2017-08-012024-05-17https://scholars.lib.ntu.edu.tw/handle/123456789/682014摘要：近年來，白色念珠球菌已成為院內感染最常見且最重要的致病菌之一。 然而在臨床上，可選擇的抗真菌藥物不多與具抗藥性真菌數量增加，造成治療上的困難。 因此，開發新的藥物與治療方式是一急迫且重要的議題。 幾丁聚醣 (chitosan) 是一種葡萄糖胺聚合的多醣體，可經由幾丁質 (Chitin) 經脫乙醯 (Deacetylation) 獲得之產物。由於具有生物活性、可生物分解與無毒性等特色，幾丁聚醣目前已被廣泛應用至農業、醫材與保健食品。過去報導發現幾丁聚醣對致病細菌與真菌具抑菌效果。然而，幾丁聚醣如何抑制或毒殺微生物的調控機制仍不清楚。 實驗室利用白色念珠球菌337個轉錄因子突變株庫進行全面性的篩選，發現有66個轉錄因子可能參與抵抗幾丁聚醣的殺菌、抑菌反應。其中，一個調控鈣離子訊息傳導路徑 (calcium-calcineurin cascade) 重要的轉錄因子Crz1，亦同時參與抵抗幾丁聚醣殺菌能力。與野生株相較，經 0.2% 幾丁聚醣處理後的crz1Δ，有顯著死亡的現象且達全殺效果。此外，由於幾丁聚醣也能刺激活化並大量表現許多MID1, CCH1, CNA1 與CNB1 等 calcineurin 相關基因。因此，本計畫研究目標一將進一步剔除調控Crz1的上游基因，了解這些鈣離子訊息傳導路徑的相關基因是否也與幾丁聚醣的抵抗有關。雖然 calcineurin 訊息傳導路徑相關的基因表現，也會被幾丁聚醣刺激活化並大量表現。有趣的是，白色念珠球菌經過 0.2% 幾丁聚醣處理後會導致菌體強力聚集，但經過0.4 M氯化鈣 (Ca2+) 處理之下，並未有此聚集情形。這些結果顯示雖然calcineurin-Crz1 傳導路徑皆參與兩者的調控反應，幾丁聚醣與鈣離子刺激白色念珠球菌的調控機制仍具有差異。因此，研究目標二將利用染色質免疫沉澱DNA片段進行高通量定序 (Chromatin Immunoprecipitation Sequencing; ChIP-seq) 與轉錄體定序 (RNA-sequencing) 等實驗方法，全面性分析白色念珠球菌在經過鈣離子與幾丁聚醣處理後，Crz1 結合至下游基因啟動子的異同與整體基因表現的情況。過去研究顯示，calcineurin 傳導路徑的相關基因，具未來發展新抗真菌藥物標地的潛力。此外，合併使用抗真菌藥物與 calcineurin 抑制劑 FK506，能有殺死白色念珠球菌的協同作用。實驗室已觀察到合併使用一抗真菌藥物 fluconazole 與幾丁聚醣，產生非常顯著的殺菌能力。因此，研究目標三將利用前述建立的技術與配方，評估幾丁聚醣合併使用其它唑類 (azoles)，多烯類 (polyenes)，氟胞嘧啶類 (flucytosines) 與棘白菌素類 (echinocandins) 的抗菌與殺菌效果。這些研究成果不僅僅能了解白色念珠球菌對幾丁聚醣的反應機制，更能提供未來進行真菌感染治療與製程配方開發更進一步的改良方式。<br> Abstract: Candida albicans has become the most predominant fungal species, in which it is responsible for half of all clinical infections among human infectious fungal diseases. However, due to the limited choices of antifungal drugs and the emergence of drug-resistant strains, it has caused huge impact in the management of this infection. Thus, development or searching new drugs or alternative therapeutic methods to control fungal infections is urgent and required. Chitosan is a linear D-glucosamine polysaccharide and it is derived from deacetylated chitin compounds. Due to its bio-activity, bio-degradable and non-toxic porperties, chitosan has been wildly applied to agriculture, medical devices, and health supplements. Although several study reports indicate that chitosan possesses antimicrobial activities, mechanistic insight into how chitosan against microorganisms remains largely unknown. Our preliminary results have identified at least 66 transcriptional regulators that are involved in the chitosan response via screening the 337 transcriptional factors deletion strains in the C. albicans mutant library. Of them, Crz1, a calcium-calcineurin-responsive transcriptional factor, was determined to be involved in the chitosan response. Comparison with the wildtype strain, crz1Δ mutant strains were unable to survive under 0.2% chitosan treatment. Furthermore, expression levels of each upstream component gene of calcineurin cascade, including MID1, CCHI, CNA1 and CNB1, were evaluated during the response to chitosan. Thus, Specific Aim 1 of this proposal will determine whether upstream components of calcineurin cascade are also involved in chitosan response. Although many calcineurin-related genes highly expressed when challenged with chitosan, treatment with 0.2% of chitosan caused dramatically aggregated in C. albicans cells, whereas cell morphologies of C. albicans remained no change when challenged with 0.4 M CaCl2 (Ca2+). These results suggested that regulatory mechanisms of C. albicans in response to chitosan and high Ca2+ are fundamentally different, even though both chitosan and Ca2+ activate the expression of calcineurin-Crz1 cascade. Specific Aim 2 will be therefore to identify what downstream genes and the differences of downstream genes is directly bound and regulated by Crz1 during the response to chitosan and Ca2+, respectively, by using chromatin immunoprecipitation sequencing (ChIP-seq). Moreover, RNA-sequencing will be conducted to determine entire genetic expressions when C. albicans challenged with chitosan and Ca2+. Several review articles have been showed that signaling components in calcineurin pathway will be potential targets for the development of new antifungal drugs. Besides, combinational treatment of fluconazole and a calcineurin inhibitor FK506 resulted in a synergistic killing effect on C. albicans. Thus, Specific Aim 3 will be based on the established method to evaluate the effectiveness of antifungal activity on C. albicans by a combinational treatment of chitosan with each antifungal drug, including azoles, polyenes, flucytosines and echinocandins,. Taken together, these studies will allow us to clarify the responding mechanisms of C. albicans to chitosan, in which it will provide a feedback for us to amend formulations or develop a better strategy to control candidiasis or even to manage other fungal infectious diseases.白色念珠球菌鈣離子訊息傳導路徑 (calcineurin)Crz1幾丁聚醣協同作用Candida albicanscalcineurinCrz1chitosansynergistic effect