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.

抽象的 细胞机械传导对于检测环境变化和压电通道非常重要， 2010 年发现的，在哺乳动物细胞的多个过程中发挥着至关重要的作用，例如 血管发育和功能、肺呼吸和感觉转导。他们的保护在 单细胞生物及其在 Ca2+ 信号传导中的功能是一个尚未开发的研究领域。 Ca2+是一种强效 用于控制刺激特定生物反应的各种下游效应器的信号。我们和 其他人多年前就发现，与宿主细胞的接触会引发宿主细胞胞质 Ca2+ 的增加。 克氏锥虫（恰加斯病的病原体）的感染性锥鞭毛体阶段。预防 细胞内 Ca2+ 螯合剂的这种 Ca2+ 增加阻止了宿主细胞的侵袭。 Ca2+ 在克氏锥虫中的作用 还提出了复制和分化。 Ca2+ 增加的机制 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 力传导过程。压电通道的刺激优先导致 Ca2+，并且在某些情况下 Na+和Ca2+进入细胞的情况。 T. cruzi 中存在两个旁系同源物 TcPiezo1 和 TcPiezo2。使用 通过 CRISPR/Cas9 技术，我们在 C 端标记了 TcPiezo1，并将其定位到 T. cruzi 血浆 膜和细胞内斑点。我们还获得了TcPiezo1-KO突变体，该突变体在宿主中存在缺陷 细胞侵袭和细胞内复制。我们建议研究两个压电通道在 Ca2+ 中的作用 克氏锥虫宿主细胞入侵和复制过程中的信号传导。我们的假设是 T. cruzi 压电通道 将在质膜上作为 Ca2+ 进入通道发挥作用，从而激活信号级联 最终导致特定的寄生虫功能，例如入侵和分化的激活。旨在 研究 T. cruzi 感染阶段的胞质 Ca2+ 浓度波动，我们建议使用 除了传统的化学指示剂外，还包括基因编码的 Ca2+ 指示剂 (GECI)。 GECI 是 强大的工具，可以对确定的细胞和区室进行无创成像。使用这些新 工具将有助于直接实时观察 T. cruzi 宿主细胞入侵和 复制，调查压电通道在这些变化的产生中的作用，以及研究 未来工作中其他潜在的 Ca2+ 作用。我们建议生成 T. cruzi 不同阶段的表达 GECI 靶向其细胞质和线粒体，以研究活寄生虫中 Ca2+ 的动态 探索锥鞭毛体入侵宿主细胞期间和过程中对 Ca2+ 信号传导的需求 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。克鲁兹 压电通道可能具有谱系特定特征，这与那些不同 哺乳动物细胞压电通道，他们的研究可能会带来对 T. cruzi 生物学和 新目标的潜在识别。