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首页> 外文期刊>mSphere >LeishIF4E1 Deletion Affects the Promastigote Proteome, Morphology, and Infectivity
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LeishIF4E1 Deletion Affects the Promastigote Proteome, Morphology, and Infectivity

机译：Leishif4e1删除影响春季蛋白质组，形态和感染性

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摘要

Leishmania parasites cycle between sand-fly vectors and mammalian hosts, adapting to changing environmental conditions by driving a stage-specific program of gene expression, which is tightly regulated by translation processes. Leishmania encodes six eIF4E orthologs (LeishIF4Es) and five eIF4G candidates, forming different cap-binding complexes with potentially varying functions. Most LeishIF4E paralogs display temperature sensitivity in their cap-binding activity, except for LeishIF4E1, which maintains its cap-binding activity under all conditions. We used the CRISPR-Cas9 system to successfully generate a null mutant of LeishIF4E1 and examine how its elimination affected parasite physiology. Although the LeishIF4E1sup–/–/sup null mutant was viable, its growth was impaired, in line with a reduction in global translation. As a result of the mutation, the null LeishIF4E1sup–/–/sup mutant had a defective morphology, as the cells were round and unable to grow a normal flagellum. This was further emphasized when the LeishIF4E1sup–/–/sup cells failed to develop the promastigote morphology once they shifted from conditions that generate axenic amastigotes (33°C, pH 5.5) back to neutral pH and 25°C, and they maintained their short flagellum and circular structure. Finally, the LeishIF4E1sup–/–/sup null mutant displayed difficulty in infecting cultured macrophages. The morphological changes and reduced infectivity of the mutant may be related to differences in the proteomic profile of LeishIF4E1sup–/–/sup cells from that of controls. All defects monitored in the LeishIF4E1sup–/–/sup null mutant were reversed in the add-back strain, in which expression of LeishIF4E1 was reconstituted, establishing a strong link between the cellular defects and the absence of LeishIF4E1 expression. IMPORTANCE Leishmania parasites are the causative agents of a broad spectrum of diseases. The parasites migrate between sand-fly vectors and mammalian hosts, adapting to changing environments by driving a regulated program of gene expression, with translation regulation playing a key role. The leishmanias encode six different paralogs of eIF4E, the cap-binding translation initiation factor. Since these vary in function, expression profile, and assemblage, it is assumed that each is assigned a specific role throughout the life cycle. Using the CRISPR-Cas9 system for Leishmania , we generated a null mutant of LeishIF4E1, eliminating both alleles. Although the mutant cells were viable, their morphology was altered and their ability to synthesize the flagellum was impaired. Elimination of LeishIF4E1 affected their protein expression profile and decreased their ability to infect cultured macrophages. Restoring LeishIF4E1 expression restored the affected features. This study highlights the importance of LeishIF4E1 in diverse cellular events during the life cycle of Leishmania .
机译：Leishmania寄生虫循环砂飞传载体和哺乳动物宿主之间，通过推动特定的基因表达程序来适应改变环境条件，这是通过翻译过程紧密调节的。 LeishMania编码六个EIF4E Orthologs（Leishif4E）和五个EIF4G候选，形成具有潜在不同功能的不同帽结合复合物。大多数Leishif4e Paralogs在其帽结合活性中显示温度敏感性，除了Leishif4e1，其在所有条件下保持其帽结合活性。我们使用CRISPR-CAS9系统成功地生成了Leishif4E1的空突变体，并检查其消除如何影响寄生虫生理学。虽然Leishif4e1 - / - / sup> null突变体是可行的，但其增长损害，这符合全球翻译的减少。作为突变的结果，Null Leishif4e1 - / - / sup>突变体具有缺陷的形态，因为细胞是圆形的，并且不能生长正常的鞭毛。当Leishif4e1 - / - / - sup>细胞未能发生突出的形态后，从产生亚酮amastigotes（33℃，pH5.5）的条件后，进一步强调了这一点，从而恢复中性pH和25°C，他们保持了他们的短鞭毛和圆形结构。最后，Leishif4e1 - / - null突变体显示出感染培养的巨噬细胞的难题。突变体的形态学变化和降低的感染性可能与来自对照的Leishif4e1 -/ - -/ sup>细胞的蛋白质组曲线的差异有关。在leishif4e1 - / - - / sup> null突变体中检测所有缺陷在加回菌株中反转，其中重建了Leishif4e1的表达，在细胞缺陷和缺乏Leishif4e1表达之间建立了强烈的联系。重要的Leishmania寄生虫是广谱疾病的致病因子。寄生虫在沙蝇载体和哺乳动物宿主之间迁移，通过推动受调节基因表达程序来调整改变环境，翻译规则发挥关键作用。 LeishManias编码六个不同的eif4e副族，帽绑定翻译开始因子。由于这些功能，表达式简介和组装中所不同，因此假设每个在整个生命周期中分配特定角色。使用莱山尼亚的CRISPR-CAS9系统，我们生成了Leishif4e1的空突变体，消除了两个等位基因。虽然突变细胞是可行的，但它们的形态被改变，并且它们合成鞭毛的能力受到损害。消除Leishif4e1影响了它们的蛋白质表达谱，并降低了感染培养巨噬细胞的能力。恢复Leishif4e1表达式恢复受影响的功能。本研究突出了Leishif4e1在Leishmania生命周期中不同的细胞事件中的重要性。

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