臺灣博碩士論文加值系統

白色念珠菌是人類主要的伺機性真菌病原菌，它會共生在健康人類的體內，一旦免疫力降低時，它就有機會侵入人體，可能造成嚴重感染，甚至會造成死亡。白色念珠菌常貼附在人類皮膚、口腔黏膜、胃腸道甚至是醫療器材的表面。貼附是白色念珠菌侵入表皮細胞時最重要的第一步驟。在本研究中，白色念珠菌的Hom6蛋白被證實可能是一個多絲胺酸去氫酵素，參與 threonine 和 methionine 的合成。在缺乏胺基酸的環境中，將HOM6基因剔除會影響蛋白質的轉譯過程。另外在研究Hom6蛋白與白色念珠菌毒性之關係，我們發現Hom6會影響細胞的貼附能力。於已知會影響細胞貼附的因子中，細胞壁蛋白扮演著極重要的角色。而本研究中，我們也發現Hom6蛋白可能會存在於細胞壁中，並影響細胞貼附。Xog1是一種存在於細胞壁的exoglucanase，並且被發現會影響細胞貼附。我們也進而發現Hom6蛋白會影響Xog1蛋白在細胞壁的表現。使用西方墨點轉漬分析，於HOM6基因剔除菌株中，Xog1蛋白的表現量在細胞壁上是增加的，但是細胞的exoglucanase活性卻是下降的。推測HOM6的剔除會造成其他exoglucanase或是製造glucan的蛋白質表現量降低，而促使Xog1蛋白被釋放到細胞壁上，來補足細胞壁的缺陷。這個研究證實了Hom6蛋白對白色念珠菌毒性的重要性，也顯示了Hom6蛋白將會是一個很好的標定蛋白，用來發展新的抗真菌藥物。除了細胞貼附，細胞的抗壓能力也很重要。在感染的過程中，白色念珠菌除了自身的氧化壓力外，也必須對抗吞噬細胞製造的ROS。Zcf29蛋白被發現與抗壓調節和藥物耐受性有關。ZCF29剔除的菌株對menadione和caffeine較敏感但是卻對抑制ergosterol合成的藥物fenpropimorph較有抵抗性。而且在one-hybrid的實驗中，Zcf29的轉錄活性在menadione作用下會上升。然而一些主要抗氧化壓力蛋白質的基因表現以及酵素活性在ZCF29剔除後卻沒看到差異。另外在ergostrol合成相關的基因表現量及細胞內ergosterol含量也都沒有差異。因此Zcf29蛋白可能會透過其他方式來調節外界壓力，而這方式並未在我們實驗中發現，還需要更多研究來發掘。

Candida albicans is the most important opportunistic fungal pathogen of humans. It is a harmless commensal in a healthy person, but can cause life-threatening invasive infections particularly in immunocompromised patients. C. albicans can adhere to the mucosal surfaces and even to the surfaces of medical devices. Cell adhesion is the first step in C. albicans infection of epithelia. In this study, we found that C. albicans Hom6p has a homoserine dehydrogenase activity and is probably involved in the pathway for threonine and methionine biosynthesis. In polysome profile analysis, deletion of HOM6 caused translational arrest in the absence of amino acid supplements. In addition, we found that C. albican Hom6p has an effect on cell adhesion, which is important for C. albicans virulence. Many cell wall proteins are known to play major roles in cell adhesion; we also demonstrate that Hom6p can localize at cell wall by western blot. Moreover, C. albicans Hom6p affects expression of the cell wall protein Xog1p which is a major exoglucanase and is found to be involved in cell adhesion. Xog1p level increased on the cell wall, however, the overall exoglucanase activity reduced in the hom6hom6 mutant. We speculated that the defect of protein synthesis attenuated expression of other exoglucanases or glucan synthesis-related proteins, and then induced release of Xog1p to the cell wall. C. albicans Hom6p thus had an effect on cell survival and cell adhesion by maintaining protein synthesis and cell wall function. Moreover, this study suggests that Hom6p would be a good target to develop new antifungal drugs. In addition to adhesion, the second part of this work was to study a transcription factor ZCF29 probably involved in stress response in C. albicans. The zcf29zcf29 mutants were sensitive to menadione and caffeine but resistant to fenpropimorph. The transcriptional activity of Zcf29p was induced after menadione treatment, revealed by one-hybrid assay. However, the gene expression and activity of antioxidative enzymes had no differences between wild type and the zcf29zcf29 mutant. In addition, the expression of ERG2, ERG24 and ERG11 genes and the ergosterol content had no differences between wild type and the mutant. Therefore, Zcf29p regulates stress response probably through its control of other genes not tested in our study.

