基于RNA-Seq技术分析氟康唑与环孢菌素A联合应用对生物膜态白念珠菌转录谱的影响

Use of RNA-Seq technology to analyze the effects of a combination of fluconazole and cyclosporine A on the transcriptome of Candida albicans and its biofilm formation

目的利用转录组测序技术(RNA-Seq技术)分析生物膜态白念珠菌不同处理组间转录谱的表达差异,寻找与白念珠菌生物膜形成、耐药等相关的功能基因。方法构建白念珠菌标准菌株的生物膜模型,将其分为生物膜态无处理组与氟康唑、环孢菌素A联合用药两个处理组,利用高通量RNA-Seq技术分析联合用药对转录组产生的影响,分析差异表达基因与生物膜形成、耐药等的相互关系,并对临床筛选的白念珠菌进行目的基因验证。结果通过产膜能力与药物敏感性试验筛选出8株与白念珠菌标准菌株产膜与药敏结果相同的临床分离菌株。测序分析显示差异表达基因共有522个,其中上调的差异基因有253个,下调的差异基因有269个。根据GO功能分类结果显示,这些基因主要涉及细胞壁、浆膜的合成,外排转运,免疫反应,菌相转换等功能;KEGG分析表明这些差异表达基因参与糖酵解,脂肪酸代谢,细胞因子受体相互作用,细胞分裂等信号通路。通过分析筛选出9个目的基因,利用临床分离株白念珠菌对目的基因验证,这些目的基因变化均与转录组结果一致。其中,als3,cdr1,cln1,cln2,rfg1,erg3,ddc1基因表达量呈不同程度的升高或降低,差异具有统计学意义(P<0.05);cpp1,efg1呈升高与降低,差异无统计学意义(P>0.05)。结论利用RNA-Seq技术初步分析得到与白念珠菌生物膜形成与耐药相关的几个基因,发现氟康唑与环孢菌素A联合用药抑制生物膜是一个多基因参与、多个生物过程协同调控的过程,从而为研发新型抗真菌药物提供了新的靶点,为临床合理使用抗生素药提供了依据。

Objective In order to identify functional genes related to biofilm formation by and the drug resistance of Candida Albicans, transcriptome sequencing technology （RNA-Seq） was used to analyze the transcriptional profile of C. albicans under different conditions. Methods Biofilm models of the reference strain SC5314 of C. albicans were constructed and treated with a combination of fluconazole and cyclosporine A or left untreated. The effect that treatment with a combination of the 2 drugs had on the transcriptome of C. albicans was analyzed using high throughput RNA sequencing technology. Differences in gene expression, biofilm formation, and drug resistance were determined by identifying target genes in clinical isolates of C. albicans. Results Examination of biofilm formation and a drug sensitivity test yielded 8 clinical isolates that behaved similar to the reference strain. The zone of inhibition had a diameter greater than 19 mm and the absorbance at 570 nm （Asro） was greater than 0. 5, which implies that clinical isolates were highly sensitive to fluconazole and cyclosporine A. RNA-Seq yielded 522 differently expressed genes, 253 of which were up-regulated and 269 of which were down-regulated. GO analysis revealed that these genes were mainly related to functions such as the synthesis of the cell wall and plasma membrane, exocytosis and transport, immune response, and phase transformation. KEGG pathway analysis indicated that these genes were involved in pathways related to glycolysis, fatty acid metabolism, cell factor receptor interactions, cytokine receptor interactions, and cell division. Screening yielded 9 target genes, and these genes were verified using clinical isolates. Changes in those target genes matched transcriptome results. Expression of 7 of those genes （als3, cdrl, clnl, cln2, rfgl, erg3, and ddcl） differed significantly different between the biofilm and planktonic states of C. albicans （P〈 0. 05）, while expression of 2 genes （cppl and efgl） differed only slightly between those two states （P〉0. 05）. Conclusion RNA-Seq technology was used to preliminarily identify several genes related to biofihn formation by and the drug resistance of C. albicans. These genes were expressed at different levels, suggesting that the inhibition of biofilm formation by a combination of fluconazole and cyclosporine A might involve multiple genes. These results thus warrant further investigation and may offer new targets for development of novel antifungal agents. These results may also provide scientific evidence to guide treatment of a C. albicans infection in clinical settings.