Article Figures & Data
Figures
- FIG. 1.
Susceptibility of different C. albicans strains and Candida species to RsAFP2. (A to E) Candida isolates and strains were cultivated in potato dextrose broth/yeast peptone dextrose broth (pH 7.0) for 24 h at room temperature (27). Yeast cells (2 × 103) were treated overnight with different concentrations (0.6 to 10 μM) of RsAFP2. The percentage of viability for each RsAFP2-treated Candida species relative to that of control treatment (water) was calculated after plating the treated yeast on brain heart infusion agar dishes for CFU counting. (F) C. glabrata and C. albicans (Ca 2A) isolates were treated with 10 μM RsAFP2. Survival rates, determined as the percentage of viability, of the different strains after treatment with 10 μM RsAFP2 are shown.
- FIG. 2.
Correlation between GlcCer content and RsAFP2 susceptibility in Candida sp. (A) Expression of GlcCer by three different strains of C. albicans (lanes 1, strain 2A; 2, strain 78; 3, strain 12A) and one C. glabrata isolate (lane 4) (+++ indicates more susceptible; ++, intermediate susceptibility; +, less susceptible). GlcCer was extracted with organic solvents, and the resulting molecules were analyzed by high-performance thin-layer chromatography. (B) Densitometric analysis of the chromatogram shown in panel A for the different C. albicans strains was performed by using Scion Image (NHI) software, and the relative content of GlcCer in each strain is shown.
- FIG. 3.
Antifungal activity of RsAFP2 after treatment with serum and its toxicity to human cells. (A) After treatment with 10 μM RsAFP2 with different concentrations of serum (% of FBS), the peptide antifungal activity against C. albicans (strain 12A) cells (2 × 103) was assessed using CFU determination. (B) Monolayers of primary human brain endothelial cells (HBMEC) and the astroglia tumor-derived cell line (U87) in 96-well plates in serum-free medium were incubated overnight with RsAFP2 (10 μM), and culture supernatants were collected for LDH determination. Positive controls consisted of supernatants of Triton X-100 (10%) and gomesin (data not shown) cultures. The negative control corresponded to supernatant from untreated cells.
- FIG. 4.
Activity of RsAFP2 in prophylactic murine models of candidiasis. Infection was performed at 1 h after (A) and 1 h before (B) RsAFP2 treatment. Mice (n = 5) were infected intravenously with C. albicans (strain 78) and treated with saline (control, n = 5), RsAFP2 (n = 5), or fluconazole (10 mg/kg) (n = 5). CFU counts in the kidneys of one representative experiment (out of three) are shown. Significant statistical differences (P < 0.01) were observed between antifungal-treated mice (RsAFP2 and fluconazole) and control mice (saline) in both experiments. Nonsignificant (ns; P = 0.152) differences were observed between CFU counts from RsAFP2-treated and fluconazole-treated mice in the prophylactic model. Procedures involving animals and their care were conducted in conformity with the local ethics committee and international recommendations.
