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： Chagas病是由原生动物寄生虫锥虫引起的一种传染病，影响了6至7 美洲的百万人及其治疗仅限于毒性相对较高且低的药物感染的慢性阶段的功效。迫切需要新的经过验证的目标来应对这种疾病。磷酸化介导的真核生物中的信号转导起着关键作用，作为蛋白质功能的调节剂。考虑到克鲁齐发育阶段报告的磷酸化模式的变化，很可能很可能蛋白激酶在细胞周期，代谢，生存，尤其是寄生虫的调节中起关键作用分化。因此，蛋白激酶代表t. cruzi基因组的2％，构成一个吸引人的蛋白激酶用于抗寄生虫干预措施的分子靶标。另外，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，阐明其本地化对于预测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在T. Cruzi中产生精确的基因组操纵。但是，当前的方法论有局限性对Cruzi的多个敲除突变体进行同时分析，这也限制了识别药物发现的分子靶标。在这里，我们将通过基因敲除和在克鲁兹t. ipimastigotes中的基因标记，以鉴定涉及生命周期进程的蛋白激酶。我们将研究了124个蛋白质的蛋白激酶，其中大多数在哺乳动物中没有明显的直系同源物。我们将分析合并敲除突变体的表型，该突变体具有独特的DNA条形码基于CRISPR/CAS9的方法。此外，我们将使用下一代测序来识别蛋白质表量生存，生长和分化为Metacyclic锥虫所需的激酶，用于T。 Cruzi感染和组织培养的宿主细胞中的增殖。总体而言，我们将进行大规模筛选评估寄生虫健身。我们还将对编码蛋白激酶的选定基因进行内源性标记确定其亚细胞定位。阐明蛋白激酶在克鲁齐的蛋白质激酶的作用将允许获得寄生虫生存，增殖和分化所需的洞察有机体相关的信号通路。