Piezo channels and calcium signaling in Trypanosoma cruzi

克氏锥虫的压电通道和钙信号传导

• 批准号: 10216716
• 负责人: ROBERTO DOCAMPO
• 金额: $ 18.88万
• 依托单位: UNIVERSITY OF GEORGIA
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-03-12 至 2023-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10216716
• 关键词: Area; Biological; Biology; Blood Vessels; Buffers; C-terminal; CRISPR/Cas technology; Calcium Channel; Calcium Signaling; Calcium ion; Cell Compartmentation; Cell membrane; Cells; Cellular Mechanotransduction; Chagas Disease; Characteristics; Chelating Agents; Chemicals; Cytosol; Dependence; Development; Electrophysiology (science); Etiology; Future; Generations; Goals; Growth; Image; Investigation; Knock-out; Lead; Leishmania; Mammalian Cell; Mitochondria; Organism; Orthologous Gene; Parasites; Physiological; Piezo ion channels; Plasmids; Process; Property; Pulmonary Ventilation; Research; Role; Sensory; Signal Transduction; Spottings; Techniques; Time; Trypanosoma; Trypanosoma brucei brucei; Trypanosoma cruzi; Tumor stage; Work; cell motility; environmental change; experimental study; extracellular; genome editing; insight; mechanotransduction; mutant; non-invasive imaging; paralogous gene; prevent; response; temporal measurement; tool

Abstract Cellular mechano-transduction is important for detecting environmental changes and Piezo channels, which were discovered in 2010, have crucial roles in several processes in mammalian cells, such as vascular development and function, pulmonary respiration, and sensory transduction. Their conservation in unicellular organisms and their function in Ca2+ signaling is an untapped area of research. Ca2+ is a potent signal for controlling a variety of downstream effectors that stimulate specific biological responses. We and others discovered years ago that contact with host cells triggers an increase in cytosolic Ca2+ of the infective trypomastigote stage of Trypanosoma cruzi, the etiologic agent of Chagas disease. Preventing this Ca2+ increase with intracellular Ca2+ chelators prevented host cell invasion. A role for Ca2+ in T. cruzi replication and differentiation was also proposed. The mechanism involved in the Ca2+ increase upon contact of trypomastigotes with host cells was never identified but its contact-dependence suggests a mechano-transduction process. Stimulation of Piezo channels preferentially leads to Ca2+ and in some cases Na+ and Ca2+ entry into cells. Two paralogs, TcPiezo1 and TcPiezo2, are present in T. cruzi. Using CRISPR/Cas9 techniques we have C-terminally tagged TcPiezo1 and localized it to the T. cruzi plasma membrane and an intracellular spot. We also obtained TcPiezo1-KO mutants, which are deficient in host cell invasion and intracellular replication. We propose to study the role of both Piezo channels in Ca2+ signaling during T. cruzi host cell invasion and replication. Our hypothesis is that T. cruzi Piezo channels will function at the plasma membrane as Ca2+ entry channels that will activate a signaling cascade culminating in specific parasite functions like activation of invasion and differentiation. With the aim of studying cytosolic Ca2+ concentration fluctuations in the infective stages of T. cruzi we propose to use genetically encoded Ca2+ indicators (GECIs), in addition to the traditional chemical indicators. GECIs are powerful tools that allow the noninvasive imaging of defined cells and compartments. The use of these new tools will facilitate direct real-time observation of Ca2+ changes during T. cruzi host cell invasion and replication, the investigation of the role of Piezo channels in the generation of these changes, and the study of other potential Ca2+ roles in future work. We propose to generate T. cruzi different stages expressing GECIs targeted to their cytosol and to their mitochondria to study the dynamics of Ca2+ in live parasites and explore the requirement for Ca2+ signaling during host cell invasion by trypomastigotes and during replication and differentiation of amastigotes, and the role of Piezo channels in these processes. T. cruzi Piezo channels will likely have lineage specific characteristics, which are different from those of mammalian cells Piezo channels, and their study could lead to new insights into the T. cruzi biology and the potential identification of new targets.

摘要 细胞机械转导对于检测环境变化和压电通道是重要的， 2010年发现的，在哺乳动物细胞的几个过程中起着关键作用， 血管发育和功能、肺呼吸和感觉传导。保护在 单细胞生物及其在Ca 2+信号传导中的功能是一个尚未开发的研究领域。Ca 2+是一种强有力的 用于控制刺激特定生物反应的多种下游效应物的信号。我们和 其他人在几年前发现，与宿主细胞接触会引起细胞内Ca 2+的增加， 克氏锥虫的感染性锥鞭毛体阶段，恰加斯病的病原体。防止 细胞内Ca 2+螯合剂引起的Ca 2+增加阻止了宿主细胞的侵入。Ca ~（2+）在T. cruzi 并提出了复制和分化的观点。Ca 2+增加的机制涉及 锥鞭毛体与宿主细胞的接触从未被发现，但其接触依赖性表明， 机械传导过程压电通道的刺激优先导致Ca 2+，并且在某些情况下， Na+、Ca ~（2+）内流细胞例。TcPiezo 1和TcPiezo 2是T.克鲁兹使用 CRISPR/Cas9技术，我们有C-末端标记的TcPiezo 1，并将其定位到T.克鲁奇等离子体 膜和细胞内点。我们还获得了TcPiezo 1-KO突变体，它们在宿主中是缺陷的， 细胞侵袭和细胞内复制。我们建议研究这两个压电通道在钙离子通道中的作用。 在T. Cruzi宿主细胞的侵袭和复制。我们假设T. cruzi压电通道 将在质膜上作为Ca 2+进入通道起作用，其将激活信号级联 最终导致特定的寄生虫功能，如入侵和分化的激活。目的是 研究了T.我们建议使用cruzi 除了传统的化学指示剂之外，还可以使用基因编码的Ca 2+指示剂（GECIs）。GECI是 强大的工具，允许非侵入性成像的定义细胞和隔室。使用这些新型 工具将有助于直接实时观察T. Cruzi宿主细胞侵袭和 复制，调查压电通道在这些变化的产生中的作用，以及研究 在未来的工作中，其他潜在的Ca 2+作用。我们建议生成T. cruzi不同阶段表达 GECIs靶向其胞质溶胶和线粒体，以研究活寄生虫中Ca 2+的动力学， 探索在锥虫侵入宿主细胞期间以及在宿主细胞内的细胞周期中对Ca 2+信号的需求。 无鞭毛体的复制和分化，以及压电通道在这些过程中的作用。T. cruzi 压电通道将可能具有谱系特异性特征，其不同于压电通道的谱系特异性特征。 哺乳动物细胞压电通道，他们的研究可能会导致新的见解T。克鲁兹生物学和 新目标的潜在识别。