| Over the last30years, the incidence of clinical fungal infection has dramatically increased due to the increased immune-compromised patients caused by organ transplantation, the application of immunosuppressive drugs, cancer chemotherapy, and widely used antibiotics. Additionally, the drug resistance appears as the antifungal agents are broadly used in clinic. With the long time treatment received by the patients, the persistence emerges, which makes the clinical therapy much more difficult. However, the antifungal drugs used are limited, especially for the fungicidal agents. The development of new antifungal drugs and therapitical strategies, investigating the mechanism of persistence and designing drugs targeting the persistence are therefore important.Natural plants, as an important source of natural medicines are used to treat human diseases. Lichens are quite a special large group in the the plant kingdom as high degree of integration of the symbiotic complex between algae and fungi. Recently, abundant secondary metabolites from lichens with multiple bioactivities have been obtained as reported in the literature. Retigeric acid B (RAB), a pentacyclic triterpenoid from the lichen species Lobaria kurokawae, has been isolated in our lab. It displays moderate antifungal action, with minimum inhibitory concentrations (MICs) ranging from8to16μg/ml. It also has synergistic antifungal activity when applied in combination with azoles including fluconazole, ketoconazole and itraconazole, especially for azoles-resistant strains. Our previous research showed the synergistic action was astributed to the inhibited activity of efflux pumps by RAB and reduced ergosterol synthesis through downregulation of ERG11. To better elucidate the antifungal mechanisms of RAB, we focused on the virulence, biofilm formation, proliferation, apoptosis and necrosis of Candida albicans affected by RAB. Besides that, the persistence of C. albicans, an important factor for the relapse of fungal infection, was also investigated.C. albicans, an opportunistic human pathogen, undergoes yeast-to-hyphal switch according to the growing conditions. Hyphae filaments, having the advantage of adhering and penetrating tissue, are apt to invade the host. The yeast to hyphae conversion significantly increases its virulence. The study showed RAB could inhibit the yeast-to-hyphal transition in hyphae stimulating growing condition. The in-vivo study showed RAB could reduce the virulence of C. albicans through blocking the hyphae formation when Caenorhabditis elegans was utilized as an infection model, which conferred the improved longevity of the hosts. To elucidate the underlying mechanism of RAB against hyphae formation, real time quantitative PCR (qPCR) was applied. The results suggested RAB downregulated Rasl-cAMP-Efgl pathway, among which cAMP plays a critical role in regulating the hyphae formation. Furthern research showed RAB reduced the intracellular level of cAMP in a dose-dependent manner revealed by cAMP detection assay. The exogenerous cAMP could restore the hyphae formation inhibited by RAB, suggesting the reduced cAMP by RAB was responsible for the inhibited hyphae formation. In C. albicans, cAMP was synthized by Cdc35. To determine whether RAB has inhibitory effect on the activity of Cdc35, two independent methods were carried out. C. albicans cells treated with RAB were lysed to detect the Cdc35activity in the cell lysates. The results showed the Cdc35activity was inhibited by RAB in a dose-dependent way. To determine whether RAB directly inhibit Cdc35, the purified catalytic domain of Cdc35was used in the adenylyl cyclase activity inhibition assays. Experimental results suggested RAB did not influence the enzymatic activity, which suggested another factor induced by RAB may inhibit the activity of Cdc35. It has reported farnesol could directly interact with Cdc35, inhibiting cAMP synthesis. GC-MS (gas chromatography-mass spectrometry) was performed to detect the amounts of farnesol secreted by C. albicans when treated by RAB. The results demonstrated RAB could stimulate farnesol synthesis. It was also found RAB could induce the expression of Dpp3, a protein synthizing farnesol. The resulsts of qPCR and protein detection assay based on GFP fluorescence showed adesins and invasins such as Als3were also inhibited by RAB. Based on the above results, it is concluded that RAB upregulates the expression of Dpp3, which inhibites the activity of Cdc35, reducing the synthesis of cAMP, a critical factor in Rasl-cAMP-Efgl signaling pathway regulating hyphae formation, and then inhibites the adhesins and invasins, the virulence factors, to promote the survival of infected hosts.Based on the synergesitic action of RAB and azoles against clinical C. albicans strains, we determine the effect of RAB and azoles on inhibiting the hyphae formation and preventing biofilm formation. Filamentation is not only increasing the virulence, but also a requirement for a mature biofilm formation. A biofilm is a protected niche for microorganisms in which they are less susceptible to antibiotic treatment and can create a source of persistent infection. When the antifungal drugs are removed, the persisters start to replicate, causing the recurrent infection, which makes biofilm-associated C. albicans infections much more difficult to treat. The in-vitro study showed low doses of RAB combined with azoles displayed additive or synergistic action against hyphae formation. The Balb/C infective mice model also showed RAB could prolong the longevity of the hosts when applied with