指導教授：林晉玄臺灣大學：生化科技學系鄭仁華Cheng, Jen-HuaJen-HuaCheng2014-11-262018-07-062014-11-262018-07-062014http://ntur.lib.ntu.edu.tw//handle/246246/261621白色念珠球菌 (Candida albicans) 是人類的伺機性病原真菌，主要棲息於人體的腸胃道以及女性的生殖道。對於免疫力正常的人，此菌並不會造成嚴重的感染，但是對於免疫力異常的患者則可能造成嚴重的真菌感染。一旦感染惡化，可能進一步引發菌血症，死亡率高達 47% 以上。C. albicans具有一種稱之為 White-Opaque的型態轉換與其致病力有很大的關聯性。研究發現 C. albicans 從 White 細胞轉換成 Opaque 細胞後被宿主巨噬細胞辨識、毒殺的比例會大幅度降低 ; 此外，相對於 White 細胞， Opaque 細胞才具有菌種交配的能力。目前發現White-Opaque 型態的轉換受許多因子調控，例如在分子調控機制的部分，主要是受Wor1 轉錄因子所調控。外在環境的部分，環境中的 PH 值、 CO2、 UV、 溫度、氧化壓力等都會對其轉換造成影響。因此，本研究將 C. albicans 調控氧化壓力的 HOG1 剔除後培養於 SC 培養基，發現性狀 a/a 與 α/α 的hog1突變株會從 White 細胞完全轉換至 Opaque 細胞，野生株則維持於 White 細胞的狀態。將實驗環境模擬體內腸胃道 (37oC + 5% CO2)，性狀 a/a 與 α/α的 hog1突變株轉換至 Opaque 細胞的比率同樣為100%，野生珠則依舊無法轉換至 Opaque 細胞。然而，hog1 / wor1雙突變株無法轉換至 Opaque 細胞，顯示Hog1 調控 White-Opaque 型態的轉換仍受到 Wor1所調控。為了尋找 Hog1 下游可能參與調控White-Opaque 型態轉換的轉錄因子，將 MSN2 與 MSN4剔除後進行型態轉換的測試，結果顯示兩者形成的菌落均維持在 White 細胞的型態，顯示 Msn2 和 Msn4與 White-Opaque型態轉換之調控無關。 hog1突變株形成的 Opaque 被發現較不穩定，僅約 80% 細胞會維持在 Opaque 細胞型態；野生株則有 95% 是 Opaque 細胞的型態。當Opaque 細胞受到α-Pheromone誘導後野生株有 48% 細胞會產生mating projection， hog1突變株僅約 15% 細胞能生成。此外， hog1突變株生成的mating projection平均長度為 10.54 μm，低於野生株的平均長度24.39 μm。菌株交配效率測試結果顯示 hog1突變株的mating efficiency 為 2.71%，相較於野生株 9.92% 與互補株 8.45%，有顯著降低，顯示 Hog1 對菌株的交配能力亦扮演重要角色。綜合以上結果，C. albicans的 Hog1不只調控環境氧化壓力，更具有調控 White-Opaque 型態的轉換與影響 Opaque 細胞的性質。Candida albicans is the opportunistic human fungal pathogen found in the gastrointestinal and urogenital tracts of healthy people. However, for the people who are immumocompromised, C. albicans can cause invasive candidiasis, which would result in systemic infection and high mortality rate. A phenotypic transition between White and Opaque states in C. albicans is associated with its virulence in different colonizing niches. Furthermore, comparised with white cells, opaque cells could escape attacks from macrophages. Besides, Opaque cells, but not white cells, have been shown to have the exclusive ability of mating. The White-Opaque phenotypic transition is majorly controlled by the Wor1 transcriptional feedback loop. Some environmental clues, including PH, oxidative stress, carbon sources, and temperature, also have influence on regulating the White-Opaque switching. Hog1 MAP kinase has been recognized as the main regulator for adapting to environmental oxidative stress of C. albicans. Therefore, we hypothesized that Hog1 MAP kinase not only has the ability to adapt to environmental stresses, but also has the ability to regulate the White-Opaque switching. In this study, knocking out the HOG1 gene in a/a and α/α cells resulted in 100% opaque cell formation on SC medium at both 25℃ and 37℃ + 5% CO2 culture condition (mimic gastrointestinal condition). However, hog1/wor1 mutants remained in the white state, suggesting