Genetically Encoded Calcium Indicators in Toxoplasma gondii

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的各种细胞功能。然而，如果Ca2+升高超过生理水平，它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，胞质中升高Ca2+的信号被降低Ca2+的机制所补偿。这些Ca2+依赖的体内平衡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。顶复合体寄生虫包括一些与医学和兽医相关的病原体。Ca2+调节和体内平衡在弓形虫感染周期中的作用已经得到了很好的证明。关于Ca2+在弓形虫生命周期中的作用也有一些研究。在弓形虫中，Ca2+信号参与刺激微素分泌、滑行运动、锥体挤压和侵袭。然而，关于这一过程的机制和相关分子的信息是碎片化或缺失的。此外，绝大多数已知的Ca2+相关基因仍未被表征。一个有趣的例子是，在疟原虫和弓形虫中存在肌醇1,4,5-三磷酸（IP3）依赖性Ca2+反应的实验证据，但在这两种基因组中都没有发现IP3 （IP3R）或ryanodine （RyR）受体同源物。我们自己的初步数据支持Ca2+进入的高度调节机制的存在，但这些调节分子的候选基因尚不清楚。几个潜在的Ca2+通道存在于弓形虫中，而不存在于疟原虫或隐孢子虫基因组中。关于顶复体中Ca2+储存和功能的现有信息，虽然碎片化，但指向这些寄生虫中存在独特的Ca2+介导途径。关于Ca2+在弓形虫溶解循环中的作用的信息已经通过间接方法获得，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。我们的工具将允许在弓形虫滑翔运动、圆锥体挤压、微素分泌和宿主细胞入侵和输出过程中进行直接实时观察Ca2+的变化。我们提议产生表达基因编码Ca2+指标（GECI）的弓形虫速殖子，靶向不同的细胞区室，以揭示活寄生虫体内Ca2+的动态，并探索Ca2+信号在宿主细胞入侵和出口中的要求。弓形虫是理想的系统，因为它的遗传易感性，克隆系的容易分离和对Ca2+的明确反应使得这些工具的验证是可行的。这些细胞将允许进行直接测量，并将对细胞内速殖子进行可行的研究。我们将能够研究Ca2+在宿主细胞出口的作用和不同Ca2+储存之间的生理Ca2+通量。

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.