Surfactin and Capric Acid Affect the Posaconazole Susceptibility of Candida albicans Strains with Altered Sterols and Sphingolipids Biosynthesis

Abstract

Infections caused by Candida spp. pose a continuing challenge for modern medicine, due to widespread resistance to commonly used antifungal agents (e.g., azoles). Thus, there is considerable interest in discovering new, natural compounds that can be used in combination therapy with conventional antibiotics. Here, we investigate whether the natural compounds surfactin and capric acid, in combination with posaconazole, enhance the growth inhibition of C. albicans strains with alterations in sterols and the sphingolipids biosynthesis pathway. We demonstrate that combinations of posaconazole with surfactin or capric acid correspond with the decreased growth of C. albicans strains. Moreover, surfactin and capric acid can independently contribute to the reduced adhesion of C. albicans strains with altered ergosterol biosynthesis to abiotic surfaces (up to 90% reduction in adhesion). A microscopic study of the C. albicans plasma membrane revealed that combinations of those compounds do not correspond with the increased permeabilization of the plasma membrane when compared to cells treated with posaconazole alone. This suggests that the fungistatic effect of posaconazole in combination with surfactin or capric acid is related to the reduction in adhesion of C. albicans.

Keywords: Candida albicans; azole resistance; capric acid; posaconazole; surfactin; synergism.

Figure 1

Chemical structure of posaconazole (PSZ).

Figure 2

Chemical structure of surfactin (SU (A)) and capric acid (CA (B)).

Figure 3

Growth (%) of C. albicans strains with the deletion of ERG11 gene or with amino acid substitutions in Erg11p in the presence of PSZ (0–0.125 µg/mL) with or without the addition of SU (0–32 µg/mL). The C. albicans SC5314 (WT (A)), KS058 (erg11Δ/Δ (B)), 10C1B1I1 (ERG11^K143R (C)), 27A5A33A (ERG11^Y132F,F145L (D)) and 9B4B34A (ERG11^Y132F,K143R (E)) strains were cultured stationary for 24 h at a temperature of 28 °C. The experiment was performed in 3 replicates and obtained data were analyzed using Student’s t-test (*, p < 0.05; **, p < 0.01; ***, p < 0.001). The data for C. albicans KS058, 10C1B1I1, 27A5A33A and 9B4B34A strains were compared to those obtained for C. albicans SC5314 (WT) in certain concentrations of PSZ or SU.

Figure 4

Growth (%) of C. albicans strains with deletion of genes involved in sphingolipids (SLs) biosynthesis in the presence of PSZ (0–0.125 µg/mL) with or without addition of SU (0–32 µg/mL). The C. albicans SN95 (WT (A)), fen1Δ/Δ (B), fen12Δ/Δ (C) and fen1Δ/Δ;fen12Δ/Δ (D) strains were cultured stationary for 24 h at a temperature of 28 °C. The experiment was performed in 3 replicates and obtained data were analyzed using Student’s t-test (*, p < 0.05; **, p < 0.01; ***, p < 0.001). The data for C. albicans fen1Δ/Δ, fen12Δ/Δ and fen1Δ/Δ and fen12Δ/Δ strains were compared to those obtained for C. albicans SN95 (WT) in certain concentrations of PSZ or SU.

Figure 5

Growth (%) of C. albicans strains with the deletion of the ERG11 gene or with amino acid substitutions in Erg11p in the presence of PSZ (0–0.125 µg/mL) with or without the addition of capric acid (0–90.60 µg/mL). The C. albicans SC5314 (WT (A)), KS058 (erg11Δ/Δ (B)), 10C1B1I1 (ERG11^K143R (C)), 27A5A33A (ERG11^Y132F,F145L (D)) and 9B4B34A (ERG11^Y132F,K143R (E)) strains were cultured stationary for 24 h at a temperature of 28 °C. The experiment was performed in 3 replicates and obtained data were analyzed using Student’s t-test (*, p < 0.05; **, p < 0.01; ***, p < 0.001). The data for C. albicans KS058, 10C1B1I1, 27A5A33A and 9B4B34A strains were compared to those obtained for C. albicans SC5314 (WT) in certain concentrations of PSZ or SU.

