Hog1 上游訊息傳導 Sln1-Ssk1-Ssk2-Pbs2 及 Sho1 滲透壓感受器與白色念珠球菌 White-Opaque 型態轉換關聯性之探討

Abstract

白色念珠球菌 (Candida albicans) 為常見的人類伺機性真菌，主要生存在人類腸道中，當人體免疫力因為年齡、疾病或醫療治療而下降時，則會引發嚴重的系統性感染。白色念珠球菌存在一種特殊的型態轉換，稱為 White-Opaque Switching。White-Opaque 表現型轉換影響了許多白色念珠球菌的特性，包含型態上的不同、生物膜 (biofilm) 的生成、細胞交配 (mating) 的能力以及其對宿主免疫系統的反應。過去的研究顯示，此種型態轉換的轉換率受到外在環境的不同因子所影響，例如：氧氣濃度、溫度、二氧化碳濃度、氧化壓力以及菌珠生長速度。Hog1 Mitogen-Activated Protein Kinase (MAPK) 是一種 Stress-Activated Protein Kinase (SAPK) 訊息傳導路徑，會對細胞外在環境的滲透壓和氧化壓力產生反應，調控 Hog1 蛋白質的活性，進而影響下游的基因表現，使細胞產生適當反應。與釀酒酵母菌 (Saccharomyce cerevisiae) 不同的是，Sho1 蛋白質雖參與了滲透壓的訊息傳導，但並不影響 Hog1 蛋白質的活化。過去本實驗室研究發現，Hog1 也參與 White-Opaque Switching 的調控。將 Mating-Type Locus (MTL) 為同型合子 (homozygote) 的野生型 (wild type) 白色念珠球菌，包含 MTLa/a 以及 MTLα/α，培養於 Synthetic complete (SC) 培養基上，觀察其 White-to-Opaque 型態轉換率小於 10-3。當剔除 MTLa/a 或 MTLα/α 菌株的 HOG1 基因後，會使得菌株在此培養條件下的型態轉換率大幅提升至 100%。然而，此現象並未在異型合子 MTLa/α 菌株中被觀察到，表示White-to-Opaque 型態轉換仍然受到蛋白質 a1/α2 異二聚體 (heterodimer) 的抑制作用。於是，本研究欲更進一步探討 Hog1 MAPK訊息傳導路徑的上游分子 (Pbs2、Ssk2、Ssk1 與 Sln1)，以及 Sho1 蛋白質在 White-Opaque 表現型轉換上扮演的角色。在同型合子菌株中剔除 PBS2 以及 SSK2 基因之後，同樣會誘導 100% 的 White-to-Opaque 型態轉換率。然而，剔除 SSK1 基因後，僅會造成 41 ± 8% 的菌落發生 White-to-Opaque 型態轉換，暗示 Ssk2 除了受到 Ssk1 的調控以外，可能還受到另一條未知的路徑的調控。剔除 SLN1 以及 SHO1 基因則不會誘發此型態轉換。西方墨點法 (Western blotting) 實驗結果顯示，Hog1 的磷酸化作用受到上游的 Pbs2、Ssk2、Ssk1 蛋白質的正向調控以及 Sln1 蛋白質的負向調控，而 Sho1 則不影響 Hog1 磷酸化。在誘導交配突出物 (mating projection) 的實驗中，發現 ssk1、ssk2 及 pbs2 突變株產生的交配突出物都較野生型菌株的短。此外，本研究意外發現，sho1 突變株在 Lee’s N-acetylglucosamine 培養基上培養數天後，其 White-to-Opaque 型態轉換率僅有 16.5 ± 3.60%，比野生型菌株的轉換率 32.7 ± 10.00% 低，表示 Sho1 蛋白質在白色念珠球菌中可能參與了一個未知的機制，調控 White-Opaque 型態轉換。根據上述實驗結果，本研究提出兩個在白色念珠球菌中調控 White-Opaque Switch 的機制，分別透過 Hog1 MAPK 路徑以及 Sho1 滲透壓訊息傳導路徑，並進一步說明 White-Opaque 表現型轉換的主要調控分子 Wor1 與這些路徑可能有交互作用。

Fungal pathogen Candida albicans is prevalent in healthy human populations, widely found in the normal gastrointestinal flora. Severe Systemic infections are commonly found in individuals with a depressed immune system, associated with aging, diseases or therapies. White-opaque switching is an epigenetic morphological change in C. albicans. This phenotypic switch regulates many properties including biofilm formation, virulence and sexual mating. The switching frequency is highly associated with different stresses, such as O2, temperature, CO2, oxidative stress and the growth rate. The osmotic response MAPK gene, HOG1, has been known for helping the cell cope with the osmotic and oxidative stress, although unlike Saccharomyce cerevisiae, Sho1, the osmosensor, does not play a central role in activation of Hog1 in C. albicans. In our previous study, Hog1 MAPK is involved in the regulation of white-opaque switching. Homozygous wild type strains (MTLa/a and MTLα/α) cannot undergo white-opaque switching on synthetic complete (SC) medium, with a switching frequency less than 10-3. Deletion of the HOG1 gene in MTLa/a or MTLα/α strains stimulates switching with a frequency of 100% on SC medium. Nevertheless, this phenomenon is not observed in a/α cells, suggesting that the phenotypic change is also inhibited by the a1/α2 complex. Therefore, in this study, I have further identified the role of four upstream components (Pbs2 MAPKK, Ssk2 MAPKKK, Ssk1 and Sln1) of the Hog1 SAPK pathway, and the osmosensor Sho1, the one that does not mediate Hog1 activation in C. albicans, in white-opaque switching. As expected, deletion of PBS2 and SSK2 in homozygous C. albicans cells induced 100% of phenotypic transition from white to opaque cells. Interestingly, inactivation of the SSK1 gene caused 41 ± 8% of colonies to form opaque cells, suggesting the existence of an alternative pathway regulating the white-opaque switch in the Hog1 MAPK pathway, given that the switching frequencies of ssk1 mutants were much lower than those of ssk2 and pbs2 mutants. On the other hand, homozygous sln1 and sho1 mutants remain white colonies on the SC medium. Western blotting revealed that the Hog1 phosphorylation is positively regulated by the upstream component, Pbs2, Ssk2 and Ssk1, but negatively regulated in both of MTLa/a and MTLa/α of sln1 mutants. Under pheromone treatment, ssk1, ssk2 and pbs2 mutants exhibited shorter mating projections compared to those of the wild-type strain. Surprisingly, the white-to-opaque switching experiment showed that sho1 mutants displayed a lower switching frequency (16.5 ± 3.60%) than those of the wild type (32.7 ± 10.00%) on Lee’s N-acetylglucosamine medium, implicating that an unknown mechanism is involved in this phenotypic change through the Sho1 pathway. Taken together, our study has provided two alternative signaling pathways (Hog1 MAPK and Sho1 osmosensing pathways) involved in this unique phenotypic switch in C. albicans and will elucidate how the interaction happens between the Wor1 and these pathways.