Mechanism Study Of Pterostilbene Against Candida Albicans Biofilm And Autophagy In Biofilm

Over the past 30 years,with the advances in biomedical science,transplantation of hematopoietic stem cell and organ,the extensive use of immunosuppressive agents and broad-spectrum antibiotics,radiotherapy or chemotherapy of cancer patients,the development of various catheter technologies and HIV spread,the incidence of invasive fungal infection morbidity and mortality in the world increase significantly,fungal infection has attracted a lot of attention.Of which the Candida albicans infection shows the highest incidence.On the one hand,effective anti-Candida albicans drugs are very limited and Candida albicans has a high propensity to develop biofilms,which brings a new challenge to clinical treatment.Biofilms are highly resistant to antifungal drugs,and biofilms formed on medical devices contribute to repeated infections and eventually lead to the treatment failure.Therefore,it is significant to find new anti-Candida albicans drugs and effective anti-biofilm drugs.Our previous study exhibited that PTE had good anti-biofilm activity,while PTE is a structural analogue of resveratrol,and resveratrol is widely used as a good health care product,which decided that resveratrol and PTE had the advantage of low toxicity.It was hopeful to become a leading compound,then to further develope into anti-biofilm drug.Therefore,we carried out the study on the structure-activity relationship of PTE against Candida albicans biofilm in this study,in order to find the anti-biofilm drug of high efficiency and low toxicity,and make the structural transformation of PTE in late time.When we found that PTE showed strong anti-biofilm activity,we also carried out the mechanism study of PTE.Chip experiment found that after the treatment of PTE,the expression of many autophagy-related genes changed.Autophagy was first discovered in the early 1960 s,which proteins and organelles that were damaged or aged in the cell were transported to lysosomes or vacuoles,and the enzymes there were used to digest and degrade these wastes to small molecules,then to be reused.Autophagy is a major research hot spot,and the research of autophagy in the yeast and mammalian cells is very mature,which makes clear that autophagy plays an important role in the cell response to external stress and adaptability to the environment.But the study of autophagy in Candida albicans has just started,and it is controversial.Glen E.Palmer believed that autophagy has no meaning role in the physiological process of Candida albicans.However,in the past two years,Li Mingchun,a professor at Nankai University,believed that autophagy of Candida albicans was very important in response to various external stimuli such as oxidative stress,osmotic pressure.And autophagy was related with the pathogenicity of Candida albicans.Very few literatures were reported about autophagy in Candida albicans,and the two groups of viewpoints were controversial.Then what role does autophagy play in Candida albicans? With the treatment of PTE,a anti-biofilm small molecule compound,the expression of autophagy-related gene changed,so whether autophagy plays a role in Candida albicans biofilm or not.So far,no related literature has been reported.Therefor it is worthy for us to carry out deep study.In the field of antifungal research,most groups are committed to discovering new small molecule compounds with antifungal activity.When we develop drugs,it is important to find small molecule compounds with defined target.Therefore,it is very important to reveal the target protein of small molecule compounds.It is important to establish and optimize the method of finding out the target of compound.There are many ways to find drug targets in mammalian cells,such as isotope tracer method,fluorescence spectroscopy,genomics,proteomics research methods,etc,which DARTS(drug affinity responsive target stability)get the attention of scientists and is widely used because of its principle is relatively simple,less practical steps,no need to transform the structure of small molecules and the results are more reliable.This experimental method has been reported to successfully applied to mammalian cells,finding out the target protein of anti-aging drugs including Rapamycin and Resveratrol.We attempt to use DARTS to carry out the target research of anti-fungal drugs.Results are below:Part?: We conducted the study of the structure-activity relationship of PTE against Candida albicans biofilms.In order to facilitate the study,we divided the structure of PTE into three parts,and investigated the ability of PTE and 28 structural analogues against biofilms in RPMI 1640 medium(including against biofilm formation and mature biofilms),which focused on 15 analogues,which the structure were more closed to the structure of PTE.We found that the hydroxyl group(-OH)of Partition A,which was once replaced by other substituents(including C1-C5,B1 and B2),the activity of the anti-Candida albicans biofilms disappeared.So we concluded that the hydroxyl group(-OH)is important for analogues to maintain the activity.When the double bond of the Partition B becomed single bond(C6),the activity becomed weaker,indicating that the double bond contributed to the optimal activity of such a compound.When the double bond becomed part of the furan ring(B3),the compound had almost the same inhibitory activity as PTE against biofilm.However,when the double bond becomed part of the unsaturated lactone(C7),or when the Partition A and the Partition B formed a cyclic