Genetically Encoded Calcium Indicators in Toxoplasma gondii

弓形虫中的基因编码钙指标

• 批准号: 8786763
• 负责人: Silvia N Moreno
• 金额: $ 22.35万
• 依托单位: UNIVERSITY OF GEORGIA
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-07-01 至 2016-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8786763
• 关键词: Animals; Binding; Calcium; Calcium Signaling; Calcium ion; Calmodulin; Candidate Disease Gene; Cell membrane; Cell physiology; Cells; Cessation of life; Chelating Agents; Cryptosporidium; Cytosol; Data; Disease; Endoplasmic Reticulum; Equilibrium; Eukaryota; Fluorescence; Fluorescent Dyes; Genes; Genetic; Genome; Green Fluorescent Proteins; Heart failure; Homeostasis; Human; ITPR1 gene; Image; Infection; Inositol; Ionophores; Label; Life; Life Cycle Stages; Lytic Phase; Measurement; Measures; Mediating; Medical; Methods; Microscopy; Monitor; Mutagenesis; Neurons; Organelles; Orthologous Gene; Parasites; Pathway interactions; Peptides; Performance; Physiological; Plasmodium; Protein Engineering; Protein Secretion; Regulation; Resolution; Role; Ryanodine; Second Messenger Systems; Signal Pathway; Signal Transduction; System; Time; Toxoplasma; Toxoplasma gondii; Vacuole; Validation; Variant; Widespread Disease; base; calcium indicator; cell motility; extracellular; interest; mutant; pathogen; public health relevance; receptor; response; second messenger; sensor; tool

DESCRIPTION (provided by applicant): Fluctuations of the cytosolic free calcium (Ca2+) concentration regulate a variety of cellular functions in all eukaryotes. However, if Ca2+ elevates over the physiological level it becomes damaging to the cell. Cells contain a sophisticated set of mechanisms to balance the cytosolic Ca2+ levels and the signals that elevate Ca2+ in the cytosol are compensated by mechanisms that reduce it. Alterations in these Ca2+-dependent homeostatic mechanisms are the cause of many prominent diseases in humans, as for example heart failure or neuronal death. Apicomplexan parasites include a number of pathogens of medical and veterinary relevance. The role of Ca2+ regulation and homeostasis in the infection cycle of Toxoplasma gondii has been very well documented. There is also some information on the role of Ca2+ in the life cycle of Plasmodium spp. In Toxoplasma, Ca2+ signaling is involved in the stimulation of microneme secretion, gliding motility, conoid extrusion and invasion. However, the information on how this happens mechanistically and the molecules involved is fragmented or missing. In addition, the vast majority of the known Ca2+-related genes remain uncharacterized. An interesting case is that the presence of experimental evidence for an inositol 1,4,5- trisphosphate (IP3)-dependent Ca2+ response in Plasmodium spp. and T. gondii, but no IP3 (IP3R) or ryanodine (RyR) receptor orthologs have been identified in either of those genomes. Our own preliminary data supports the presence of a highly regulated mechanism for Ca2+ entry but the candidate genes for these regulatory molecules are not known. Several potential Ca2+ channels are present in T. gondii but not in Plasmodium spp. or Cryptosporidium spp. genomes. The available information on Ca2+ storage and function in Apicomplexans, although fragmented, point toward the presence of unique Ca2+-mediated pathways in these parasites. The information on the role of Ca2+ in the lytic cycle of T. gondii has been obtained using indirect methods such as labeling of extracellular parasites with fluorescent dyes or using intracellular Ca2+ chelators and/or ionophores. Our tools will allow performing direct real-time observations of Ca2+ changes during T. gondii gliding motility, conoid extrusion, microneme secretion and host-cell invasion and egress. We propose to generate T. gondii tachyzoites expressing Genetically Encoded Ca2+ Indicators (GECI) targeted to different cellular compartments to reveal the dynamics of Ca2+ in live parasites and explore the requirements for Ca2+ signaling in host-cell invasion and egress. Toxoplasma is the ideal system because of its genetic tractability, the easiness of isolating clonal lines and its clear-cut response to Ca2+ making validation of these tools feasible. These cells will allow performing direct measurements and will make feasible studies in intracellular tachyzoites. We will be able to study the role of Ca2+ in host-cell egress and physiological Ca2+ fluxes between different Ca2+ stores.

描述（由申请人提供）： 胞质游离钙 (Ca2+) 浓度的波动调节所有真核生物中的多种细胞功能。然而，如果 Ca2+ 升高超过生理水平，就会对细胞造成损害。细胞包含一套复杂的机制来平衡胞质 Ca2+ 水平，并且胞质中升高 Ca2+ 的信号会通过降低 Ca2+ 的机制得到补偿。这些 Ca2+ 依赖性稳态机制的改变是人类许多突出疾病的原因，例如心力衰竭或神经元死亡。顶复门寄生虫包括许多具有医学和兽医相关性的病原体。 Ca2+ 调节和稳态在弓形虫感染周期中的作用已得到充分记录。还有一些关于 Ca2+ 在疟原虫属生命周期中的作用的信息。在弓形虫中，Ca2+信号传导参与刺激微线体分泌、滑行运动、圆锥体挤出和入侵。然而，关于这是如何发生的机制以及所涉及的分子的信息是支离破碎或缺失的。此外，绝大多数已知的 Ca2+ 相关基因仍未被表征。一个有趣的案例是，疟原虫属中存在肌醇 1,4,5-三磷酸 (IP3) 依赖性 Ca2+ 反应的实验证据。和弓形虫，但在这两个基因组中均未发现 IP3 (IP3R) 或兰尼碱 (RyR) 受体直系同源物。我们自己的初步数据支持 Ca2+ 进入的高度调控机制的存在，但这些调控分子的候选基因尚不清楚。弓形虫中存在几种潜在的 Ca2+ 通道，但疟原虫属中不存在。或隐孢子虫属。基因组。关于顶复门中 Ca2+ 储存和功能的可用信息，尽管不完整，但表明这些寄生虫中存在独特的 Ca2+ 介导的途径。关于 Ca2+ 在弓形虫裂解周期中的作用的信息已通过间接方法获得，例如用荧光染料标记细胞外寄生虫或使用细胞内 Ca2+ 螯合剂和/或离子载体。我们的工具将允许直接实时观察刚地弓形虫滑行运动、圆锥体挤出、微线体分泌和宿主细胞入侵和流出过程中的 Ca2+ 变化。我们建议生成表达针对不同细胞区室的基因编码 Ca2+ 指示剂 (GECI) 的弓形虫速殖子，以揭示活寄生虫中 Ca2+ 的动态，并探索宿主细胞入侵和流出中 Ca2+ 信号传导的要求。弓形虫是理想的系统，因为它具有遗传易处理性、易于分离克隆系及其对 Ca2+ 的明确反应，使得这些工具的验证变得可行。这些细胞将允许进行直接测量，并将在细胞内速殖子中进行可行的研究。我们将能够研究 Ca2+ 在宿主细胞流出和不同 Ca2+ 储存之间的生理 Ca2+ 通量中的作用。