The role of polyphosphate in Toxoplasma gondii

聚磷酸盐在弓形虫中的作用

• 批准号: 10681078
• 负责人: Silvia N Moreno
• 金额: $ 21.28万
• 依托单位: UNIVERSITY OF GEORGIA
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-07-13 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10681078
• 关键词: AIDS-Related Opportunistic Infections; Address; Alkalinization; Bacteria; Binding; Bioinformatics; Calcium; Catalogs; Catalytic Domain; Cells; Cellular Metabolic Process; Cellular Stress; Client; Complex; Cryptosporidiosis; Cryptosporidium; Cyst; Cytolysis; Cytosol; Data; Diatoms; Disease; Electrostatics; Energy-Generating Resources; Enzymes; Eukaryota; Experimental Designs; Future; Gene Expression; Genome; Heat Stress Disorders; Homologous Gene; Human; Immune response; Immunocompromised Host; Infection; Ions; Knowledge; Link; Liquid substance; Lytic Phase; Malaria; Mammalian Cell; Modification; Molecular Chaperones; Morbidity - disease rate; Names; Orthologous Gene; Parasites; Pathogenesis; Pathogenicity; Pathway interactions; Persons; Phase; Plasmodium; Play; Polymers; Polyphosphates; Polyps; Population; Post-Translational Protein Processing; Process; Proteins; Proteome; Regulation; Role; Rupture; Saccharomyces cerevisiae; Signal Transduction; System; Testing; Tissues; Toxoplasma; Toxoplasma gondii; Toxoplasmosis; Vacuole; Vertebrate Biology; Vertebrates; Virulence; Virulence Factors; Yeasts; acute infection; biological adaptation to stress; chronic infection; cohort; forgetting; fungus; human disease; inorganic phosphate; knock-down; member; metal chelator; mortality; mutant; novel; response; scaffold; validation studies

PROJECT SUMMARY/ABSTRACT Apicomplexan parasites cause persistent mortality and morbidity worldwide through diseases like malaria, toxoplasmosis, and cryptosporidiosis. The phylum member Toxoplasma gondii alone infects approximately one third of the world population. The pathogenesis of T. gondii is reliant on its capacity to replicate within host cells and spread to other host cells. The acute infection is ultimately controlled by the host immune response and the parasite establishes a chronic infection characterized by the presence of tissue cysts containing slow replicating bradyzoites. Tissue cyst rupture releases bradyzoites that convert into tachyzoites that replicate fast and if the host is immunocompromised, could cause severe and even fatal tissue damage. We propose to study how polyphosphate (polyP), a “forgotten molecule”, exerts its role in pathogenicity and virulence of T. gondii. PolyP is a ubiquitous polymer of three to hundreds of phosphate residues linked by high-energy phosphoanhydride bonds and can reach millimolar levels in protozoan parasites, while the concentration in host cells is at the micromolar level. T. gondii stores polyP at molar levels in acidocalcisomes which are hydrolyzed upon alkalinization followed by release of Ca2+ into the cytosol. It is puzzling how little is known about the function of polyP in the T. gondii infection cycle and/or virulence. However, characterizing how polyP functions will impact our knowledge of the mechanism by which T. gondii causes disease. Both in bacteria and eukaryotes polyP is involved in a large number of diverse and apparently unrelated functions. However, these apparently unrelated functions may be the result of polyP’s ability to interact with proteins either electrostatically or covalently and we propose to test this for T. gondii. Our hypothesis is that polyP exerts its essential function in the lytic cycle of T. gondii through its interaction with proteins and/or their covalent modification by polyphosphorylation. We propose two strategies to determine how polyP exerts its role in virulence. To define candidate proteins that interact with polyP we will use Thermal Proteome Profiling and expose intact parasites and/or lysates to heat stress to underscore the role of polyP as a protein-like chaperone. We will also use a bioinformatics approach to discover potential proteins that are covalently modified by polyP. We propose to use polyP mutants (that do not make polyP) already available for the characterization of the role of polyP interaction with target proteins. The synthesis of polyP represents a novel target as it is essential, and the enzymes are absent in the mammalian host. The proteins involved in polyP synthesis are conserved in other apicomplexan parasites. The polyP target proteins will be specific for T. gondii virulence pathways and could have orthologs in other apicomplexans. Additionally, findings from this project could have wider impact for vertebrate biology since the mammalian polyP synthesis machinery is unknown and our findings may uncover novel functions and proteins.

项目概要/摘要 顶复门寄生虫通过疟疾、 弓形虫病和隐孢子虫病仅由弓形虫门成员感染。 世界三分之一人口的弓形虫发病机制取决于其在体内复制的能力。 宿主细胞并扩散到其他宿主细胞，急性感染最终由宿主免疫控制。 反应和寄生虫建立以组织囊肿为特征的慢性感染 含有缓慢复制的缓殖子 组织囊肿破裂释放缓殖子，并转化为速殖子。 复制速度快，如果宿主免疫功能低下，可能会导致严重甚至致命的组织损伤。 我们建议研究“被遗忘的分子”多磷酸盐（polyP）如何发挥其致病作用 PolyP 是一种普遍存在的由三至数百个磷酸残基连接的聚合物。 通过高能磷酸酐键，在原生动物寄生虫中可以达到毫摩尔水平，而 宿主细胞中的浓度为微摩尔水平，弓形虫在酸钙体中以摩尔水平储存多聚磷。 碱化后会水解，然后将 Ca2+ 释放到细胞质中，令人费解的是，其含量是如此之少。 然而，polyP 在弓形虫感染周期和/或毒力中的功能是已知的。 PolyP 的功能将如何影响我们对弓形虫致病机制的了解。 在细菌和真核生物中，polyP 参与大​​量不同且明显不相关的过程 然而，这些看似不相关的功能可能是 PolyP 相互作用的结果。 蛋白质通过静电或共价键作用，我们建议对弓形虫进行测试。 PolyP 通过与蛋白质的相互作用在弓形虫的裂解循环中发挥其重要功能 和/或它们通过多磷酸化的共价修饰。 我们提出了两种策略来确定polyP如何发挥其毒力作用来定义候选蛋白质。 与polyP相互作用，我们将使用热蛋白质组分析并将完整的寄生虫和/或裂解物暴露于 热应激来强调 PolyP 作为类蛋白质伴侣的作用，我们还将使用生物信息学。 发现被polyP共价修饰的潜在蛋白质的方法我们建议使用polyP。 突变体（不产生polyP）已经可用于表征polyP与 PolyP 的合成代表了一个新的靶标，因为它是必需的，并且酶不存在。 在哺乳动物宿主中，参与polyP合成的蛋白质在其他apicomplexan中是保守的。 PolyP 靶蛋白对弓形虫毒力途径具有特异性，并且可能具有直向同源物。 此外，该项目的发现可能对脊椎动物生物学产生更广泛的影响。 由于哺乳动物多聚蛋白合成机制尚不清楚，我们的发现可能会揭示新功能 和蛋白质。