Genetically Encoded Calcium Indicators in Toxoplasma gondii

描述(申请人提供)：在所有真核生物中，细胞内游离钙浓度的波动调节多种细胞功能。然而，如果钙离子升高超过生理水平，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浆中的钙离子水平，而升高胞浆中钙离子的信号被降低钙离子的机制所补偿。这些依赖钙离子的动态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。Apicomplexan寄生虫包括一些与医学和兽医相关的病原体。钙离子调节和动态平衡在弓形虫感染循环中的作用已经得到了很好的证明。也有一些关于钙离子在疟原虫生命周期中的作用的信息。在弓形虫中，Ca~(2+)信号参与刺激微丝分泌、滑动运动、锥体挤出和侵袭。然而，关于这种现象是如何发生的以及所涉及的分子的信息是支离破碎的或缺失的。此外，绝大多数已知的与钙离子相关的基因仍未确定其特征。一个有趣的案例是，在疟原虫中，存在依赖于肌醇1，4，5-三磷酸(IP3)的钙反应的实验证据。和弓形虫，但在这两个基因组中都没有发现IP3(IP3R)或Ryanodine(RyR)受体同源基因。我们自己的初步数据支持存在一种高度调控的钙离子进入机制，但这些调控分子的候选基因尚不清楚。在弓形虫中存在几种潜在的钙通道，而在疟原虫中不存在。或隐孢子虫。基因组。关于复合体中钙离子储存和功能的现有信息，虽然是片断的，但表明在这些寄生虫中存在独特的钙离子介导的途径。有关Ca~(2+)在弓形虫裂解循环中作用的信息是通过间接方法获得的，如用荧光染料标记胞外寄生虫或使用胞内Ca~(2+)螯合剂和/或离子载体。我们的工具将允许对弓形虫滑动运动、锥形挤出、微丝分泌和宿主细胞入侵和外出过程中的钙变化进行直接实时观察。我们建议建立针对不同细胞室的表达基因编码的钙指示物(GECI)的弓形虫速殖子，以揭示活体寄生虫中钙的动态变化，并探索宿主细胞入侵和输出中对钙信号的要求。弓形虫是理想的系统，因为它的遗传易操纵性，容易分离克隆系，以及它对钙离子的明确反应，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，并将在细胞内速殖子中进行可行的研究。我们将能够研究钙离子在宿主细胞出口中的作用以及不同钙库之间的生理钙离子流动。

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.