Dmc1a對調控囊體蛋白基因及對梨形鞭毛蟲分化的影響

Abstract

梨形鞭毛蟲(Giardia lamblia)是一種全球性廣泛存在之致病性腸內原蟲寄生蟲，感染途徑為飲用受汙染之水源。其滋養體寄生於小腸，而後隨著腸內環境的改變進行囊體化作用(encystation)形成具有囊壁之感染型 ── 囊體。囊壁是梨形鞭毛蟲在抵禦惡劣環境下生存所需之特殊構造，其主要由蛋白質與多醣構成。梨形鞭毛蟲的生活史有兩種型態，一為擁有兩個核並具有鞭毛運動性之滋養體(trophozoite)以及四個核的成熟囊體(cyst)。在囊體化的過程中，囊體蛋白質(cyst wall protein, cwp)的表現量會增加，而滋養體的雙核在此刻也有機會彼此交換訊息(gene exchange)，進一步的研究指出，有減數分裂基因同源物(meiotic gene homologs)像Dmc1a會參與在雙核之間的同源重組(homologous recombination)過程。 已知在梨形鞭毛蟲的基因庫分析數據中有兩個Dmc1之同源物：Dmc1a及Dmc1b (orf 13104及13346)。推測Dmc1a或Dmc1b或許在梨形鞭毛蟲的囊體化過程扮演不可或缺的角色之一，為了釐清與證實，我們先利用即時定量PCR與西方墨點法分別偵測到Dmc1a的RNA與蛋白質表現量在囊體化時都是上升，而Dmc1b則是在滋養體時表現量上升。而免疫螢光染色發現Dmc1a位於細胞核，而與CWP1共染色結果也確定Dmc1a可促進CWP1基因表現。 我們建立過量表現Dmc1a的細胞株pPDmc1a，發現大量表現Dmc1a時促使CWP1表現與促進生成囊體。我們也建立三個Dmc1a的突變株，針對Dmc1a的ATP酶活性位與N端結構位進行突變，也證實突變位置對Dmc1a促進梨形鞭毛蟲的CWP1蛋白質表現與囊體形成扮演重要的功能。為了與Dmc1a比較，我們同時建立了過量表現的Dmc1b細胞株，結果顯示比起Dmc1a，Dmc1b的過量表現對於促進CWP1蛋白質表現與囊體的形成僅微幅上升。我們推測Dmc1b在滋養體時其可能有未知的調控。另外，有鑑於Dmc1a與DNA的鍵結活性，我們利用染色質免疫沉澱(ChIP)分析發現Dmc1a會與cwp1、cwp3、myb2和wrky的啟動子鍵結，又從免疫沉澱(IP)證實Dmc1a會與囊體化轉錄因子MYB2 、WRKY有交互作用。在梨形鞭毛蟲根據目前實驗結果可推得，Dmc1a參與了囊體化相關基因的調控以促進囊體化進行；比起Dmc1b更顯著的參與囊體化的調控。因此，我們也推測Dmc1a除了參與DNA修復與梨形鞭毛蟲基因多樣性(genome diversity)的增加以外，也與促進囊體化有關。

Giardia lamblia is one of the most common protozoan parasites causing waterborne intestinal infections in humans. G. lamblia have two stages in the life cycle: a flagellated trophozoite with 2 nuclei and an inert cyst with 4 nuclei. During encystation, differentiation from a trophozoite into a cyst, the cyst wall protein (CWPs) is highly synthesized in a concerted manner. Research indicates that the 2 nuclei have chance to exchange the information during the life cycle and the meiotic gene homologs, such as Dmc1a, are involved in mediating homologous recombination between the 2 nuclei. Two genes encoding homologs of Dmc1, Dmc1a and Dmc1b (orf 13104 and 13346), are present in G. lamblia genome. We speculated that the Dmc1a and Dmc1b might play a role in G. lamblia encystation. Using quantitative RT-PCR and Western blots, we found an up-regulation of Dmc1a gene during the encystation stage, but we also found the Dmc1b gene is up-regulated during the trophozoite stage. Immunofluorescence assay also revealed that Dmc1a was localized to the nuclei. We performed the Dmc1a overexpression assay in a stable transfection system and found an induction of the expression of cwp1 gene and cyst formation. Also, we created three Dmc1a mutants, in which mutations located in the ATPase active site and N-terminal domain. We confirmed the ATPase active site and N-terminal domain of Dmc1a are important for cyst formation and CWP1 protein production of G. lamblia. For comparison, we established a Dmc1b overexpression cell line and the result shows a slightly increase but not as significant as Dmc1a. We suggest Dmc1b might have an unknown regulation during trophozoite stage. Furthermore, we found Dmc1a has DNA binding activity. Using chromatin immunoprecipitation assay, we found Dmc1a can bind to the promoters of cwp1, cwp3, myb2 and wrky genes. We also found Dmc1a can interact with encystation transcription factors, MYB2 and WRKY, using immunoprecipitation assay. Taken together, Dmc1a plays much more important role in induction of the cwp genes, which is key to Giardia differentiation into cysts, compared with Dmc1b. Dmc1a might be involved in not only homologous recombination for increasing the diversity of genome of G. lamblia, but also play as transcription factor in induction of Giardia encystation.