Giardia lamblia causes waterborne diarrhoea by transmission of infective cysts. Three cyst wall proteins are highly expressed in a concerted manner during encystation of trophozoites into cysts. However, their gene regulatory mechanism is still largely unknown. DNA topoisomerases control topological homeostasis of genomic DNA during replication, transcription and chromosome segregation. They are involved in a variety of cellular processes including cell cycle, cell proliferation and differentiation, so they may be valuable drug targets. Giardia lamblia possesses a type IA DNA topoisomerase (TOP3β) with similarity to the mammalian topoisomerase IIIβ. We found that TOP3β was upregulated during encystation and it possessed DNA-binding and cleavage activity. TOP3β can bind to the cwp promoters in vivo using norfloxacin-mediated topoisomerase immunoprecipitation assays. We also found TOP3β can interact with MYB2, a transcription factor involved in the coordinate expression of cwp1-3 genes during encystation. Interestingly, overexpression of TOP3β increased expression of cwp1-3 and myb2 genes and cyst formation. Microarray analysis confirmed upregulation of cwp1-3 and myb2 genes by TOP3β. Mutation of the catalytically important Tyr residue, deletion of C-terminal zinc ribbon domain or further deletion of partial catalytic core domain reduced the levels of cleavage activity, cwp1-3 and myb2 gene expression, and cyst formation. Interestingly, some of these mutant proteins were mis-localized to cytoplasm. Using a CRISPR/Cas9 system for targeted disruption of top3β gene, we found a significant decrease in cwp1-3 and myb2 gene expression and cyst number. Our results suggest that TOP3β may be functionally conserved, and involved in inducing Giardia cyst formation.

中文翻译：

---

DNA拓扑异构酶IIIβ通过诱导兰氏贾第鞭毛虫中的囊壁蛋白基因表达来促进囊肿的产生。

贾第鞭毛虫通过传播感染性囊肿引起水源性腹泻。在滋养体进入囊肿的过程中，三个囊壁蛋白以一致的方式高表达。但是，它们的基因调控机制仍然是未知的。DNA拓扑异构酶控制复制，转录和染色体分离过程中基因组DNA的拓扑稳态。它们参与多种细胞过程，包括细胞周期，细胞增殖和分化，因此它们可能是有价值的药物靶标。贾第鞭毛虫具有与哺乳动物拓扑异构酶IIIβ相似的IA型DNA拓扑异构酶（TOP3β）。我们发现TOP3β在融合过程中被上调，并具有DNA结合和裂解活性。使用诺氟沙星介导的拓扑异构酶免疫沉淀测定法，TOP3β可在体内与cwp启动子结合。我们还发现TOP3β可以与MYB2相互作用，MYB2是在侵入过程中参与cwp1-3基因协调表达的转录因子。有趣的是，TOP3β的过表达增加了cwp1-3和myb2基因的表达以及囊肿的形成。微阵列分析证实了TOP3β对cwp1-3和myb2基因的上调。催化重要的Tyr残基的突变，C末端锌带结构域的缺失或部分催化核心结构域的进一步缺失降低了切割活性，cwp1-3和myb2基因表达以及囊肿形成的水平。有趣的是，其中一些突变蛋白被错误地定位到细胞质。使用CRISPR / Cas9系统定向破坏top3β基因，我们发现cwp1-3和myb2基因表达及囊肿数目显着减少。我们的结果表明，TOP3β可能在功能上是保守的，并参与诱导贾第鞭毛虫囊肿的形成。