Department of Life Sciences, Ben-Gurion University of the Negev, Beer Sheva, Israel.
Centre for Cellular and Molecular Biology, Hyderabad, India.
PLoS Negl Trop Dis. 2021 Mar 24;15(3):e0008352. doi: 10.1371/journal.pntd.0008352. eCollection 2021 Mar.
Leishmania parasites cycle between sand-fly vectors and mammalian hosts adapting to alternating environments by stage-differentiation accompanied by changes in the proteome profiles. Translation regulation plays a central role in driving the differential program of gene expression since control of gene regulation in Leishmania is mostly post-transcriptional. The Leishmania genome encodes six eIF4E paralogs, some of which bind a dedicated eIF4G candidate, and each eIF4E is assumed to have specific functions with perhaps some overlaps. However, LeishIF4E2 does not bind any known eIF4G ortholog and was previously shown to comigrate with the polysomal fractions of sucrose gradients in contrast to the other initiation factors that usually comigrate with pre-initiation and initiation complexes. Here we deleted one of the two LeishIF4E2 gene copies using the CRISPR-Cas9 methodology. The deletion caused severe alterations in the morphology of the mutant cells that became round, small, and equipped with a very short flagellum that did not protrude from its pocket. Reduced expression of LeishIF4E2 had no global effect on translation and growth, unlike other LeishIF4Es; however, there was a change in the proteome profile of the LeishIF4E2(+/-) cells. Upregulated proteins were related mainly to general metabolic processes including enzymes involved in fatty acid metabolism, DNA repair and replication, signaling, and cellular motor activity. The downregulated proteins included flagellar rod and cytoskeletal proteins, as well as surface antigens involved in virulence. Moreover, the LeishIF4E2(+/-) cells were impaired in their ability to infect cultured macrophages. Overall, LeishIF4E2 does not behave like a general translation factor and its function remains elusive. Our results also suggest that the individual LeishIF4Es perform unique functions.
利什曼原虫寄生虫在沙蝇媒介和哺乳动物宿主之间循环，通过伴随蛋白质组谱变化的阶段分化来适应交替的环境。翻译调控在驱动基因表达的差异程序中起着核心作用，因为在利什曼原虫中，基因调控的控制主要是转录后。利什曼原虫基因组编码六个 eIF4E 同源物，其中一些与特定的 eIF4G 候选物结合，并且每个 eIF4E 都假定具有特定的功能，可能有些重叠。然而，LeishIF4E2 不与任何已知的 eIF4G 直系同源物结合，并且先前的研究表明，它与蔗糖梯度的多核糖体部分共迁移，而其他起始因子通常与起始前和起始复合物共迁移。在这里，我们使用 CRISPR-Cas9 方法删除了两个 LeishIF4E2 基因拷贝之一。该缺失导致突变细胞的形态发生严重改变，细胞变成圆形、小而短，鞭毛很短，无法从其口袋中伸出。与其他 LeishIF4E 不同，LeishIF4E2 的表达减少对翻译和生长没有全局影响；然而，LeishIF4E2(+/-)细胞的蛋白质组谱发生了变化。上调的蛋白质主要与一般代谢过程有关，包括参与脂肪酸代谢、DNA 修复和复制、信号转导和细胞运动活性的酶。下调的蛋白质包括鞭毛杆和细胞骨架蛋白，以及参与毒力的表面抗原。此外，LeishIF4E2(+/-)细胞感染培养的巨噬细胞的能力受损。总的来说，LeishIF4E2 不像一般的翻译因子那样发挥作用，其功能仍然难以捉摸。我们的结果还表明，单个 LeishIF4E 执行独特的功能。
