The Impact Of A Combination Of Fluconazole And CyclosporineA On Biofilm Formation Of Candida Albicans Evaluated By Transcriptome Analysis Using A RNA-seq Technology

Objective The goal of present study is to reveal and verify functional genes relevant to the biofilm formation and drug resistance of Candida albicans,by analyzing the impact of a combination of fluconazole and calcineurin inhibitors cyclosporine A on biofim formation of C.albicans using the transcriptome sequencing technology(RNA-Seq).Methods Biofilm models of C.albicans reference strain SC5314 were constructed and the viability of bacteria against a combination of fluconazole and cyclosporine A was ascertained by an XTT method.Biofilm cells cultured in media with or without a combination of fluconazole and cyclosporine A were collected and used transcriptome analysis using a high-throughput RNA sequencing technology.The differentially expressed genes related to biofilm formation and drug-resistance were analyzed and verified.The expression of selected reference genes were further validated in C.albicans clinical isolates by a qRT-PCR assay.Results By capacity of biofilm formation testing,we selected 8 clinical isolates whose behavior were in accordance with reference strain,and all isolates were sensitive to anti-fungi drugs.We found the biofilm was matured after 48 h culture in biofilm formation test.The results showed that there was a limited effect of cyclosporine A and fluconazole in biofilm formation when they were used alone as compared to the control group.However,a combination of cyclosporine A(75?g/ml)and fluconazole(32?g/ml)showed a strongly synergistic anti-biofilm effect,which was statistically significant.231 new genes and 522 differentially expressed genes were detected by RNA-Seq,among them 253 were up-regulated and 269 were down-regulated.We screened 9 candidate genes that mainlyrelated to functions including the synthesis of cell wall,plasma membranes,exocytosis transport,immune response,phase transformation after the GO analysis.The KEGG pathway analysis showed that these genes were involved in pathways related to glycolysis,fatty acid metabolism,cytokine receptor interactions,cell division.These genes were further verified using clinical isolates,the results of clinical isolates were in agreement with the RNA-Seq results.Among these genes,the expression of als3,cdr1,cln1,cln2,rfg1,erg3,ddc1 were statistically different between the biofilm and control group,which was statistically significant,while genes of cpp1 and efg1 only showed a moderate alteration,which had no significant difference.Then we interfered the carbohydrate metabolism which were focused by differentially expressed genes,and found that there was a strong connection between glyoxylate cycle and C.albicans infection.The result showed that there had a more expression of biofilm related genes in glyoxylate cycle when the acetaldehyde dehydrogenase was inhibited.However,the expression of biofilm related genes was down-regulated in the presence of ethanol.The underlying mechanism between biofilm formation and drug resistence thus warrants further investigation.Conclusions 1.In the process of C.albicans biofilm formation,the yeast cell was predominant in the beginning and gradually turned into hypha cell along with the time.When they were cultured 48 h,the hypha cell interweaved together and secrete more extracellular for biofilm maturation.2.From the drug sensitivity test,we can concluded that the C.albicans displayed resistant to fluconazole when biofilm was formed.Fluconazole showed a strongly anti-biofilm effect when it was combined with cyclosporine A.3.231 new genes and 522 differentially expressed genes were detected by RNA-Seq.After annotated function analysis and even verified by clinical isolates on the differentially expressed genes,we examined 9 candidate genes.In this process,their functions have been verified,and also suggesting the reliability of the RNA-Seq results by als3,cdr1.4.The carbohydrate metabolism was found to be the most reliable after analyzed the difference of metabolize pathway.The results of glyoxylate cycle study showed that ethanol had a great potential against C.albicans,dispite a controversial result was observed in disulfiram study.Therefore the mechanism of biofilm formation of C.albicans is far from be completely understood,which needs further exploration.