Mub1蛋白質調控隱球菌有性生殖與酵母細胞形態發生

Abstract

隱球菌 (Cryptococcus neoformans)，為一伺機性人類病原真菌，在分類地位上屬於擔子菌，具有酵母菌與菌絲兩種形態。隱球菌存在MATa與MATα兩種交配型細胞，在氮素源缺乏的環境下，MATa與MATα 細胞產生接合管進行細胞融合，並產生雙核生殖菌絲，擔子柄及擔孢子完成有性生活史。本實驗室過去的研究發現，隱球菌藉由Cwc1與Cwc2兩蛋白質形成之複合體，在光照環境下，感應藍光抑制雙核生殖菌絲的生長。為了進一步瞭解藍光抑制雙核菌絲生長的機制，我們利用農桿菌轉殖系統 (Agrobacterium-mediated insertional mutagenesis)，針對CWC1過度表現株進行逢機突變，並於持續光照環境下，篩選表現型由雙核生殖菌絲完全被抑制回復為可生長雙核生殖菌絲的突變株，藉以找出與藍光調控菌絲生長有關的下游基因。其中一株回復突變株EE24，進一步分析發現T-DNA插入破壞隱球菌MUB1基因啓動子序列。啤酒酵母菌 (Saccharomyces cerevisiae) MUB1基因為隱球菌MUB1之同源基因，在細胞中參與蛋白酶體轉錄因子Rpn4之泛素化，並且突變株呈現多聚體細胞 (multi-budding) 之性狀。本研究嘗試探討MUB1基因在隱球菌中所扮演之角色，結果發現MUB1基因突變，會造成隱球菌37°C生長之缺陷，且酵母細胞亦呈現多聚體之性狀，深入研究發現隱球菌mub1突變株酵母細胞在出芽分裂上發生缺失。此外，突變株亦無法維持有性生殖菌絲之正常形態發育，無法產生子代。有趣的是，本研究發現，隱球菌MUB1基因，亦參與負向調控MATα與MATa隱球菌單性有性生殖 (same sex mating) 之初期發育。除此之外，研究顯示MUB1基因亦和隱球菌致病因子莢膜之形成調控有關。綜合本研究之結果，MUB1基因為隱球菌酵母細胞形態發生及有性生殖發育重要之調控因子。

Cryptococcus neoformans is a heterothallic basidiomycete that grows vegetatively as yeast cells and produces mating filaments in the sexual state. Mating initiates when MATa and MATα cells conjugate and fuse, and then dikaryotic sexual filaments are subsequently produced. Generation of final fruiting structures basidia and meiotic progeny basidiospores leads to completion of the sexual cycle. Prior studies have revealed that C. neoformans Cwc1 and Cwc2 proteins are two central photoregulators which form a complex to inhibit the production of sexual filaments upon blue light irradiation. To reveal the detailed light response networks, a genome wide mutagenesis screen was conducted and components involved in light-mediated filamentation pathway have been identified. In this study, a suppressor mutant EE24 was characterized and T-DNA was found to insert at the upstream regulatory region of C. neoformans MUB1 gene, a homologue of Saccharomyces cerevisiae MUB1 (multi-budding) gene. In S. cerevisiae, Mub1p is a MYND domain-containing protein required for ubiquitination and turnover of Rpn4p, a transcription factor of proteasome genes. Deletion of C. neoformans MUB1 gene caused compromised growth at 37°C. C. neoformans mub1 mutants similarly displayed a multiple-budding phenotype and altered structures of bud scars were observed. Furthermore, morphogenesis of dikaryotic sexual filaments and generation of basidiospores were defective in the mub1 bilateral cross. Interestingly, early stages of same sex mating was negatively regulated by Mub1. Our studies demonstrate that C. neoformans MUB1 is an important gene that regulates yeast cell morphogenesis and mating differentiation.