白色念珠菌是一種伺機性感染真菌，在健康人體的口腔及胃腸道等黏膜的表面上可以發現此種共生真菌。然而，隨著近年來免疫功能缺乏病人的增加，白色念珠菌經常造成嚴重的院內感染，並且有著很高的死亡率。探討其致病原因，是因為白色念珠菌擁有許多獨特的致病因子，例如：細胞黏附和生物膜形成。而此兩因子與菌體細胞壁結構和組成息息相關，更重要的是，這些特徵最後可能與抗藥性相關聯。白色念珠菌致病因子之合成與否經常受外界壓力及環境狀況所調控。根據先前的研究，白色念珠菌Rhb1是屬於Ras家族的小型G蛋白(Small G-protein)，在TOR (target of rapamycin) 訊息傳導路徑上扮演著正向調控的角色，並且參與細胞壁及菌絲生長。本研究更進一步探討Rhb1對細胞壁、生物膜及抗藥性的影響，並研究其相關訊號傳導路徑。本研究發現將RHB1基因剃除後會造成細胞壁組成改變並活化許多細胞壁修補基因，而Mkc1激酶的活化也顯示Rhb1參與細胞壁完整性訊號的傳遞。使用caspofungin (glucan synthesis inhibitor)作為外在壓力的實驗，Rhb1確實藉由其下游的Mkc1來影響菌株的藥物感受性。另外，已知生物膜形成與細胞壁改變有密切的關係，本研究發現RHB1 在細胞黏附和生物膜形成扮演著負向調控的角色，這結果可能是RHB1 突變株降低了TOR激酶的活性並活化Mkc1所導致。雖然RHB1 突變株對細胞壁相關藥物感受性比較高，但有趣的是，它在針對細胞膜作用的藥物(如：Fluconazole)卻呈現抗藥性。白色念珠菌抗藥機制通常是藉由改變藥物作用目標，利用幫浦將藥物打出或啟動特殊訊號和路徑。我們發現RHB1突變株的幫浦基因表現量有所增加而且其Mkc1也有活化的現象，而這些結果可能和TOR訊號傳遞及細胞壁完整性有關。最後，以臨床菌株為實驗對象，抑制TOR的活性呈現出與RHB1突變株一樣的藥物耐受性，顯示氮源攝取和調控與白色念珠菌抗藥機制有相當大的關聯性，同時為白色念珠菌未來臨床治療提供嶄新的觀點。

Candida albicans is an important fungal pathogen of humans. It is generally harmless in healthy individuals, but can cause invasive and life-threatening infections particularly in immunocompromised patients. The first step for C. albicans to establish infection is to adhere the surface of host cells. Moreover, C. albicans adhesion is also the key step of biofilm formation on mucosal surfaces and medical devices. The cell wall of C. albicans plays a critical role in cell adhesion and pathogen-host interaction, and is essential for C. albicans to withstand environmental stresses, such as antifungal drugs. In this study, we focus on Rhb1, a member of the small G-protein of Ras superfamily. The RHB1 gene deletion mutant was sensitive to cell wall disrupting agents, Congo red and Calcofluor white, whereas was resistant to the antifungal drug, fluconazole. Moreover, our results showed that the RHB1 deletion causes the alteration of C. albicans cell wall components and activation of the Mkc1 kinase, the key component of the cell wall integrity (CWI) signaling pathway. Finally, we also demonstrated that RHB1 is closely related to cell adhesion and biofilm formation. Together, this study reveals new roles of RHB1 and enriches our understanding in pathogenesis of C. albicans, suggesting new strategies for the future treatment of fungal diseases.

中文摘要 I
Abstract II
致謝辭 III
Table of Contents IV
List of Tables VI
List of Figures VII


Chapter 1: Introduction 1
1.1. Candida albicans is an important fungal pathogen 2
1.2. The cell wall of C. albicans 3
1.2.1. Cell wall structure of C. albicans 3
1.2.2. Cell wall integrity in C. albicans 4
1.3. The virulence factors of C. albicans 4
1.3.1. C. albicans cell adhesins 5
1.3.2. C. albicans biofilm formation 5
1.3.3. C. albicans invasion and host response 6
1.4. C. albicans antifungal drug resistance 7
1.4.1. Antifungal drugs against C. albicans 7
1.4.2. Mechanisms of antifungal drug resistance in C. albicans 7
1.5. Rhb1 signaling pathway in C. albicans 9
1.5.1. The Rheb subfamily of small GTPase 9
1.5.2. Rhb1 in C. albicans 9
1.6. The aims of this study 10


Chapter 2: Materials and Methods 11
2.1. Strains and growth conditions 12
2.2. Antifungal susceptibility testing 12
2.3. Zymolyase assay 13
2.4. Propidium iodide staining 14
2.5. RNA isolation, RT-PCR and real-time quantitative PCR 14
2.6. Protein extraction and western blot 15
2.7. Cell surface hydrophobicity 16
2.8. Measuring carbohydrate content of the cell wall 17
2.9. Extraction and quantification of ergosterol content 18
3.0. Strain construction 18
3.1. Cell flocculation 19
3.2. Measurement of cell adhesion to polystyrene and biofilm formation 20
3.3. Biofilm observation by using scanning electron microscopy 20
3.4. Macrophage response 21


Chapter 3: Results 23
Part I: The Small GTPase Rhb1 modulates cell wall integrity, biofilm formation in Candida albicans 24
3.1.1. Rhb1 regulates cell wall integrity of C. albicans 25
3.1.2. Rhb1 induces cell wall damage responses. 26
3.1.3. Rhb1 influences caspofungin susceptibility through its regulation of the cell wall integrity pathway. 27
3.1.4. Rhb1 also affects cell adhesion and biofilm formation 29
Part II : Candida albicans small G-protein Rhb1 is involved in regulation of fluconazole susceptibility 32
3.2.1. Deletion of RHB1 confers resistance to cell membrane perturbing agents 33
3.2.2. Fluconazole resistance of the RHB1 deletion mutant may be due to the increased activity of efflux pumps 34
3.2.3. Alternation of ergosterol biosynthesis is not related to fluconazole resistance in the RHB1 deletion mutant 35
3.2.4. Rhb1 regulates fluconazole susceptibility through the Mkc1 MAPK pathway 37
3.2.5. Nutrient signaling influences fluconazole resistance of C. albicans 38


Chapter 4: Discussion 42
RHB1 and C. albicans cell wall integrity 43
RHB1 and C. albicans biofilm formation 46
C. albicans cell wall and its interaction with host cells 47
RHB1 and C. albicans fluconazole resistance 48
Final remarks 51
Reference 53
Results for Part I 67
Results for Part II 76
Appendix 84

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