白色念珠球菌 Hog1 MAP kinase 對 White-Opaque 型態的轉換與穩定性之探討

Abstract

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.