Department of Life Sciences, Ben-Gurion University of the Negev, Beer-Sheva, Israel.
Department of Computer Sciences, Ben-Gurion University of the Negev, Beer-Sheva, Israel.
mSphere. 2019 Sep 4;4(5):e00450-19. doi: 10.1128/mSphere.00450-19.
The genomes of and trypanosomes encode six paralogs of the eIF4E cap-binding protein, known in other eukaryotes to anchor the translation initiation complex. In line with the heteroxenous nature of these parasites, the different LeishIF4E paralogs vary in their biophysical features and their biological behavior. We therefore hypothesize that each has a specialized function, not limited to protein synthesis. Of the six paralogs, LeishIF4E-3 has a weak cap-binding activity. It participates in the assembly of granules that store inactive transcripts and ribosomal proteins during nutritional stress that is experienced in the sand fly. We investigated the role of LeishIF4E-3 in promastigotes using the CRISPR-Cas9 system. We deleted one of the two LeishIF4E-3 alleles, generating a heterologous deletion mutant with reduced LeishIF4E-3 expression. The mutant showed a decline in protein synthesis and growth kinetics, altered morphology, and impaired infectivity. The mutant cells were rounded and failed to transform into the nectomonad-like form, in response to purine starvation. Furthermore, the infectivity of macrophage cells by the LeishIF4E-3(+/-) mutant was severely reduced. These phenotypic features were not observed in the addback cells, in which expression of LeishIF4E-3 was restored. The observed phenotypic changes correlated with the profile of transcripts associated with LeishIF4E-3. These were enriched for cytoskeleton- and flagellum-encoding genes, along with genes for RNA binding proteins. Our data illustrate the importance of LeishIF4E-3 in translation and in the parasite virulence. species are the causative agents of a spectrum of diseases. Available drug treatment is toxic and expensive, with drug resistance a growing concern. parasites migrate between transmitting sand flies and mammalian hosts, experiencing unfavorable extreme conditions. The parasites therefore developed unique mechanisms for promoting a stage-specific program for gene expression, with translation playing a central role. There are six paralogs of the cap-binding protein eIF4E, which vary in their function, expression profiles, and assemblages. Using the CRISPR-Cas9 system for , we deleted one of the two LeishIF4E-3 alleles. Expression of LeishIF4E-3 in the deletion mutant was low, leading to reduction in global translation and growth of the mutant cells. Cell morphology also changed, affecting flagellum growth, cell shape, and infectivity. The importance of this study is in highlighting that LeishIF4E-3 is essential for completion of the parasite life cycle. Our study gives new insight into how parasite virulence is determined.
和锥虫的基因组编码六种 eIF4E 帽结合蛋白的同源物，在其他真核生物中，这种蛋白将翻译起始复合物锚定在起始位置。与这些寄生虫的异宿主性质一致，不同的 LeishIF4E 同源物在其生物物理特性和生物学行为上存在差异。因此，我们假设每个同源物都具有专门的功能，而不仅仅局限于蛋白质合成。在这六种同源物中，LeishIF4E-3 与帽结构的结合活性较弱。它参与了在沙蝇中经历的营养胁迫下储存无活性转录本和核糖体蛋白的颗粒的组装。我们使用 CRISPR-Cas9 系统研究了 LeishIF4E-3 在 中的作用。我们敲除了两个 LeishIF4E-3 等位基因中的一个，产生了一个 LeishIF4E-3 表达降低的异源缺失突变体。该突变体表现出蛋白质合成和生长动力学下降、形态改变和感染能力受损。突变体细胞呈圆形，无法响应嘌呤饥饿转化为似动体形态。此外，LeishIF4E-3(+/-)突变体对巨噬细胞的感染能力严重降低。在表达 LeishIF4E-3 的添加细胞中没有观察到这些表型特征。观察到的表型变化与与 LeishIF4E-3 相关的转录本谱相关。这些转录本富含细胞骨架和鞭毛编码基因以及 RNA 结合蛋白基因。我们的数据说明了 LeishIF4E-3 在翻译和寄生虫毒力中的重要性。 物种是一系列疾病的病原体。现有的药物治疗既有毒性又昂贵，而且耐药性问题日益严重。 寄生虫在传播沙蝇和哺乳动物宿主之间迁移，经历着不利的极端条件。因此，寄生虫发展出了独特的机制来促进特定阶段的基因表达程序，其中翻译起着核心作用。eIF4E 帽结合蛋白有六个同源物，它们在功能、表达谱和组装方面存在差异。我们使用 CRISPR-Cas9 系统在 中敲除了两个 LeishIF4E-3 等位基因中的一个。LeishIF4E-3 在缺失突变体中的表达较低，导致全局翻译减少和突变体细胞生长减慢。细胞形态也发生了变化，影响了鞭毛生长、细胞形状和感染能力。这项研究的重要性在于强调 LeishIF4E-3 对于完成寄生虫生命周期是必不可少的。我们的研究为寄生虫毒力的决定因素提供了新的见解。
