| Fungal infection is a common disease,which seriously threatens to human health.As the limitation of the existing antifungal drugs and the drug resistance of the pathogenic fungi to these drugs,it is necessary to find new antifungal drugs.Natural medicines(including traditional Chinese herbal Medicines)mainly refer to the single or multi components from animals,plants and other organisms which with a clear therapeutic effect.These drugs have various advantages including novel structure,high activity and low cost,which make them important for the research and development of new drugs in the pharmaceutical industry.Honokiol(HNK),one of the main medicinal components in Magnolia officinalis,possesses antimicrobial activity against a variety of pathogenic bacteria and fungi,including yeasts.However,little is known on the molecular mechanisms underpinning the antimicrobial activity.In this study,we investigated the protein targets and anti-fungal molecular mechanism of HNK in the model fungus Saccharomyces cerevisiae through the genome-wide mutant library screening,gene microarray analysis and drug affinity responsive target stability(DARTS)technique,which may lay a foundation for the application of HNK to other pathogenic fungi in the future.The following main results are obtained:1.Screening the HNK-sensitive and-resistant mutants from the yeast genome-wide mutant library indicates that honokiol affects multiple physiological processes.Yeast mutant library has two collections,one is YSC5095(a Yeast DAmP Library of 878 essential genes),the other is YSC1053(a Yeast MATa Deletion Collection of 5153 nonessential genes).In this study,using the sensitivity of wild type yeast BY4741 to honokiol as a control,we systematically screened the HNK-sensitive and-resistant mutants from the yeast mutant library.It was found that 276 mutant strains were resistant to HNK and 175 mutant strains were sensitive to HNK compared with wild-type.Then these genes corresponding to the mutant strains were functionally classified according to the physiological processes involved by these genes.The resistant strains are mainly related to mitochondrial metabolism and respiration,protein modification and transport,transcription,metal ion metabolism,DNA and RNA metabolism,etc.Nearly 36%of the resistant strains were dysfunctional in mitochondria and formed petite.The sensitive strains are mainly related to vacuolar proton ATPase activity maintenance,cell cycle regulation,transcription and translation,protein transport,RNA metabolism,drug response,mitochondria metabolism,eell wall and cell membrane maintenance,etc.These results indieate that honokiol may affect RNA metabolism,mitochondrial function,vesicle transport,vacuolar ATPase activity,cell wall and membrane structure.2.The HNK-resistant mitochondria mutation petite mutants are able to pump out HNK to increase resistance through increased expression of ABC transporter Pdr5.Among the resistant mutants,we found that 98 mutants strains were petite which are related to the mitochondria mutation.Further studies showed that the expression of ABC transporter Pdr5 in the representative petite strains(atp7? and atp15?)were significantly higher than that in wild type.Accordingly,the effluxes of rhodamine 6G and HNK were higher in atp7? and atp15? than in wild type,while the retention of HNK was lower in aip7? and atp15? than in wild type.Meanwhile,in other petite mutants(cbs2?,Pcp1? etc.),the efflux of rhodamine 6G were also higher than in wild type.More importantly,the deletion of PDR5 conferred sensitivity to HNK and over-expression of PDR5 enhanced resistance to HNK.These results suggest that the resistance of mitochondria mutation petite mutants to HNK is associated with the induced expression of ABC transporter Pdr5 to pump out HNK.3.HNK destroyed mitochondria and induced the formation of supersized lipid droplets.Among the INK-sensitive mutants,we have found that several are mutated in mitochondrial related genes(TAH18,YME2,MIMI,MAS1,MIA40).Further analysis showed that HNK impaired mitochondrial function by directly destroying the integrity of mitochondria.Meanwhile,the mitochondrial shape was changed from tubular network to fragmentation and aggregation,while the aggregation shaped mitochondria and ER membrane structure were suspectly wrapped on the outside of lipid droplets(LDs).Furthermore,it was found that the formation of supersized LDs was caused by the increased synthesis of triacylglycerol which was induced by HNK.Further study showed that the formation of supersized lipid droplets were arised from the fusion of regular LDs or the growth of a LD wrapped with mitochondria after HNK treatment.Meanwhile,different jfrom the normal lipid droplets in wild type without HNK treatment,the sizes and numbers of lipid droplets in HNK-sensitive miml-DAmP,masl-DAmP,mia40-DAmP mutants also increased.Therefore,we speculated that HNK treatment increased the size of lipid droplets by destroying the function of mitochondria.In addition,HNK had fungistatic and fungicidal effects on Candida albicans and induced the supersized lipid droplets in C.albicans,mammalian cells and other eukaryotic cells.These results indicate that the effect of HNK in interfering lipid metabolism in eukaryotic cells is conservative.4.Microarray analysis results showed that HNK mainly affected the gene expression of iron metabolism and efflux pump.To explore the molecular mechanism of HNK's anti-fungal activity,we determined the effects of HNK on the mRNA expression profile of S.cerevisiae using a DNA microarray approach.We found that 202 genes were up-regulated more than 2-fold and 52 genes were down-regulated more than 50%.Functional classification of up-and down-regulated genes according to their physiological processes showed that the up-regulated genes are involved in metal ion metabolism,oxidative stress response,RNA metabolism,transcription,ribosome synthesis,and drug response;the down-regulated genes are involved in amino acid metabolism,respiration and carbohydrate metabolism,stress response,protein metabolism.Among them,the up-regulation of iron metabolism related genes and the down-regulation of iron consumption related genes involved in TCA cycle and respiration were caused by the iron depletion in which HNK reacted with iron to produce iron-complexes.In addition,after HNK treatment,the yeast cells induced the expression of ABC transporter Pdr5 to pump out HNK to increase resistance to HNK.5.Drug affinity responsive target stability(DARTS)to determine the target proteins of HNKIn order to examine the molecular mechanism of HNK,Fas2,Pdcl,Atp2,Hsc82,Eftl were identified as the HNK target proteins by DARTS and mass spectrometry.We also found that the target proteins which combined to HNK in the HNK-resistant petite mutant of atp7? were reduced,which may be a result of more expression of Pdr5 in the petite mutants to pump out HNK.In this study,we examined the fungicidal effect,potential targets and antifungal mechanism of HNK in S.cerevisiae.We found that HNK may function in inducing iron starvation and the formation of supersized lipid droplets by destroying mitochondrial function to inhibit cell growth.Meanwhile,HNK treatment could induce the expression of the ABC transporter Pdr5 which pumped out intracellular HNK to increase the resistance to HNK.Therefore,honokiol may be a multi targets drug.This study paved the role for further research on the mechanism of HNK,and provides useful references for studying the mechanism of HNK on anti-cancer cells. |
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