白念珠菌铁通透酶基因CaFTH1的功能研究

Functional characterization of the CaFTH1 gene in Candida albicans

【目的】白念珠菌CaFTH1是一种铁通透酶编码基因。为了研究CaFTH1对胞内铁代谢和液泡功能的影响,构建fth1/单基因缺失菌株和fth1/fet33/双基因缺失菌株。【方法】利用生物信息学软件对CaFTH1进行序列比对和分析;通过实时荧光定量PCR技术研究铁离子丰度对CaFTH1表达的影响;利用PCR介导的同源重组方法构建基因缺失菌株;利用原子吸收光谱方法测定基因缺失菌株胞内铁含量的变化,并对基因缺失菌株在缺铁条件和菌丝诱导条件下的生长状况进行研究;通过代谢转换实验,研究CaFTH1对细胞液泡功能的影响。【结果】序列比对结果表明白念珠菌CaFth1蛋白属于铁通透酶Ftr1超家族,与酿酒酵母液泡膜蛋白ScFth1具有最高的同源性。铁匮乏条件会诱导CaFTH1的表达,而富铁条件则会抑制其表达。白念珠菌CaFTH1的缺失会导致胞内铁含量的降低,fth1/突变菌株基础上CaFET33的缺失则会进一步降低胞内铁含量。在缺铁条件下,fth1/fet33/双基因缺失菌株在一定程度上表现出代谢转换能力的缺陷。另外,在某些固体菌丝诱导培养条件下,fth1/fet33/缺失菌株菌落表面形成褶皱能力显著增强;而在液体菌丝诱导条件下,则表现为增强的菌丝聚集能力。【结论】CaFTH1是一种低铁应答基因,在维持白念珠菌胞内铁离子稳态及液泡功能方面具有重要作用。CaFTH1和CaFET33基因的双缺失会对白念珠菌的菌落形态和菌丝聚集产生影响。

[Objective] CaFTH1 gene encodes a putative iron permease in Candida albicans. To investigate the role of CaFTH1 in cellular iron metabolism and vacuolar function, we con- structedfthl△/△ single gene deletion andfihlA/Afet33△/△ double deletion mutants. [Methods] Sequence alignment and analysis were performed by the relevant bioinformatic software. Ef- fect of iron avaibility on the expression of CaFTH1 was conducted by real-time PCR. We con- structed the fihl△/△ and fihl△/△fet33△/△mutants by PCR-mediated gene disruption. We monitored the change of cellular iron content of the mutants by using atomic absorption spec- troscopy （AAS）, and explored the growth status under iron-limited and hyphal-inducing con- ditions. Furthermore, we also investigated the effect of gene deletions on vacuolar function by metabolic shift experiment. [Results] Sequence alignment revealed that C. albicans CaFthl protein is a member of Ftrl iron permease superfamily, with the highest homology with S. cerevisiae vacuolar membrane proteins ScFthl. Iron starvation induced the expression of CaFTH1, while iron repletion decreased its mRNA levels. Deletion of CaFTH1 resulted in de- creased cellular iron content, and deletion of CaFET33 on the basis offihlA/A mutant further decreased cellular iron content. For the metabolic shift experiment, the fthlA/Afet33△/△ mu- tant also exhibited growth defect under iron-limited conditions. In addition, the fthlA/Afet33△/△ mutant showed distinct wrinkled colonies in some solid hyphal-inducing conditions, while the fihlA/Afet33A/A mutant displayed increased aggregation ability in the presence of liquid-inducing cues. [Conclusion] CaFTH1 is induced in response to iron limita- tion, and plays an important role in maintaining C. albicans cellular iron homoeostasis and vacuolar function. Double deletion of CaFTH1 and CaFET33 genes affects the colonial morphology and hyphal aggregation.