The role of polyphosphate in Toxoplasma gondii

项目摘要/摘要 Apicomplexan寄生虫通过疟疾等疾病在全球范围内造成持续的死亡和发病， 弓形虫病和隐孢子虫病。仅弓形虫一门成员就可感染约 占世界人口的三分之一。弓形虫的致病机制依赖于其在体内复制的能力 宿主细胞并传播到其他宿主细胞。急性感染最终由宿主免疫控制。 反应和寄生虫建立了一种慢性感染，其特征是组织囊的存在 含有复制缓慢的缓殖子。组织囊肿破裂会释放缓殖子，并转化为速殖子 这种复制速度很快，如果宿主的免疫功能受损，可能会导致严重的甚至致命的组织损伤。 我们建议研究聚磷酸盐(Polyp)，一种“被遗忘的分子”如何在致病中发挥作用。 和弓形虫的致病力。息肉是一种普遍存在的聚合物，由三到数百个连接的磷酸盐残基组成。 通过高能的磷酸酸酐键，在原生动物寄生虫中可以达到毫米尔级，而 宿主细胞中的浓度处于微摩尔水平。弓形虫在酸性钙体中储存摩尔水平的息肉 它们在碱化后被水解，然后钙离子释放到胞浆中。令人费解的是，如此之少 已知息肉在弓形虫感染循环和/或毒力中的作用。然而，刻画 息肉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，息肉都涉及大量不同的显然无关的 功能。然而，这些明显无关的功能可能是息肉相互作用的能力的结果 蛋白质或静电或共价，我们建议测试弓形虫的这一点。我们的假设是 息肉通过与蛋白质的相互作用在弓形虫的裂解周期中发挥重要作用 和/或通过多聚磷酸化修饰它们的共价键。 我们提出了两种策略来确定息肉如何在毒力中发挥作用。确定候选蛋白质 与息肉相互作用的人，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 热应激以强调息肉作为蛋白质样伴侣的作用。我们还将使用生物信息学 发现息肉共价修饰的潜在蛋白质的方法。我们建议使用息肉 突变体(不会导致息肉)已经可以用来表征息肉与 靶蛋白。息肉的合成是一个新的靶点，因为它是必不可少的，而且没有酶。 在哺乳动物的宿主中。参与息肉合成的蛋白质在其他顶端复合体中是保守的 寄生虫。这些息肉目标蛋白将是弓形虫毒力途径的特异性蛋白，并可能具有同源基因。 在其他尖端复合体中。此外，该项目的发现可能会对脊椎动物生物学产生更广泛的影响。 由于哺乳动物的息肉合成机制尚不清楚，我们的发现可能会揭示新的功能 和蛋白质。

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.