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Establishment of quantitative RNAi-based forward genetics in Entamoeba histolytica and identification of genes required for growth
bioRxiv - Microbiology Pub Date : 2021-10-28 , DOI: 10.1101/2020.05.28.121780
Akhila Bettadapur , Samuel S. Hunter , Rene L. Suleiman , Maura C. Ruyechan , Wesley Huang , Charles G. Barbieri , Hannah W. Miller , Tammie S.Y. Tam , Matthew L. Settles , Katherine S. Ralston

While Entamoeba histolytica remains a globally important pathogen, it is dramatically understudied. The tractability of E. histolytica has historically been limited, which is largely due to challenging features of its genome. To enable forward genetics, we constructed and validated the first genome-wide E. histolytica RNAi knockdown mutant library. This library allows for Illumina deep sequencing analysis for quantitative identification of mutants that are enriched or depleted after selection. We developed a novel analysis pipeline to precisely define and quantify gene fragments. We used the library to perform the first RNAi screen in E. histolytica and identified slow growth (SG) mutants. Among genes targeted in SG mutants, many had annotated functions consistent with roles in cellular growth or metabolic pathways. Some targeted genes were annotated as hypothetical or lacked annotated domains, supporting the power of forward genetics in uncovering functional information that cannot be gleaned from databases. While the localization of neither of the proteins targeted in SG1 nor SG2 mutants could be predicted by sequence analysis, we showed experimentally that SG1 localized to the cytoplasm and cell surface, while SG2 localized to the cytoplasm. Overexpression of SG1 led to increased growth, while expression of a truncation mutant did not lead to increased growth, and thus aided in defining functional domains in this protein. Finally, in addition to establishing forward genetics, we uncovered new details of the unusual E. histolytica RNAi pathway. These studies dramatically improve the tractability of E. histolytica and open up the possibility of applying genetics to improve understanding of this important pathogen.

中文翻译:

在溶组织内阿米巴中建立基于定量 RNAi 的正向遗传学并鉴定生长所需的基因

虽然溶组织内阿米巴仍然是一种全球重要的病原体,但对它的研究却很少。E. histolytica的易处理性历来受到限制,这主要是由于其基因组具有挑战性的特征。为了实现正向遗传学,我们构建并验证了第一个全基因组E. histolytica RNAi 敲低突变文库。该文库允许 Illumina 深度测序分析,用于定量鉴定选择后富集或耗尽的突变体。我们开发了一种新颖的分析流程来精确定义和量化基因片段。我们使用该文库在溶组织大肠杆菌中进行了第一次 RNAi 筛选并确定了缓慢生长 (SG) 突变体。在 SG 突变体中靶向的基因中,许多具有与细胞生长或代谢途径中的作用一致的注释功能。一些目标基因被注释为假设的或缺少注释的域,支持正向遗传学在揭示无法从数据库中收集的功能信息方面的力量。虽然通过序列分析无法预测 SG1 和 SG2 突变体中靶向蛋白质的定位,但我们通过实验表明 SG1 定位于细胞质和细胞表面,而 SG2 定位于细胞质。SG1 的过度表达导致生长增加,而截断突变体的表达不会导致生长增加,因此有助于定义该蛋白质的功能域。最后,除了建立正向遗传学,E.histolytica RNAi途径。这些研究极大地提高了溶组织大肠杆菌的易处理性,并开辟了应用遗传学来提高对这一重要病原体的了解的可能性。
更新日期:2021-10-31
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