Effect of loureirin A against Candida albicans biofilms.

Loureirin A is a major active component of Draconis sanguis, a traditional Chinese medicine. This work aimed to investigate the activity of loureirin A against Candida albicans biofilms. 2, 3-Bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT)reduction assay and scanning electron microscopy were used to investigate the anti-biofilm effect. Minimal inhibitory concentration testing and time-kill curve assay were used to evaluate fungicidal activity. Cell surface hydrophobicity (CSH) assay and hyphal formation experiment were respectively carried out to investigate adhesion and morphological transition, two virulence traits of C. albicans. Real-time RT-PCR was used to investigate gene expression. Galleria mellonella-C. albicans and Caenorhabditis elegans-C. albicans infection models were used to evaluate the in-vivo antifungal effect. Human umbilical vein endothelial cells and C. elegans nematodes were used to evaluate the toxicity ofloureirin A. Our data indicated that loureirin A had a significant effect on inhibiting C. albicans biofilms, decreasing CSH, and suppressing hyphal formation. Consistently, loureirin A down-regulated the expression of some adhesion-related genes and hypha/biofilm-related genes. Moreover, loureirin A prolonged the survival of Galleria mellonella and Caenorhabditis elegans in C. albicans infection models and exhibited low toxicity. Collectively, loureirin A inhibits fungal biofilms, and this effect may be associated with the suppression of pathogenic traits, adhesion and hyphal formation.

Activity of Sanguinarine against Candida albicans Biofilms.

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 MIC50 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 (IC50) 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.

In vitro and in vivo activities of pterostilbene against Candida albicans biofilms.

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.

Effect of tetrandrine against Candida albicans biofilms.

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.

Using Galleria mellonella-Candida albicans infection model to evaluate antifungal agents.

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 × 10(5) 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.