2013-08-012024-05-18https://scholars.lib.ntu.edu.tw/handle/123456789/698776摘要：隱球菌(Cryptococcus neoformans)，為全球性分布的人類病原真菌，亦為研究真菌致病性、生理、分化及演化的模式系統。隱球菌為異宗交配型之擔子菌具有MATa 及 MATα 兩種交配型，在適當的環境條件下可進行 a-α 雙性有性生殖。此外，在隱球菌中發現一種特殊的 α-α 單性有性生殖，並且研究顯示該有性生殖產生的高致病性重組子代，為近年於北美地區引發隱球菌腦膜炎，並造成死亡之主要病原菌。而目前對單性有性生殖之發生及分子調控機制，所知仍相當有限。本實驗室先前的研究顯示，a-α 雙性有性生殖及 α-α 單性有性生殖受藍光調控，外在光線的刺激透過演化保守之真菌藍光反應蛋白質 Cwc1及Cwc2，抑制隱球菌有性生殖菌絲之形成與生長。為瞭解光反應調控菌絲生長之訊息網絡，本實驗室進行全基因體之突變篩選，找到 mediator複合體成員 SSN8，染色質修飾基因 SET2，以及減數分裂調控基因 CRK1等，參與其相關過程之調控。Ssn8 及 Ssn3分別是 C-type Cyclin 及協同作用的 Cyclin-dependent kinase (CDK)，二者為mediator 複合體 free module 的成員，分析顯示在傳統 a-α 雙性有性生殖中，Ssn8 及 Ssn3扮演負向調控有性生殖的角色;有趣的是，相較於野生菌株，二基因之突變株不僅呈現更為快速明顯的 α-α 單性有性生殖菌絲生長之性狀，MATa突變株亦呈現明顯的 a-a 單性有性生殖之結果。此外，Ssn8 證實亦參與負向調控侵入菌絲生長，以及莢膜與黑色素之形成，並且調控細胞壁完整性及致病性。SET2為 histone H3 lysine 36 methyl- transferase 基因，其突變亦可回復CWC1基因過度表現株受光照抑制菌絲生長的性狀。Crk1為減數分裂調控蛋白質，具有保守性 Ser/Thr kinase 功能區塊及 TXY dual phosphorylation 位點，並且負向調控 a-α 雙性有性生殖的角色，突變株有性生殖細胞融合效率增加，並且雙核生殖菌絲、擔子柄，及擔孢子皆提早形成。本2年期研究計劃之主要目標包括，1) 探討隱球菌單性有性生殖調控路徑，找尋受到 Ssn8 蛋白質調控的下游標的基因，2) 探討隱球菌 histone H3 lysine 36 methyl- transferase SET2 基因如何參與有性生殖、菌絲生長及其他生理之調控，3) 找尋減數分裂調控蛋白質 Crk1 在隱球菌有性生殖過程的上下游標的。希望藉由本計畫之研究，能進一步瞭解隱球菌單性有性生殖的調控網絡，以及染色質修飾與 Crk1 減數分裂調控蛋白質如何協同其他基因，進行隱球菌有性生殖、菌絲生長及其他生理之調控。<br> Abstract: Cryptococcus neoformans is an important human fungal pathogen with worldwide distribution and also a model for studying fungal virulence, physiology, differentiation and evolution. C. neoformans is a heterothallic basidiomycetous yeast with MATa and MATα mating types that can undergo a-α opposite-sex mating under appropriate conditions. α-α same-sex mating is an unusual type of sexual differentiation which has been recently discovered in C. neoformans and found to contribute to the generation of hypervirulent recombinant propagules for recent fungal meningoencephalitis outbreak and death in Canada. Although its recognized features and importance, the molecular mechanisms of initiation and regulation in same-sex mating process are not well understood. Our previous studies revealed that both opposite-sex and same-sex mating processes are regulated by light and blue light represses the production of sexual filaments via the evolutionally conserved fungal blue light response proteins Cwc1 and Cwc2. To reveal how the signaling network is executed, we conducted a genome wide screen and identified mediator component SSN8, chromatin modification gene SET2, and meiotic regulatory gene CRK1 involved in these light-mediated filamentation processes. Ssn8 and Ssn3, a C-type Cyclin, and its interacting Cyclin-dependent kinase (CDK), are members of the free module in the Mediator complex. Our characterization revealed that Ssn8 and Ssn3 functions as negative regulators in traditionally heterothallic a-α mating. Interestingly, same-sex α-α and also a-a mating processes are dramatically de-repressed by the mutation of SSN8 or SSN3 gene. Additionally, Ssn8 is involved in the regulation of other physiological processes including invasive growth, production of capsule and melanin, maintenance of cell wall integrity, and virulence. C. neoformans Set2, a histone H3 lysine 36 methyltransferase (H3K36me), was also identified and confirmed to suppress CWC1 light-dependent overexpression phenotype. CRK1 is a meiosis-specific gene with the conserved Ser/Thr kinase domain and TXY dual phosphorylation site. Similarly, we found that C. neoformans Crk1 plays a negative role in the mating process. Mating efficiency is increased and dikaryotic filaments, basidia, and basidiospores are produced earlier in the crk1 mutant crosses. The goals of this two-year grant proposal are divided into the following aims. The first aim is to dissect the same-sex mating pathway and identify the molecular targets of Ssn8 in this process. The second aim is to determine how SET2 gene regulates mating process and filamentous growth in C. neoformans. The final aim is to determine the upstream and downstream components of Crk1 kinase in sexual development of C. neoformans. We hope to understand the regulatory network of same-sex mating and reveal the roles of chromatin remodeling and Crk1 kinase in mating and filamentous growth the sexual development and other physiological processes in C. neoformans.隱球菌單性有性生殖菌絲生長致病性分子機制SSN8SSN3SET2CRK1Cryptococcus neoformanssame-sex matingfilamentous growthvirulencemolecular mechanismSSN8SSN3SET2CRK1