Ai-Xia Cheng

Plagiochin E, an antifungal active macrocyclic bis(bibenzyl), induced apoptosis in Candida albicans through a metacaspase-dependent apoptotic pathway.

BACKGROUND Plagiochin E (PLE) is an antifungal active macrocyclic bis(bibenzyl) isolated from liverwort Marchantia polymorpha L. To elucidate the mechanism of action, previous studies revealed that the antifungal effect of PLE was associated with the accumulation of ROS, an important regulator of apoptosis in Candida albicans. The present study was designed to find whether PLE caused apoptosis in C. albicans. METHODS 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. RESULTS Cells treated with PLE showed typical markers of apoptosis: G(2)/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 G(2)/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. CONCLUSIONS PLE induced apoptosis in C. albicans through a metacaspase-dependent apoptotic pathway. GENERAL SIGNIFICANCE In this study, we reported for the first time that PLE induced apoptosis in C. albicans through activating the metacaspase. These results would conduce to elucidate its underlying antifungal mechanism.

Plagiochin E, an antifungal bis(bibenzyl), exerts its antifungal activity through mitochondrial dysfunction-induced reactive oxygen species accumulation in Candida albicans.

BACKGROUND Plagiochin E (PLE) is an antifungal macrocyclic bis(bibenzyl) isolated from liverwort Marchantia polymorpha L. Its antifungal mechanism is unknown. To elucidate the mechanism of action, its effect on mitochondria function in Candida albicans was studied. METHODS We assayed the mitochondrial membrane potential (mtDeltapsi) using rhodamine 123, measured ATP level in mitochondria by HPLC, and detected the activities of mitochondrial F(0)F(1)-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 Exposure to PLE resulted in an elevation of mtDeltapsi, and a decrease of ATP level in mitochondria. The ATP depletion owed to PLE-induced enhancement of mitochondrial F(0)F(1)-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. CONCLUSIONS PLE exerts its antifungal activity through mitochondrial dysfunction-induced ROS accumulation in C. albicans. GENERAL SIGNIFICANCE The effect of PLE on the mitochondria function in C. albicans was assayed for the first time. These results would conduce to elucidate its underlying antifungal mechanism.

A lysosomal-mitochondrial death pathway is induced by solamargine in human K562 leukemia cells.

Solamargine (SM), a steroidal alkaloid glycoside from Solanum nigrum L., displayed a superior cytotoxicity to many human tumor cells. Further investigation with human K562 leukemia cells found that SM could induce an early lysosomal rupture within 2h as assessed by acridine-orange relocation and alkalinization of lysosomes. Intracellular lysosomal rupture is also confirmed with the release of cathepsin B to cytosol detected by western blot. Subsequent mitochondrial damage including mitochondrial membrane permeabilization detected by decrease membrane potential as well as the release of cytochrome c from mitochondria was also observed. The cellular Ca(2+) overload is more pronounced in SM-treated cells. Cells exposed to 10 microM SM for 30 min showed a maximum 7-fold increase in intracellular calcium concentration compared with vehicle-treated controls. The down-expression of Bcl-2, up-regulation of Bax, caspase-3 and caspase-9 activities followed by above changes revealed that the cytotoxicity of SM was involved in a lysosomal-mitochondrial death pathway induced by SM.

In Vitro Activities of Retigeric Acid B Alone and in Combination with Azole Antifungal Agents against Candida albicans

ABSTRACT The vitro antifungal activity of retigeric acid B (RAB), a pentacyclic triterpenoid from the lichen species Lobaria kurokawae, was evaluated alone and in combination with fluconazole, ketoconazole, and itraconazole against Candida albicans using checkerboard microdilution and time-killing tests. The MICs for RAB against 10 different C. albicans isolates ranged from 8 to 16 μg/ml. A synergistic action of RAB and azole was observed in azole-resistant strains, whereas synergistic or indifferent effects were observed in azole-sensitive strains when interpreted by a separate approach of the fractional inhibitory concentration index and ΔE model (the difference between the predicted and measured fungal growth percentages). In time-killing tests, we used both colony counts and a colorimetric assay to evaluate the combinational antifungal effects of RAB and azoles, which further confirmed their synergistic interactions. These findings suggest that the natural product RAB may play a certain role in increasing the susceptibilities of azole-resistant C. albicans strains.

