Characterization of plant-derived saponin natural products against Candida albicans

Abstract

Candida albicans is an opportunistic fungal pathogen capable of life-threatening disseminated infections particularly in immunocompromised patients. Resistance to many clinically used antifungal agents has created a need to identify and develop a new generation of compounds for therapeutic use. A compound screen to identify potential antifungal natural products was undertaken, identifying 12 saponins, some of which have not been previously described. In the Caenorhabditis elegans model, some saponins conferred nematode survival comparable to that of amphotericin B. Of the 12 antifungal saponins identified, two were selected for further analysis. C. albicans isolates were inhibited by these compounds at relatively low concentrations (16 and 32 microg mL(-1)) including isolates resistant to clinically used antifungal agents. C. albicans hyphae and biofilm formation were also disrupted in the presence of these natural products, and studies demonstrate that fungal cells in the presence of saponins are more susceptible to salt-induced osmotic stress. Although saponins are known for their hemolytic activity, no hemolysis of erythrocytes was observed at three times the minimal inhibitory concentration for C. albicans, suggesting the saponins may have a preference for binding to fungal ergosterol when compared to cholesterol. Importantly, when used in combination with photosensitizer compounds, the fungus displayed increased susceptibility to photodynamic inactivation due to the ability of the saponins to increase cell permeability, thereby facilitating penetration of the photosensitizers. The large proportion of compounds identified as antifungal agents containing saponin structural features suggests it may be a suitable chemical scaffold for a new generation of antifungal compounds.

Figure 1

Structures of the natural product saponins identified in the C. elegans-C. albicans antifungal drug discovery screen (all structural representations were provided by Analyticon Discovery). For each of the compounds the maximum nematode survival (%) is indicated.

Figure 2

Dose response of select compounds identified in the C. elegans-C. albicans assay. (A) Two saponins (A7 and A20) were as effective as amphotericin B in promoting C. elegans survival. The decrease in nematode survival for A7 at the highest concentration tested suggests the saponin maybe toxic to the nematode (B) Dose response of two saponin compounds (A16 and A19) used in further studies.

Figure 3

Biofilm formation for two saponin family members identified in the screen compared to untreated silicone pads and caspofungin, a compound able to inhibit C. albicans biofilm formation. Standard deviations are depicted and based on 5–11 silicone pad measurements.

Figure 4

Phototoxicity in C. albicans DAY185 after incubation with or without 4 μg/ml A16 and (A) 100 μM RB, (B) 100 μM ce6, and (C) 10μM PEI- ce6. Fungal cells were incubated with the PS for 30 min, washed and then illuminated and survival fractions were determined as described in the methods. Values are means of three separate experiments and bars are SEM. *** P<0.001 compared to PS alone

Figure 5

Confocal laser scanning microscopy of C. albicans cells after incubation with (A) 100μM ce6 and in combination with 4 μg/ml A16 for (B) 1hr and (C) 24hrs. Scale bar = 20 μm for (A) and (B) and 14 μm for (C).