Restor Dent Endod.  2016 May;41(2):91-97. 10.5395/rde.2016.41.2.91.

Antifungal effects of synthetic human β-defensin 3-C15 peptide

• Lim SM
• Ahn KB
• Kim C
• Kum JW
• Perinpanayagam H
• Gu Y
• Yoo YJ
• Chang SW
• Han SH
• Shon WJ
• Lee W
• Baek SH
• Zhu Q
• Kum KY

• KMID: 2163290
• DOI: http://doi.org/10.5395/rde.2016.41.2.91

Abstract

OBJECTIVES
The purpose of this ex vivo study was to compare the antifungal activity of a synthetic peptide consisting of 15 amino acids at the C-terminus of human β-defensin 3 (HBD3-C15) with calcium hydroxide (CH) and Nystatin (Nys) against Candida albicans (C. albicans) biofilm.
MATERIALS AND METHODS
C. albicans were grown on cover glass bottom dishes or human dentin disks for 48 hr, and then treated with HBD3-C15 (0, 12.5, 25, 50, 100, 150, 200, and 300 µg/mL), CH (100 µg/mL), and Nys (20 µg/mL) for 7 days at 37℃. On cover glass, live and dead cells in the biomass were measured by the FilmTracer Biofilm viability assay, and observed by confocal laser scanning microscopy (CLSM). On dentin, normal, diminished and ruptured cells were observed by field-emission scanning electron microscopy (FE-SEM). The results were subjected to a two-tailed t-test, a one way analysis variance and a post hoc test at a significance level of p = 0.05.
RESULTS
C. albicans survival on dentin was inhibited by HBD3-C15 in a dose-dependent manner. There were fewer aggregations of C. albicans in the groups of Nys and HBD3-C15 (≥ 100 µg/mL). CLSM showed C. albicans survival was reduced by HBD3-C15 in a dose dependent manner. Nys and HBD3-C15 (≥ 100 µg/mL) showed significant fungicidal activity compared to CH group (p < 0.05).
CONCLUSIONS
Synthetic HBD3-C15 peptide (≥ 100 µg/mL) and Nys exhibited significantly higher antifungal activity than CH against C. albicans by inhibiting cell survival and biofilm.

Keyword

Antifungal activity; C. albicans; Confocal laser scanning microscopy; Dentin disk; Human β-defensin-3 peptide; LIVE/DEAD Biofilm viability assay

MeSH Terms

Amino Acids
Biofilms
Biomass
Calcium Hydroxide
Candida albicans
Cell Survival
Dentin
Glass
Humans*
Microscopy, Confocal
Microscopy, Electron, Scanning
Nystatin
Amino Acids
Calcium Hydroxide
Nystatin

Figure

• 

  Figure 1 HBD3-C15 and Nystatin (Nys) inhibited C. albicans biofilm on dentin. C. albicans (6 × 106 cells/mL) was incubated for 48 hr and treated with either HBD3-C15 (0, 12.5, 25, 50, 100, 150, 200, or 300 µg/mL), aqueous calcium hydroxide (CH, 100 µg/mL), and Nys (20 µg/mL) for 7 days. Field-emission scanning electron microscopy (FE-SEM) showed C. albicans biofilm decreased by HBD3-C15 in a dose-dependent manner (×30,000, ×5,000, ×1,000). Cells that appeared normal (saline), smaller (25 µg/mL), bloated (50, 100 µg/mL) and ruptured (150, 200, 300 µg/mL) were seen at high magnification (×30,000).

• 

  Figure 2 HBD3-C15 and Nystatin (Nys) showed fungicidal activity against C. albicans. C. albicans (6 × 106 cells/mL) was incubated for 48 hr on cover glass and treated with either HBD3-C15 (0, 12.5, 25, 50, 100, 150, 200, 300 µg/mL), aqueous calcium hydroxide (CH, 100 µg/mL), and Nys (20 µg/mL) for 7 days. (a) FilmTracer LIVE/DEAD Biofilm Viability staining was performed and observed under CLSM; (b) The numbers of dead cells (PI stained red) were increased in the group of HBD3-C15 in a dose-dependent manner; (c) The dead cell biomass were significantly higher in the groups of HBD3-C15 (≥ 100 µg/mL) and Nys than CH group (p < 0.05). #Indicates no significant difference between HBD3-C15 (300 µg/mL) and Nys (p < 0.05). *Significant difference compared with CH and HBD3-C15 (below 50 µg/mL) groups (p < 0.05). PI, propidium iodide; 3D, 3 dimensional.

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