Retigeric Acid B Attenuates the Virulence of Candida albicans via Inhibiting Adenylyl Cyclase Activity Targeted by Enhanced Farnesol Production

Candida albicans, the most prevalent fungal pathogen, undergoes yeast-to-hyphal switch which has long been identified as a key fungal virulence factor. We showed here that the lichen-derived small molecule retigeric acid B (RAB) acted as an inhibitor that significantly inhibited the filamentation of C. albicans, leading to the prolonged survival of nematodes infected by C. albicans. Quantitative real-time PCR analysis and intracellular cAMP measurement revealed RAB regulated the Ras1-cAMP-Efg1 pathway by reducing cAMP level to inhibit the hyphae formation. Confocal microscopic observation showed RAB induced the expression of Dpp3, synthesizing more farnesol, which was confirmed by gas chromatography-mass spectroscopy detection. An adenylyl cyclase activity assay demonstrated RAB could repress the activity of Cdc35 through stimulating farnesol synthesis, thus causing a decrease in cAMP synthesis, leading to retarded yeast-to-hyphal transition. Moreover, reduced levels of intracellular cAMP resulted in the inhibition of downstream adhesins. Together, these findings indicate that RAB stimulates farnesol production that directly inhibits the Cdc35 activity, reducing the synthesis of cAMP and thereby causing the disruption of the morphologic transition and attenuating the virulence of C. albicans. Our work illustrates the underlying mechanism of RAB-dependent inhibition of the yeast-to-hyphal switch and provides a potential application in treating the infection of C. albicans.

Effect of plagiochin E, an antifungal macrocyclic bis (bibenzyl), on cell wall chitin synthesis in Candida albicans

AbstractAim:To investigate the effect of plagiochin E (PLE), an antifungal macrocyclic bis(bibenzyl) isolated from liverwort Marchantia polymorpha L, on cell wall chitin synthesis in Candida albicans.Methods:The effect of PLE on chitin synthesis in Candida albicans was investigated at the cellular and molecular levels. First, the ultrastructural changes were observed under transmission electron microscopy (TEM). Second, the effects of PLE on chitin synthetase (Chs) activities in vitro were assayed using 6-O-dansyl-N-acetylglucosamine as a fluorescent substrate, and its effect on chitin synthesis in situ was assayed by spheroplast regeneration. Finally, real-time RT–PCR was performed to assay its effect on the expression of Chs genes (CHS).Results:Observation under TEM showed that the structure of the cell wall in Candida albicans was seriously damaged, which suggested that the antifungal activity of PLE was associated with its effect on the cell wall. Enzymatic assays and spheroplast regeneration showed that PLE inhibited chitin synthesis in vitro and in situ. The results of the PCR showed that PLE significantly downregulated the expression of CHS1, and upregulated the expression of CHS2 and CHS3. Because different Chs is regulated at different stages of transcription and post-translation, the downregulation of CHS1 would decrease the level of Chs1 and inhibit its activity, and the inhibitory effects of PLE on Chs2 and Chs3 would be at the post-translational level or by the inhibition on the enzyme-active center.Conclusion:These results indicate that the antifungal activity of PLE would be attributed to its inhibitory effect on cell wall chitin synthesis in Candida albicans.

