Characterizing the unique endocytic organelle of Trypanosoma cruzi

表征克氏锥虫独特的内吞细胞器

• 批准号: 9808880
• 负责人: RONALD DREW ETHERIDGE
• 金额: $ 18.85万
• 依托单位: UNIVERSITY OF GEORGIA
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-05-21 至 2021-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9808880
• 关键词: 3-Dimensional; Age; Americas; Amino Acids; Basic Science; Belief; Biology; Biotin; Blood Cells; Bovine Serum Albumin; CRISPR/Cas technology; Catabolism; Cell membrane; Cells; Cellular Structures; Cessation of life; Chagas Disease; Clinical; Complex; Conflict (Psychology); Consumption; Cytoplasm; Cytosol; Diet Habits; Digestion; Disease; Eating; Endocytosis; Endoplasmic Reticulum; Ensure; Environment; Epitopes; Etiology; Family; Genes; Genetic; Glean; Goals; Golgi Apparatus; Growth; Harvest; Immune system; Infection; Insect Vectors; Insecta; Investigation; Knock-out; Knowledge; Label; Leishmania; Life; Life Cycle Stages; Ligase; Lysosomes; Mass Spectrum Analysis; Mediating; Metabolic Control; Mitochondria; Molecular; Molecular Analysis; Nutrient; Organelles; Parasites; Parasitic infection; Pathogenicity; Populations at Risk; Positioning Attribute; Proliferating; Proteins; Resources; Role; Specificity; Structure; Surface; Techniques; Technology; Therapeutic; Time; Transferrin; Trypanosoma brucei brucei; Trypanosoma cruzi; Tubular formation; Vesicle; Work; acute infection; base; chronic infection; electron tomography; experimental study; extracellular; feeding; fighting; obligate intracellular parasite; parasitism; pathogen; pathogenic bacteria; protein degradation; reconstruction; tool; uptake; virtual

PROJECT SUMMARY The etiological agent of Chagas disease, Trypanosoma cruzi, is an obligate intracellular parasite that infects an estimated 10 million people in the Americas, with an at-risk population of 70 million. While the acute infection by the parasite is effectively controlled by the immune system, a chronic infection can persist for the lifetime of the host. Despite its recognition as the highest impact parasitic infection of the Americas, Chagas disease remains underreported, understudied and underfunded. Basic research into the biology of T. cruzi has been previously hindered by a lack of efficient genetic tools, but the advent of CRISPR/Cas9 gene editing technology has cleared the way for more in-depth molecular analyses. The primary interface between T. cruzi and its mammalian hosts is at the level of the replicative intracellular amastigotes in the cytoplasm of infected host cells. Trypanosoma cruzi is one of only a few protozoan pathogens that live and replicate directly in the cytosol of nucleated cells. Of the (mostly bacterial) pathogens that can inhabit the host cytosol, many have been shown to actively induce host cytosolic protein degradation in order to obtain energy and amino acids which are normally limiting in the cytoplasm. A long-standing mystery is how T. cruzi is able to extract necessary nutrients from this impoverished environment? Unlike other parasites, T. cruzi has the ability to endocytose and digest host cytosolic material via a long tubular invagination (cytopharynx) starting at a surface plasma membrane pore (cytostome). This structure, referred to here as the cytostome/cytopharynx or CSP complex, is present only in replicating forms of the T. cruzi and disassembles during the transition to trypomastigote stages. The CSP structure, which has been examined extensively using electron tomography techniques, has resisted molecular analysis, as the protein components comprising it have remained elusive. We have recently identified the first protein targeted solely to the CSP structure, CP1, and our preliminary work has shown that this protein colocalizes with endocytosed cargo in the CSP. In this proposal, we will use CP1 to carry out the first in-depth characterization of the T. cruzi CSP. We will fuse CP1 to the BioID biotin ligase and perform proximity labeling of the CSP to identify its protein components. A selected subset of the identified components will be validated by endogenous tagging and assessed for their role in parasite feeding, replication and survival. Additionally, we will extend our analysis of the host cell components taken up in the CSP to determine the selectively with respect to host proteins or organelles during intracellular amastigote replication. Completion of this study will allow us to begin analyzing in detail a unique, but crucial, aspect of T. cruzi parasitism which previously resisted in-depth investigation.

项目摘要 查加斯病的病原体克氏锥虫是一种专性细胞内寄生虫， 据估计，美洲有1000万人，其中7000万人处于危险之中。虽然急性感染 由寄生虫有效地控制免疫系统，慢性感染可以持续一生， 主持人尽管它被认为是美洲影响最大的寄生虫感染， 仍然没有得到充分的报道、研究和资助。对T.克鲁兹一直是 以前由于缺乏有效的遗传工具而受到阻碍，但CRISPR/Cas9基因编辑的出现 技术为更深入的分子分析扫清了道路。T. cruzi 和其哺乳动物宿主是在水平的复制细胞内无鞭毛体在细胞质中的感染 宿主细胞克氏锥虫是仅有的几种直接在环境中生活和复制的原生动物病原体之一。 有核细胞的胞浆。在可以栖息在宿主细胞质中的病原体（大多数是细菌）中， 已显示出主动诱导宿主胞质蛋白质降解以获得能量和氨基酸 通常限制在细胞质中。一个长期存在的谜团是T。cruzi能够提取 从这贫瘠的环境中获取必需的养分与其他寄生虫不同，T.克鲁兹有能力 通过从表面开始长管状内陷（细胞咽）内吞和消化宿主胞质物质 质膜孔（细胞口）。这种结构，在这里被称为细胞口/细胞咽或CSP 复合体，只存在于T. cruzi和拆卸在过渡到 锥鞭毛体阶段CSP结构，已被广泛使用电子断层扫描检查 技术，一直抵制分子分析，因为组成它的蛋白质成分仍然难以捉摸。 我们最近发现了第一个仅针对CSP结构的蛋白质CP 1，以及我们的初步工作 已经表明这种蛋白质与CSP中的内吞货物共定位。在本提案中，我们将使用CP1来 对T. cruzi CSP。我们将CP1与BioID生物素连接酶融合， 对CSP进行邻近标记以鉴定其蛋白质组分。已识别的 成分将通过内源性标记进行验证，并评估它们在寄生虫摄食、复制和繁殖中的作用。 和生存此外，我们将扩展我们对CSP中宿主细胞组分的分析， 确定选择性相对于宿主蛋白质或细胞器在细胞内无鞭毛体复制。 这项研究的完成将使我们开始详细分析T. cruzi 寄生，以前抵制深入调查。