白色念珠菌 Hog1 路徑及轉錄因子 Cap1 抵抗光動力殺菌之機制探討

Abstract

白色念珠菌 (Candida albicans) 為常見伺機性感染人類的真菌，此真菌平常共生在人類的腸道中，當人體因老化、疾病或是藥物治療導致免疫力下降時，則容易引發嚴重的系統性感染。近年來菌株產生抗藥性的問題日益嚴重，光動力殺菌 (antimicrobial photodynamic inactivation) 便成為新興替代性治療微生物感染的方式，光動力殺菌是透過光感物質 (photosensitizer) 經特定波長光源激發後進行光化學反應，產生高活性的單態氧及自由基並毒殺細胞。將光動力療法應用於癌症細胞，所引發的細胞抵抗機制在過去已經有相當多研究，然而，以光動力殺菌治療真菌感染，其下游抵禦機制尚未明瞭。因此，本研究旨在瞭解當白色念珠菌經光感物質甲苯胺藍 (toluidine blue O, TBO) 之光動力殺菌刺激後，會啟動哪些相關抵禦氧化壓力的機制。過去研究顯示，Hog1 stress-activated protein kinase (SAPK) 訊息傳導路徑會對細胞外在環境的氧化壓力產生反應，當受到氧化壓力刺激後，Hog1 蛋白質會進行磷酸化並活化。本研究首先以西方墨點法分析，結果顯示受到光動力殺菌刺激後，Hog1 蛋白質的磷酸化程度升高，以不同濃度 TBO 進行光動力殺菌後計算菌數，發現 Hog1 路徑之突變株 (ssk1Δ、pbs2Δ、hog1Δ) 相較於野生株，其生存率顯著下降；此外，Hog1 蛋白質磷酸化位點之點突變株 (HOG1T174A、HOG1Y176F) 經光動力殺菌後生存率亦顯著降低，顯示 Hog1 蛋白質在對抗光動力殺菌的重要性。前人研究指出轉錄因子 Cap1 同樣參與白色念珠菌抗氧化壓力的訊息傳導，氧化態之 Cap1 會進入細胞核調控相關抗氧化基因表現；本研究利用即時聚合酶鏈式反應分析 CAP1 基因之表現量，結果顯示經光動力殺菌刺激後 CAP1 表現量顯著上升，以不同濃度 TBO 進行光動力殺菌後計算菌數，發現 cap1 突變株與野生株相比，其生存率同樣降低。根據上述實驗結果，顯示光動力殺菌會促使 Hog1蛋白質活化及 CAP1 表現量升高，推測 Hog1 SAPK 路徑及轉錄因子 Cap1 在白色念珠菌抵抗光動力殺菌中應扮演了重要的角色，幫助我們了解白色念珠菌面臨光動力殺菌下之防護機制。

Candida albicans is the most important fungal pathogen afflicting humans, particularly in immunocompromised patients. However, current available antifungal drugs are limited and are ineffective against drug-resistant strains. Development of new drugs or alternative therapeutic approaches to control fungal infection is urgent and necessary. Photodynamic inactivation (PDI) is a new promising therapy for eradicating microorganism infection through combining visible light, photosensitizer and oxygen to generate reactive oxygen species (ROS). Although cytoprotective responses induced by photodynamic therapy have been well studied in cancer cells, mechanisms as to how C. albicans in response to PDI remains large unknown. In this study, we first demonstrated that PDI can induce C. albicans Hog1p activation. Deletion of SSK2, PBS2, or HOG1 caused a significantly decreased survival rate after photochemical reactions, indicating that the Hog1 SAPK pathway is required for tolerance to PDI. Furthermore, a basic leucine zipper transcription factor Cap1 regulating several downstream antioxidant genes was highly expressed during the response to PDI, and loss of the CAP1 also resulted in decreased C. albicans survival rates. Together, this study demonstrates the importance of the Hog1 SAPK and the Cap1 regulator in resistance to PDI-mediated oxidative stress in C. albicans. Understanding the mechanisms of how C. albicans responds to PDI leading to scavenge ROS will provide a great feedback for further therapeutic development to control fungal infectious diseases, particular of the skin and mucosal infections.