The Toxoplasma apicoplast and calcium signaling

顶复门寄生虫包括许多具有医学和兽医重要性的病原体， 长期目标是鉴定这些微生物中的新分子和途径， 化疗、诊断应用或疫苗。 这些寄生虫中的许多依赖于在残余质体中发生的生物合成途径来生存 称为顶质体。大量的这些合成代谢途径对寄生虫的生活至关重要。 我们的实验室最近发现了一个双孔通道（TPC），它定位于顶质体膜， 对刚地弓形虫的生长至关重要。双孔通道属于电压门控离子超家族 通道，其特征在于存在两组六跨膜结构域（TMD）。这是 这是一个重要的发现，因为它揭示了一个完全未开发的问题，即像Ca2+这样的离子的作用， 对细胞器的必需合成代谢酶的活性以及这如何影响寄生虫适应性的影响。在 此外，更重要的是，离子通道是许多治疗上有用的药剂的靶标， 作为治疗靶点，甚至作为抗寄生虫剂，仍然显著开发不足。 顶质体是次级内共生过程的产物，是一种被 细胞膜来源于蓝细菌，是质体、内共生菌和 宿主内体区室。金属离子对三分之一以上的活性起着至关重要的作用， 酶和考虑到大量的必需活动存在于顶质体，它很可能是离子 会在细胞器中受到高度调控在顶质体中还没有离子转运体或通道的报道 到现在 这使得我们最近发现的一个位于顶质体的双孔通道成为一个独特的发现， 这也是开发新疗法的一个意想不到的机会。TPC在动物和哺乳动物中均存在， 在植物细胞中，它们定位于酸性细胞器，如内体、溶酶体和液泡。TPCs 在哺乳动物细胞中作为Ca2+通道起作用，并被第二信使烟酸激活 腺嘌呤二核苷酸磷酸（NAADP）。弓形虫TPC定位于质体是唯一的， Apicomplexa和突出了一个新的和潜在的具体作用的细胞器的多种功能。 我们让T沉默了弓形虫TgTPC基因，发现突变体显示出严重的生长缺陷， 通过与功能通道互补来拯救。我们的假设是TPC是一种非选择性的 在电压门控时能够传导阳离子的通道，但在细胞的环境中， 偏好Ca2+。这一假设意味着顶质体含有Ca2+，初步研究结果支持这一观点。 证据我们提出了一个模型，其中顶质体通过TPC和Ca 2+与其他细胞通讯， 细胞器通过其他潜在的Ca2+通道。将研究另外两个候选Ca2+通道。 我们的目标是使用复杂的生物物理学方法来定义TgTPC的生理功能。 在几个模型系统的范围内的方法。我们将联合收割机与活细胞研究相结合， 确定它们的活性如何影响顶质体和其他细胞内 细胞器和寄生虫的适应性。

Apicomplexan parasites include a number of pathogens of medical and veterinary importance and our long- term goal is to identify novel molecules and pathways in these microorganisms that could be targets for chemotherapy, diagnostic applications, or vaccines. Many of these parasites rely for their survival on biosynthetic pathways that occur in a remnant plastid known as the apicoplast. A large number of these anabolic pathways are essential for the life of the parasite. Our laboratory recently found a two-pore channel (TPC) that localizes to the apicoplast membrane(s) and is essential for growth of Toxoplasma gondii. Two-pore channels belong to the superfamily of voltage-gated ion channels and are characterized by the presence of two sets of six-transmembrane domains (TMDs). This was an important discovery because it exposed a completely untapped question, which is the role of ions like Ca2+ on the activity of essential anabolic enzymes of the organelle and how this impacts parasite fitness. In addition, and more importantly, ion channels are targets of many therapeutically useful agents and they remain significantly under-exploited as therapeutic targets, even more so as antiparasitic agents. The apicoplast, the product of a secondary endosymbiosis process, is an organelle surrounded by membranes derived from the cyanobacterium that gave origin to the plastid, the endosymbiotic alga, and the host endosomal compartment. Metal ions play essential roles for the activity of more than one third of all enzymes and considering the large number of essential activities present in the apicoplast, it is likely that ions will be highly regulated in the organelle. No ion transporters or channels have been reported in the apicoplast until now. This makes our recent discovery of a two-pore channel localized to the apicoplast a unique finding and also an unexpected opportunity for the development of new therapies. TPCs are found in both animal and plant cells where they localize to acidic organelles such as endosomes, lysosomes and vacuoles. TPCs function as Ca2+ channels in mammalian cells and are activated by the second messenger nicotinic acid adenine dinucleotide phosphate (NAADP). The Toxoplasma TPC localization to the plastid is unique to Apicomplexa and highlights a novel and potentially specific role for the organelle multiple functions. We silenced the T. gondii TgTPC gene and found that the mutants display a severe growth defect that can be rescued by complementation with a functional channel. Our hypothesis is that the TPC is a non-selective channel that upon voltage gating is able to conduct cations but in the environment of the cell it probably prefers Ca2+. This hypothesis implies that the apicoplast contains Ca2+, an idea supported by preliminary evidence. We propose a model in which the apicoplast through the TPC and Ca2+ communicates with other organelles via other potential Ca2+ channels. Two other candidate Ca2+ channels will be studied. Our goal is to define the physiological function of TgTPC using sophisticated biophysical approaches in the context of several model systems. We will combine this with live cell studies to determine how their activity influences the physiology of the apicoplast and other intracellular organelles and the fitness of the parasite.