| Objective: In order to obtain drug resistant isolates of major prevalent types of Candida albicans and to provide evidence for diagnosis and treatment of clinical Candida albicans infections,isolation and purification clinical Candida albicans strains was performed firstly,then,antifungal drug susceptibility testing for 5 antifungal agents like fluconazole(FLU),itraconazole(ITR),ketoconazole(KETO),5-Flucytosine(5-FC)and Amphotericin B(Am B),and the Random Amplified Polymorphic(RAPD)genotyping analysis were performed.Next,screening differentially expressed genes(DEGs)in a durg resistant isolate 5508(resistant to FLU,ITR,KETO and 5-FC)disposed by different antifungal drugs(ITR,5-FC and ITR combine 5-FC)compare with no drug treated control using RNA-seq analysis.Finally,to explore preliminary function of these DEGs(such as QDR1 gene)and the correlation between DEGs and durg-resistance of Candida albicans,gene deleted mutants were constructed using fusion PCR mediated homologous recombination gene knock out strategy,whereafter,antifungal drug susceptibility testing,spot assay,and growth curves experiment were performed.Methods: 1.Isolated and purificated the clinical strains using CHROMagar Medium and multiple PCR to obtain Candida albicans isolates.2.Susceptibility testing of 116 randomly selected Candida albicans to the five commonly used antifungal agents,fluconazole(FLU),itraconazole(ITR),ketoconazole(KETO),amphotericin B(Am B)and 5-flucytosine(5-FC),were performed according to “Method for Extracting Micro-Dilute Antifungal Sensitivity of Yeast”(M27-A3),recommended by the Clinical and Laboratory Standards Institute(CLSI).3.RAPD based on DNA sequencing was used forgenotyping of 116 Candida albicans isolates,clustering analysis was performed by the NTsys 2.10 e software.4.Total RNA of isolate 5508 disposed by different antifungal drugs and no drug treated were isolated using liquid nitrogen and Tri Pure Isolation Reagent,and then screening DEGs by RNA-seq analysis.5.Gene deleted mutant was constructed using fusion PCR mediated LEU-HIS-ARG gene knock out strategy,then antifungal drug susceptibility texting,spot assay,and growth curves experiment were performed to explore preliminary function of these DEGs and the correlation between DEGs and durg-resistance of Candida albicans.Results: 1.In this study,751 Candida species isolates were obtained by isolation,purification and identification.There were 260(34.62%)Candida albicans,230(30.63%)Candida krusei,122(16.25%)Candida glabrata,133(17.71%)Candida Tropicalis and 6(0.80%)other Candida species.These clinical isolates were isolated from Sputum(247,32.89%),Feces(170,22.54%),Vagina(157,20.91%),Urine(6.03%),Blood(69,9.19%)and Other sites(36,4.79%).2.The resistance rates of 116 Candida albicans isolates against FLU,ITR,KETO,5-FC and Am B were 3.45%、4.31%、1.72%、0.86% and 0.00%,respectively.The clustering analysis of RAPD genetyping showed that 116 Candida albicans isolates were divided into 10 branches with a 0.75 coefficient of similarity,and clusterⅠhad the highest number of strains(59,50.86%),followed by clusterⅡ(17,14.66%),cluster Ⅲ(14,12.07%),clusterⅣ(9,0.78%),clusterⅤ(5,0.43%),clusterⅥ(4,0.34%),cluster Ⅶ(4,0.34%),cluster Ⅷ(3,0.26%),cluster Ⅸ(1,0.07%)and clusterⅩ(1,0.07%).ATCC90028 and 5508 were classified into Class I.The most drug-resistant isolateds were found in Class I,including drug resistant isolate 5508,but the isolate 17515(resistance to ITR and KETO)was classified into Class III.3.The RNA-seq analysis showed that:(1)no drug treated group compared with 5-FC treated group(N vs C),7 DEGs were discovered,3 were up-regulation and 4 were down-regulation.(2)No drug treated group compare with ITR treated group(N vs I),371 DEGs were discovered,301 were up-regulation and 70 were down-regulation.(3)No drug treated group compare with 5-FC combine ITR treated group(N vs L),367 DEGs were discovered,290 were up-regulation and 77 were down-regulation.(4)A total of 407 genes were discovered by summarizing all the differential genes(see Appendix 1 for details),of which 318 genes were upregulated(269 newly identified genes that may be associated with Candida albicans resistance,such as ERG5,ERG7,ERG9,QDR1,and POT1,etc.),and there were 89 down-regulated genes(74 newly identified genes that may be associated with Candida albicans resistance,such as NAG3,NAG4,SOD1,and SOD2,etc.).4.The GO(Gene Ontology)analysis of DEGs showed that:(1)the group N vs C was different from the other two groups,GO classification of DEGs was mainly distributed in the Biological Process(BP)and the Molecular Function(MF);in BP classification,cell process,metabolic process,single-organism process,multi-organism process and response to stimulate were mainly involved,and down-regulate genes more than up-regulate genes;in MF classification,the DEGs were mainly distributed in binding,suggesting that the composition and metabolic activity of bacterial cells changed after 5-FC treatment.(2)The classification results of group N vs I and N vs L are approximately the same,in BP classification,the single-organism process,metabolic process and cell process are the most common GO functional categories;in Cellular Component(CC)classification,the most widely distributed classifications are cells,cell parts,membrane and membrane components;in MF classification,catalytic activity and binding were the most.It indicated that the cell composition and membrane components changed after ITR treatment.5.The KEGG(Kyoto Encyclopedia of Genes and Genomes)pathway analysis of DEGs showed that:(1)in group N vs C,a gene PUT1 was enriched to Arginine and proline metabolism.(2)In group N vs I,DEGs are mainly enriched in steroid biosynthesis(including ERG2,ERG5,ERG6,ERG7,ERG9,ERG24,ERG25,ERG26,and ERG27,of which ERG5 I,ERG7,ERG9,ERG25,ERG26,and ERG27 are newly discovered drug-resistant related genes),peroxisomes(including SOD1,SOD2,SOD3,PXP2,CAT1,CTN3,FAA22,IDP2,POX1,POT1,PEX6 and ECI1 genes,among them SOD3,PXP2,CTN3,FAA22,IDP2,POX1,POT1,PEX6 and ECI1 are newly discovered drug-resistant related genes),Fatty acid degradation,Biosynthesis of amino acids,ABC transporter(SNQ2 genes are newly discovered drug-resistance related genes),MFS transporter(QDR1 gene is a newly discovered possible drug-resistance related gene).(3)The group N vs L was almost identical to the group N vs I,but the group N vs L was also enriched for the Phospholipase D signaling pathway,Oxidative phosphorylation,and NOD-like receptor signaling pathways,Oxytocin signaling pathways and Adherens junctions.6.The QDR1 gene of Candida albicans was important for a moderate increase of Candida albicans to ITR and KETO,and no significantly correlation was found between QDR1 and the growth of Candida albicans in growth curve experiment.Conclusions: 1.Candida albicans remains the predominant reasons for the fungal infections,and remained highly susceptible to the five antifungal regents.2.Some drug-resistance related genes were discouvered using RNA-seq analysis,such as ERG5,ERG7,ERG9,ERG25,ERG26,ERG27,SOD3,PXP2,CTN3,FAA22,IDP2,POX1,POT1,PEX6,ECI1,SNQ2 and QDR1,involve in Steroid biosynthesis,Peroxisome,ABC transporter and MFS transporter pathways,but further validationis was required.3.The gene QDR1 was important for a moderate increase of Candida albicans to ITR and KETO. |
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