The Toxoplasma apicoplast and calcium signaling

Apicomplexan寄生虫包括一些具有医学和兽医重要性的病原体，以及我们的长期... 学期目标是确定这些微生物中可能成为靶点的新分子和途径。 化疗、诊断应用或疫苗。 这些寄生虫中的许多依靠残存的质体中的生物合成途径生存。 被称为质外体。大量这样的合成代谢途径对寄生虫的生命是必不可少的。 我们实验室最近发现了一个位于质外体膜上的双孔通道(S)，它是 对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，其特征是存在两组六跨膜结构域(TMD)。这是 一个重要的发现，因为它揭示了一个完全没有被利用的问题，那就是像钙离子这样的离子的作用 关于细胞器中必需的合成代谢酶的活性以及这如何影响寄生虫的适合性。在……里面 此外，更重要的是，离子通道是许多治疗有用的药物的靶标，它们 作为治疗目标仍未得到充分利用，作为抗寄生虫剂更是如此。 顶体是次级内生共生过程的产物，是一个细胞器，周围有 从蓝藻衍生的膜，它起源于叶绿体、内生共生藻类和 宿主内胚体隔室。金属离子对三分之一以上的生物活性起着至关重要的作用。 酶，考虑到质外体中存在大量的基本活动，很可能离子 将在细胞器中受到高度调控。在质外体中没有离子转运体或离子通道的报道。 直到现在。 这使得我们最近发现的位于质外体上的两孔通道是一个独特的发现和 这也是开发新疗法的一个意想不到的机会。在动物和动物体内都发现了TPC 定位于酸性细胞器的植物细胞，如内小体、溶酶体和液泡。TPC 在哺乳动物细胞中起钙通道的作用，并被第二信使烟酸激活 腺嘌呤二核苷酸磷酸(NAADP)。弓形虫TPC定位于叶绿体是独一无二的 Apicomplexa，并强调了细胞器多种功能的新的和潜在的特定作用。 我们沉默了弓形虫TgTPC基因，发现突变株表现出严重的生长缺陷，可以 通过功能渠道的补充而被拯救。我们的假设是，TPC是非选择性的 在电压门控时能够传导阳离子的通道，但在电池的环境中，它可能 更喜欢钙。这一假说表明，质外体含有钙，这一观点得到了初步的支持 证据。我们提出了一个模型，在该模型中，顶生质体通过TPC和钙离子与其他 细胞器通过其他潜在的钙通道。另外两个候选的钙通道将被研究。 我们的目标是利用复杂的生物物理学来确定TgTPC的生理功能 在几个模型系统的背景下的方法。我们将把这与活细胞研究结合起来 确定它们的活性如何影响质外体和其他细胞内的生理 细胞器和寄生虫的适合性。

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.