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首页> 外文期刊>Infection and immunity >Development of RNA Interference Trigger-Mediated Gene Silencing in Entamoeba invadens
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Development of RNA Interference Trigger-Mediated Gene Silencing in Entamoeba invadens

机译：入侵变形虫中RNA干扰触发介导的基因沉默的发展。

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Entamoeba histolytica, a protozoan parasite, is an important human pathogen and a leading parasitic cause of death. The organism has two life cycle stages, trophozoites, which are responsible for tissue invasion, and cysts, which are involved in pathogen transmission. Entamoeba invadens is the model system to study Entamoeba developmental biology, as high-grade regulated encystation and excystation are readily achievable. However, the lack of gene-silencing tools in E. invadens has limited the molecular studies that can be performed. Using the endogenous RNA interference (RNAi) pathway in Entamoeba, we developed an RNAi-based trigger gene-silencing approach in E. invadens. We demonstrate that a gene's coding region that has abundant antisense small RNAs (sRNAs) can trigger silencing of a gene that is fused to it. The trigger fusion leads to the generation of abundant antisense sRNAs that map to the target gene, with silencing occurring independently of trigger location at the 5′ or 3′ end of a gene. Gene silencing is stably maintained during development, including encystation and excystation. We have used this approach to successfully silence two E. invadens genes: a putative rhomboid protease gene and a SHAQKY family Myb gene. The Myb gene is upregulated during oxidative stress and development, and its downregulation led, as predicted, to decreased viability under oxidative stress and decreased cyst formation. Thus, the RNAi trigger silencing method can be used to successfully investigate the molecular functions of genes in E. invadens. Dissection of the molecular basis of Entamoeba stage conversion is now possible, representing an important technical advance for the system.
机译：变形虫Entomoeba histolytica，是一种原生动物寄生虫，是一种重要的人类病原体，也是主要的寄生死亡原因。该生物有两个生命周期阶段：滋养体（负责组织入侵）和囊肿（参与病原体传播）。 Entamoeba invadens是研究Entamoeba发育生物学的模型系统，因为可以很容易地实现高级调控的入侵和激发。然而，在入侵大肠杆菌中缺乏基因沉默工具限制了可以进行的分子研究。使用Entamoeba中的内源性RNA干扰（RNAi）途径，我们在入侵大肠杆菌中开发了基于RNAi的触发基因沉默方法。我们证明了具有大量反义小RNA（sRNA）的基因编码区可以触发与之融合的基因的沉默。触发融合导致映射到靶基因的大量反义sRNA的产生，而沉默独立于基因5'或3'端的触发位置而发生。基因沉默在发育过程中被稳定地保持，包括侵入和激发。我们已经使用这种方法成功沉默两个入侵大肠杆菌基因：一个假定的菱形蛋白酶基因和一个SHAQKY家族Myb基因。 Myb基因在氧化应激和发育过程中被上调，并且如预测的那样，其下调导致氧化应激下的生存力降低和囊肿形成减少。因此，RNAi触发沉默方法可用于成功调查入侵大肠杆菌中基因的分子功能。现在可以解剖Entamoeba阶段转化的分子基础，代表该系统的一项重要技术进步。

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