Investigation of molecular types, mating types, antifungal susceptibilities and virulence factors in clinical isolates of Cryptococcus neoformans

新型隱球菌(Cryptococcus neoformans)，為具有莢膜的黴菌，可行出芽生殖，乃臨床上重要的伺機性病原菌，尤其好發於免疫低下者如愛滋病患者及接受器官移植或癌症化療等的族群中。新型隱球菌的感染大多藉由吸入空氣中的孢子或隱球菌細胞所造成，肺部則是初期感染的部位。新型隱球菌可透過血行性擴散至中樞神經系統，嚴重時可引發隱球菌性腦膜炎、甚至造成死亡，乃愛滋病患者常見的死因之一。新型隱球菌可根據莢膜抗原特異性區分為不同血清型，血清型A、D及AD屬於Cryptococcus neoformans；而血清型B、C則於近年獨立為新的一個種(species) Cryptococcus gattii。Cryptococcus neoformans的感染遍及全世界，且多與鴿糞及免疫低下的族群相關，而Cryptococcus gattii則被認為分佈於熱帶及亞熱帶地區，近年更有感染免疫健全者(immunocompetent)的病例出現，而新型隱球菌的發生率及各種型別的盛行率調查，在世界各地均有之。有鑑於此，本篇論文即欲探討臺灣地區造成感染的隱球菌菌株類別及其分子特性，進行包括分型(typing)、毒力因子表現，與藥物感受性之研究。我們發現，目前造成臺灣臨床上感染的菌株仍以Cryptococcus neoformans血清型A/ VNI/ VN6, 交配型α為主流，且多項毒力因子表現各有差異，而藥物感受性大致上仍屬良好，無抗藥性菌株出現。所有的臨床菌株皆可產生黑色素及具備尿素酶活性。在本研究中亦發現臨床菌株與環境菌株及實驗室標準菌株相較之下，具有較高的磷脂酶活性。我們選用磷脂酶活性最高的臨床菌株59及磷脂酶活性最低的臨床菌株24，進一步分析其他毒力因子表現與所引起的宿主反應是否有差異。菌株59與24於37℃的生長速率相似，在細胞模式中發現，菌株59對肺部上皮細胞A549具有較高的吸附性(Cell adhesion ability)，並可造成較大的細胞毒性(cytotoxicity)，但59的莢膜基因表現量則低於24。顯示磷脂酶活性可能在引起肺部細胞感染的過程中扮演關鍵的角色，可增加對細胞的吸附性，而其對細胞吸附性的影響甚至可能凌駕於莢膜的影響之上，是一項重要的毒力因子。The encapsulated budding yeast, Cryptococcus neoformans, has become a critically important opportunistic pathogen in individuals who are immunocompromised in all parts of the world. Cryptococcosis is initiated by fungal spores or cells deposited in the alveoli of the lungs and life-threatening for immunocompromised individuals when Cryptococcus cells disseminate from the lungs to major organs of the body, such as central nervous system (CNS). Cryptococcal meningitis is the most common fungal infection of the CNS in AIDS patients and the etiologic agent of 99% of these cases is Cryptococcus neoformans var. grubii /serotype A. The serotype classification is based on the agglutination reactions of the capsular polysaccharide antigens, which can be determined by using absorbed rabbit sera. The serotype A, D and the hybrid AD strains belong to C. neoformans whereas serotype B and C have been classdied as Cryptococcus gattii. C. neoformans is primarily found worldwide associated with excreta from pigeons and in tree hollows. C. gattii was primarily found in tropical and subtropical regions for years until it cause endemic outbreak in immunocompetent humans on Vancouver Island in Canada. According to molecular methods, such as URA5-RFLP, M13 PCR fingerprinting, C. neoformans and C. gattii are further classified into four molecular types for each species: VNI-VNIV and VGI-VGIV respectively. There are several virulence factor which have been explored including 37℃ growth ability, melanin production, capsule formation, phospholipase and urease, etc. n this study, we analyze the molecular types and mating type of 125 clinical isolates in Taiwan from 1999 to 2004. We also investigate the virulence factors, antifugal susceptibilities, and host response in different clinical isolates. 123 of the 125 isolates were C. neoformans serotype A while the other two were C. gattii serotype B.122 of the 123 serotype A strains belongs to VNI genotype and the remain one is VNII. Two C. gattii strains are both VGI genotype. All isolates were susceptible to antifungal drugs and exhibited mating typeα. Virulence factors expression was different among these clinical isolates. All of the clinical isolates all were urease and melanin positive. Most of them were hyperphospholipase. We further analyzed the host response to hyperphospholipase strain 59 and hypophospholipase strain 24 in cell-line model and find that 59 presented greater cell adhesion ability to lung epithelial cell A549 and induced more cytotoxicty.誌謝……………………………………………………………………i文摘要………………………………………………………………Ⅰ文摘要………………………………………………………………Ⅱ錄……………………………………………………………………一目錄…………………………………………………………………三目錄…………………………………………………………………四錄目錄………………………………………………………………六一章 緒論……………………………………………………………1一節 新型隱球菌(Cryptococcis neoformans)介紹………………………1二節 研究動機與目的……………………………………………………5三節 實驗流程……………………………………………………………6二章 研究方法與材料………………………………………7一節 實驗材料……………………………………………………………7二節 實驗方法……………………………………………………………10一) 以分生方法調查新型隱球菌臨床菌株之分型(molecular typing)………………………………10二) 新型隱球菌臨床菌株之毒力因子分析……………………17三) 藥物感受性試驗…………………22四) 宿主反應試驗(host response)……………26三章 實驗結果……………………29一節 新型隱球菌臨床菌株之來源……………………………29二節 以分生方法調查新型隱球菌臨床菌株之分型……………………30三節 新型隱球菌臨床菌株之毒力因子分析……………………………33四節 新型隱球菌臨床菌株對抗真菌藥物之感受性……………………36四章 結論與討論……………………………………………37一節 調查新型隱球菌臨床菌株之分型與分類……………………37二節 新型隱球菌臨床菌株之毒力因子分析…………………………41三節 新型隱球菌臨床菌株對抗真菌藥物之感受性……………43四節 結論…………………………………………………………………44五章 表………………………………………………………………46六章 圖……………………………………………………59七章 附錄…………………………………72八章 參考文獻…………………………………7