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

项目摘要/摘要 弓形虫具有食源性、人畜共患病和先天性传播途径的能力，是一种 顶端复合体寄生虫，可在免疫受损的人群中引起严重的传染病。 市面上有几种抗生素可以用来治疗弓形虫感染。然而，它们的强烈副作用 致畸性限制了它们在某些人群中的使用。抑制基本营养代谢 这种寄生虫的特异性将定义新的药物靶点，并帮助开发新药来管理T。 弓形虫感染。 我们先前的研究表明，弓形虫编码了一个抗氯喹疟原虫的同源基因 转运蛋白(TgCRT)，并定位于消化液泡，称为液泡室/类植物 液泡(VAc/PLV，以下简称VAc)。我们的初步数据发现，缺乏TgCRT的寄生虫使他们的 Vacs~15倍。这些结果导致了我们的中心假设：(1)TgCRT作为一种多特异性转运蛋白 调节弓形虫VAC的生理和功能；(2)TgCRT与多药协同 VAC中的抗性转运蛋白介导营养输出，从而调节微环境 在VAC内。对于这些假说，我们已经揭示了肿胀的VAC破坏了寄生虫的 内溶体系统，并降低VAC相关蛋白酶的转录本和蛋白质丰度。我们 发现在TgCRT缺失突变体中，抑制VAC内的蛋白分解会缩小其肿胀表型。我们 还发现了弓形虫多药耐药转运蛋白(TgMDR)的同源基因是 定位于VAC，当TgCRT被激活时，寄生虫的转录水平显著增加 缺席。最后，我们确定TgCRT通过在酵母中异源表达TgCRT来运输氯喹， 提示TgCRT确实是一个功能转运体，并提供了一个顺从的系统来理解 TgCRT和其他VAC定位转运蛋白的天然功能。在我们令人信服的初步研究的指导下， 我们提出了三个具体目标来表征TgCRT和TgMDR的固有功能，以及它们是如何 作为营养转运体，VAC在功能上相互作用，共同调节生理和功能：(1) 量化VAC内的生理环境；(2)通过以下方法测量小营养溶质的传输 确定TgCRT和TgMDR在VAC调节中的功能关系 形态和生理学。 我们提议的研究将通过表征分子机制来广泛影响该领域 弓形虫调节其消化液泡的生理和功能。我们的学习也会对我们有所帮助 理解TgCRT和TgMDR的原生功能，这些知识可以推广到扩展 了解它们在其他顶端复合体寄生虫和生物体中的同源基因。

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.