國立臺灣大學植物病理與微生物學系暨研究所沈偉強2006-07-262018-06-292006-07-262018-06-292002http://ntur.lib.ntu.edu.tw//handle/246246/17683在過去二十年中，因愛滋病毒感染， 組織或器官移植以及癌症化學治療等原 因，所導致免疫系統缺陷之個體大量增 加，因而使得真菌性感染成為新興之醫療 問題。隱球菌(Cryptococcus neoformans) 為 一伺機性人體病原真菌，對免疫缺陷之族 群，具致命性真菌性腦膜炎之威脅。隱球 菌之病原性研究顯示，其主要致病因子包 括，莢膜(capsule)、黑色素(melanin)，以及 該菌之生殖型基因位點(mating type locus)。 經由自然環境及臨床的調查，以及動 物病原性的實驗等結果顯示，交配型á 基 因位點可能與環境流行及致病性有關。據 此，交配型á 基因位點被選殖出，其上之 基因已被陸續分析，其中包含三個費洛蒙 基因。經分析三個費洛蒙基因之突變株後 發現，在交配型á 的菌株中，可能存有費 洛蒙自我調控之機轉存在，並可能為交配 型á 的菌株具較高病原性之成因。本計畫 之目的乃在進一步瞭解隱球菌血清型D 型 菌之費洛蒙反應機制，探討交配型基因位 點與致病機制及其他生理調控之關係，主 要分為兩個方向，一為針對隱球菌血清型 D 型菌之G 蛋白質複合體β次單元，進行 其生理角色之探討，二為找出費洛蒙反應 機制之下游基因STE6，以進一步確認費洛 蒙自我調控之機轉與有性生殖、致病性之 關係。 90 年度為執行第一年，在血清型D 型 隱球菌之G 蛋白質複合體β次單元分析 上，已篩選及確認G 蛋白質β次單元基因 變異種。性狀分析顯示，其參予有性生殖 及單價菌絲生長之調控，至於是否參予調 控致病性，則有待建構好之變異種回復菌 株後，進行動物試驗後始能確認。在尋找 費洛蒙反應機制之下游基因STE6 上，已自 史丹福大學隱球菌基因體計劃中確認， STE6 基因變異株亦已篩選獲得，初步分析 之結果顯示，其亦參予有性生殖之調控， 而在單價菌絲生長及致病性上之角色，有 待進一步證明。另外，在測試與調整細胞 生長及RNA 分離等條件及步驟後， subtraction PCR 篩選其他參與訊息傳導基 因的實驗，亦將於近期進行。Fungal infection has drawn lots of attention due to dramatically increased number of immunocompromised patients caused by HIV infection, organ and tissue transplantation, and cancer chemotherapy in the past two decades. Cryptococcus neoformans, a human pathogenic basidiomycetous yeast, causes the life-threatening meningoencephalitis mainly in such individuals with compromised immune functions. Studies of the pathogenesis in C. neoformans have revealed several important virulence factors such as capsule, melanin, and interestingly mating type locus. Environmental and clinical prevalence of MATα strains and virulence 2 studies of the congenic pair of C. neoformans serotype D strains have suggested that MAT α locus might involve in regulating the virulence in C. neoforman. Following these observations, MAT α locus has been identified and characterized. Three copies of pheromone precursor genes were identified in the MATα locus. Characterization of three pheromone genes deletion mutant strains have suggested an autocrine signaling loop may function and contribute to the virulence of the MATá cells (Shen et al., 2002). The purpose of this proposal is to characterize the components in the pheromone response pathway of C. neoforman serotype D strain and further address how mating type locus regulates the virulence and autocrine signaling loop functions in C. neoforman. C. neoformans serotype D strain GPB1 gene were identified and disrupted in the MATa strain. MATa gpb1 mutants were also identified in the progeny of cross between the MATαgpb1 mutant and MATa strain. The MATαgpb1 and MATagpb1mutant strains were mating impaired but not sterile when coincubated with the wild-type strain of opposite mating type on V8 mating medium. Haploid fruiting was reduced, but not completely abolished, in the MATαgpb1 mutant strains, similar to the mfα 1,2,3 pheromoneless mutant. To further address how pheromones act in the autocrine signaling loop, we have identified STE6 homologue, a pheromone transporter, in the C. neoformans genome project at Stanford Genome Technology Center and begun to dissect its function. By disrupting the STE6, we found that ste6 mutants in either MATá or MATa background showed partially impaired mating function, although slight differences were noticed. However, when ste6 MATá and MATa mutants cross with each other, the mating process was nearly completely abolished. Our data indicates that the STE6 functions bilaterally and is required but not essential for mating in C. neoformans. Currently we are constructing the gpb1 and ste6 reconstituted strains, virulence test will be conducted when verified those strains. We are also conducting epistasis and functional analysis on these two genes, and hoping to claify their role in virulence and other physiological processes. To identify novel targets in the downstream of the signaling pathway, we have optimized growth conditions and RNA extraction procedures. We will start the subtractive PCR screen shortly.application/pdf769744 bytesapplication/pdfzh-TW國立臺灣大學植物病理與微生物學系暨研究所隱球菌費洛蒙反應機制G 蛋白質β次單元費洛蒙傳送蛋白質Cryptococcus neoformanspheromone response pathwayheterotrimeric GTP binding protein ß subunitpheromone transporter[SDGs]SDG3otherhttp://ntur.lib.ntu.edu.tw/bitstream/246246/17683/1/902311B002062.pdf