The Antifungal Effect Against Virulence Of Bisbibenzyls

Candida albicans, which exsist in normal oral, upper respiratory tract, bowel and vagina, normally commensal with human body. Once the external environment changes into pathogenic conditions, the delicate commensal balance between the host and pathogens is broken and converted into the parasitic relationship. Thus Candida albicans causes disease by infecting the human intestinal tract or broken the immune system disease. Azoles and polyene antibiotics are the main clinical used antifungal agents, but they all have different side effects to human body, and the emergency of drug resistance to zoles causes great difficulties for clinical treatment, so it is more and more important that we discover new antifungal drugs. The rich secondary metabolites of bryophytes provide great help for new drug research and development.Bryophyte is the second largest terrestrial plant group just behind angiosperms, widely distributed in nature, including the liverworts, mosses and hornworts. From ancient times there are some records about the application of bryophytes in medicine and pharmacy, bryophyte is widely used in the treatment of traumatic burns infection, inflammation, tuberculosis, cardiovascular disease, tonsillitis, bronchitis and otitis media. In recent years, botanists have isolated more than250kinds of aromatic compounds from bryophyte. Bisbenzyls display very good antifungal, antitumor and antioxidant effects. The purpose of this paper is to clarify the antifungal mechanism of bisbenzyls through a series of experiments.With relatively comprehensive investigation on the structure and mechanism of fluorescent protein, GFP has been widely applied. In order to explore new potential antifungal drugs and their mechanism, we established a visualization technology for pathogen research. Using the synchronous expression of marker genes and GFP, we judged the expression of purpose genes, and then helped to explore the development of new antifungal agents. Further more, GFP also had very important significance to the colonization activities and mechanism of microbe beneficial to agriculture or the environment. After test of multiple genes with GFP-labeled, we discovered that DPP3as the related gene to farnesol had a very good inhibitory effect on the convertion of yeast to hyphal. This gave us a prompt that farnesol and the related genes are very important mediums for the antifungal virulence of bibenzyls.Farnesol is a quorum sensing molecules. Many studies suggest that farnesol could inhibit the conversion of candida albicans from yeast to hyphal and formation of biofilm. DPP3, encoding phosphatase, has been demonstrated to be involved in farnesol synthesis by converting farnesyl pyrophosphate to farnesol. We used a GFP labelled Candida albicans strain BWP17-DPP3-GFP to detect antifungal activity of the compounds from genes levels. We measured the farnesol production by HPLC-MS and quantified Dpp3expression by detecting the fluorescent intensity of green fluorescent protein tagged strain using Confocal Laser Scanning microscopy and Multifunction Microplate Reader. The DPP3transcripts were determined by real-time PCR. The data indicated that the bisbibenzyls exerted antifungal effects through stimulating the synthesis of farnesol via upregulation of Dpp3, suggesting a potential antifungal application of bisbibenzyls. In addition, our assay provides a novel, visual and convenient method to measure active compounds against morphogenesis switch.Caenorhabditis elegans as a model organism with a short life cycle, could multiply in a short time. The worm was very tiny and easily operated. We just needed a few medicines to achieve its related experiment, so it is an ideal internal experimental models for antifungal study. We did the in vivo study to the clinical resistant strains, clinical sensitive plant and wild strains, and the results showed that plaginchin F could increase the survival rate of nematodes to almost50%at8-16μg/ml.This subject demonstrated that bisbibenzyls can inhibit hyphae growth and biofilms formation in vitro, and also could prolong animal life through inhibiting the growth of fungal growth and hyphal in vivo. Our work provided a new approach for screening the compounds possessing the antifunfal activity.