白色念珠球菌 Hog1 蛋白質內的兩個磷酸化點對於 White-Opaque 型態轉換、交配能力及費洛蒙誘導細胞附著之重要性

Two Conserved Phosphorylation Sites of The Candida albicans Hog1 Protein Are Important for White-Opaque Switching, Mating Response and Pheromone-Stimulated Cell Adhesion

張雯涵(Wen-Han Chang)

指導教授 ： 林晉玄

國立臺灣大學/生命科學院/生化科技學系/碩士(2015年)

https://doi.org/10.6342/NTU.2015.02301

摘要

白色念珠球菌 (Candida albicans) 為人類伺機型致病真菌，在免疫不全的患者身上易引發系統性感染。白色念珠球菌存在一種特殊的型態轉換，稱之為 white - opaque switching。white 細胞與 opaque 細胞具有不同的型態特徵、致病能力、交配能力、生物膜形成及對寄主免疫系統的反應等。white-opaque 型態轉換會受到外界環境因子的調控，例如二氧化碳濃度、 N-acetylglucosamine 及氧化壓力等。過去本實驗室發現，剔除掉 SSK2、PBS2 或 HOG1 基因會使 white 細胞在 SC 培養基上 100% 轉換成 opaque 細胞，並使交配能力下降。Hog1 蛋白質具有兩個重要的保守磷酸化點，分別位於 Thr-174 及 Tyr-176 位置上，當受到外界環境壓力刺激時，會使得 Hog1 蛋白質上的兩個磷酸化點被磷酸化，使 Hog1 蛋白質具有活性並做出適當反應。本次研究首先發現 Hog1 蛋白質上的兩個磷酸化點對於 white-opaque 型態轉換、交配能力及費洛蒙誘導細胞附著皆扮演重要的角色。實驗採用六種 Hog1 點突變株，包含無法磷酸化的HOG1T174A、HOG1Y176F 及 HOG1T174A,Y176F 突變株，以及模擬磷酸化的HOG1T174D、 HOG1Y176D及 HOG1T174D,Y176D 突變株。將同型合子菌株的 Hog1 蛋白質 Thr-174 及 Tyr-176 位置上的胺基酸突變與剔除掉 HOG1 基因結果一樣，皆會使 white 細胞在 SC 培養基上 100% 轉換成 opaque 細胞。所有 Hog1 點突變株皆會增加對高滲透壓力及氧化壓力的敏感性，與 hog1 突變株結果相似。西方墨點法分析結果顯示，所有 Hog1 點突變株的 Hog1 蛋白質皆可被表現出來，但在受到 hydrogen peroxide 的刺激下，皆無法被磷酸化。而剔除掉可以將 Hog1 蛋白質去磷酸化的 PTP2 或是 PTP3 基因，在 Lee’s NAG 固態培養基上皆無法去抑制 white-to-opaque 型態轉換。此外，與野生株相比，Hog1 點突變株的交配突出物 (mating projections) 有明顯縮短的趨勢；而 Hog1 點突變株的交配能力也有明顯的下降。另外，所有 Hog1 點突變株的 white 細胞在受到費洛蒙刺激誘導下，與野生株相比，細胞附著量有明顯減少的趨勢。根據上述研究結果，本研究發現將 Hog1 蛋白質上的兩個重要的磷酸化點 Thr-174 及 Tyr-176 位置作改變，會使菌株如同 hog1 突變株去影響 white-opaque 型態轉換、交配能力及費洛蒙誘導細胞之附著。

關鍵字

白色念珠球菌 ； White-Opaque 型態轉換 ； 費洛蒙

並列摘要

Candida albicans is an opportunistic human fungal pathogen and able to cause life-threatening infections in immunocompromised patients. C. albicans has a unique morphological transition between white and opaque phases. These two cell forms have different properties in virulence, mating capability, biofilm formation and host-cell interaction. White-opaque transition is regulated by several external stimuli, such as CO2, N-acetylglucosamine and oxidative stress. Our previous study revealed that deletion of SSK2, PBS2 or HOG1 gene resulted in 100% white-to-opaque switching on SC medium, and suppressed the mating response. Hog1 protein has two important phosphoacceptors, Thr-174 and Tyr-176, and will be activated when phosphorylated in response to stimuli. In this study, we first demonstrated that two conserved phosphorylation sites are not only required for stress response, but also involved in white-opaque switching, mating and pheromone-stimulated cell adhesion. Six Hog1 point-mutated strains were generated, including non-phosphorylated strains (HOG1T174A, HOG1Y176F and HOG1T174A,Y176F) and mimic phosphorylated strains (HOG1T174D, HOG1Y176D and HOG1T174D,Y176D). Point mutation on Thr-174, Tyr-176 or in combination of Hog1 protein in MTL homozygous strains of C. albicans would stimulate opaque cell formation in a frequency of 100% on SC medium. Similar to the hog1 mutant, all of the point mutation strains were sensitive to both osmotic and oxidative stresses. Western blotting showed that Hog1 proteins were expressed in these mutants, but could not be phosphorylated when treated with H2O2. Deletion of PTP2 or PTP3 gene would not impact white-to-opaque switching on Lee’s NAG medium. Furthermore, mating projections of point-mutated strains were significantly shorter and their mating efficiencies were lower than those of the wild-type. In addition, numbers of pheromone-induced cell adhesion of white cells in all point-mutated strains reduced. Taken together, our study demonstrated that mutation on either Thr-174 or Tyr-176 of Hog1 resulted in similar characters with hog1 mutants in white-opaque transition, sexual mating and pheromone-induced cell adhesion in C. albicans.

並列關鍵字

Candida albicans ； White-Opaque Switching ； Mating Response ； Pheromone-Stimulated Cell Adhesion

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