隱球菌Mediator Ssn8蛋白質負向生理調控及致病性之探討

Mediator protein Ssn8 negatively regulates diverse physiological processes and is required for virulence in Cryptococcus neoformans

王綾霙(Lin-Ing Wang)

指導教授 ： 沈偉強

國立臺灣大學/生物資源暨農學院/植物病理與微生物學研究所/碩士(2010年)

https://doi.org/10.6342/NTU.2010.01719

摘要

隱球菌 (Cryptococcus neoformans) 為一伺機性之人體病原真菌，亦為一種重要模式真菌。過去的實驗發現，隱球菌具有藍光反應，亦即隱球菌透過藍光調控蛋白質Cwc1和Cwc2形成複合體，感受光線並抑制生殖菌絲的形成。為進一步瞭解隱球菌Cwc複合體抑制生殖菌絲的調控機制，本實驗室利用農桿菌轉殖系統(Agrobacterium-mediated insertional mutagenesis)，針對CWC1過度表現株於光照下抑制生殖菌絲之性狀，進行突變篩選；而AY18為一會部分回復生殖菌絲，並產生大量產生同性有性生殖(monokaryotic fruiting)菌絲之突變株，經證明其表現性狀乃因為T-DNA插入隱球菌SSN8同源基因所導致。啤酒酵母菌(Saccharomyces cerevisiae) SSN8基因，為一Mediator，負責調控真核細胞在開始進行轉錄的過程。在啤酒酵母菌中，SSN8基因又稱SRB11/UME3/RYE2/CYCC，參與糖類利用、減數分裂及逆境生長等生理之調控。在本篇的研究中，透過SSN8突變株的建構及分析，瞭解SSN8基因在隱球菌中，負向調控異宗交配生殖、同性有性生殖、侵入菌絲生長(invasive growth)，以及黑色素與莢膜的生成，並且影響細胞壁結構及完整性，參與致病性之調控。此外，利用上位性分析(epistasis analysis)、酵母菌雙雜合分析(yeast two-hybrid system)，以及基因表現分析，證明Ssn8可能作用於Cpk1費洛蒙調控路徑，以及Cwc複合體調控光反應路徑之下游，負向調控生殖菌絲的形成。綜合本研究之結果，證明隱球菌保守性Mediator同源基因SSN8為一重要負向調控因子，並廣泛地調控隱球菌生理、發生及致病性。

關鍵字

隱球菌 ； SSN8同源基因 ； Mediator ； 有性生殖 ； 光反應

並列摘要

Cryptococcus neoformans is an important human pathogen and also a model for studying fungal physiology and differentiation. Our prior studies have showed that blue light negatively regulates filamentous growth during sexual differentiation via the Cwc1/Cwc2 complex. An Agrobacterium T-DNA insertional mutant AY18 identified in a suppressor screen restores mating filamentation and also dramatically derepress the production of monokaryotic filaments in the overexpressed CWC1 background. Further characterization determined that the disruption of a Saccharomyces cerevisiae SSN8 homologue is responsible for the phenotypes. Ssn8 is a member of the Mediator complex which connects RNA polymerase II and transcriptional regulators during the initiation process. S. cerevisiae SSN8, also named SRB11/UME3/RYE2/CYCC, is widely involved in diverse physiological processes, including sugar utilization, meiosis, and gene regulation in response to different stresses. To determine its roles in C. neoformans, ssn8 deletion mutants were generated in different strain backgrounds. In addition to mating and monokaryotic filamentation, Ssn8 also negatively regulates other processes including invasive growth, and in vitro production of capsule and melanin. Ssn8 is also required for the maintenance of cell wall structure and integrity and virulence. Based on the epistasis analysis, yeast two hybrid assay and gene expression study, we conclude that C. neoformans Ssn8 functions downstream the Cpk1-pheromone response pathway and also resides at one of major branch downstream the Cwc complex along the light-mediated filamentation pathway. Taken together, our studies demonstrate that the conserved Mediator protein Ssn8 functions as a global regulator which negatively regulates diverse physiological and developmental processes and is also required for the virulence in C. neoformans.

並列關鍵字

Cryptococcus neoformans ； SSN8 ； Mediator ； sexual development ； light response

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