Figure 6

Growth (%) of C. albicans strains with the deletion of genes involved in sphingolipids (SLs) biosynthesis in the presence of PSZ (0–0.125 µg/mL) with or without the addition of capric acid (0–90.60 µg/mL). The C. albicans SN95 (WT (A)), fen1Δ/Δ (B), fen12Δ/Δ (C) and fen1Δ/Δ;fen12Δ/Δ (D) strains were cultured stationary for 24 h at a temperature of 28 °C. The experiment was performed in 3 replicates and obtained data were analyzed using Student’s t-test (*, p < 0.05; **, p < 0.01; ***, p < 0.001). The data for C. albicans fen1Δ/Δ, fen12Δ/Δ and fen1Δ/Δ;fen12Δ/Δ strains were compared to those obtained for C. albicans SN95 (WT) in certain concentrations of PSZ or SU.

Figure 7

Growth curves of C. albicans strains with deletion of ERG11 gene or with amino acid substitutions in Erg11p under control conditions (YPD), in the presence of PSZ (0.0156 µg/mL) with or without the addition of surfactin (4 µg/mL; PSZ = 0.0156 µg/mL) or capric acid (45.3 µg/mL; PSZ = 0.0078 µg/mL). The C. albicans SC5314 (WT (A)), KS058 (erg11Δ/Δ (B)), 10C1B1I1 (ERG11^K143R (C)), 27A5A33A (ERG11^Y132F,F145L (D)) and 9B4B34A (ERG11^Y132F,K143R (E)) strains were cultured for 24 h (or 48 h in the case of KS058 strain) at a temperature of 28 °C, with shaking (120 rpm). The experiment was performed in 3 replicates and the figure represents mean absorbance (λ = 600 nm) ± SD.

Figure 8

Growth curves of C. albicans strains with deletion of genes involved in sphingolipids (SLs) biosynthesis in the presence of PSZ (0.0156 µg/mL) with or without the addition of surfactin (4 µg/mL) or capric acid (22.7 µg/mL or for fen1Δ/Δ and fen12Δ/Δ strain 11.35 µg/mL). The C. albicans SN95 (WT (A)), fen1Δ/Δ (B), fen12Δ/Δ (C) and fen1Δ/Δ;fen12Δ/Δ (D) strains were cultured for 24 h (or 48 h in case of fen1Δ/Δ and fen12Δ/Δ strain) at a temperature of 28 °C, with shaking (120 rpm). The experiment was performed in 3 replicates and the figure represents mean absorbance (λ = 600 nm) ± SD.

Figure 9

The adhesion (%) of C. albicans strains with altered ergosterol (SC5314 (WT), KS058, 10C1B1I1, 27A5A33A and 9B4B34A (A)) or sphingolipids biosynthesis (SN95 (WT), fen1Δ/Δ, fen12Δ/Δ and fen1Δ/Δ;fen12Δ/Δ) to abiotic surface under control conditions (PBS) or in the presence of surfactin (SU, 4 μg/mL) or capric acid (CA = 45.3 μg/mL for strains at (A); 22.7 μg/mL for strains at (B) or 11.35 μg/mL for C. albicans fen1Δ/Δ;fen12Δ/Δ strain). The data obtained for C. albicans strains incubated with SU or CA were compared to those under control conditions. The experiment was performed in 6 replicates and obtained data were analyzed using Student’s t-test (*, p < 0.05; **, p < 0.01; ***, p < 0.001).

Figure 10

The permeabilization of the plasma membrane (PM) of C. albicans strains SC5314 (WT), KS058, 10C1B1I1, 27A5A33A and 9B4B34A after 24 h of culturing with or without the presence of PSZ (0.0156 μg/mL), PSZ with SU (0.0156 with 4 μg/mL, respectively), or PSZ with CA (0.0078 with 45.3 μg/mL, respectively). The cells were stained with propidium iodide (PI, 4 μg/mL) for 5 min and then analyzed for PM permeabilization. The figure contains the representative microphotographs (scalebar = 50 µm). For every strain under each condition, at least 150 cells were analyzed.

Figure 11

The permeabilization of the plasma membrane (PM) of C. albicans strains SN95 (WT), fen1Δ/Δ, fen12Δ/Δ and fen1Δ/Δ;fen12Δ/Δ after 24 h culture with or without the presence of PSZ (0.0156 μg/mL), PSZ with SU (0.0156 with 4 μg/mL, respectively) or PSZ with CA (0.0156 with 22.7 μg/mL or 0.0156 μg/mL with 11.35 μg/mL for fen1Δ/Δ and fen12Δ/Δ strain, respectively). The cells were stained with propidium iodide (PI, 4 μg/mL) for 5 min and then analyzed for PM permeabilization. The figure contains representative microphotographs (scalebar = 50 µm). For every strain under each condition, at least 150 cells were analyzed.