【作者】 郑长龙；

【导师】 蒋伶活；

【作者基本信息】 天津大学 ， 应用化学， 2007， 博士

【摘要】 白色念珠菌是临床上一种最重要的条件致病真菌。目前,人们对白色念珠菌的研究主要集中在弄清其致病机理和发现新的药物作用靶点。通过用酿酒酵母基因编码的氨基酸序列对斯坦福大学白色念珠菌基因组数据库进行比对,我们得到白色念珠菌基因CaTCO89和CaPTC1的序列。基因CaTCO89的ORF长为2127 bp,编码708个氨基酸。CaTCO89与酿酒酵母ScTCO89所编码蛋白氨基酸序列的相似性为27.5%。我们发现CaTCO89基因可以互补ScTCO89在雷帕霉素和氯化锂抗性以及保持细胞壁完整性方面的功能。我们以白色念珠菌菌株RM1000为背景,构建了CaTCO89的双缺失菌株TJU4(tco89/tco89)。与RM1000相比,TJU4对雷帕霉素和氯化锂的敏感性增加。在TJU4细胞中异位表达CaTCO89基因能够恢复TJU4对雷帕霉素抗性到野生型水平,然而却显著增强TJU4细胞对氯化锂和过氧化氢的抗性以及对Cd2+的敏感性。此外,在Spider和SD-Ura液体培养基中,CaTCO89基因在TJU4中的异位表达能引起细胞凝集形成絮状沉淀。在Spider固体培养基上,TJU4的菌落形态与野生型菌株相同,为边缘不整齐扁平状、表面凸凹不平,且有周边菌丝形成,但是异位表达CaTCO89的TJU4菌落形态表现为表面光滑的面包状。基因CaPTC1的ORF长度为1128 bp,编码375个氨基酸,与酿酒酵母ScPtc1p的相似性为52%。CaPTC1的双缺失菌株SYY4(ptc1/ptc1)对Li+、Na+和K+离子敏感,异位表达CaPTC1能够抑制SYY4细胞的缺失表型。我们克隆并在细菌中表达了CaPtc1p的催化区域,发现纯化的重组蛋白具有去磷酸化活性,这种活性还依赖于Mn2+或Mg2+的存在,并且受到丝氨酸-苏氨酸去磷酸酯酶抑制剂NaF的抑制。这些结果表明CaPtc1p是一种PP2C类蛋白磷酸酯酶。更多还原

【Abstract】 Candida albicans is the most important fungal pathogen in humans. Recent studies on C. albicans have been focused on understanding its pathogenesis and on identifying therapeutic targets for the development of novel antifungal drugs. From Stanford C. albicans genomic database, we identified CaTCO89 and CaPTC1 gene sequences. CaTCO89 has an ORF of 2127 bp, which encodes a protein of 708 amino acids sharing 17% identity and 28% similarity with ScTco89p at the amino acids level. We found that CaTCO89 could complement the functions of ScTCO89 in rapamycin and lithium tolerance as well as in cellular integrity in S. cerevisiae. We constructed the deletion mutant of CaTCO89 (TJU4) in RM1000 background. In comparison with RM1000, TJU4 cells showed rapamycin and lethium sensitivity. Ectopic expression of CaTCO89 recovered the tolerance of TJU4 cells to rapamycin to the level of the wild-type RM1000 cells, but significantly increased the tolerance of TJU4 cells to LiCl and H2O2 and the sentivity to CdCl2 above the levels of RM1000 cells. Deletion of CaTCO89 did not affect the morphological development. However, ectopic expression of CaTCO89 caused cell aggregation of C. albicans in Spider and SD-Ura liquid medium as welll as formation of smooth and bread-shaped coloneys on solid spider medium.CaPTC1 has an ORF of 1128 bp in length, which encodes a protein of 375 amino acids sharing 41% identity and 52% similarity with ScPtc1p. We disrupted the two alleles of CaPTC1 from RM1000, which resulted in the strain SYY4. SYY4 cells showed sensitivity to LiCl, NaCl and KCl but not the osmolar agent sorbitol. Ectopic expression of CaPTC1 recovered the tolerance of SYY4 to these salt stresses. The DNA sequence encoding the catalytic domain of CaPtc1p was cloned and expressed in E. Coli cells. The purified protein exhibited dephosphorylation activity, which was dependent on the presence of Mg2+ or Mn2+ ion and inhibited by the protein Ser/Thr phosphatase inhibitor NaF. These results indicate CaPTC1 encodes a type 2C Ser/Thr phosphatase (PP2C).更多还原