Screening And Mechanism Study Of Compounds From TCM Medicine 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. The aim of the present work was to screen natural compounds against C. albicans biofilm and to investigate the anti-biofilm activities of selected compounds as well as their mechanism of action in vitro and in vivo. Of the29compounds were tested in our study, tetrandrine (TET) and pterostilbene (PTE) were found to exert antibiofilm activity when when applied to biofilm modes of growth alone. XTT reduction assay was performed to investigate the anti-biofilm activity of TET and PTE. The results showed that TET and PTE dramatically inhibit C. albicans biofilm formation and significantly reduced mature (24h) biofilm metabolic activity. These findings were visually confirmed by confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM), which showed that incubated with32μg/ml TET or16μg/ml PTE, the typical compact three-dimensional structure of biofilms were destructed and resulted in scant or nonexistent biofilms. Measurement of biofilm biomass assay results showed that TET reduced the biomass at the concentration of32μg/ml significantly (P<0.01), so did1μg/ml PTE (P<0.001), and a negative correlation was observed between compounds concentrations and the biomass of compounds treated biofilms. To clarify the underlying mechanism, cellular surface hydrophobicity (CSH) assay was performed to investigate the effects of TET or PTE on adherence, CSH results showed that TET or PTE decreased CSH of C. albicans biofilm significantly in a dose dependent manner, and a negative correlations was observed between CSH and TET or PTE concentrations. Time-kill curve assay was used to examine the growth of C. albicans when incubated with TET or PTE, and the results indicated that neither16μg/ml TET nor8μg/ml PTE could not affect the growth of C. albicans significantly. Next, C. albicans were grown in four media (solid and liquid) known to induce the morphological switch to study the role of TET or PTE in morphological transition from yeast to hyphae,the results showed that in liquid and on solid Spider meidia, TET affected germ tube formation obviously, while low concentration of PTE totally disrupted the formation of true hyphae in all the media tested. Then, cDNA microarray assay and real-time RT-PCR assay were conducted to study and research the genes expression profiles of C. albicans biofilm treated or untreated with PTE, change in C. albicans biofilm gene expression after PTE treatment was observed and some most important biofilm/filamentation-related genes were down-regulated, including RAS signal transduction GTPase gene RAS1and cell elongation protein gene ECE1. Moreover, ergosterol synthetic process-related genes and glycolysis/gluconeogenesis-related genes were down-regulated, while5heat shock protein genes were up-regulated in C. albicans after PTE exposure. These results indicated that inhibition of PTE on biofilm formation possibily depends on impairing the ability of C. albicans to change its morphology via down-regulating the expression of Ras/cAMP pathway. Real-time RT-PCR assay was performed to investigate the effect of TET, and the results showed that TET treatment resulted in a striking down-regulation of hypha-specific genes including HWP1, ALS3, ECE1. The GTP-ase gene RASI and the adenylate cyclase gene CYR1were also significantly down-regulated and a marked down-regulation of transcriptional factor EEG1in C. albicans biofilms, which is directly linked to inhibit hyphae formation.. Finally, C. albicans-C. elegans assay was used to study the antifungal antivity of TET in vivo and a rat central venous catheter biofilm model was adopted to investigate the anti-biofilm activity of PTE in vivo. C. elegans-C. albicans assay suggested that TET possessed antifungal activity in vivo and a rat central venous catheter biofilm model revealed that PTE inhibited C. albicans biofilm formation in the central venous catheter. Collectively, TET and PTE have a strong anti-biofilm effect against C. albicans in vitro and in vivo, and further translational research is required to determine whether the anti-biofilm effect of TET or PTE is applicable in a clinical setting.