fluconazole. The kidney fungal burden examination showed the combination treatment significantly reduced the pathogenesis of C. albicans. Histopathological analysis revealed that C. albicans under the combination treatment displayed only in yeast form, while the organisms in control group or drug-alone treated group grew mainly in hyphae form. It is concluded that the combination treatment could promote the longevity of the infected host through inhibiting the hyphae formation and reducing the pathogenesis. The in vitro co-culture of C. albicans cells and hosts’cells reveals the combination of RAB and fluconazole significantly inhibit the invasiveness and pathogenesis. To dermine the effect of the combination treatment of RAB and fluconazole on biofilm formation, checkerboard microdilution assay was performed. FICI (fractional inhibitory concentration index) method used in the assay showed RAB displayed syngergistic manner against the biofilm formation together with azoles. Furtherly, the biofilm structure stained by fluorescein diacetate and propidium iodide was observed using confocal scanning laser microscope. The biofilm under single drug treatment is thinner compared with that in control. They all are mainly composed of filaments. However, no biofilm formes and only clusters of yeast cells adhere to the substratum when cells are exposed to combination treatment. Of the interest is the fungicidal effect under the combination treatment. Three dimentional scanning results showed non-treated C. albicans forms a thick biofilm with a tight structure, while single treated C. albicans forms a thinner one. Above results showed the combination of RAB and azoles could synergistically inhibit the biofilm formation through preventing the hyphae formation. qPCR results showed the downregulation of MDR1, one of drug pump, by RAB facilitates the antifungal effect of azoles.For C. albicans, the yeast form, as a disseminating form, plays a critical role in the early stage of Candida infectious process for the suitable dimensions and physical properties to get access to the host’s bloodstream. So inhibiting the replication is also a strategy for overcoming the Candida infection. RAB suppresses the proliferative rate of cells in C. albicans by regulating the cell cycle. Low doses of RAB treatment leads to accumulation of cells at the G2/M phase, whereas high concentrations of RAB intriguingly caused cells accumulated in G1phase of the cell cycle. The finding that RAB causes ROS accumulation in C. albicans suggestes the extra ROS could damage DNA and prevent the G2/M transition at the cell cycle checkpoint, and subsequently leading to G2/M cell cycle arrest. RAB treatment could also decrease the levels of cAMP in a dose-dependent manner, resulting in G1cell cycle arrest, which is in line with a previous report that the antifungal agents with an ability of reducing the cAMP level could prolong the cell cycle through delaying the expression of G1cyclins. This notion is further supported by the recovery of RAB-induced G1cell cycle arrest with addition of exogenous cAMP.C. albicans cells exhibit features of death revealed by apoptosis and necrosis detection when they are exposed to32μg/ml of RAB. Transmission electron micrographs showed chromatin condensation, dispersion and nuclear fragmentation and plasma membrane breakage and organelle swelling in RAB-treated cells. RAB induced cells death in C. albicans is due to the enhanced ROS formation, which consistent with published studies that some antifungal agents exert the antifungal activity via ROS. RAB increases the mt△ψ, but decreases ATP level in a dose-dependent manner. The drop of ATP level is likely attributed to the mt△ψ hyperpolarization that is induced by enhanced ROS accumulation, which together contributes to the cell death.To better understand the persistence of C. albicans, an effective model needs to be established to screen the persisters. The strain with TDH3tagged by GFP is treated by high dose of amphotericin B, then cells with GFP positive and PI negative are considered as persisters. This model revealed RAB had no action to eliminate the persister, in consistent with the conventional method. To isolate the persister for transcriptional profiling, different cultural conditions were screened to achieve high persister rate.To construct a molecular tool used in C. albicans genetic research or screening antifungal agents, a plasmid backbone was inserted with NATl sequence (resistant to nourseothricin), an origin sequence ORC984or CEN4, and inverted telomeres at both ends. The constructed plasmids were linearized and transformed into C. albicans cells. The contour-clamped homogeneous electric field gel electrophoresis plus southern blot revealed some transformants have free plasmids in Candida cells, which are not integrative to the genomes. And the plasmids in the transformants could stably replicate in different generations. The plasmid carring a functional gene could also makes the tranformants display the phenotype, suggesting it is a good genetic tool for Candida research.In this thesis, we described the antifungal mechanism of RAB, which is the basis for the mechanism of triterpene analogs. The combination treatment provides a possible application in overcoming clinical fungal infection. The investigation of persistence paves the way for understanding the mechanism of persistence and designing new drugs targeting the persister formation. The plasmid constructed for C. albicans works as an effective molecular tool in Candida genetic research and screening antifungal agents. |
