A Chimeric Cationic Peptide Composed of Human β-Defensin 3 and Human β-Defensin 4 Exhibits Improved Antibacterial Activity and Salt Resistance

Abstract

Human beta-defensins (hBDs) play an important role in the host defense against various microbes, showing different levels of antibacterial activity and salt resistance in vitro. It is of interest to investigate whether can chimeric hBD analogs enhanced antibacterial activity and salt resistance. In this study, we designed a chimeric human defensin, named H4, by combining sequences of human beta-defensin-3 (hBD-3) and human beta-defensin-4 (hBD-4), then evaluated its antibacterial activity, salt resistance, and cytotoxic effects. The result showed that the antibacterial activity of H4 against most tested strains, including Klebsiella pneumonia, Enterococcus faecalis, Staphyloccocus aureus, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumonia, and Acinetobacter baumannii was significantly improved compared to that of hBD-3 and hBD-4. Notably, H4 exhibited significantly better antibacterial activity against multidrug resistant isolate A. baumannii MDR-ZJ06 than commonly used antibiotics. Chimeric H4 still showed more than 80% antibacterial activity at high salt concentration (150 μM), which proves its good salt tolerance. The cytotoxic effect assay showed that the toxicity of H4 to Hela, Vero, A549 cells and erythrocytes at a low dose (<10 μg/ml) was similar to that of hBD-3 and hBD-4. In conclusion, given its broad spectrum of antibacterial activity and high salt resistance, chimeric H4 could serve as a promising template for new therapeutic antimicrobial agents.

Keywords: antibacterial activity; chimeric human defensin; human beta-defensins; multidrug resistant; salt resistance.

Figure 1

Salt resistance of hBD-3, hBD-4, and H4. Antibacterial activity at increasing concentrations of NaCl of hBD-3, hBD-4, and H4 against (A) Escherichia coli American Type Culture Collection (ATCC) 25922, (B) Klebsiella pneumoniae ATCC 700603, (C) Pseudomonas aeruginosa ATCC 15442, (D) Staphyloccocus aureus ATCC 29213, (E) Enterococcus faecium ATCC 29212, (F) Enterococcus faecalis ATCC 6057, (G) Acinetobacter baumannii ATCC 19606, and (H) multidrug resistant Acinetobacter baumannii strain MDR-ZJ06. Error bars show the SDs of experiments performed in triplicate. Non-parametric tests were used in the statistical analysis. ^**Significantly different (p < 0.001) from the activity of hBD-3. ^##Significantly different (p < 0.001) from the activity of hBD-4.

Figure 2

Cytotoxic activity at different concentrations of hBD-3, hBD-4, and H4. Detection of the cytotoxicity of H4 to Hela, Vero, and A549 cells. The ratio of absorbance of cells treated with different concentrations of hBD analogs to that of the control group in (A) Hela cells, (B) Vero cells, and (C) A549 cells. Error bars show the SDs of experiments performed in triplicate. **Significantly different (p < 0.001) from the activity of hBD-3. ##Significantly different (p < 0.001) from the activity of hBD-4.

Figure 3

Hemolysis assays of hBD-3, hBD-4 and H4. Detection of the hemolysis rate of hBD-3, hBD-4, and H4. Error bars show the SDs of experiments performed in triplicate. Non-parametric tests were used in the statistical analysis. ^**Significantly different (p < 0.001) from the activity of hBD-3. ^##Significantly different (p < 0.001) from the activity of hBD-4.

Figure 4

The antibacterial activity of H4 against intracellular bacteria infecting A549 cells. Detection of the antibacterial activity of H4 to infected A549 cells. The ratio of bacterial survival treated with PBS and 10 μg/ml H4. (A) The antibacterial activity of H4 against A. baumannii ATCC 19606 infecting A549 cells. (B) The antibacterial activity of H4 against K. pneumoniae ATCC 700603 infecting A549 cells. Error bars show the SDs of experiments performed in triplicate. **Significantly different (p < 0.001) between the treated with H4 and PBS.