ester(B4),the activity was almost lost.In partition C,we changed the number and position of the methoxy group(-OCH3),or replaced the methoxy group(-OCH3)with the hydroxyl group(-OH),the activity of the compound was weakened,indicating that the meta-methoxy(-OCH3)helped to maintain the best anti-biofilm activity of analogues.The hyphal formation assay in Spider and RPMI 1640 liquid media further confirmed the results of the biofilm.The effect of PTE analogues on hyphal formation in Spider + 2 g / L glucose medium was also investigated,finding that PTE,C4,C6,B3,B5 and B6 showed concentration-dependent hyphal inhibition in these three media.In a word,the hydroxyl group(-OH)was superior to other substituents.Double bond and meta-dimethoxy(-OCH3)helped to maintain the optimal activity of PTE against Candida albicans biofilms.Part II: We examined the effects of autophagy in Candida albicans biofilm.In this study,we used Saccharomyces cerevisiae as a positive control and investigated the autophagy in Candida albicans by using of rapamycin(autophagy inducing agent),bafilomycin A1(autophagy inhibitor)and some gene-knockout strains atg8?/? and ccz1?/?.The presence of autophagy in Candida albicans was investigated by detecting a series of indexes,such as the count of autophagy bodies under phase contrast microscope,the positive rate of cells after acridine orange(AO)staining and alkaline phosphatase activity,confirming that like Saccharomyces cerevisiae,Candida albicans existed the physiological process of autophagy,the autophagy tool agents in mammalian cells were also applicable to Candida albicans.Then we focued on the presence of autophagy in Candida albicans biofilms.In the same way,by detecting autophagy indicators,we confirmed that the level of autophagy in biofilm was greatly improved.The XTT assay showed that there was no significant difference in biofilms formed between the parental and knockout strains at 24 h,but significant difference were observed between the two groups at 48 h,72 h and 96 h.The results of Real-Time RT PCR showed that the expression of autophagy-related genes CCZ1,ATG1-10,ATG12,ATG13,ATG16-18 and CHK1 were up-regulated,confirmed this event.At the same time,we investigated the growth tendency,hyphal formation ability and adhesion ability of the parent and knockout strains,and found that there was no significant difference between them,indicating that the difference in the ability of forming biofilm was independent of growth,hyphal formation and adhesion,but relevant with the existence of autophagy process.Biofilms of atg8?/? and ccz1?/? formed on silicon squares at 48 h were more easily destroyed.The results of CLSM showed that the internal structure of biofilm formed by the parental and knockout strains was significantly different.Meanwhile,we used BFM to perform all assays to confirm our results.The results of BFM were consistent with the results of gene-knockout strains,indicating that autophagy affected the maintenance and the internal structure of biofilms.We explored the relationship between autophagy and drug resistance of biofilms by using antifungal agents and gene-knockout strains.Drug susceptibility tests showed that the knockout strains were more sensitive to the commonly used antifungal drugs,indicating that the resistance of the biofilms wsa related with autophagy.Finally,we used the nematode infection model to study the pathogenicity of knockout strains and found that the pathogenicity was greatly reduced.Part ?: We identified the strong inhibitory effect of PTE on Candida albicans biofilm and completed the preliminary study of structure-activity relationship of PTE.We further wanted to clarify the specific mechanism of PTE.In this study,we successfully constructed the DARTS(drug affinity responsive target stability)system by improving the experimental conditions of each link.We used the advanced experimental system to successfully find the differential bands of Rap and Res The differential protein bands were then analyzed by mass spectrometry,and the targets FKB12 and eIF4 A were reported.Then we used the system to testify the target of FLU,a positive and target-known anti-Candida albicans drug.And we got the same results.Next we used the improved system to find the target of PTE,and trid to r extract the membrane protein and change the concentration of the separation gel,it was expected to clarify the target protein of PTE in the following study,provided a new way to discover the target of other anti-fungal small molecule drugs.Collectively,SAR study in this work demonstrated that the activity of PTE analogues against biofilms is strongly associated with their structures.Of which the para-hydroxyl(-OH)in Partition A exhibited better activity than other substituents,the double bond of the Partition B and the meta-dimethoxy group(-OCH3)of the Partition C were beneficial to the activity of this kind of compound against Candida albicans biofilms.As a chance pathogen,Candida albicans did exist autophagy process.And autophagy played a pivotal role in Candida albicans biofilms,which autophagy played an important role in keeping the maintenance and the internal structure of the mature biofilm,and was closely related to the high drug resistance of biofilms.DARTS experimental system could be used to find and confirm the target of drugs against Candida albicans.Due to the specificity of Candida albicans cells and small molecule compounds,some experimental conditions might need to be changed during the specific experiment to obtain the target protein of more small molecule compound..