Cloning and functional characterization of a 4-coumarate CoA ligase from liverwort Plagiochasma appendiculatum

Plant phenylpropanoids represent a large group of secondary metabolites which have played an important role in terrestrial plant life, beginning with the evolution of land plants from primitive green algae. 4-Coumarate: coenzyme A ligase (4CL) is a provider of activated thioester substrates within the phenylpropanoid synthesis pathway. Although 4CLs have been extensively characterized in angiosperm, gymnosperm and moss species, little is known of their functions in liverworts. Here, a 4CL homolog (designated as Pa4CL1) was isolated from the liverwort species Plagiochasma appendiculatum. The full-length cDNA sequence of Pa4CL1 contains 1644bp and is predicted to encode a protein with 547amino acids. The gene products were 40-50% identical with 4CL sequences reported in public databases. The recombinant protein was heterologously expressed in Escherichia coli and exhibited a high level of 4CL activity, catalyzing formation of hydroxycinnamate-CoA thioesters by a two-step reaction mechanism from corresponding hydroxycinnamic acids. Kinetic analysis indicated that the most favorable substrate for Pa4CL1 is p-coumaric acid. The transcription of Pa4CL1 was induced when P. appendiculatum thallus was treated with either salicylic acid or methyl jasmonate.

The Function and Catalysis of 2-Oxoglutarate-Dependent Oxygenases Involved in Plant Flavonoid Biosynthesis

Flavonoids are secondary metabolites derived from phenylalanine and acetate metabolism. They fulfil a variety of functions in plants and have health benefits for humans. During the synthesis of the tricyclic flavonoid natural products in plants, oxidative modifications to the central C ring are catalyzed by four of FeII and 2-oxoglutarate dependent (2-ODD) oxygenases, namely flavone synthase I (FNS I), flavonol synthase (FLS), anthocyanidin synthase (ANS) and flavanone 3β-hydroxylase (FHT). FNS I, FLS and ANS are involved in desaturation of C2–C3 of flavonoids and FHT in hydroxylation of C3. FNS I, which is restricted to the Apiaceae species and in rice, is predicted to have evolved from FHT by duplication. Due to their sequence similarity and substrate specificity, FLS and ANS, which interact with the α surface of the substrate, belong to a group of dioxygenases having a broad substrate specificity, while FNS I and FHT are more selective, and interact with the naringenin β surface. Here, we summarize recent findings regarding the function of the four 2-ODD oxygenases and the relationship between their catalytic activity, their polypeptide sequence and their tertiary structure.

Synergistic mechanisms of retigeric acid B and azoles against Candida albicans

Aims:  To clarify the underlying synergistic antifungal mechanisms of retigeric acid B (RAB) in combination with azoles against Candida albicans.

Retigeric acid B exerts antifungal effect through enhanced reactive oxygen species and decreased cAMP

BACKGROUND Retigeric acid B (RAB), a triterpene acid isolated from Lobaria kurokawae exerts antifungal effect. The present study was designed to elucidate the underlying mechanisms by which RAB regulates the proliferation and cell death of Candida albicans. METHODS We measured the metabolic activity of C. albicans with WST1 Cell Proliferation and Cytotoxicity Assay Kit, analyzed the cell cycle by flow cytometry, visualized the ultrastructure by transmission electron microscopy (TEM) and investigated the apoptosis and necrosis induced by RAB using confocal microscopy. The reactive oxygen species (ROS) accumulation was determined by spectrophotometry, flow cytometry and fluorescent microscopy. The mtΔψ was detected using flow cytometry. And the levels of intracellular cAMP and ATP were measured with cAMP ELISA and ATP Assay Kits, respectively. RESULTS The proliferation of the yeasts was blocked in G(2)/M phase by a low dose of RAB treatment and in G(1) phase at high concentration. When cultured in phosphate buffered saline (PBS) deprived of energy source, yeasts displayed the phenotype of death caused by accumulated ROS, mtΔψ hyperpolarization and dramatic decrease in ATP level in the presence of high dose of RAB. GENERAL SIGNIFICANCE RAB inhibits the growth of C. albicans by stimulating ROS production and reducing intracellular cAMP. The ROS accumulation, mtΔψ hyperpolarization, ATP depletion and damaged plasma membrane integrity together mediate cell death of C. albicans induced by RAB. Our findings provide a novel molecular mechanism for exploring possible applications of lichen derived metabolites in fighting fungal infection in humans.