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

克氏锥虫生命周期进展中必需蛋白激酶的条形码筛选

基本信息

批准号：
10726233
负责人：
Miguel A Chiurillo
金额：
$ 20.25万
依托单位：
UNIVERSITY OF CINCINNATI
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-08-15 至 2025-07-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10726233
关键词：
Affect Americas Antiparasitic Agents Atlases Bar Codes Biological Biological Process Blood Transfusion C-terminal CRISPR/Cas technology Cell Cycle Progression Cell Cycle Regulation Cell physiology Cells Chagas Disease Chronic Phase Classification Communicable Diseases Complex Country Coupled Cyclic AMP-Dependent Protein Kinases DNA DNA Repair Pathway Detection Development Differentiation and Growth Disease Drug Targeting Equipment and supply inventories Etiology Eukaryota Fluorescence Microscopy Gene Deletion Gene Silencing Genes Genetic Genetic Drift Genome HIV Human Individual Infection Insect Vectors Intervention Invaded Investigation Knock-out Latin America Leishmania Libraries Life Cycle Stages Ligands Malaria Mammals Mediating Metabolism Methodology Molecular Target Morphology Nonhomologous DNA End Joining Organ Transplantation Organelles Organism Orthologous Gene PRKR gene Parasites Pattern Persons Pharmaceutical Preparations Phase II Clinical Trials Phenotype Phosphorylation Phosphotransferases Play Population Proliferating Protein Kinase Proteins RNA Interference Regulation Reporting Research Project Grants Role Scaffolding Protein Signal Pathway Signal Transduction Specificity System Testing Toxic effect Trypanosoma brucei brucei Trypanosoma cruzi chemotherapy co-infection combat community based participatory research deletion library design drug discovery druggable target experimental study fitness genetic approach homologous recombination improved inhibitor insight knockout gene migration molecular drug target mutant nanomolar next generation sequencing pathogen phosphoproteomics prevent protein function response reverse genetics screening side effect tissue culture vaccine access

项目摘要

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.

概括：查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 岁美洲有数百万人，其治疗仅限于毒性相对较高、较低的药物感染慢性期的疗效。迫切需要新的经过验证的靶点来对抗这种疾病。真核生物中磷酸化介导的信号转导在蛋白质功能调节中发挥着关键作用。考虑到克氏锥虫发育阶段报道的磷酸化模式的变化，很可能蛋白激酶在细胞周期、代谢、生存的调节中发挥着关键作用，特别是在寄生虫中差异化。因此，占 T. cruzi 基因组近 2% 的蛋白激酶符合一个有吸引力的抗寄生虫干预的一组分子靶标。此外，蛋白质的亚细胞定位激酶决定其活性和功能。因此，阐明它们的定位对于预测至关重要他们参与特定的细胞过程。可编程CRISPR/Cas9系统提高了我们的能力对克氏锥虫进行精确的基因组操作。然而，目前的方法有局限性，无法进一步同时分析 T. cruzi 中的多个敲除突变体，这也限制了鉴定药物发现的分子靶标。在这里，我们将通过基因敲除来应用蛋白激酶筛选，对克氏锥虫上鞭毛体进行基因标记，以识别参与生命周期进展的蛋白激酶。我们将研究了 T. cruzi 的 124 种蛋白激酶，其中大多数在哺乳动物中没有明显的直系同源物。我们将分析汇集的敲除突变体的表型含有独特的 DNA 条形码，这些条形码将由基于 CRISPR/Cas9 的方法。此外，我们将使用下一代测序来鉴定蛋白质上鞭毛体存活、生长和分化为后循环锥鞭毛体以及 T 所需的激酶。 cruzi 感染和组织培养宿主细胞的增殖。总体而言，我们将进行大规模筛选评估寄生虫的适应性。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。阐明克氏锥虫中蛋白激酶的作用将有助于获得洞察寄生虫生存、增殖和分化所需的细胞器相关信号通路。