidis and lower hemolytic activity than the parent peptide LfcinB17-31. These peptides permeabilized the outer and inner membranes of S. enteritidis; however, Lfcin5 did not permeabilize the inner membrane of S. aureus. Gel retardation and circular dichroism spectra showed that Lfcin4 and Lfcin5 bound to bacterial genomic DNA. Lfcin4 inhibited DNA, RNA and protein synthesis. Both peptides induced the peeling of membranes and the lysis of S. enteritidis. At doses of 10 and 15 mg/kg, Lfcin4 and Lfcin5 reduced the bacterial counts in infected thigh muscles by 0.03‒0.10 and 0.05‒0.63 log₁₀ CFU/g of tissue, respectively, within 10 h. Lfcin4 and Lfcin5 enhanced the survival rate of endotoxemic mice; reduced serum IL-6, IL-1β and TNF-α levels; and protected mice from lipopolysaccharide-induced lung injury. These data suggest that Lfcin4 and Lfcin5 may be antimicrobial and anti-endotoxin peptides that could serve as the basis for the development of dual-function agents.

Figure 1. Hemolysis and CD spectra of the secondary structures of Lfcin4 and Lfcin5.

(A) Hemolytic activity of Lfcin4 and Lfcin5 against mouse erythrocytes. The results are presented as the mean ± SEM (n = 3). (B,C) CD spectra of the secondary structures of Lfcin4 (B) and Lfcin5 (C).

Figure 2. Effects of Lfcin4 and Lfcin5 on the cell surface hydrophobicity and cell morphology of bacteria.

(A,B) Effects of Lfcin4 and Lfcin5 on the cell surface hydrophobicity of S. aureus ATCC25923 (A) and S. enteritidis CVCC3377. (B) The results are presented as the mean ± SEM (n = 3). (C) SEM photographs of S. aureus ATCC25923 and S. enteritidis CVCC3377 cells with or without 1 × MIC Lfcin4 and Lfcin5.

Figure 3. Effects of Lfcin4 and Lfcin5 on the biological membrane.

(A) Leakage from POPC/POPG (1:3) vesicles induced by different concentrations of Lfcin4 and Lfcin5. Large unilamellar vesicles (LUVs) loaded with calcein were incubated with Lfcin4 and Lfcin5 and leakage of calcein was monitored for 10 min on a Tecan Infinite M200 PRO plate reader. (B,C) Time-response curve of the outer membrane permeabilization of S. aureus ATCC25923 (B) and S. enteritidis CVCC337 (C) cells treated with Lfcin4 and Lfcin5 in the presence of NPN. PBS treatment was used as a negative control. Treatment with ampicillin and colistin were used as positive controls. Amp: ampicillin; Coli: colistin. (D–G) Fluorescence-activated cell sorting (FACS) analysis of PI staining in S. aureus ATCC25923 (D,E) and S. enteritidis CVCC337 (F,G) cells treated with Lfcin4 (D,F) and Lfcin5 (E,G).

Figure 4. In vitro binding of Lfcin4 and Lfcin5 to bacterial genomic DNA.

(A) Gel retardation analysis of the binding of Lfcin4 and Lfcin5 to genomic DNA. M: DNA marker λDNA/HindIII. Lanes 1–6: genomic DNA from E. coli CICC21530; Lanes 7–12: genomic DNA from S. aureus ATCC25923; Lanes 13–18: genomic DNA from S. enteritidis CVCC3377. The mass ratios of peptide to genomic DNA were 10, 5, 2.5, 1, 0.5, and 0. Full-length gels are presented in Supplementary Figure S2. (B–D) CD spectra of genomic DNA from E. coli CICC21530 (B), S. aureus ATCC25923 (C) and S. enteritidis CVCC3377 (D) in the presence of Lfcin4 and Lfcin5. The mass ratios of peptide to DNA were 5 and 10.

Figure 5. Effects of Lfcin4 and Lfcin5 on macromolecular synthesis in S. aureus ATCC25923.

Incorporation of ³H-glucosamine hydrochloride (peptidoglycan) (A), ³H-leucine (protein) (B), ³H-thymidine (DNA) (C), and ³H-uridine (RNA) (D) was determined in cells treated with 1 × MIC Lfcin4 and Lfcin5. Van: vancomycin (2 × MIC); Ery: erythromycin (2 × MIC); Cip: ciprofloxacin (8 × MIC); Rif: rifampicin (4 × MIC). Antibiotics were used as controls. Results are presented as the mean ± SEM (n = 3).

Figure 6. Efficacy of Lfcin4 and Lfcin5 against S. aureus ATCC25923 in the neutropenic murine thigh infection model.

(A) Lfcin4 treatment. (B) Lfcin5 treatment. Control: mice treated with saline only; Van (10 mg/kg): mice treated with a single intravenous (tail) dose of vancomycin (10 mg/kg); Lfcin4/Lfcin5 (10 mgkg) and Lfcin4/Lfcin5 (15 mg/kg): mice treated with a single intravenous (tail) dose of 10 or 15 mg/kg Lfcin4 or Lfcin5, respectively. Results are presented as the mean ± SEM. Differences between groups were determined by one-way ANOVA followed by SPSS analysis (n = 4 per group). *p < 0.05 compared to the control group.

Figure 7. Effects of Lfcin4 and Lfcin5 on LPS-induced responses in vivo.

(A) Groups of eight C57BL/6 mice were intraperitoneally injected with LPS from E. coli O111:B4 (30 mg/kg of body weight). Thirty minutes after LPS injection, Lfcin4, Lfcin5, colistin, or saline was administered intraperitoneally. Survival was recorded every 12 h and followed for up to 7 d. (B,C) C57BL/6 mice were intraperitoneally injected with 10 mg/kg LPS followed by intraperitoneal administration of 15 mg/kg Lfcin4, Lfcin5 or colistin 30 min later. Mice treated with buffer only served as a control. Cytokines were measured in blood from mice sacrificed at 2 h, 8 h or 20 h after LPS injection. The results are presented as the mean ± SEM. Differences between groups were determined by one-way ANOVA followed by SPSS analysis (n = 3 per group). ^#p < 0.05 compared to the control group and *p < 0.05 compared to the LPS-treated group (B). Light microscopy images (10 × magnification, scale bar: 100 μm) of lung tissue from representative mice sacrificed at 8 h, 1 d, 4 d, and 7 d after LPS injection (C).