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 细胞机械转移对于检测环境变化和压电通道很重要， 这是在2010年发现的，在哺乳动物细胞的多个过程中具有至关重要的作用，例如 血管发育和功能，肺呼吸和感觉转导。他们的保护 单细胞生物及其在Ca2+信号传导中的功能是未开发的研究领域。 CA2+是有效的 信号用于控制刺激特定生物学反应的各种下游效应子。我们和 其他人几年前发现，与宿主细胞的接触会触发胞质Ca2+的增加 Cruzi锥虫的感染性锥虫阶段，Crypanosoma，这是Chagas疾病的病因学剂。预防 这种Ca2+随着细胞内Ca2+螯合剂的增加而增加，可阻止宿主细胞侵袭。 Ca2+在T. Cruzi中的角色 还提出了复制和分化。 Ca2+增加的机制增加 从未发现锥虫与宿主细胞的接触，但其接触依赖性表明 机械转移过程。压电通道的刺激优先导致Ca2+，在某些方面 病例Na+和Ca2+进入细胞。 T. Cruzi出现了两个旁系同源物TCPIEZO1和TCPIEZO2。使用 CRISPR/CAS9技术我们具有C端标记的TCPIEZO1，并将其定位到T. Cruzi等离子体 膜和细胞内斑。我们还获得了TCPIEZO1-KO突变体，这些突变体在宿主中不足 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 在克鲁兹的宿主细胞侵袭和复制期间的信号传导。我们的假设是Cruzi Piezo通道 将在质膜上起作用，作为Ca2+进入通道，将激活信号级联 在特定的寄生虫功能中达到顶点，例如激活入侵和分化。目的 在T. Cruzi的感染阶段研究胞质Ca2+浓度波动，我们建议使用 除传统的化学指标外，遗传编码的Ca2+指标（GECIS）。 Gecis是 强大的工具，可以对定义的单元和隔室进行无创成像。这些新的使用 工具将促进在T. cruzi宿主细胞入侵和 复制，对压电通道在这些变化产生中的作用的研究以及研究 未来工作中其他潜在的CA2+角色。我们建议生成Cruzi的T. Cruzi不同阶段表达 GECI针对其细胞质和线粒体，以研究活寄生虫和 探索锥虫和期间宿主细胞入侵期间Ca2+信号传导的要求 杂物的复制和分化，以及压电通道在这些过程中的作用。 T. Cruzi 压电通道可能具有特定特定特征，这与 哺乳动物细胞压电通道，他们的研究可能导致对克鲁兹生物学的新见解和 新目标的潜在识别。