中文摘要 I
Abstract II
誌謝辭 III
Table of contents IV
List of Tables VI
List of Figures VII
List of Supplemental Tables and Figures VIII
1. Introduction 1
1.1. Candida albicans is an important fungal pathogen of humans 1
1.2. The major virulence factors in C. albicans 1
1.2.1. Secreted hydrolytic activity 2
1.2.2. C. albicans cell wall and adhesins 2
1.2.3. Morphogenesis in C. albicans 4
1.2.4. Phenotypic switching of C. albicans 4
1.3. Drug resistance of C. albicans 5
1.3.1. The emergence of drug resistance in C. albicans 5
1.3.2. Developing new antifungals and identifying new drugs targets 6
1.3.3. Homoserine dehydrogenase in S. cerevisiae and C. albicans 7
1.4. Stress responses in C. albicans 8
1.4.1. Antioxidant is important for C. albicans virulence 8
1.4.2. Ergosterol biosynthesis plays a role in stress resistance 10
1.4.3. C. albicans ZCF29 is a potential transcriptional regulator between ergosterol biosynthesis and oxidative response 11
1.5. The aims of this study 11
2. Materials and Methods 13
2.1. Strains and growth conditions 13
2.2. Isolation of C. albicans genomic DNA 13
2.3. Gene deletion in C. albicans 14
2.4. Yeast transformation 15
2.5. Construction of Hom6-GFP fusion protein 16
2.6. Construction of HOM6-overexpression strain 17
2.7. Spot assay of C. albicans 17
2.8. Reverse transcription (RT)-PCR 18
2.9. Southern blot analysis 19
2.10. Assay for homoserine dehydrogenase activity 19
2.11. Amino acid analysis 20
2.12. Polysome profile 21
2.13. Cell wall protein extraction 22
2.14. Western blot 23
2.15. Glucanase activity assay 23
2.16. Cell adhesion to polystyrene 24
2.17. Candida One-hybrid assay 24
2.18. Antioxidative activity assay 26
2.19. Quantification of Ergosterol content 27
3. Results 28
3.1. Studying the functions of Hom6p in C. albicans 28
3.1.1. C. albicans Hom6p is a homoserine dehydrogenase and involves in biosynthesis of threonine and methionine. 28
3.1.2. C. albicans Hom6p affected the concentrations of intracellular amino acid and translational activity in depletion of amino acids. 30
3.1.3. Hom6p attenuated the ability of cell adhesion in C. albicans. 32
3.1.4. Hom6p in C. albicans not only was a cytosolic protein but can also localize on the cell wall. 32
3.1.5. The hom6-deleted mutant had an effect on the exoglucanase activity. 33
3.2. Study of the functions of Zcf29 in C. albicans 34
3.2.1. Zcf29p functions as a transcriptional activator. 34
3.2.2. The zcf29-deleted mutants were sensitive to menadione and caffeine but resistant to fenpropimorph. 35
3.2.3. Zcf29p was involved in neither cell wall integrity nor response to H2O2 and fluconazole. 36
3.2.4. The transcriptional activity of Zcf29p was induced after menadione treatment. 37
3.2.5. Zcf29p affected neither the expression of genes associated with antioxidant nor the activity of antioxidative enzymes. 38
3.2.6. Zcf29p also did not affect the biosynthesis of ergosterol. 38
4. Discussion 39
4.1. For the study of Hom6p 39
4.2. For the study of Zcf29p 43
5. References 46

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