Proteomic mapping of differential secretion in Toxoplasma gondii

弓形虫差异分泌的蛋白质组图谱

基本信息

批准号：
9228917
负责人：
Marc-Jan Gubbels
金额：
$ 7.83万
依托单位：
BOSTON COLLEGE
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-02-18 至 2019-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9228917
关键词：
Acute Alleles Amino Acids Antigens Cells Clinical Communities Complex Mixtures Congenital Abnormality Contracts Cytoplasmic Granules DAB2 gene Data Data Set Detection Disease Dominant-Negative Mutation Drug Design Drug Targeting Encephalitis Environment Goals Growth Human Immunocompromised Host Impairment Infection Ionophores Kinetics Life Ligands Lye Lytic Mass Spectrum Analysis Mediating Minor Modeling Mutation Opportunistic Infections Organelles Parasites Pathologic Pathology Pharmaceutical Preparations Population Process Protein Secretion Proteins Proteome Proteomics Resolution Stable Isotope Labeling Symptoms Temperature Time Tissues Toxoplasma Toxoplasma gondii Toxoplasmosis Transmembrane Domain Vacuole Variant cell motility conditional mutant extracellular immunosuppressed improved insight lytic replication mutant novel novel therapeutics overexpression permissiveness protein function public health relevance rhoptry temperature sensitive mutant transmission process

项目摘要

DESCRIPTION (provided by applicant): The apicomplexan parasite Toxoplasma gondii is the causative agent of life-threatening encephalitis in immunocompromised patients and in addition can cause a variety of birth defects if the infection is contracted congenitally. The pathology associated with disease originates in fast rounds of lytic intracellular replication cycles. The lyic replication cycle is comprised of rounds of host cell invasion, replication in the intracellular vacuole, and egress from the host cell. The essential host cell invasion step has been studied for its potential as a novel and specific drug target. During this process, sequential secretion of three secretory organelles (micronemes, rhoptries, and dense granules) occurs. Of these, it is the microneme proteins that contribute most to egress, gliding motility, and host cell invasion. These diverse functions have led to speculation as to whether there are micronemes with different contents secreted at different times, or whether microneme protein function is regulated by differences in parasite environment. Observations in support and against both models abound but there is no overwhelmingly convincing data to settle the argument in either direction. To contribute data that will inform this important debate, this proposal exploits the differential microneme secretion kinetics of two different conditional mutants in the Ca2+-dependent secretion machinery used by the micronemes. One mutant has a temperature sensitive (ts) mutation in TgDOC2 (ts-DOC2), and the other harbors a dominant negative allele of Ferlin-like protein TgFLP (DN-FLP) generated by conditional overexpression (ligand-controlled) of a TgFLP allele lacking the transmembrane domain. ts-DOC2 secretes no micronemes at all and will yield a background free of proteins secreted through the micronemes. DN-FLP has functional constitutive microneme secretion but is impaired in Ca2+-ionophore induced secretion. This mutant will facilitate differentiation between microneme proteins secreted at different points along the parasite's egress-motility-invasion journey. Thus, these data will contribute to the discussion of whether or not there is differential microneme protein secretion as outlined above. Excretory and secretory antigens (ESA) will be collected from mutant ts-DOC2 and DN-FLP grown under both the permissive and restrictive conditions. We will apply Stable Isotope Labeling by Amino Acids in Culture (SILAC). Most pertinent to the goals of this project, SILAC is quantitative and permits the detection of minor differences in protein abundance in complex mixtures across different mutants and under permissive versus restrictive conditions. Overall, the combination of different secretion mutants with the power of SILAC will result in an unprecedented level of ESA resolution and will either boost our confidence in, or disprove the possibility of differential microneme secretion. Hence, these will be valuable data sets for the Toxoplasma community in understanding the uniquely parasitic process of host cell invasion, and the insights could be of use in rational drug design.

描述（由适用提供）：Apicomplexan寄生虫弓形虫Gondii是免疫功能低下的患者威胁生命的脑炎的病因，如果先天感染签约，可能会导致多种出生缺陷。与疾病相关的病理起源于裂解的细胞内复制周期。抒情复制周期由宿主细胞侵袭，细胞内液泡中的复制和从宿主细胞出口中的复制组成。基本的宿主细胞侵袭步骤已经研究了其作为一种新颖和特定的药物靶标的潜力。在此过程中，顺序分泌发生了三个秘密细胞器（微生物，犀牛和密集颗粒）。其中，是微原蛋白对流出，滑行运动和宿主细胞侵袭的最大贡献。这些不同的功能导致人们对是否存在不同时间分泌不同含量的微元素，或者微生物蛋白函数是否受寄生虫环境中的差异调节。支持和反对这两个模型的观察都充满了，但没有压倒性的令人信服的数据可以朝任何方向解决这一论点。为了贡献这一重要辩论的数据，该提案探讨了Microcenes使用的Ca2+依赖性秘密机械中两个不同条件突变体的差异微神分泌动力学。一个突变体在TGDOC2（TS-DOC2）中具有温度敏感性（TS）突变，而另一个突变体具有由条件过表达（配体控制）产生的Ferlin样蛋白TGFLP（DN-FLP）的显性负等位基因，该等位基因缺乏TGFLP等位基因缺乏跨膜的tgflp等位基因。 TS-DOC2根本没有微分分泌，并且会产生不含微分子分泌的蛋白质的背景。 DN-FLP具有功能性组成型微元分泌，但在Ca2+离子载体诱导的分泌中受损。该突变体将促进在寄生虫的出口渗透率侵入性旅程的不同点分泌的微元蛋白之间的分化。这就是这些数据将有助于讨论上述差异微蛋白蛋白分泌。排泄物和秘密抗原（ESA）将从在允许和限制性条件下生长的突变体TS-DOC2和DN-FLP收集。我们将在培养物（SILAC）中通过氨基酸进行稳定的同位素标记。与该项目的目标最相关，SILAC是定量的，允许检测不同突变体以及允许性和限制性条件下的复杂混合物中蛋白质丰度的微小差异。总体而言，不同的分泌突变体与SILAC的功能的组合将导致前所未有的ESA分辨率水平，并可以提高我们对差异微原质分泌的可能性。因此，这些对于托克斯玛群岛群落的宝贵数据集将是理解宿主细胞侵袭的独特寄生过程的宝贵数据集，并且可以在理性的药物设计中使用这些见解。