研究白色念珠菌依賴 GCN4-THR1 在 TOR 訊息路徑中之營養反應

白色念珠菌是一種常存於人體內的伺機性真菌，當體內免疫力下降時其致病力會增加；而白色念珠菌的型態轉換與其致病力有高度相關。先前實驗室得知 CaCDC4 為菌絲負調控者，當 CaCDC4 deletion 時會抑制菌絲的生成，而又利用親和力找出與 CDC4 有相關的基因，分別為 THR1 與 GPH1。CaTHR1 是蘇胺酸生合成路徑 (threonine biosynthesis pathway) 中的其中一員，含有 homoserine kinase，會將 threonine 生合成的中間有毒物質 homoserine 代謝掉，使細胞不會因為累積過多的 homoserine 而導致死亡；在釀酒酵母菌中，THR1 會受到轉錄因子 GCN4 的調控，但在白色念珠菌中 THR1 會受到 GCN4 與 GCN2 的共同調控，但調控機制上不明確。而白色念珠菌的 GCN4 是個轉錄因子，會調控大部分的胺基酸生合成路徑，當細胞遭受到營養缺乏時 GCN4 的表現量會增加，藉由結合到目標基因啟動子上的 GCRE element (general control response element) 來調節下游基因。因此在本研究中，我們想要知道當營養缺乏的環境下，GCN4 與THR1 之間的交互關係，並利用昆蟲模式大蠟蛾來看注射白色念珠菌野生型與其他改造品系後，大蠟蛾的生長情況與存活率比較。藉由 RT-PCR 我們發現，在 rapamycin 處理白色念珠菌野生型 SC5314品系 3 小時下只有 GCN4 有增加表現，但 THR1卻沒有；而加入 3-AT 10 mM 與 40 mM 後處理 2 小時則發現 GCN4 與 THR1 兩者 mRNA 都有明顯增加。而 GCN4 轉錄活化 THR1 表現則建立分別帶有 THR1 兩個等位基因上游的報導系統，在 3-AT 誘導下的結果顯示，THR1 兩個等位基因的其中一個才具備完整的功能性；另外，透過 spot assay 則發現 thr1Δ/Δ 的生長主要經由 homoserine 累積而有明顯受到抑制，此抑制情形在 thr1Δ/Δ gcn4Δ/Δ有減緩，暗指白色念珠菌失去 GCN4 會影響 threonine biosynthesis 因 homoserine 累積而死亡之過程；而注射白色念珠菌相關品系進入大蠟蛾後發現，注射 thr1Δ/Δ 的大蠟蛾存活率比其他的品系包括 gcn4Δ/Δ 與 thr1Δ/Δ gcn4Δ/Δ 的存活率相比較高。 綜合以上，白色念珠菌存在 GCN4-THR1 依賴方式的 TOR 路徑之營養反應，而 TOR 路徑與型態變化是透過 THR1-CDC4 交互連結，而與同在 TOR pathway 路徑下的 GCN4 不同的是 THR1 的確是白色念珠菌致病力所需，顯示 THR1 致病力獨立於 TOR 路徑。

Candida albicans is a major human fungal pathogen. Its virulence is associated with the ability to undergo reversible morphological transitions among yeast, pseudohyphal and hyphal growth forms. We have previously identified THR1 as an interactor of CaCDC4, known to suppress yeast-to-filament transition in C. albicans. THR1 encodes a homoserine kinase, cells lost of which are attenuated in virulence and die upon threonine starvation and serum incubation. In C. albicans, GCN4 encodes a bZIP transcription factor that is required for amino acid control response. As THR1 is one of targets of nutrient response TOR signaling pathway mediated by GCN4 in Saccharomyces cerevisiae and presence of general response elements (GCREs) of amino acid biosynthetic pathways are known to be the target of GCN4 in the upstream of C. albicans THR1 gene, we have sought to assess dependency of THR1 on GCN4 in various nutrient depleted and stress conditions in C. albicans. The insect model Galleria mellonella was introduced to determine the survival rate upon host infection.By RT-PCR, we showed that GCN4 but not THR1 was up-regulated upon induced by TOR pathway-repressing agents rapamycin at 3 hrs. However, both THR1 and GCN4 were up-regulated upon induction with either 10 mM or 40 mM 3-AT in SC medium at 2 hrs. The reporter system where each of the two alleles of THR1 was incorporated showed that only one allele was likely to be functional. Also, spot assay revealed growth inhibition in thr1 homozygous null mutant (thr1Δ/Δ) was mainly due to the accumulation of homoserine. The growth inhibition could be alleviated by simultaneously deleting the GCN4 (thr1Δ/Δ gcn4Δ/Δ), suggesting that lost of GCN4 may release the homoserine accumulation in C.albicans cells. The Galleria mellonella infection assessment showed that thr1Δ/Δ strain had higher survival rate compared with thoses strains of WT, gcn4Δ/Δ, and thr1Δ/Δ gcn4Δ/Δ.In summary, our results imply that a GCN4-THR1 dependent nutrient response in C. albicans TOR signaling and that a cross-talk between TOR signaling and morphogenesis is mediated by THR1-CDC4. Unlike GCN4 that is also on the TOR signaling, only THR1 is required for virulence, suggesting that the virulence caused by THR1 is independent of TOR signaling pathway.