【作者】 张冰；

【导师】 李明春；

【作者基本信息】 南开大学 ， 微生物学， 2015， 硕士

【摘要】 白念珠菌是临床上最为重要的条件性致病真菌之一。由于免疫缺陷人群的不断增加,白念珠菌引起的感染备受关注。在与宿主相互作用的过程中,白念珠菌进化出极为精细的形态发生及压力应答系统,能够有效定殖并抵御宿主免疫系统攻击。磷脂酰肌醇衍生物是真核细胞极为重要的一类小分子物质,与细胞的信号转导、生长代谢调节紧密相关。在酿酒酵母及许多哺乳动物细胞中,磷脂酰肌醇磷酸酶Sac1及其同源蛋白介导了磷脂酰肌醇一磷酸或磷脂酰肌醇二磷酸向磷脂酰肌醇的转化,在肌动蛋白组装、细胞极性确立、细胞壁功能维持、胞吞与胞吐等过程中扮演重要角色。然而,白念珠菌的Sacl蛋白及其功能至今尚未明确。本研究即对白念珠菌的Sacl蛋白进行分子鉴定及功能分析。主要结果如下：(1)通过BLASTP分析,从白念珠菌基因组数据库中鉴定出一种与酿酒酵母ScSac1同源的蛋白,命名为Sacl。序列比对分析发现,该蛋白属于SAC家族成员,具有保守的CX5R(T/S)基序及SAC结构域。通过构建白念珠菌SAC1基因在酿酒酵母中的回补菌株,发现该基因能够回补酿酒酵母ScSAC1缺失造成的肌醇营养缺陷,提示白念珠菌Sacl蛋白参与了磷脂酰肌醇代谢。通过GFP定位技术,发现Sac1蛋白主要定位于内质网膜,提示该蛋白与内质网功能紧密相关。(2)采用两步PCR介导的同源重组法,构建白念珠菌SAC1基因的缺失菌株,在此基础上构建回补菌株。采用固体点板实验,发现SAC1基因的缺失造成白念珠菌对内质网压力及细胞壁压力药物高度敏感；酸性磷酸酶分析实验表明,SAC1基因的缺失导致白念珠菌的糖基化能力发生缺陷；几丁质染色及透射电镜观察结果显示,该基因缺失导致细胞壁几丁质分布与含量发生改变,以及细胞壁超显微结构异常；采用GFP报告系统及实时定量PCR技术,发现该基因的缺失导致非折叠蛋白应答(UPR)基因及细胞壁完整性(CWI)基因表达的上调。上述结果表明Sac1蛋白在内质网及细胞壁完整性维持方面发挥重要作用。(3)SAC1基因缺失导致白念珠菌菌丝发育能力降低,细胞仅能形成弯曲的菌丝,提示sac1Δ/Δ缺失株菌丝的极性生长能力受阻。肌动蛋白染色及Hwp1定位分析表明,SAC1基因缺失导致肌动蛋白斑在菌丝顶端的定位发生紊乱,以及菌丝特异性蛋白的极性转运过程存在缺陷,表明Sacl通过维持肌动蛋白骨架的顶端定位,调控菌丝特异性蛋白的极性转运过程。(4)SAC1基因缺失导致白念珠菌侵染宿主上皮细胞及耐受巨噬细胞攻击的能力显著下降,并且丧失了系统性感染小鼠的能力,表明Sac1与白念珠菌的毒力紧密相关,是一种重要的毒力相关因子更多还原

【Abstract】 Candida albicans is one of the most important opportunistic fungal pathogens in clinical practice. Candida albicans infections, especially systemic infections, are urgent due to the increase of immunocompromised individuals. During the interaction of C. albicans with the host, this pathogen evolved elaborate systems functioning in morphogenesis and stress response, which are important for efficient colonization and survival in host immune attacks. Phosphatidyl inositol phosphates (PIs) are one type of important small molecules involved in signal transduction and regulation of growth and metabolism. In Saccharomyces cerevisiae and mammalian cells, the phosphatidyl inositol phosphatase Sacl and its homologs mediate the transmission of PIP and PIP2 to PI, functioning in organization of actin skeleton, determination of cell polarization, maintenance of cell wall integrity (CWI), endocytosis and exocytosis. However, the characterization and function of Sacl in C. albicans remains to be investigated. This study aims to identify Sacl and to explore its function in this pathogen. The main results are demonstrated as follows.(1) BLASTP analysis revealed a homolog of ScSacl in Candida albicans genome database, named Sacl. Sequence analysis showed that this protein is a member of proteins containing SAC domain, which is characterized by a conserved CXsR(T/S) motif. Complementatin tests found that SAC1 can rescue the growth defect in inositol metabolism of ScsaclA. Sacl-GFP localization revealed that this protein is an endoplasmic reticulum (ER)-localized membrane protein.(2) Using two-step PCR mediated homogenous recombination and the integration system, SAC1 deletion strain and its reconstituted strain of C. albicans were constructed. Solid dotting experiments showed that deletion of SAC1 caused high-sensitivity to ER stress and cell wall stress. Acid phosphatase assays showed that this deletion led to a defect in glycosylation. Chitin staining and transmission electron microscopy revealed that this deletion caused abnormal distribution and increased content of cell wall chitin, and abnormal cell wall ultramicroscopic structure. GFP reporting systems and real-time PCR assays further demonstrated that SAC1 deletion up-regulated the expression of unfolded protein response (UPR) genes and cell wall integrity (CWI) genes. Hence, Sacl has an important role in maintenance of both ER function and CWI.(3) Deletion of SAC1 caused decreased ability of hyphal development, and the mutant formed short and curved hyphae, suggesting that the mutant had a defect in polarized growth. Actin staining and Hwpl localization revealed that this deletion led to abnormal localization of actin patches at the hyphal tip, and a defect in polarized transport of Hwp1, indicating that Sacl regulates polarized transport of hypha-specific proteins by maintaining the tip localization of actin cytoskeleton.(4) Deletion of SAC] led to significant decrease in resistance ability against macrophage attacks, and in invasion of host epithelial cells and systemic infection to mice, suggesting that Sacl is involved in C. albicans pathogenicity, and is an impotant virulence-related factor.更多还原