Pterostilbene (PTE) is a stilbene-derived phytoalexin that originates from several natural plant sources. In this study, we evaluated the activity of PTE against Candida albicans biofilms and explored the underlying mechanisms. In 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) reduction assays, biofilm biomass measurement, confocal laser scanning microscopy, and scanning electron microscopy, we found that ≤16 μg/ml PTE had a significant effect against C. albicans biofilms in vitro , while it had no fungicidal effect on planktonic C. albicans cells, which suggested a unique antibiofilm effect of PTE. Then we found that PTE could inhibit biofilm formation and destroy the maintenance of mature biofilms. At 4 μg/ml, PTE decreased cellular surface hydrophobicity (CSH) and suppressed hyphal formation. Gene expression microarrays and real-time reverse transcription-PCR showed that exposure of C. albicans to 16 μg/ml PTE altered the expression of genes that function in morphological transition, ergosterol biosynthesis, oxidoreductase activity, and cell surface and protein unfolding processes (heat shock proteins). Filamentation-related genes, especially those regulated by the Ras/cyclic AMP (cAMP) pathway, including ECE1 , ALS3 , HWP1 , HGC1 , and RAS1 itself, were downregulated upon PTE treatment, indicating that the antibiofilm effect of PTE was related to the Ras/cAMP pathway. Then, we found that the addition of exogenous cAMP reverted the PTE-induced filamentous growth defect. Finally, with a rat central venous catheter infection model, we confirmed the in vivo activity of PTE against C. albicans biofilms. Collectively, PTE had strong activities against C. albicans biofilms both in vitro and in vivo , and these activities were associated with the Ras/cAMP pathway.

Candida albicans is the most common fungal pathogen. Galleria mellonella is widely used as an infection model host. Nevertheless, the G. mellonella–C. albicans infection model had not been optimized for drug evaluation before this study. In this work, we revealed that 5×105 colony forming unit (CFU)/larva was a suitable inoculum to optimize the G. mellonella–C. albicans infection model in order to evaluate antifungal agents. Using our optimized model, the antifungal effect of fluconazole, amphotericin B and flucytosine, and the synergy between amphotericin B and flucytosine were successfully verified. Thus, this study provides a rapid, inexpensive and reliable way to evaluate antifungals in vivo.

It was found in our previous study that berberine (BBR) and fluconazole (FLC) used concomitantly exhibited a synergism against FLC-resistant Candida albicans in vitro . The aim of the present study was to clarify how BBR and FLC worked synergistically and the underlying mechanism. Antifungal time-kill curves indicated that the synergistic effect of the two drugs was BBR dose dependent rather than FLC dose dependent. In addition, we found that BBR accumulated in C. albicans cells, especially in the nucleus, and resulted in cell cycle arrest and significant change in the transcription of cell cycle-related genes. Besides BBR, other DNA intercalators, including methylene blue, sanguinarine, and acridine orange, were all found to synergize with FLC against FLC-resistant C. albicans . Detection of intracellular BBR accumulation by fluorescence measurement showed that FLC played a role in increasing intracellular BBR concentration, probably due to its effect in disrupting the fungal cell membrane. Similar to the case with FLC, other antifungal agents acting on the cell membrane were able to synergize with BBR. Interestingly, we found that the efflux of intracellular BBR was FLC independent but strongly glucose dependent and associated with the drug efflux pump Cdr2p. These results suggest that BBR plays a major antifungal role in the synergism of FLC and BBR, while FLC plays a role in increasing the intracellular BBR concentration.

Candida albicans biofilms show resistance to many clinical antifungal agents and play a considerable contributing role in the process of C. albicans infections. New antifungal agents against C. albicans biofilms are sorely needed. The aim of this study was to evaluate sanguinarine (SAN) for its activity against Candida albicans biofilms and explore the underlying mechanism. The MIC ₅₀ of SAN was 3.2 μg/ml, while ≥0.8 μg/ml of SAN could suppress C. albicans biofilms. Further study revealed that ≥0.8 μg/ml of SAN could decrease cellular surface hydrophobicity (CSH) and inhibited hypha formation. Real-time reverse transcription-PCR (RT-PCR) results indicated that the exposure of C. albicans to SAN suppressed the expression of some adhesion- and hypha-specific/essential genes related to the cyclic AMP (cAMP) pathway, including ALS3 , HWP1 , ECE1 , HGC1 , and CYR1 . Consistently, the endogenous cAMP level of C. albicans was downregulated after SAN treatment, and the addition of cAMP rescued the SAN-induced filamentation defect. In addition, SAN showed relatively low toxicity to human umbilical vein endothelial cells, the 50% inhibitory concentration (IC ₅₀ ) being 7.8 μg/ml. Collectively, the results show that SAN exhibits strong activity against C. albicans biofilms, and the activity was associated with its inhibitory effect on adhesion and hypha formation due to cAMP pathway suppression.

Candida albicans is the most common human fungal pathogen and has a high propensity to develop biofilms that are resistant to traditional antifungal agents. In this study, we investigated the effect of tetrandrine (TET) on growth, biofilm formation and yeast-to-hypha transition of C. albicans. We characterized the inhibitory effect of TET on hyphal growth and addressed its possible mechanism of action. Treatment of TET at a low concentration without affecting fungal growth inhibited hyphal growth in both liquid and solid Spider media. Real-time RT-PCR revealed that TET down-regulated the expression of hypha-specific genes ECE1, ALS3 and HWP1, and abrogated the induction of EFG1 and RAS1, regulators of hyphal growth. Addition of cAMP restored the normal phenotype of the SC5314 strain. These results indicate that TET may inhibit hyphal growth through the Ras1p-cAMP-PKA pathway. In vivo, at a range of concentrations from 4 mg/L to 32 mg/L, TET prolonged the survival of C. albicans-infected Caenorhabditis elegans significantly. This study provides useful information for the development of new strategies to reduce the incidence of C. albicans biofilm-associated infections.