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

弓形虫消化液泡生理和功能的调节

• 批准号: 10530610
• 负责人: Zhicheng Dou
• 金额: $ 36.62万
• 依托单位: CLEMSON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-12-10 至 2024-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10530610
• 关键词: Ablation; Acute; Affect; Amino Acids; Antibiotics; Autophagosome; Chloroquine; Chloroquine resistance; Chronic; Communicable Diseases; Consumption; Data; Defect; Development; Digestive Physiology; Disease; Endocytosis; Environment; Exhibits; Family; Future; Genetic Transcription; Glutathione; Goals; Growth; Human; Immune system; Immunocompromised Host; In Vitro; Infection; Ingestion; Knowledge; Learning; Lysosomes; Malaria; Measures; Mediating; Modeling; Molecular; Morphology; Multi-Drug Resistance; Multidrug Resistance Gene; Nutrient; Organelles; Organism; Orthologous Gene; Osmotic Pressure; Oxidation-Reduction; Parasite Control; Parasites; Parasitic infection; Pathogenesis; Peptide Hydrolases; Persons; Phenotype; Physiological; Physiology; Plants; Plasmodium; Plasmodium falciparum; Play; Population; Proteins; Proteolysis; Regulation; Reporting; Repression; Research; Role; Route; Solid; Swelling; System; Teratogens; Testing; Therapeutic Intervention; Time; Toxoplasma; Toxoplasma gondii; Toxoplasmosis; Transcript; Vacuole; Virulence; Work; Yeasts; Zoonoses; acute infection; chronic infection; foodborne; in vivo; innovation; insight; member; mouse model; mutant; new therapeutic target; novel therapeutics; nutrient metabolism; pathogen; proteoliposomes; side effect; solute; therapeutic target; transmission process

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.

项目摘要/摘要 具有使用食源性，人畜共患病和先天性传播途径的弓形虫弓形虫是一种 免疫受损人群中可能引起严重传染病的apicomplexan寄生虫。 商业上可以使用一些抗生素来治疗毒质量感染。但是，它们的副作用很强 变性性限制了它们在某些人群中的使用。抑制基本营养代谢 该寄生虫的特异性将定义新的药物靶标，并帮助开发新的药物来管理T。 Gondii感染。 我们先前的研究表明，毒质量编码氯喹抗性的直系同源 转运蛋白（TGCRT），并将其定位在消化液真空中，称为液泡隔室/植物样 液泡（vac/plv，vac，selter）。我们的初步数据发现，TGCRT缺陷寄生虫膨胀了 VACS 〜15倍。这些结果导致了我们的中心假设：（1）TGCRT用作多性转运蛋白 调节弓形虫的VAC生理学和功能，（2）TGCRT与多药合作 VAC中的抗性转运蛋白样蛋白介导营养出口，从而调节微环境 在VAC内。针对这些假设，我们揭示了肿胀的Vac破坏了寄生虫的 内溶性系统并降低与VAC相关蛋白酶的转录本和蛋白质丰度。我们 发现VAC中抑制蛋白水解会在TGCRT-NULL突变体中收缩其肿胀的表型。我们 还已经确定了疟原虫多药耐药性转运蛋白（TGMDR）的毒品直系同源物为 定位于VAC中，在TGCRT为寄生虫的转录水平上显着增加 缺席的。最后，我们确定TGCRT通过TGCRT在酵母中的异源表达传递氯喹， 表明TGCRT确实是一种功能性转运蛋白，并提供了一个可正常的系统来理解 TGCRT和其他VAC-定位转运蛋白的天然功能。在我们引人入胜的初步研究的指导下 我们提出了三个特定的目的，以表征TGCRT和TGMDR的天然功能，以及它们如何 在功能上相互作用以通过作为营养转运蛋白来调节VAC生理和功能：（1） 量化VAC内的物理环境； （2）测量小营养溶液的运输 CRT; （3）确定在VAC调节中TGCRT和TGMDR之间的功能关系 形态和生理学。 我们提出的研究将通过表征分子机制来广泛影响该领域 弓形虫寄生虫调节其消化液的生理和功能。我们的研究也会有所帮助 理解TGCRT和TGMDR的本地功能，并且可以将这些知识推广到扩展 在其他Apicomplexan寄生虫和生物体中了解它们的直系同源物。