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.

项目摘要/摘要 恰加斯病是由克氏锥虫寄生虫引起的，这种寄生虫慢性感染心肌细胞， 在宿主的一生中导致严重的心脏病理。这是一个根本性的差距 了解克氏毛滴虫对人体心肌的亲和力。对非心肌细胞和啮齿动物的研究 暗示脂代谢是克氏锥虫细胞内维持的一个重要因素，但数据特定 缺乏对心肌细胞代谢和寄生虫-宿主相互作用产生的脂类产物。 将焦点转移到心脏组织的作用上，对于理解人类克氏锥虫的自然历史至关重要。 感染。本研究的目的是确定人类心肌细胞特有的代谢因子， 是感染克鲁兹毛滴虫所必需的。中心假设是特定的心脏脂质或脂质代谢物 克鲁兹毛滴虫细胞内复制和维持的必要营养来源，导致这种寄生虫 组织亲和力和随后的心脏病理学。我们的长期目标是利用对宿主的这种理解- 寄生虫相互作用开发精确的心脏诊断并改进这种隐匿性疾病的治疗选择 和衰弱的感染。了解人体心肌代谢在克氏毛滴虫调控中的作用 感染测试将有两个特定的目的：1)评估感染克氏毛滴虫的脂体微扰性 心肌细胞和2)确定人类心脏代谢调节器的毛滴虫感染。目标1将 采用由申请人建立的基于质谱学的脂肪组学工作流程来量化数百种 诱导多能干细胞来源的克氏毛虫感染周期中生物学相关的脂质 心肌细胞(iPS-CMS)。培养液和细胞裂解物中显著失调的脂类 用单个稳定同位素标记的脂类标准验证以确定重标记物的掺入 寄主细胞裂解后收获的克鲁兹毛虫体内关键脂类代谢途径的成分。AIM 2将聘请一名 人类心脏代谢相关基因的首次CRISPR相互参照(CRISPRi)筛查 以评估影响克氏毛滴虫复制和维持的生理相关宿主因素。 这些实验将利用带有CRISPRi机制的iPS-CMS，该机制通过聚焦的引导RNA进行转导 克隆人类代谢基因的文库。用绿色荧光蛋白(GFP)标记的T. CRUZI，我们将对含有高、正常和低寄生虫负担的iPS-CM群体进行排序。这些 实验将揭示每个复制表型中的相关基因敲除靶点，以提供洞察力 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技术 脂质组学和CRISPR筛查技术研究宿主-病原菌的界面 生理相关心脏模型中的新陈代谢。这项拟议的研究意义重大，因为它将 为克氏毛滴虫心脏趋向性中脂代谢的特定方面提供生物学证据，这将 为开发精确的心脏诊断和新的循证疗法提供基础。