隱球菌BUD16同源基因之研究

Abstract

隱球菌 (Cryptococcus neoformans) 為一人體伺機性病原真菌，主要感染免疫功能有缺陷之患者，如AIDS、癌症或長期服用類固醇等病患。隱球菌之感染由呼吸道吸入，經血液循環造成系統性感染，並可進一步導致真菌性腦膜炎，如未及時治療，可導致死亡。隱球菌在真菌分類地位上，屬於異宗交配型之擔子菌，一般培養條件下，為出芽生殖形式的酵母細胞，當環境中缺氮時，兩種不同交配型之細胞可進行交配，形成雙核菌絲，菌絲具有擔子菌之典型扣子體，並於菌絲末端產生擔子柄及四串擔孢子。本實驗室過去的研究，發現藍光可藉由Cwc1及Cwc2蛋白質抑制生殖菌絲之生成，並且為了解Cwc1及Cwc2如何調控光反應路徑，利用農桿菌轉殖系統 (Agrobaterium tumefaciens-mediated transformation, ATMT)，隨機插入CWC1基因過度表現株之基因體中，篩選交配過程中可回復菌絲生長的轉殖株。其中，EG30轉殖株經反向聚合酶連鎖反應 (inverse PCR) 及定序比對後，發現T-DNA插入於隱球菌與酵母細胞出芽位置選擇相關蛋白質 (bud site selection-related protein) 基因上游的啟動子區域，此基因經序列比對後，發現為啤酒酵母菌 (Saccharomyces cerevisiae) BUD16的同源基因。在啤酒酵母菌的研究中發現，bud16突變株會造成雙倍體之酵母菌細胞，無法正常進行雙極性出芽生殖 (bipolar budding)，並且多數細胞可於隨機位置進行出芽生殖；Bud16p為吡多醛激酶 (pyridoxal kinase)，於維生素B6生合成路徑中，將吡多醛 (pyridoxal) 轉變成生物可利用的5’-磷酸吡多醛 (pyridoxal 5’-phosphate, PLP)。在隱球菌中，本研究建構了BUD16同源基因之過度表現株，發現在交配過程中，其生殖菌絲不論在照光及黑暗之情形下均受到抑制。此外，bud16突變株，在光照情形下，與野生型菌株比較，其生殖菌絲較早產生且生成量較野生型菌株為多；進一步比較細胞融合情形，不同交配型bud16突變株細胞融合率較野生型菌株為高。此外，在隱球菌單一交配型進行有性生殖 (monokaryotic fruiting) 的性狀方面，交配型α之bud16突變株仍具有與野生型菌株相似的單核菌絲形成之能力，而過度表現株卻完全抑制單核菌絲之生成。為探討有性生殖中BUD16與CWC1兩基因間之作用關係，本研究在CWC1過度表現背景下建構bud16突變株，發現其表現型與bud16突變株相似；而在cwc1突變株中過度表現BUD16基因，其表現型與BUD16過度表現株相似。由此觀察結果顯示，BUD16基因作用於CWC1基因之下游，並且於有性生殖過程中，扮演負向調控之角色。

Light regulates the physiology, development and behavior of many organisms including fungi. Cryptococcus neoformans, a heterothallic basidiomycetous yeast, can sense blue light and negatively regulate the production of sexual filaments via the Cwc1 and Cwc2 proteins. To dissect this pathway, we conducted a suppressor screen utilized Agrobacterium tumefaciens-mediated transformation (ATMT) technique to identify mutants suppressing the mating phenotype of the CWC1 overexpression strain, which displayed no filaments under light illumination. Here, we report that the EG30 suppressor strain restored the filamentation to the wild-type level and T-DNA was confirmed to insert at the promoter of a gene homologous to the Saccharomyces cerevisiae BUD16. S. cerevisiae BUD16 gene was identified due to the random budding pattern in the homozygous diploid mutant. The Bud16p protein is a predicted pyridoxal kinase which converts pyridoxal into pyridoxal 5’-phosphate (PLP), the biologically active form of vitamin B6. In C. neoformans, overexpression of the BUD16 gene slightly reduced and delayed the production of dikaryotic filaments when compared to the wild-type strain. Meanwhile, we also found that the bud16 mutants produced mating filaments earlier and more than the wild-type strain and higher cell fusion efficiencies were also verified. On the other hand, monokaryotic filamentation, another sexual process involved the same sex of the MATα cells, appeared unaffected in the MATα bud16 mutants; however, overexpression of the BUD16 gene blocked this differentiation. In epistasis analysis, we found that the mating phenotype of the bud16 mutant under CWC1 overexpression background was similar to the bud16 mutant; while the phenotype of cwc1 gene deletion under the BUD16 overexpression strain was resemble to the BUD16 overexpression strain. These results suggested that BUD16 may be a negative regulator functioning downstream of the Cwc complex during the light-regulated sexual differentiation process.