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

• 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
• 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

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.

总结： 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， 它的治疗仅限于毒性相对较高和毒性较低的药物， 在感染的慢性阶段有效。迫切需要新的有效目标来防治这一疾病。 磷酸化介导的信号转导在真核生物中起着调节蛋白质功能的关键作用。 考虑到T. cruzi发育阶段，很可能， 蛋白激酶在细胞周期、代谢、存活的调节中起关键作用，特别是在寄生虫中 分化因此，占T. cruzi基因组，符合一个有吸引力的 一组抗寄生虫干预的分子靶点。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，阐明它们的局限性对于预测 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.克鲁兹然而，目前的方法在进一步研究方面存在局限性。 同时分析T. cruzi也限制了 药物发现的分子靶点。在这里，我们将通过基因敲除应用蛋白激酶筛选， T. cruzi epimastigotes，以鉴定参与生命周期进程的蛋白激酶。我们将 对124种T. cruzi，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 具有独特DNA条形码的合并敲除突变体的表型，所述DNA条形码将通过 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、生长和分化成亚周期锥鞭毛体所需的激酶，以及T. Cruzi感染和组织培养的宿主细胞中的增殖。总的来说，我们将进行大规模的筛选， 评估寄生虫适应性我们还将进行内源性标记选定的基因编码蛋白激酶 以确定它们的亚细胞定位。阐明蛋白激酶在T.克鲁兹将允许获得 深入了解寄生虫生存、增殖和分化所需的细胞器相关信号通路。