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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.

简介: