Antifungal Activities Of Terpenoids And Metallic Copper

Recently,the incidence and mortality of invasive fungal infections are increasing ever year.Additonaly,the emergence and rapid spread of drug resistance further reduces the efficacy of traditional antifungal drug treatment,bring great challenge to clinical therapy.Therefore,the development of new antifungal drugs and more effective strategies to overcome fungal infection and resistance is of paramount importance at present.The bryophytes are widely distributed in nature,and the vitality is stubborn.As a traditional medicinal resource,lots of compounds with good antifungal activity have been screened out in recent years.Terpenoids are the most common chemical constituents found in bryophytes,exhibiting a wide range of biological activities and have great application value.Through the determination of minimum inhibitory concentration(MIC),we have screened 106 terpenoid natural products in the compound library constructed by our laboratory,and five small molecular compounds with good antifungal activity were found,including three sesquiterpenoides and two diterpenoids,MICs of which were all less than 128 ug/ml against Candida albicans.Among them,Chiloscyphenol A(CA),a sesquiterpenoid with a novel rearranged calamenene skeleton isolated from two Chinese liverworts,Chiloscyphus polyanthus(L.)and Bazzania albifolia Horik.,shows good antifungal activity and the amount is sufficient and thus is used to do further research.We found that CA was effective against five tested Candida species with the MIC of 8～32μg/ml and exhibited fungicidal activity against C.albicans in both the planktonic state and mature biofilms.Furher observations showed that in the presence of CA,the mitochondrial membrane potential and ATP content of Candida albicans were significantly changed,and the mitochondrial outer membrane protein Tom70 was abnormally distributed,accompanied by ROS accumulation,resulting in mitochondrial dysfunction and oxidative damage to other cellular structures.In addition,CA caused perturbation of the cell membrane and increased membrane permeability,as demonstrated by specific staining and confocal microscopic and transmission electron microscopy(TEM)observations and by calcein-leakage measurements.Therefore,we conclude that CA exerts fungicidal activity by eliciting both mitochondrial dysfunction and plasma membrane destruction in C.albicans.Copper surfaces possess efficient and rapid killing activity on bacteria,fungi,and viruses.The clinical application of copper can be taken as an effective strategy for preventing the fungal infections.Studies have shown that copper and copper alloys could completely kill the planktonic C.albicans cells within 90 min.By measuring the killing kinetics,we found that copper surface also has a good fungicidal effect on C.albicans mature biofilms.The ICP-AES analysis revealed that the copper ion released from copper surfaces is much lower than the effective concentration of copper sulfate,indicating that the ions of cupric copper automatic released are not the key determinant responsible for the contact killing.The fluorescence staining showed that intracellular ROS in biofilms cells was rapidly stimulated following a short-time exposure to copper surfaces and this effect was time-dependent in a given time range.In addition,we found that the biofilms of alsl/alsl and als3/als3 displayed higher resistance to the copper surface toxicity and the genes ALS1 and ALS3 encode cell-surface associated glycoproteins that are considered to be important for C.albicans biofilm formation.An FDA-PI staining assay was performed and the relationship between cell morphology and cell death was not observed.Further research showed that the intracellular copper contents in ALS mutant was significantly lower than that of wild strain.CRP1 and CUP1 were two genes related to copper homeostasis in C.albicans and the qPCR results showed the expression of CRP1 in ALS mutant was dramatically increased after copper treatment,whereas the expression of CUP1 did not change significantly.In summary,we considered that the proximity between the copper surface and cellular membrane promoted production of high concentrations of copper ions in the contact site and damaged the cell membrane,then a large number of copper ions immediately influx into the cell,induce ROS accumulation which cause protein oxidation or DNA damage and ultimately led to cell death.Als1 and Als3 proteins can promote the process by inhibiting the expression of the P-type ATPase copper transporter CRP1.In this thesis,five terpenoids with antifungal activity were found through mass screening,then the antifungal activity and mechanism of CA were elucidated,which presented a theoretical basis for the development of sesquiterpenes as new antifungal drugs and provided insights into the modes of action of other sesquiterpenoid natural products.In addition,we confirmed the antifungal effect of metallic copper and made an intensive study of the copper surface on C.albicans biofilms and found two genes ALS1 and ALS3 ralated to tolerance to copper,which provided a theoretical basis for promoting the application of copper materials in clinic.