摘要:真菌能藉由有性生殖產生對環境逆境或宿主免疫系統適性的子代。白色念珠球菌 (Candida albicans) 為人體重要的致病真菌之一,尤其好發於免疫不全的患者。 此真菌具有一種獨特的轉換,稱之為 White-Opaque 表現型的轉換。 此型態的轉變影響許多特性;例如: opaque 細胞才能進行菌種間的交配,而white 細胞與生物膜的生成有關。 此外,opaque 細胞不會分泌化學引誘劑 (chemoattractant),而能夠躲避免疫系統的攻擊與白血球的吞噬,顯示 White-Opaque 的轉換對於真菌-宿主之間的交互作用扮演重要的角色。 許多環境外在的因子會影響 White-Opaque 的轉換,而此分子機制主要受到 Wor1 轉錄因子的回路所調控。 實驗室初步研究結果已發現全新但廣為人知的訊息傳導路徑能調控此獨特的轉換- Hog1 SAPK (Stress-activated protein kinase; Sln1 支線) 與另一Sho1滲透壓感受器 (osmosensor)。 剔除 HOG1 基因後,導致 C. albicans MTLa/a 同型合子 (homozygote) 的white細胞完全轉換成 opaque 細胞。且此突變株所產生的交配突觸 (mating projections; 18.1 m) 比野生株短 (32.2 m)。 因此,本計畫研究目標一將深入探討 HOG1 基因是否會影響白色念珠球菌另一同型合子 (MTL/) 與 MTLa/ 異型合子 (heterozygote) White-Opaque 細胞之間的轉換與菌體交配的情況。 除此之外,Hog1 與 Wor1 轉錄因子回路之間的關聯性亦會進一步驗證。 研究目標二將研究能調控 Hog1 活化的上游基因與Hog1 蛋白質內 2 個重要的磷酸化胺基酸 (Thr 174 與 Tyr 176) 是否與 White-Opaque 轉換有關。 白色念珠球菌 Sho1 滲透壓感受器所扮演的角色與酵母菌不同,並無法調控 Hog1 的活化。 有趣的是,Δsho1 突變株會大幅降低 opaque 細胞的形成。 因此,研究目標三將探討為何 Sho1 支線能夠調控 White-Opaque 細胞間的轉換。 我們更觀察到 Δhog1突變株會加強費洛蒙誘導生物膜 (pheromone-induced biofilms) 的生成;此現象可能是由於低量表現的磷酸酶Cpp1,進而影響Cek1/Cek2 的活化所導致。 因此,研究目標四將深入探討Hog1 SAPK 路徑影響費洛蒙誘導生物膜生成的機制。 這些結果將能釐清為何滲透壓傳導路徑參與白色念珠球菌獨特的轉換,並了解細胞如何透過一些因子的作用下,進行訊息傳導間的交互對話 (signaling cross-talks)。
Abstract: Sexual reproduction is one of the fascinating phenomena in most fungi. This leads to new progenies that are highly adapted to a specific environment or host immune system. An opportunistic pathogen, Candida albicans, responsible for half of all clinical fungal infections in the immunocompromised patients, exhibits unusual phenotypes, the white and opaque states. The epigenetic transition between white and opaque states regulates many respects. In particular, opaque cells are mating competent, whereas white cells do not mate but generate biofilms in response to pheromone. Furthermore, unlike white cells, opaque cells are less susceptible to phagocytosis due to no secretion of chemoattractant for leukocytes, indicating that this switching plays a crucial role in the fungus-host interaction. Switching between white and opaque cells is associated with many external stimuli and is directly regulated by the Wor1 transcriptional feedback loops. My preliminary studies have identified a novel but well-known signaling pathway involved in the white-opaque switching as Hog1 SAPK (Stress-activated protein kinase; Sln1 branch) and another osmosensor (Sho1 branch). Deletion of HOG1 gene in C. albicans MTLa/a (homozygote) cells resulted in 100% opaque cell formations, compared to those of the wild-type (< 10-3). However, the mechanism of how Δhog1 mutants involved in opaque cell formations remain obscure. Additionally, Δhog1 mutants exhibited shorter mating projection (18.1 m) as the shmoos’ lengths of the wild-type reached to 32.2 m. Hence, Specific Aim 1 will investigate the frequency of white-opaque switching in C. albicans MTL/ and MTLa/(heterozygote) cells, as well as the mating efficiency. The relationship between the Hog1 and Wor1 transcriptional feedback circuits will be addressed, too. Activation of Hog1 is directly regulated by several upstream components through phosphorylation from two-component systems to MAP kinases. My experiment outlined in the Specific Aim 2 will then determine upstream components and two conserved phosphorylation sites (Thr 174 and Tyr 176) of Hog1 in regulation of white-opaque switching. Unlike Saccharomyces cerevisiae, Sho1, the osmosensor, does not play a central role in activation of Hog1 in C. albicans. Interestingly, the white-to-opaque switching experiments showed that Δsho1 mutants displayed lower switching frequency, implicating that an unknown mechanism is required for the phenotypic change through Sho1 pathway. Thus, Specific Aim 3 will determine the mechanism by which white-opaque transition is involved in the Hog1-independent branch. Finally, I hypothesize that the increases of pheromone-induced biofilms in Δhog1 mutants are due to lower expression of Cpp1, a putative phosphatase that is required for inhibition of Cek1/Cek2. Specific Aim 4 will examine how Hog1 SAPK cascades affect the formation of pheromone-induced biofilms. Taken together, these studies will elucidate how a novel but widely known pathway directs the epigenetic transition and will reveal how cells evolve a delicate mechanism for signaling cross-talks via potential connected factors.