C. albicans growth, transition, biofilm formation, and gene expression modulation by antimicrobial decapeptide KSL-W

Abstract

Background: Antimicrobial peptides have been the focus of much research over the last decade because of their effectiveness and broad-spectrum activity against microbial pathogens. These peptides also participate in inflammation and the innate host defense system by modulating the immune function that promotes immune cell adhesion and migration as well as the respiratory burst, which makes them even more attractive as therapeutic agents. This has led to the synthesis of various antimicrobial peptides, including KSL-W (KKVVFWVKFK-NH2), for potential clinical use. Because this peptide displays antimicrobial activity against bacteria, we sought to determine its antifungal effect on C. albicans. Growth, hyphal form, biofilm formation, and degradation were thus examined along with EFG1, NRG1, EAP1, HWP1, and SAP 2-4-5-6 gene expression by quantitative RT-PCR.

Results: This study demonstrates that KSL-W markedly reduced C. albicans growth at both early and late incubation times. The significant effect of KSL-W on C. albicans growth was observed beginning at 10 μg/ml after 5 h of contact by reducing C. albicans transition and at 25 μg/ml by completely inhibiting C. albicans transition. Cultured C. albicans under biofilm-inducing conditions revealed that both KSL-W and amphotericin B significantly decreased biofilm formation at 2, 4, and 6 days of culture. KSL-W also disrupted mature C. albicans biofilms. The effect of KSL-W on C. albicans growth, transition, and biofilm formation/disruption may thus occur through gene modulation, as the expression of various genes involved in C. albicans growth, transition and biofilm formation were all downregulated when C. albicans was treated with KSL-W. The effect was greater when C. albicans was cultured under hyphae-inducing conditions.

Conclusions: These data provide new insight into the efficacy of KSL-W against C. albicans and its potential use as an antifungal therapy.

Figure 1

KSL-W inhibited C. albicans growth. The yeast was cultured in Sabouraud supplemented medium with or without KSL-W at various concentrations. The cultures were maintained for 5, 10, and 15 h at 37°C, after which time an MTT assay was performed for each culture condition. The growth was plotted as means ± SD of the absorbance at 550 nm. (A)C. albicans growth with KSL-W for 5 h; (B)C. albicans growth with KSL-W for 10 h; and (C)C. albicans growth with KSL-W for 15 h. The levels of significance for C. albicans growth in the presence or not of KSL-W or amphotericin B (10 μg/ml) were considered significant at P < 0 · 05.

Figure 2

KSL-W inhibited C. albicans yeast-to-hyphae transition.C. albicans was cultured in Sabouraud medium containing 10% fetal bovine serum with or without KSL-W at various concentrations and was maintained for 4 and 8 h at 37°C. After each time point, the cultures were observed under an inverted microscope and photographed. Representative photos of the morphological changes after 4 h of culture are presented.

Figure 3

Scanning electron microscope analyses of the biofilm formation.C. albicans was cultured in Sabouraud medium with or without KSL-W at various concentrations for 4 days in a porous 3D collagen scaffold. Cultures in the presence of amphotericin B (10 μg/ml) were used as the positive controls. Following incubation, the samples were prepared as described in the Methods section and were observed under a scanning electron microscope. Negative control refers to the non-seeded scaffolds.

Figure 4

Quantitative measurement of the reduced biofilm formation with KSL-W.C. albicans was cultured on a 3D porous scaffold in the presence of KSL-W for 2, 4, and 6 days. After each culture period, the samples were supplemented with XTT solution and incubated for 5 h at 37°C. The absorbance at 450 nm was measured to quantify XTT metabolic product intensity proportional to the number of viable cells. (A) 2 days; (B) 4 days; (C) 6 days. Results are means ± SD for three different separate experiments.

Figure 5

Biofilm ultrastructure following KSL-W treatment.C. albicans was cultured in Sabouraud medium without KSL-W for 6 days to promote biofilm formation and maturation. The resulting biofilms were then treated or not with KSL-W or amphotericin B for 6 days, with medium and peptide refreshing every 2 days. Following incubation, the samples were prepared as described in the Methods section and observed under a scanning electron microscope.

Figure 6

Decrease of biofilm mass following KSL-W treatment.C. albicans was cultured in a 3D porous scaffold in Sabouraud medium for 6 days to promote biofilm formation and maturation. The resulting biofilms were exposed or not to KSL-W or amphotericin B for 2, 4, and 6 days. Medium and peptide were refreshed every 2 days. Following each treatment period, the samples were supplemented with XTT solution and subsequently incubated for 5 h at 37°C. Absorbance at 450 nm was measured to quantify XTT metabolic product intensity proportional to the number of viable cells. (A) 2 days; (B) 4 days; (C) 6 days. Results are means ± SD for three separate experiments.