that Wor1 still plays an important role in opaque cell formation. To indentify the potential downstram transcritption factors regulated by Hog1, deletion of both MSN2 and MSN4 did not induce the White-to-Opaque switching. The opaque cells forming from hog1 mutants are less stable than those of the wild-type, only 80% of the colonies remained in opaque phenotype, whereas the stability of opaque cells in the wild-type and complementary strain is near 95%. Besides, only 15% of the hog1 mutant cells formed pheromone-induced mating projection, whereas the shmoo formation of the wild-type reached to 48%. The average length of mating projections generated from hog1 mutants is 10.54 μm, which is shorter than those of the wild-type (24.39 μm). As the expectation, the mating efficiency of hog1 mutants significantly decreased to 2.71% compared to the wild-type (9.92%) and complementary strain (8.15%). In summary, my studies revealed that the Hog1 MAP kinase of C. albicans not only is required for oxidative stress, but also is involved in White-Opaque switching and the characteristics of opaque cells.口試委員會審定書 # 誌謝 ii 中文摘要 iii ABSTRACT v 目錄 vii 圖目錄 x 表目錄 xi 附錄 xii 前言 1 念珠菌感染 (Candidiasis ) 1 白色念珠菌 (Candida albicans) 1 White-Opaque 型態轉換 2 交配 (Mating) 3 Mitogen activated protein kinase (MAP kinase) 訊息傳遞途徑 3 High osmotic glycerol 1 (Hog1) MAP kinase 4 研究的動機與目標 4 材料與方法 5 引子、質體、菌株、藥品、培養基 5 DNA 聚合酶鏈式反應 (polymerase chain reaction, PCR) 5 DNA Clean Up 與 Gel Recovery 5 洋菜膠體電泳分析 (Agarose gel electrophoresis) 5 DNA 接合反應 (Ligation) 6 大腸桿菌勝任細胞 (Competent cell) 之製備 6 大腸桿菌轉形作用 (Transformation) 6 質體DNA之抽取 8 C. albicans 轉形作用 (Transformation) 8 環境高滲透壓與氧化壓力敏感度之測試 11 White-Opaque 型態轉換之測試 11 SDS-PAGE (polyacrylamide) 蛋白質電泳分析 12 西方墨點法 (Western blot) 12 Opaque 細胞穩定度之測試 13 α-Pheromone 誘導 Opaque 細胞產生 mating projection 之測試 13 Mating Efficiency之測試 13 Fluorescence-activated cell sorting (FACS) 14 結果 15 構築 C. albicans 之 hog1突變株與 hog1::HOG1互補株 15 hog1突變株對環境高滲透壓與氧化壓力具有高度敏感性 15 於25oC環境性狀為a/a 與 α/α的 hog1突變株在SC 培養基有 White-to-Opaque 型態的轉換。 16 於37oC + 5% CO2 培養環境，性狀為a/a 與 α/α的 hog1突變株有 White-to-Opaque 型態的轉換。 16 螢光顯微鏡下鑑定 hog1突變株形成的橢圓形長條狀細胞為 Opaque 細胞 16 野生株之 White細胞與 Opaque 細胞均無偵測到Hog1-P磷酸化 17 構築 C. albicans wor1突變株 17 Wor1 參與 Hog1調控 White-Opaque型態的轉換。 18 構築 C. albicans msn2突變株與 msn2 / msn4雙突變株 18 Hog1 下游轉錄因子Msn2與 Msn4均無參與 White-Opaque 型態轉換之調控 18 hog1突變株形成之Opaque 細胞較不穩定 18 hog1突變株之Opaque 細胞對α-Pheromone較不敏感並產生長度較短之 mating projection。 19 構築帶有 nutrition marker (∆arg4、∆leu2) 之 C. albicans 野生株、 hog1突變株與hog1::HOG1互補株 19 hog1突變株Opaque 細胞交配能力降低 20 討論 21 未來研究方向 24 圖表 25 附錄 46 參考資料 482003344 bytesapplication/pdf論文公開時間：2016/07/29論文使用權限：同意有償授權(權利金給回饋本人)白色念珠球菌White-Opaque 型態轉換Hog1Wor1Msn2Msn4交配[SDGs]SDG3thesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/261621/1/ntu-103-R01b22049-1.pdf