Host-parasite lipid metabolism in Trypanosoma cruzi-infected cardiac myocytes

克氏锥虫感染心肌细胞中宿主寄生虫的脂质代谢

• 批准号: 10249356
• 负责人: Jeffrey Dominick Whitman
• 金额: $ 11.35万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-08-28 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10249356
• 关键词: Address; Affect; Affinity; Arrhythmia; Biological; Biology; CRISPR interference; CRISPR screen; Cardiac; Cardiac Myocytes; Cell Culture Techniques; Cell Separation; Cell surface; Cells; Chagas Disease; Chronic; Clinical; Clinical Management; Clustered Regularly Interspaced Short Palindromic Repeats; Culture Media; Cytolysis; Data; Development; Diagnostic; Dilated Cardiomyopathy; Disease; Disease Management; Emerging Technologies; Evaluation; Flow Cytometry; Foundations; Frequencies; Functional disorder; Gene Targeting; Genes; Genomics; Goals; Green Fluorescent Proteins; Guide RNA; Harvest; Health; Heart Diseases; Human; Individual; Infection; Integration Host Factors; Knowledge; Label; Lead; Lipids; Maintenance; Mass Spectrum Analysis; Measurement; Metabolic; Metabolism; Mission; Modeling; Myocardial; Myocardium; Natural History; Nutrient; Organ; Parasites; Parasitic Diseases; Parasitic infection; Pathology; Pathway interactions; Phase; Phenotype; Physiological; Population; Prognostic Marker; Proteomics; Protozoan Infections; RNA library; Regulation; Research; Research Methodology; Rodent; Role; Serodiagnoses; Signal Transduction; Site; Source; Specificity; Specimen; Stable Isotope Labeling; System; Techniques; Technology; Testing; Therapeutic; Tissues; Toxoplasma gondii; Tropism; Trypanosoma cruzi; United States National Institutes of Health; Validation; base; biomarker development; biomarker discovery; chronic infection; evidence base; experimental study; heart metabolism; human tissue; improved; induced pluripotent stem cell; innovation; insight; knock-down; lipid metabolism; lipidomics; loss of function; metabolome; metabolomics; novel; pathogen; prevent; prognostic; screening; tissue culture; tissue tropism; treatment strategy

PROJECT SUMMARY/ABSTRACT Chagas disease is caused by the parasite Trypanosoma cruzi, which chronically infects cardiac myocytes, leading to significant cardiac pathology over the lifetime of the host. There is a fundamental gap in the understanding of T. cruzi’s affinity for the human myocardium. Studies in non-cardiac cells and rodents have implicated lipid metabolism as an important factor in the intracellular maintenance of T. cruzi, but data specific to cardiac myocyte metabolism and the lipid products resulting from the parasite-host interaction are lacking. Shifting the focus to the role of cardiac tissue is crucial for understanding the natural history of human T. cruzi infection. The objective of this research is to identify metabolic factors specific to human cardiac myocytes that are necessary for T. cruzi infection. The central hypothesis is that specific cardiac lipids or lipid metabolites are essential nutrient sources for intracellular replication and maintenance of T. cruzi, leading to this parasite’s tissue affinity and subsequent cardiac pathology. The long-term goal is to use this understanding of host- parasite interaction to develop precision cardiac diagnostics and improve therapeutic options for this insidious and debilitating infection. Understanding the role of human myocardial metabolism in the regulation of T. cruzi infection will be tested with two specific aims: 1) evaluation of lipidomic perturbances in T. cruzi-infected cardiac myocytes and 2) determining human cardiac metabolic regulators of T. cruzi infection. Aim 1 will employ a mass spectrometry-based lipidomic workflow, established by the applicant, to quantify hundreds of biologically relevant lipids during a 6-day T. cruzi infection cycle in induced pluripotent stem cell-derived cardiac myocytes (iPS-CMs). Significantly dysregulated lipids in culture media and cell lysates will be validated with individual stable isotope-labeled lipid standards to determine incorporation of heavy-labeled components into key lipid metabolism pathways in T. cruzi, harvested post host cell lysis. Aim 2 will employ a first-of-its-kind CRISPR interreference (CRISPRi) screen of human metabolism-related genes in cardiac myocytes to evaluate physiologically relevant host factors affecting T. cruzi replication and maintenance. These experiments will utilize iPS-CMs harboring CRISPRi machinery transduced with a focused guide RNA library to knock down human metabolic genes. After infection with green fluorescent protein (GFP)-tagged T. cruzi, we will sequence iPS-CM populations harboring high, normal, and low parasite burdens. These experiments will reveal the relevant gene knockdown targets in each replication phenotype to provide insight into key cardiac regulators of infection. This approach is innovative because it combines state-of-the-art technologies in lipidomics and CRISPR-based screening to investigate the interface of host-pathogen metabolism in a physiologically relevant cardiac model. The proposed research is significant because it will provide biological evidence for specific aspects of lipid metabolism in the cardiac tropism of T. cruzi, which will provide a foundation on which to develop precision cardiac diagnostics and novel, evidence-based therapies.

项目总结/摘要 恰加斯病是由寄生虫克氏锥虫引起的，它慢性感染心肌细胞， 导致宿主一生中严重的心脏病。有一个基本的差距， 对T. cruzi对人类心肌的亲和力在非心肌细胞和啮齿类动物中的研究 提示脂质代谢是T. cruzi，但具体数据 心肌细胞代谢和寄生虫-宿主相互作用产生的脂质产物缺乏。 将焦点转移到心脏组织的作用对于理解人类T细胞的自然史至关重要。cruzi 感染本研究的目的是鉴定人心肌细胞特异性代谢因子， 是T.克氏感染中心假设是，特定的心脏脂质或脂质代谢物是 细胞内复制和维持T. cruzi，导致这种寄生虫的 组织亲和性和随后的心脏病理学。长期目标是利用对宿主的这种理解- 寄生虫的相互作用，以开发精确的心脏诊断，并改善这种阴险的治疗选择。 和使人衰弱的感染了解人体心肌代谢在T调节中的作用。cruzi 感染将以两个特定目的进行测试：1）评估T.克氏病感染者 心肌细胞; 2）测定T.克氏感染目标1将 采用由申请人建立的基于质谱的脂质组学工作流程来定量数百种脂质组学。 生物学相关的脂质在6天的T。诱导多能干细胞衍生的克氏感染周期 心肌细胞（iPS-CM）。培养基和细胞裂解物中的显著失调的脂质将被抑制。 使用单个稳定同位素标记的脂质标准品进行验证，以确定重标记的 组分进入T. Cruzi，在宿主细胞裂解后收获。目标2将采用 首次在心脏中筛选人类代谢相关基因的CRISPR相互参照（CRISPRi） 肌细胞，以评估影响T. cruzi复制和维护。 这些实验将利用带有用聚焦的指导RNA转导的CRISPRi机制的iPS-CM 来敲除人类代谢基因。用绿色荧光蛋白（GFP）标记的T. cruzi，我们将对具有高、正常和低寄生虫负荷的iPS-CM群体进行测序。这些 实验将揭示每个复制表型中的相关基因敲除靶点， 转化为感染的关键心脏调节因子。这种方法是创新的，因为它结合了最先进的 脂质组学和基于CRISPR的筛选技术，以研究宿主-病原体的界面 在生理学相关心脏模型中的代谢。这项研究意义重大，因为它将 为T.克鲁兹，这将 为开发精确的心脏诊断和新的循证疗法提供了基础。