| With the progress of science and technology,the development of medicine and the change of modern life style,infections caused by conditioned pathogenic fungi get severer in clinic,among which Candida albicans is the most common conditional pathogen.The shortage of clinical antifungal drugs as well as the rapid emergence and spread of drug-resistant strains has increased the difficulties of treating C.albicans infection.Therefore,it is of great significance to illuminate the pathogenic mechanism of C.albicans for the prevention,diagnosis,treatment and prognosis of clinical C.albicans infection.The presence of virulence factor is the main cause of C.albicans infection.Compared with traditional drugs,virulence-targeted drugs can't threaten the survival of C.albicans and greatly reduce the incidence of drug resistance.For this reason,researchers have focused on using fungal virulence as a new target of drug action in the process of searching for new anti-fungal strategies in recent years.The yeast-to-hyphae transition is an important virulence factor for C.albicans.In the process of tissue invasion,yeast form is conducive to reproduction and transmission,while hyphae to adhesion and invasion,both of which are frequently transformed to promote the colonization and infection of C.albicans.Therefore,inhibiting the morphogenesis of C.albicans can prevent its transformation to pathogenic morphology,and then reduce its infection ability,which can be used as an effective anti-fungal strategy.As reported in the literature,the deletion of Flo8 transcription factor in C.albicans in vitro completely blocked the hyphae development and mycelial specific gene expression,and it is proved that FLO8-deficient-strains were avirulence in the mouse model of systemic C.albicans infection.In order to find potential drug targets related to morphological transformation,we selected Flo8 as the research object.Furthermore,to investigate how the transcription factor Flo8 regulated the hyphae formation of C.albicans,chemical analysis was used to compare the differences of chemical constituents between the deficient strains and their complemented strains in various important synthetic pathways.Through analysis of the chemical constituents in the flo8A/A and wild type strains by high performance liquid chromatography(HPLC),we found that methylthioadenosine(MTA)in flo8?/? crude extract increased significantly.According to the results of gas chromatography mass spectrometry(GC-MS)analysis,there is twice as much farnesol in flo8?/? crude extract as in SC5314,besides,the exocytosis of famesol in flo8?/? increased.More than 98%of MTA is produced through the polyamine synthesis pathway,and MTA can be converted into S-adenosyl methionine(SAM)through the methionine salvage pathway,which is involved in the biosynthesis of polyamines.Polyamines synthesis pathway is closely related to hyphae growth,virulence and drug resistance of C.albicans.Documents reported that polyamines could induce mycelial transformation in C.albicans,while suppressing the synthesis of polyamines could inhibit hyphae formation.In this study,we used external standard method to analyze the content of related components in polyamine synthesis pathway.After exploring different extraction and detection methods,MTA and SAM in C.albicans were extracted by perchloric acid under ultrasonic condition,and their contents were determined by high performance liquid chromatography-diode array detector(HPLC-DAD)method.Pre-column derivatization of fluorene methoxycarbonyl chloride(FMOC-Cl)combined with high performance liquid chromatography-fluorescence detector(HPLC-FLD)method was carried out to detect the level of putrescine(PUT),spermidine(SPD)and spermidine(SPM).The results showed that the content of MTA and PUT increased,while the level of SPD,SPM and polyamines decreased in FLO8-deficient strains,indicating that the pathway of polyamines synthesis was inhibited.Sterols,as an important component of cell membrane lipids,are also closely associated to the morphological transformation of C.albicans.The inhibition of sterol synthesis pathway can curb the hyphae formation ability of C.albicans.It has been reported that inhibiting the expression of ERG11 in C.albicans can lead to the defect of hyphae formation ability.In this study,the contents of related components in sterol synthesis pathway of FLO8-deficient strains and their complemented strains were analyzed by internal standard method.Zymosterol,fecoserol,ergosterol,lanosterol and 4,4-dimethylzymosterol were analyzed by GC-MS.The results showed that the content of sterol intermediates zymosterol,fecosterol,lanosterol and 4,4-dimethylsterol increased in the sterol synthesis pathway,while the level of ergosterol remained almost unchanged.The massive accumulation of sterol intermediates reduced the proportion of ergosterol in the cell membrane and inhibited hyphae formation.In addition,the increase of farnesol content in the ergosterol synthesis pathway of FLO8-deficient strains inhibited the activity of Cdc35,which hindered the synthesis of cAMP,suggesting that Flo8 could affect the cAMP-PKA pathway and hyphae formation ability by regulating the sterol synthesis pathway.Interestingly,the transformation from zymosterol to fecosterol requires the participation of SAM in the sterol synthesis pathway,so the polyamine synthesis pathway and the sterol synthesis pathway can jointly regulate hyphae formation through the common substrate SAM.In order to further verify and analyze the experimental results,real-time fluorescence quantitative PCR(qPCR)was used to detect the expression of genes related to polyamine synthesis pathway and sterol synthesis pathway in FLO8-deficient and complemented strains.The results revealed that in the polyamine synthesis pathway,the expression of ornithine decarboxylase coding gene SPE1,SAM decarboxylase coding gene SPE2,polyamine oxidase coding gene FMS1 was up-regulated,and the expression of MTA phosphorylase coding gene MEU1 was down-regulated,while the expression of SPD synthase coding gene SPE3 and SPM synthase coding gene SPE4 subjected to negative feedback was up-regulated,which was consistent with the content detection results.The ERG6 gene,encoding the sterol C-24 methyltransferase in the sterol synthesis pathway,was also up-regulated.What's more,the expression of DPP3 gene encoding farnesol synthase increased.These results indicated that the weakening of hyphae formation ability in FLO8-deficient strains was related to the inhibition of polyamine synthesis pathways,accumulation of sterol intermediate in sterol synthesis pathway as well as relative decrease of ergosterol content.In summary,the FLO8-deficient C.albicans usually presented as yeast form and reduced its virulence mainly through the following three aspects:(1)the knock-out FL08 gene blocked polyamine synthesis pathway and reduced the overall level of polyamines,which inhibited the yeast-to-hyphae conversion;(2)the accumulation of sterol intermediates after the FL08 gene was knocked out,reduced the proportion of ergosterol,resulting to the weakening of the ability for hyphae formation and virulence;(3)the up-regulated expression of DPP3 and increased synthesis of farnesol in FLO8-deficient strains suppressed the Rasl-cAMP-Efg1 signaling pathway,inhibiting the hyphae formation and reducing virulence.Polyamine synthesis pathway,sterol synthesis pathway and cAMP-PKA signaling pathway constitute a complex,interconnected and mutually restrictive regulatory network,where transcription factor Flo8 regulates hyphae formation of C.albicans.This study focused on the new strategy of targeting virulence against fungi.It has adopted the chemical analysis method to analyze the changes of chemical components in the related pathways and investigated how the deletion of transcription factor Flo8 reduced the virulence of C.albicans.In the one hand,it has provided us with a new thought on antifungal toxicity that intervention of chemical constituent synthesis can inhibit hyphae formation and reduce virulence and functions as a theoretical basis for the study of targeting virulence.In the other hand,the mechanism of mycelial regulation was investigated by chemical composition changes accompanying with their regulating gene changes,which provided a new strategy to study and solve biological problems through chemical analysis. |
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