Antifungal Mechanism Of Plagiochin E, An Antifungal Active Bis(Bibenzyls), Against Candida Albicans

Over the last decade, the incidence of fungal infections has increased dramatically, causing high morbidity and mortality. Patients with defective immune systems, due to AIDS (Acquired Immune Deficiency Syndrome), cancer chemotherapy, or immunosuppressive drugs, are significantly affected by fungi. Additionally, with the large scale application of broad-spectrum antifungal agents and the introduction of protocols for antifungal prophylaxis in patients at risk, there has been a notable increase in drug resistance. The development of more effective antifungal therapies is therefore of paramount importance.Natural products are still major sources of innovative therapeutic agents in various conditions, including infectious diseases. The bryophytes [Musci (mosses), Marchantiophyta (liverworts) and Anthocerotae (hornworts)], are morphologically placed between the algae and the pteridophytes (fern). Liverworts have been used in various remedies of folk medicine to treat illnesses of the cardiovascular system, tonsillitis, bronchitis, tympanitis, cystitis, as well as skin diseases and burns in China and Japan, In the past decades, various types of lipophilic terpenoids and aromatic compounds, which show significant biological activities, are isolated from the bryophytes. Now the bryophytes have been well know as a source of biologically active, naturally occurring compounds.Macrocyclic bis(bibenzyls) isolated from liverworts were found to have a wide range of biological activities such as antioxidation, antifungi, antivirus, antibacteria and cytotoxicity. As a result, they are attracting more and more attention. Plagiochin E (PLE), a macrocyclic bis(bibenzyl) isolated from liverwort Marchantia polymorpha L. (Marchantiaceae), has been found to have the reversal effect on multidrug resistance in adriamycin-induced resistant K562/A02 cells at concentration of 2 to 12μg/ml, with little cytotoxicity to normal cells. Furthermore, it exhibited in vitro antifungal activity against Candida albicans, and also found to reverse fungal resistance to fluconazole by increasing accumulation of fluconazole in the C. albicans. However, the underlying mechanism of action is unknown. The present study was designed to investigate the antifungal mechanism of PLE in C. albicans.The genome-scale cDNA microarray platform provides an effective research tool to investigate the antifungal target of PLE. To get a panoramic view of the responses of yeast cells to the PLE at the molecular level, cDNA microarray analysis was conducted. The global gene expression changes in C. albicans responding to PLE treatment were measured. A lot of genes significantly up-regulated or down-regulated by PLE were related to mitochondria, which are important organelles in fungi. Mitochondria play very important roles in the life cycle of cells, which are involved in a range of processes, such as the oxygen consumption, ATP production, and calcium mobilization. The results suggested that the mitochondria are a potential target of PLE. As a consequence, the effects of PLE on mitochondria function in C. albicans were determined. We assayed the mitochondrial membrane potential (mt△Ψ) using rhodamine 123, measured ATP level in mitochondria by HPLC, and detected the activities of mitochondrial F0F1-ATPase and dehydrogenases. Besides, the mitochondrial dysfunction-induced reactive oxygen species (ROS) production was determined by a fluorometric assay, and the effects of antioxidant L-cysteine on PLE-induced ROS production and the antifungal effect of PLE on C. albicans were also investigated. Results showed that exposure to PLE resulted in an elevation of mtA△Ψ, and a decrease of ATP level in mitochondria. The ATP depletion owed to PLE-induced enhancement of mitochondrial F0F1-ATPase and inhibition of the mitochondrial dehydrogenases. These dysfunctions of mitochondria caused ROS accumulation in C. albicans, and this increase in the level of ROS production and PLE-induced decrease in cell viability were prevented by addition of L-cysteine, indicating that ROS was an important mediator of the antifungal action of PLE. In summary, PLE exerts its antifungal activity through mitochondrial dysfunction-induced ROS accumulation in C. albicans.ROS are a key regulator to yeast apoptosis. As a consequence, the further study was designed to investigate whether PLE induced apoptosis in C. albicans. We assayed the cell cycle by flow cytometry using PI staining, observed the ultrastructure by transmission electron microscopy, studied the nuclear fragmentation by DAPI staining, and investigated the exposure of phosphatidylserine at the outer layer of the cytoplasmic membrane by the FITC-annexin V staining. The effect of PLE on expression of CDC28, CLB2, and CLB4 was determined by RT-PCR. Besides, the activity of metacaspase was detected by FITC-VAD-FMK staining, and the release of cytochrome c from mitochondria was also determined. Furthermore, the effect of antioxidant L-cysteine on PLE-induced apoptosis in C. albicans was also investigated. The results showed that cells treated with PLE showed typical markers of apoptosis: G2/M cell cycle arrest, chromatin condensation, nuclear fragmentation, and phosphatidylserine exposure. The expression of CDC28, CLB2, and CLB4 was down-regulated by PLE, which may contribute to PLE-induced G2/M cell cycle arrest. Besides, PLE promoted the cytochrome c release and activated the metacaspase, which resulted in the yeast apoptosis. The addition of L-cysteine prevented PLE-induced nuclear fragmentation, phosphatidylserine exposure, and metacaspase activation, indicating the ROS was an important mediator of PLE-induced apoptosis. In summary, PLE induced apoptosis in C. albicans through a metacaspase-dependent apoptotic pathway.The fungal cell wall plays an important role in the growth and viability of fungi. Chitin is indispensable for the construction of cell wall, and therefore, for fungal survival. Inhibition of chitin polymerization may affect cell wall maturation, septum formation and bud ring formation, damaging cell division and cell growth. Observation under the TEM showed the structure of cell wall and cell division in C. albicans were seriously damaged by PLE, which suggested the antifungal activity of PLE was associated with its effect on chitin synthesis. As a result, the effect of PLE on chitin synthesis in C. albicans was investigated at cellular and molecular levels. The effect of PLE on chitin synthetases (Chs) activities in vitro were assayed using 6-O-dansyl-N-acetylglucosamine (DNAG) as a fluorescent substrate, and its effect on chitin synthesis in situ was assayed by spheroplast regeneration. Reverse transcription-PCR (RT-PCR) was performed to assay its effect on expression of chitin synthetase genes (CHS). Enzymatic assays and spheroplast regeneration showed PLE inhibited chitin synthesis in vitro and in situ. Results of RT-PCR showed PLE significantly down-regulated the expression of CHS1, and up-regulated the expression of CHS2 and CHS3. Because different Chs is regulated at different stages of transcription and posttranslation, the down-regulation of CHS1 would decrease the level of Chsl and inhibit its activity, and the changes on CHS2 and CHS3 would not affect the activities of Chs2 and Chs3. These results indicate that the antifungal activity of PLE would be attributed to its inhibitory effect on chitin synthesis of cell wall in C. albicans. The inhibition of PLE on cell wall biosynthesis affects the continuity of fungal cell wall, makes fungi sensitive about osmotic pressure, causing fungi death. This PLE-induced cell wall damage would contribute to the antifungal action of PLE, and also can promote the inflow of fluconazole into fungal cell, resulting in reversal of fungal resistance.Above results indicated that PLE inhibited cell wall chitin synthesis and induced apoptosis in C. albicans through activating the metacaspase by ROS accumulation. These results would conduce to elucidate its underlying antifungal mechanism.