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Molecular processes essential for parasite sporogony.

寄生虫孢子发生所必需的分子过程。

基本信息

批准号：
BB/V006428/1
负责人：
Johannes Dessens
金额：
$ 57.77万
依托单位：
London School of Hygiene & Tropical Medicine
依托单位国家：
英国
项目类别：
Research Grant
财政年份：
2021
资助国家：
英国
起止时间：
2021 至无数据
项目状态：
未结题

来源：
https://gtr.ukri.org/projects?ref=BB%2FV006428%2F1
关键词：
Molecular processes essential parasite sporogony.

项目摘要

Apicomplexan parasites are widespread protozoan parasites of animals, which include major pathogens of humans, domestic animals and livestock. Many existing measures against these parasites remain insufficient for disease control, and new strategies for prophylaxis, treatment and control of transmission are urgently needed. Sporogony is defined as the production of sporozoites by repeated divisions of a zygote. It is an essential part of the life cycles of many apicomplexan parasites including Plasmodium, the causative agent of malaria, where sporogony takes place in the mosquito within extracellular encysted forms named oocysts. In Plasmodium, sporogony is critically dependent on a unique organelle named the crystalloid, which is exclusively found in the ookinete (a motile form of the zygote) and young oocyst stages. Many of the crystalloid proteins thus far identified in Plasmodium are conserved in and unique to the Apicomplexa, supporting the hypothesis that the molecular processes underlying sporogony are at least partly conserved across the phylum. In this proposal, we will use an integrated cross-disciplinary approach to study the crystalloids of Plasmodium berghei aimed at increasing our fundamental knowledge of the essential cellular and molecular processes underlying sporogony. This will be achieved via the following specific objectives:Objective 1: Study crystalloid loss in the oocyst by microscopy. Using various parasites lines expressing different crystalloid proteins tagged with fluorescent protein as markers of the crystalloid organelle, combined with superresolution confocal microscopy, the destination of the crystalloid and its protein cargo will be investigated in live oocysts during sporogony. This will help elucidate the processes involved in loss of the organelle at the subcellular level, and our understanding of crystalloid function.Objective 2: Refine the crystalloid protein interactome. Three transgenic parasite lines expressing different crystalloid proteins (LAP3, NTH and TPM2) each fused to a twin fluorescent protein/affinity tag (GFP/Strep) will be generated and used to harvest protein complexes from ookinetes by Strep-based affinity purification, followed by quantitative mass spectrometry-based protein identification. These analyses will produce a high confidence crystalloid protein interaction network (interactome).Objective 3: Determine the crystalloid proteome. Parasite lines expressing the GFP/Strep-tagged crystalloid membrane proteins NTH and TPM2 will be used to purify intact organelles from mechanically lysed ookinetes by Strep-based affinity purification, followed by quantitative mass spectrometry-based protein identification. This analysis will help determine the entire crystalloid protein repertoire (proteome) irrespective of protein interactions.Objective 4: Establish the crystalloid metabolome. Whole ookinetes and Strep-based affinity-purified crystalloid organelles from parasites expressing GFP/Strep-tagged NTH (generating NADPH), and a parasite line expressing an enzymatically inactive NTH version, will be subject to mass spectrometry-based global metabolite analysis. These analyses will allow differences in the metabolite repertoire (metabolome) of NTH-deficient parasites to be identified, which will help inform which NADPH-dependent biosynthetic processes are taking place in the organelle. Objective 5. Validate newly identified crystalloid proteins. Select proteins identified from the above objectives will be validated by GFP tagging and gene knockout studies in transgenic parasite lines to make sure they are genuinely linked with crystalloid function and sporogony.The collective results will provide new basic knowledge of the essential functions of the crystalloid in sporogony, and help to identify new molecules and molecular pathways for rational intervention strategies against development and transmission of malaria and related apicomplexan parasites

顶复门寄生虫是广泛分布于动物体内的寄生原虫，是人类、家畜和家畜的主要病原体。针对这些寄生虫的许多现有措施仍然不足以控制疾病，迫切需要预防、治疗和控制传播的新战略。孢子生殖是指受精卵通过多次分裂产生子孢子的过程。它是许多顶复门寄生虫生命周期的重要组成部分，包括疟原虫，疟疾的病原体，其中孢子生殖发生在蚊子中称为卵囊的细胞外包囊形式内。在疟原虫中，孢子生殖严重依赖于一种名为晶体的独特细胞器，它只存在于动合子（受精卵的运动形式）和年轻的卵囊阶段。许多晶体蛋白质，迄今已确定在疟原虫是保守的，独特的顶复门，支持的假设，孢子生殖的分子过程至少部分保守的门。在这个建议中，我们将使用一个综合的跨学科的方法来研究伯氏疟原虫的晶体，旨在增加我们的基本知识的基本细胞和分子过程的孢子生殖。这将通过以下具体目标来实现：目标1：通过显微镜研究卵囊中的晶体损失。使用不同的寄生虫线表达不同的晶体蛋白标记的荧光蛋白作为标记的晶体细胞器，结合超分辨率共聚焦显微镜，目的地的晶体和它的蛋白货物将在活卵囊孢子生殖过程中进行调查。这将有助于在亚细胞水平上阐明细胞器丢失的过程，以及我们对晶体功能的理解。将产生表达各自融合到双荧光蛋白/亲和标签（GFP/Strep）的不同晶体蛋白（LAP 3、NTH和TPM 2）的三个转基因寄生虫系，并用于通过基于Strep的亲和纯化从动合子收获蛋白质复合物，随后进行基于定量质谱的蛋白质鉴定。这些分析将产生一个高置信度的晶体蛋白质相互作用网络（interactome）。目的3：确定晶体蛋白质组。表达GFP/Strep标记的晶体膜蛋白NTH和TPM 2的寄生虫系将用于通过基于Strep的亲和纯化从机械裂解的动合子中纯化完整的细胞器，然后进行基于定量质谱的蛋白质鉴定。这一分析将有助于确定整个晶体蛋白库（蛋白质组），而不考虑蛋白质相互作用。将对来自表达GFP/Strep标记的NTH（产生NADPH）的寄生虫的完整动卵细胞和基于Strep的亲和纯化晶体细胞器以及表达无酶活性NTH版本的寄生虫系进行基于质谱的全局代谢物分析。这些分析将允许在NTH缺陷型寄生虫的代谢物库（代谢物组）的差异被确定，这将有助于通知NADPH依赖的生物合成过程中发生的细胞器。目标5.新发现的晶体蛋白。从上述目标中鉴定出的蛋白质将通过GFP标记和转基因寄生虫系中的基因敲除研究进行验证，以确保它们真正与晶体功能和孢子生殖相关。集体结果将为晶体在孢子生殖中的基本功能提供新的基础知识，并帮助确定新的分子和分子途径，以制定合理的干预战略，防止疟疾和相关顶复体的发展和传播。寄生虫