| The number of patients who have been infected by systemic Candida albicans is increasing,and the mortality rate reaches up to 30%.Natural drug-resistance and acquired drug-resistance have brought about great difficulties in the treatment of Candida albicans infections.Clinically,the treatment of invasive Candida albicans infections faces a dilemma that there are few species of drugs,strong drug-resistance and many side effects,and the progress about the research and development of antifungal drugs is very slow.In the past ten years,new anti-drug resistant fungal drugs based on new targets still have not been discovered.Therefore,the reserach of highly effective and low-toxic anti-drug resistant new drugs has become a scientific problem that needs to be solved.In the earlier period of time,our research group has found a number of natural small molecule compounds with synergistic resistance to Candida albicans from the perspective of synergistic anti-resistance strategies against Candida albicans,and that their common efficacy groups were discovered through structure-activity relationship studies,but the specific target proteins and mechanism of action are not yet clear.In this study,we intend to use iTRAQ-based quantitative proteomics in combination with previous experimental results to study the specific differentially expressed proteins and target proteins of the active compound baicalein against Candida albicans,and use gene knock-out technology to construct target protein gene-deficient strains,then compare the phenotypic differences between the mutant strain and the wild type after the interaction of active compounds,and analyse the biological function of the target proteins and study the molecular mechanisms of the active compound against the drug-resistance Candida albicans in depth in order to provide a theoretical basis and experimetal basis for the study of new types of high-efficiency and low-toxic anti-drug resistant fungal drugs.First of all,in the experiment of extracting total Candida albicans proteins,we have analyzed and summarized the experimental system about the high-quality extraction of total protein of Candida albicans through the stability of protein concentration,the clear and uniform protein band of SDS-PAGE electrophoresis and the requirement of protein in iTRAQ experiments.In the iTRAQ experiment,we successfully screened the fluconazole target protein,and confirmed the feasibility of iTRAQ technology for screening target proteins of small molecule drugs.Next,we screened 112 different differential proteins of Candida albicans after being affected by baicalein,and 16 of which were common differential proteins.GO analysis and Pathway analysis of the differential proteins revealed that the molecular functions of the differential proteins were mainly L-malate dehydrogenase activity,ATP binding,and metal ion binding,and that the main cellular locations were: cytoplasm,peroxisomes,and that the main biological processes were: tricarboxylic acid cycle,fatty acid β-oxidation.KEGG Pathway analysis showed that there are 14 possible pathways,mainly including biosynthesis of secondary metabolites,antibiotic carbon,and pyruvate metabolism.Then,by analyzing the repeated differential proteins one by one,the genes of CPD2,SNQ2,CYS3,DBP2,PMA1,and SLA2 were selected for knocking out to further examine whether their function is related to the effect of baicalein against C.albicans.Next,we used the HIS1-LEU2-ARG4 knockout strategy to knockout these six genes.We successfully construct CPD2 gene deletion strain and the reventant strain.Phenotypic experiments showed that CPD2 gene deletion do not affect the growth and reproduction of Candida albicans,mycelial formation,biofilm formation,and cell surface hydrophobicity,and only result in increased susceptibility of Candida albicans to H2O2,LiCl,and caspofungin,and decreased susceptibility to baicalein and terbinafine.The experimental results of survival rate of mice with systemic fungal infection and the amount of fungal liver and kidneys burden showed that CPD2 gene deletion had no significant effect on the virulence of Candida albicans,but the susceptibility to baicalein decreased.The vitro susceptibility test also showed that the susceptibility of Candida albicans to baicalein was decreased after the deletion of CPD2 gene.The effect of baicalein against drug-resistance C.albicans may be related to the function of CPD2 gene.To study the mechanism of baicalein against drug-resistance Candida albicans,we investigated the effects of baicalein on the efflux function of drug transporters,and the induction of fungal apoptosis,and a series of indicators related to apoptosis.The rhodamine 6G efflux experiment showed that the deletion of CPD2 gene affected the function of the drug efflux protein Mdr1 p or down-regulate the expression of MDR1 gene;whereas baicalein against drug-resistance Candida albicans had no relation with drug efflux protein,and fluconazole up-regulated the expression of CDR1 gene.Apoptosis ratio analysis,Caspase enzyme activity assay,ROS detection assay and mitochondrial membrane potential detection and a series of apoptosis-related experiments showed that baicalein may induce apoptosis of Candida albicans by damaging the mitochondrial Caspase pathway,while the deletion of CPD2 gene can inhibit this function of baicalein.In summary,based on iTRAQ quantitative proteomics technology,this project successfully screened the differential protein Cpd2 p when baicalein function in Candida albicans,and construct CPD2 gene deletion strain.Through phenotype investigation and research on the mechanisms of baicalein inducing apoptosis of Candida albicans,we found that the function of CPD2 gene is related to baicalein inducing apoptosis of Candida albicans. |
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