梨形鞭毛蟲拓樸異構酶IIIβ在囊體化過程中所扮演的角色

Abstract

梨形鞭毛蟲為一種常見的腸道單細胞寄生性原蟲，在其生活史中為了適應宿主體外惡劣的環境，囊體化的過程是必須的，此時期細胞會大量表現囊體壁的組成因子，囊體壁蛋白質 ( cyst wall protein, CWP ) 與多醣類，形成囊體以抵抗外界逆境，目前已知的囊體壁蛋白質有CWP1、CWP2、CWP3。在細胞進行囊體化的過程中染色體套數由4倍體的滋養體擴張為16倍體的囊體，且大量的基因活動參與其中，在這一個基因體高度變化的過程中，基因體的穩定性是相當重要的。先前的研究指出，type IA topoisomerases參與細胞DNA複製、染色體分離、轉錄作用、DNA重組、以及DNA修復等基因活動，我們分析梨形鞭毛蟲的一個type IA topoisomerase，topoisomerase III β ( gTOP3β )基因的啟動子，發現內含一個調控梨形鞭毛蟲囊體化的轉錄因子Ｍyb2的結合序列，顯示梨形鞭毛蟲gTOP3β可能參與其囊體化的過程中。 本研究想了解，梨形鞭毛蟲gTOP3β在囊體化過程中所扮演的角色。我們由定量RT-PCR與西方點墨法分析得知囊體化時期梨形鞭毛蟲內生性gTOP3β基因 的表現量略高於滋養體時期。由免疫螢光染色法得知gTOP3β主要分佈於細胞核，核膜的位置。電泳位移分析實驗與DNA切割活性分析得知gTOP3β具有與囊體壁蛋白質基因啟動子結合及切割DNA的能力。由定量RT-PCR，西方點墨法與囊體計數得知，大量表現topoisomerase III β會增加囊體化相關基因cwp1-3以及myb2基因的表現及囊體形成的能力。然而當我們將topoisomerase III β的topoisomerase 活性區域中的酵素活性胺基酸Y328以及C端的Zinc ribbon區域進行突變後，發現酵素與DNA結合的能力與切割DNA的能力都受到了抑制，且降低梨形鞭毛蟲囊體化相關基因之表現與囊體的形成。經由免疫共沉澱的分析我們也發現了gTOP3β與調控梨形鞭毛蟲囊體化的轉錄因子Ｍyb2和WRKY結合的現象。 我們的結果顯示，梨形鞭毛蟲gTOP3β會在囊體化時期大量表現，並與囊體化相關基因啟動子結合以活化其基因表現，進而促使梨形鞭毛蟲的囊體化。我們認為gTOP3β可能與調控囊體化的轉錄因子共同作用，藉由解除囊體壁基因表現時所產生的拓樸學障礙以利轉錄作用的進行。

Giardia lamblia is one of the most important protozoan parasites causing intestinal infections in humans. It differentiates into infective cysts for disease transmission. G. lamblia cysts can survive in hostile environments, such as fresh water and gastric acid, during infection as they have a protective wall composed of proteins and polysaccharides. Three known cyst wall proteins (CWPs) are highly synthesized in a concerted manner during differentiation into cysts (encystation). However, little is known of their gene regulation. During encystation, a trophozoite may differentiate into a cyst by dividing 2 nuclei and by replicating DNA, generating a cyst with 4 nuclei. The maintenance of genome stability and to resolve the topological problems of DNA might be a great event in G. lamblia encystation. DNA topoisomerases solve all the topological problems such as replication, chromosome segregation, transcription, recombination, and DNA repair. A putative topoisomerase IIIβ (gTOP3β) gene has been identified in the G. lamblia genome. Analysis of the 5’-flanking region of the gTOP3β gene, we found that a binding site of the encystation-induced transcription factor, Myb2, is present in the gTOP3β promoter region. This indicates that gTOP3β might play a role in G. lamblia encystation. To understand whether gTOP3β is involved in Giardia encystation or not, we used quantitative RT-PCR and Western blots to detect the gTOP3β RNA and protein in G. lamblia cells. Data show that gTOP3β gene expression was up-regulated during the encystation stage. Immunofluorescence assay revealed that it was localized to nuclear envelope. Electrophoretic mobility shift assay and DNA cleavage assay indicate that, like other type IA topoisomerases, gTOP3β is able to bind to the cwp1-3 promoters and to cleave the single-stranded DNA. This suggests the gTOP3β might be involved in transcriptional activation of encystation genes in G. lamblia. To confirm this hypothesis we performed the overexpression and mutation analysis in a stable transfection system. We found that overexpression of gTOP3β induced the expression of cwp1–3 and myb2 genes, and cyst formation. Mutation analysis revealed that the catalytic important Tyr residue, the topoisomerase domain and the C-terminal zinc ribbon domain are important for gTOP3β function. We also performed co-immunoprecipitation assay found that the gTOP3β can interact with encystation-induced transcription activators, Myb2 and WRKY. Taken together, this study suggests that gTOP3β plays an important role in induction of the cwp genes, which are key to Giardia differentiation into cysts.