Regulation of the physiology and function of the digestive vacuole in Toxoplasma gondii

项目总结/摘要 弓形虫具有食源性、人畜共患性和先天性传播途径的能力， 顶复体寄生虫，可在免疫功能低下的人群中引起严重的传染病。 一些抗生素可用于治疗弓形虫感染。然而，它们强烈的副作用 和致畸性限制了它们在某些人群中的使用。基本营养物质代谢的抑制 这种寄生虫的特异性将确定新的药物靶点，并有助于开发新的药物来管理T。 弓形虫感染 我们以前的研究表明，弓形虫编码疟原虫氯喹抗性的直系同源物 TgCRT定位于消化泡中，被称为植物样/植物样蛋白 通气孔（VAC/PLV，以下简称VAC）。我们的初步数据发现，TgCRT缺陷型寄生虫的 VAC ~15倍。这些结果导致了我们的中心假设：（1）TgCRT作为一个多特异性转运蛋白 调节弓形虫的VAC生理和功能，以及（2）TgCRT与多药协同作用 VAC中的抗性转运蛋白样蛋白介导营养输出，从而调节微环境 在VAC。对于这些假设，我们已经揭示了肿胀的VAC破坏了寄生虫的 内溶酶体系统，并降低VAC相关蛋白酶的转录物和蛋白质丰度。我们 发现抑制VAC内的蛋白水解可以缩小TgCRT无效突变体中的肿胀表型。我们 还鉴定了疟原虫多药耐药转运蛋白（TgMDR）的弓形虫直系同源物， 定位于VAC，当TgCRT被激活时，在寄生虫的转录水平上显著增加。 无托叶最后，我们通过在酵母中异源表达TgCRT来确定TgCRT转运氯喹， 这表明TgCRT确实是一种功能性转运蛋白，并提供了一个可靠的系统来了解 TgCRT和其他VAC定位转运蛋白的天然功能。在我们引人注目的初步研究的指导下， 我们提出了三个具体的目标，以表征TgCRT和TgMDR的天然功能，以及它们如何 在功能上相互作用，通过充当营养转运蛋白来调节VAC生理学和功能：（1） 量化VAC内的生理环境;（2）通过以下方式测量小营养溶质的运输： 确定TgCRT和TgMDR在VAC调节中的函数关系 形态学和生理学。 我们提出的研究将通过表征分子机制来广泛影响该领域， 弓形虫调节其消化泡的生理和功能。我们的研究也将有助于 理解TgCRT和TgMDR的固有功能，这些知识可以被概括以扩展 了解它们在其他顶复门寄生虫和生物中的直系同源物。

PROJECT SUMMARY/ABSTRACT Toxoplasma gondii with the ability to use foodborne, zoonotic, and congenital routes of transmission is an apicomplexan parasite that can cause severe infectious disease in the immunocompromised human population. A few antibiotics are commercially available to treat Toxoplasma infections. However, their strong side effects and teratogenicity limit their use in certain human populations. Inhibition of fundamental nutrient metabolism specific to this parasite will define new drug targets and assist the development of novel drugs to manage T. gondii infection. Our previous studies revealed that Toxoplasma encodes an ortholog of Plasmodium chloroquine resistance transporter (TgCRT), and localized it in the digestive vacuole, termed the Vacuolar Compartment/Plant-Like Vacuole (VAC/PLV, VAC hereafter). Our preliminary data found that the TgCRT-deficient parasites swelled their VACs ~15-fold. These results led to our central hypotheses: (1) the TgCRT serves as a polyspecific transporter to regulate the VAC physiology and function in Toxoplasma, and (2) the TgCRT cooperates with a multidrug resistance transporter-like protein in the VAC to mediate nutrient export, thereby adjusting the microenvironment within the VAC. Towards these hypotheses, we have revealed that the swollen VAC disrupts the parasite’s endolysosomal system and decreases transcript and protein abundances of VAC-associated proteases. We discovered that inhibition of proteolysis within the VAC shrinks its swollen phenotype in TgCRT-null mutant. We have also identified that a Toxoplasma ortholog of Plasmodium multidrug resistance transporter (TgMDR) is localized in the VAC and significantly increased at the level of transcription in the parasites when TgCRT is absent. Last, we determined that TgCRT transports chloroquine by heterologous expression of TgCRT in yeast, suggesting that TgCRT is indeed a functional transporter and providing an amenable system to understand the native functions of TgCRT and other VAC-localizing transporters. Guided by our compelling preliminary studies, we propose three specific aims to characterize the native functions of TgCRT and TgMDR, and how they functionally interact together to regulate VAC physiology and function by serving as nutrient transporters: (1) Quantify the physiological environment within the VAC; (2) Measure the transport of small nutrient solutes by CRT; and (3) Determine the functional relationship between TgCRT and TgMDR in the regulation of the VAC morphology and physiology. Our proposed research will broadly impact the field by characterizing the molecular mechanisms by which Toxoplasma parasites regulate the physiology and function of their digestive vacuoles. Our studies will also help comprehend the native functions of TgCRT and TgMDR, and such knowledge can be generalized to expand understanding of their orthologs in other apicomplexan parasites and organisms.