白念珠菌GDT1基因对胞内钙离子稳态、细胞壁胁迫、菌丝发育和小鼠毒力的调控

Candida albicans is one of the most important human fungal pathogens. Regulation of intracellular calcium homeostasis and cell wall integrity are associated with the pathogenesis of C. albicans cells. Saccharomyces cerevisiae ScGdt1 and its functional homolog, human transmembrane protein 165 (TMEM165), belong to a well-conserved family of membrane proteins named UPF0016 (Uncharacterized Protein Family0016; Pfam PF01169), which exist in 919 bacterial species and 409 eukaryotic species. Mutations of TMEM165 are linked to a subtype of inborn metabolic diseases affecting the glycosylation pathway. Both ScGdt1 and TMEM165 are cation/Ca2+ exchangers contributing to Ca2+ and pH homeostasis. In this study, we have identified CaGdt1, the C. albicans homolog of S. cerevisiae Gdt1, and will characterize its functions in the response to calcium and antifungal drugs as well as in the cell wall integrity, the filamentation and the virulence in a mouse model of systemic candidiasis. In addition, we will study the genetic interactions between CaGdt1 and other regulators of calcium homeostasis. These studies might discover novel targets for new antifungal drug development. Furthermore, we will investigate the cellular functions of ScGdt1 and ScPmr1 and their regulation of cellular processes in S. cerevisiae cells. Together, our results would provide important clues for functional studies of other members of the UPF0016 membrane proteins family.

白念珠菌是人体上最重要的人体病原真菌之一。白念珠菌的胞内钙离子稳态调控和细胞壁完整性与它的致病性相关。酿酒酵母ScGdt1和和它的人体同功蛋白，跨膜蛋白TMEM165，是高度保守的膜蛋白家族（UPF0016）成员，这个家族包括919个细菌蛋白和409个真核细胞蛋白。TMEM165的突变与影响糖基化途径的人体先天性亚型代谢疾病相关联。最近研究证明，ScGdt1是酿酒酵母细胞中位于高尔基体膜上的钙离子/质子交换器。但是，ScGDT1的白念珠菌同源基因（C2_04540C）的功能目前还未被鉴定。本研究拟对该基因进行系统的细胞功能研究，并研究它对白念珠菌胞内钙离子稳态、细胞壁完整性、菌丝发育和小鼠毒力的调控。此外，还将研究它和已知的钙稳态调控因子之间的相互作用。研究结果将为抗真菌药物新靶标的发现提供重要依据。同时，本项目还拟深入研究ScGDT1和ScPMR1调控酿酒酵母相关细胞过程的机理。

白念珠菌是人体上最重要的人体病原真菌之一。白念珠菌的胞内钙离子稳态调控和细胞壁完整性与它的致病性相关。酿酒酵母ScGdt1和和它的人体同功蛋白，跨膜蛋白TMEM165，是高度保守的膜蛋白家族（UPF0016）成员，这个家族包括919个细菌蛋白和409个真核细胞蛋白。TMEM165的突变与影响糖基化途径的人体先天性亚型代谢疾病相关联。最近研究证明，ScGdt1是酿酒酵母细胞中位于高尔基体膜上的钙离子/质子交换器。但是，ScGDT1的白念珠菌同源基因（C2_04540C）的功能目前还未被鉴定。通过本研究，我们证明了白念珠菌CaGDT1基因是酿酒酵母ScGDT1基因在应对细胞壁胁迫方面的功能同源基因。在白念珠菌细胞内，CaGdt1定位于高尔基体，与CaPmr1的位置不同。CaGDT1基因的缺失导致缺失CaPMR1基因的细胞对细胞壁和内质网胁迫的敏感性增加。单独缺失CaGDT1或者同时缺失CaGDT1和CaPMR1基因会增加钙离子的摄入并激活钙/钙调神经磷酸酶信号。转录组分析表明，CaGdt1和CaPmr1共同调控的核心功能参与调控细胞内金属离子和氨基酸的转运。然而，CaGdt1具有与CaPmr1不同的功能。几丁质合酶基因CHS2在gdt1/gdt1、pmr1/pmr1和gdt1/gdt1 pmr1/pmr1突变体中都上调，而CHS3只在pmr1/pmr1和gdt1/gdt1 pmr1/pmr1突变体中上调。糖基化途径的5个基因（DIE2、STT3、OST3、PMT1和PMT4）和细胞壁完整性（CWI）途径的1个基因（SWI4）由于CaGDT1和/或CaPMR1的缺失而上调。和以上结果一致，我们发现CaPMR1或CaGDT1的缺失导致CaCek1介导的CWI信号的激活，但是这与细胞壁应激无关。钙调神经磷酸酶的功能是同时缺失CaGDT1和CaPMR1基因的细胞的细胞壁和液泡腔室的完整性所必要的，因此，CaPMR1是调节钙稳态和细胞壁应激的主要因素，而CaGDT1在白念珠菌高尔基体中对CaPMR1起到代偿作用。