Chauhan Neeraj, Inglis Diane, Roman Elvira, Pla Jesus, Li Dongmei, Calera Jose A, Calderone Richard
Department of Microbiology and Immunology, Georgetown University School of Medicine, Washington, District of Columbia 20057, USA.
Eukaryot Cell. 2003 Oct;2(5):1018-24. doi: 10.1128/EC.2.5.1018-1024.2003.
Ssk1p of Candida albicans is a putative response regulator protein of the Hog1 two-component signal transduction system. In Saccharomyces cerevisiae, the phosphorylation state of Ssk1p determines whether genes that promote the adaptation of cells to osmotic stress are activated. We have previously shown that C. albicans SSK1 does not complement the ssk1 mutant of S. cerevisiae and that the ssk1 mutant of C. albicans is not sensitive to sorbitol. In this study, we show that the C. albicans ssk1 mutant is sensitive to several oxidants, including hydrogen peroxide, t-butyl hydroperoxide, menadione, and potassium superoxide when each is incorporated in yeast extract-peptone-dextrose (YPD) agar medium. We used DNA microarrays to identify genes whose regulation is affected by the ssk1 mutation. RNA from mutant cells (strain CSSK21) grown in YPD medium for 3 h at 30 degrees C was reverse transcribed and then compared with similarly prepared RNA from wild-type cells (CAF2). We observed seven genes from mutant cells that were consistently up regulated (three-fold or greater compared to CAF2). In S. cerevisiae, three (AHP1, HSP12, and PYC2) of the seven genes that were up regulated provide cells with an adaptation function in response to oxidative stress; another gene (GPH1) is regulated under stress conditions by Hog1p. Three other genes that are up regulated encode a cell surface protein (FLO1), a mannosyl transferase (MNN4-4), and a putative two-component histidine kinase (CHK1) that regulates cell wall biosynthesis in C. albicans. Of the down-regulated genes, ALS1 is a known cell adhesin in C. albicans. Verification of the microarray data was obtained by reverse transcription-PCR for HSP12, AHP1, CHK1, PYC2, GPH1, ALS1, MNN4-4, and FLO1. To further determine the function of Ssk1p in the Hog1p signal transduction pathway in C. albicans, we used Western blot analysis to measure phosphorylation of Hog1p in the ssk1 mutant of C. albicans when grown under either osmotic or oxidative stress. We observed that Hog1p was phosphorylated in the ssk1 mutant of C. albicans when grown in a hyperosmotic medium but was not phosphorylated in the ssk1 mutant when the latter was grown in the presence of hydrogen peroxide. These data indicate that C. albicans utilizes the Ssk1p response regulator protein to adapt cells to oxidative stress, while its role in the adaptation to osmotic stress is less certain. Further, SSK1 appears to have a regulatory function in some aspects of cell wall biosynthesis. Thus, the functions of C. albicans SSK1 differ from those of S. cerevisiae SSK1.
白色念珠菌的Ssk1p是Hog1双组分信号转导系统的一种假定应答调节蛋白。在酿酒酵母中，Ssk1p的磷酸化状态决定了促进细胞适应渗透胁迫的基因是否被激活。我们之前已经表明，白色念珠菌的SSK1不能互补酿酒酵母的ssk1突变体，并且白色念珠菌的ssk1突变体对山梨醇不敏感。在本研究中，我们发现白色念珠菌的ssk1突变体对几种氧化剂敏感，包括过氧化氢、叔丁基过氧化氢、甲萘醌和超氧化钾，当它们分别添加到酵母提取物-蛋白胨-葡萄糖（YPD）琼脂培养基中时。我们使用DNA微阵列来鉴定其调控受ssk1突变影响的基因。将在30℃下于YPD培养基中培养3小时的突变细胞（菌株CSSK21）的RNA进行反转录，然后与野生型细胞（CAF2）类似制备的RNA进行比较。我们观察到突变细胞中有7个基因持续上调（与CAF2相比上调三倍或更多）。在酿酒酵母中，上调的7个基因中的3个（AHP1、HSP12和PYC2）赋予细胞对氧化应激的适应功能；另一个基因（GPH1）在应激条件下受Hog1p调控。另外3个上调的基因编码一种细胞表面蛋白（FLO1）、一种甘露糖基转移酶（MNN4-4）和一种假定的双组分组氨酸激酶（CHK1），后者在白色念珠菌中调节细胞壁生物合成。在下调的基因中，ALS1是白色念珠菌中一种已知的细胞粘附素。通过对HSP12、AHP1、CHK1、PYC2、GPH1、ALS1、MNN4-4和FLO1进行反转录PCR来验证微阵列数据。为了进一步确定Ssk1p在白色念珠菌Hog1p信号转导途径中的功能，我们使用蛋白质印迹分析来测量白色念珠菌ssk1突变体在渗透或氧化应激下生长时Hog1p的磷酸化情况。我们观察到白色念珠菌的ssk1突变体在高渗培养基中生长时Hog1p被磷酸化，但在过氧化氢存在下生长时ssk1突变体中的Hog1p未被磷酸化。这些数据表明，白色念珠菌利用Ssk1p应答调节蛋白使细胞适应氧化应激，而其在适应渗透应激中的作用尚不确定。此外，SSK1似乎在细胞壁生物合成的某些方面具有调节功能。因此，白色念珠菌SSK1的功能与酿酒酵母SSK1的功能不同。
