光滑念珠菌之轉錄輔激活因子ADA2控制溫度耐受性、藥物耐受性及毒性

Abstract

光滑念珠菌(Candida glabrata)是造成念珠菌症的重要菌種，僅次於白色念珠菌(Candida albicans)，其為一伺機性的人體病原真菌，由於其常對藥物產生抗性，不僅造成治療上的困難，同時使免疫功能低落的病患死亡率提升。為了找尋有潛力的藥物標靶，我們利用光滑念珠菌的突變株庫對抗真菌藥物micafungin與fluconazole進行篩選，發現ada2突變株對兩藥物皆敏感，因此我們假設光滑念珠菌的Ada2蛋白(轉錄輔激活因子)會控制其藥物耐受性。為了驗證此假設，我們從光滑念珠菌野生株CBS138製造ada2突變株及互補株。除了確認ada2突變株對於micafungin及fluconazole敏感之外，同時我們也發現ada2突變株具有多種不同表現型，例如對於細胞壁擾動劑、三唑類藥劑(posaconazole與voriconazole)、echinocandins類藥劑(caspofungin與anidulafungin)、amphotericin B及內質網壓力誘導物質dithiothreitol敏感，但對於另一內質網壓力誘導物質tunicamycin則較具抗性。有趣的是，ada2突變株在攝氏40度時會有嚴重的生長缺陷，證明Ada2對於溫度耐受性扮演重要角色。根據RNA-sequencing的實驗結果，對比於野生株，有43個基因在ada2突變株中表現量降低，其中HSP30及SSB1可能與溫度耐受性有關，而ERG6及ERG13則可能與藥物耐受性有關。出乎意料之外，我們發現ada2突變株在小鼠系統性感染模式中具有比野生株較強的毒力。這些發現提供了光滑念珠菌Ada2蛋白在溫度耐受性、藥物耐受性及毒力中的新視野。

Candida glabrata, the second most frequent cause of candidiasis after Candida albicans, is an emerging human fungal pathogen and commonly drug tolerant. Currently, few antifungal armamentaria are effective against C. glabrata, and thus result in treatment difficulties and higher mobidity or mortality in immunocompromised patients. To search for potential drug targets, we screened a C. glabrata deletion mutant library and found ada2 mutant susceptible to micafungin and fluconazole. We therefore hypothesized that C. glabrata Ada2, a transcription adaptor, controls drug tolerance. To test this hypothesis, we generated two independent ada2 mutants and a complementary strain of C. glabrata. In addition to confirming that newly generated C. glabrata ada2 mutants were susceptible to micafungin and fluconazole, we found that ada2 mutants exhibit pleiotropic phenotypes. For example, the ada2 mutants are susceptible to cell-wall perturbing agents (calcofluor white and sodium dodecyl sulfate), triazoles (posaconazole and voriconazole), echinocandins (caspofungin and anidulafungin), amphotericin B and endoplasmic reticulum stress chemical dithiothretol but resistant to tunicamycin. Interestingly, the ada2 mutants exhibit severe growth defects at 40°C, suggesting that Ada2 plays a critical role on controlling thermotolerance. According to RNA sequencing analyzes, we found 43 genes are down regulated in ada2 mutant compared to the wild type CBS138. Two genes associated with thermotolerance might be HSP30 and SSB1, while genes linked with drug tolerance might be ERG6 and ERG13. Surprisingly, we found that Ada2 negatively regulated virulence in immunocompromised murine model of systemic infection. These findings provide novel perspective to Ada2 controlling thermotolerance, drug tolerance and virulence in C. glabrata.