Barcode screening of essential protein kinases in the life cycle progression of Trypanosoma cruzi

摘要： 恰加斯病是一种由原生动物寄生虫克氏锥虫引起的传染病，影响6至7人 在美洲有100万人，其治疗仅限于毒性相对较高和较低的药物 在慢性感染阶段的疗效。迫切需要新的有效靶标来抗击这种疾病。 在真核生物中，磷酸化介导的信号转导作为蛋白质功能的调节起着关键作用。 考虑到在克氏锥虫发育阶段报告的磷酸化模式的变化，很可能是 蛋白激酶在调节细胞周期、新陈代谢、生存，特别是在寄生虫中起着关键作用。 差异化。因此，占克氏锥虫基因组近2%的蛋白激酶形成了一个有吸引力的 一组用于抗寄生虫干预的分子靶标。此外，蛋白质的亚细胞定位 激酶是其活性和功能的决定因素。因此，阐明它们的定位对预测至关重要。 它们在特定细胞过程中的参与。可编程CRISPR/CAS9系统提高了我们的能力 在克氏锥虫中进行精确的基因组操作。然而，当前的方法有局限性，无法进一步 同时分析克氏锥虫的多个基因敲除突变体，这也限制了鉴定 药物发现的分子靶点。在这里，我们将应用一种通过基因敲除和 克氏锥虫表鞭毛体中的基因标记，以确定参与生命周期进程的蛋白激酶。我们会 研究了124个克氏毛滴虫的蛋白激酶，其中大多数在哺乳动物中没有明显的同源基因。我们将分析 包含独特DNA条形码的混合基因敲除突变体的表型将由 基于CRISPR/CAS9的方法。此外，我们将使用下一代测序来识别蛋白质 表鞭毛体存活、生长和分化为亚环类鞭毛虫所需的激酶，以及T。 克鲁兹病毒在组织培养宿主细胞中的感染和增殖。总体而言，我们将进行大规模的筛选， 评估寄生虫的适合性。我们还将对编码蛋白激酶的选定基因进行内源标记 以确定它们的亚细胞定位。阐明蛋白激酶在克氏锥虫中的作用将允许获得 Insight细胞器相关信号通路是寄生虫生存、增殖和分化所必需的。

Summary: Chagas disease, an infectious disease caused by the protozoan parasite Trypanosoma cruzi, affects 6 to 7 million people in the Americas, and its treatment has been limited to drugs with relatively high toxicity and low efficacy in the chronic phase of the infection. New validated targets are urgently needed to combat this disease. Phosphorylation-mediated signal transduction in eukaryotes plays key roles as regulator of protein function. Considering the changes in phosphorylation patterns reported in T. cruzi developmental stages, it is likely that protein kinases play a pivotal role in the regulation of cell cycle, metabolism, survival, and particularly in parasite differentiation. Therefore, protein kinases, which represent near 2% of the T. cruzi genome, conform an attractive group of molecular targets for antiparasitic interventions. In addition, the subcellular localization of protein kinases is determinant for their activity and function. Therefore, elucidating their localization is crucial to predict their participation in specific cellular processes. Programmable CRISPR/Cas9 systems have improved our ability to produce precise genome manipulations in T. cruzi. However, current methodologies have limitations to further perform the simultaneous analysis of multiple knockout mutants in T. cruzi, which also restricts the identification of molecular targets for drug discovery. Here we will apply a protein kinase screening by gene knockout and gene tagging in T. cruzi epimastigotes, to identify protein kinases involved in life cycle progression. We will investigate 124 protein kinases of T. cruzi, most of them with no apparent orthologs in mammals. We will analyze the phenotype of pooled knockout mutants harboring unique DNA barcodes that will be generated by CRISPR/Cas9-based methodology. Furthermore, we will use next-generation sequencing to identify protein kinases required for epimastigote survival, growth and differentiation into metacyclic trypomastigotes, and for T. cruzi infection and proliferation in tissue-cultured host cells. Overall, we will perform a large-scale screening to assess parasite fitness. We will also perform endogenous tagging of selected genes encoding protein kinases to determine their subcellular localization. Elucidating the role of protein kinases in T. cruzi will allow to gain insight organelle-associated signaling pathways required for parasite survival, proliferation and differentiation.