LAP function in apicomplexan parasite development

LAP 在 apicomplexan 寄生虫发育中的功能

• 批准号: BB/M001598/1
• 负责人: Johannes Dessens
• 金额: $ 46.82万
• 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: 2015
• 资助国家: 英国
• 起止时间: 2015 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FM001598%2F1
• 关键词: LAP; function; apicomplexan; parasite; development

Apicomplexan parasites are widespread protozoan parasites and 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. This research proposal focuses on an Apicomplexa-specific conserved family of modular proteins (named LAPs) containing domains implicated in lipid, carbohydrate and protein interaction. In Plasmodium, the LAP family has six conserved members that form a protein complex, localize to distinctive multivesicular organelles named crystalloids, and have essential roles in crystalloid biogenesis and sporozoite development. The uniqueness, complex architectures and conservation of the LAPs strongly point to a conserved function in all apicomplexan parasites, but thus far it is unknown what that function is. Determining the function and mode of action of the LAPs is of much interest both from a parasite cell biological perspective, and as potential molecular targets for antiparasitic chemotherapy. This proposal aims to investigate the function of the LAPs in parasite development and test the hypothesis that the conserved function of the LAP complex in apicomplexan parasites is the formation of specific transport vesicles. We will do this using the rodent malaria parasite species P. berghei as a model, because this is a suitable parasite for genetic modification and the culture and purification of relevant life stages. Moreover, we already possess a range of transgenic parasite lines expressing genetically modified LAPs, which provide unique tools for the proposed studies. The specific objectives are: (1) Test the functional requirement of crystalloids in parasite development. We will use inhibitors of vesicle transport to chemically disrupt crystalloid assembly in parasites. The effects of this on downstream parasite development will allow us to test whether the LAPs function downstream of crystalloid formation, and whether crystalloid assembly is required for downstream parasite development; (2) Define the LAP 'interactome'. We possess genetically modified parasite lines that express LAPs tagged with green fluorescent protein (GFP). We will pull-down LAP complexes from these parasites using magnetic beads that can bind to the GFP, and these complexes will then be analyzed by mass spectrometry (MS) to determine their protein composition. The identification of other proteins in the LAP complex (the LAP interactome) will shed new light on its mode of action; (3) Define the crystalloid proteome. We possess transgenic parasite lines that do not possess crystalloids. Using MS we will compare the total protein content of crystalloid-positive (wildtype) vs. crystalloid-negative parasites to identify the other crystalloid constituents. As a parallel approach we will use MS analysis of purified crystalloid fractions. The identification of other proteins in the crystalloids (the crystalloid proteome) will shed new light on its mode of action and that of the LAPs; (4) Investigate the subcellular localization, function and mode of action of novel LAP interactome and crystalloid proteome components. Newly identified proteins of the LAP interactome and crystalloid proteome identified from objectives 2 and 3 will be functionally characterized using fluorescent protein tagging and gene knockout approaches in genetically modified parasites. The results will provide new mechanistic insight into LAP function and mode of action.The proposed research will build a comprehensive picture of the molecules that are part of the LAP complex and of the crystalloid organelle, which will lead to better understanding of the functions and modes of action of the LAPs. This will form a platform for developing new and rational intervention strategies to combat diseases caused by apicomplexan parasites in humans and domestic animals.

Apicomplexan寄生虫是一种分布广泛的原生动物寄生虫，包括人类、家畜和牲畜的主要病原体。许多针对这些寄生虫的现有措施仍然不足以控制疾病，迫切需要新的预防、治疗和控制传播战略。这项研究建议集中在ApicomplexA特异性保守的模块蛋白家族(称为LAPS)，包含与脂类、碳水化合物和蛋白质相互作用有关的结构域。在疟原虫中，LAP家族有6个保守成员，它们形成一个蛋白质复合体，定位于独特的多囊细胞器，称为晶体，在晶体生物发生和子孢子发育中发挥重要作用。LAPS的独特性、复杂的结构和保守性强烈地表明，在所有的顶复合体寄生虫中都有一种保守的功能，但到目前为止，人们还不知道该功能是什么。从寄生虫细胞生物学的角度和作为抗寄生虫化疗的潜在分子靶点，确定LAPS的功能和作用方式是非常有意义的。本研究旨在研究LAPS在寄生虫发育中的作用，并验证顶端复合体中LAPS复合体的保守功能是形成特定的运输小泡的假说。我们将以啮齿动物疟疾寄生虫P.berghei为模型进行这项工作，因为这是一种适合进行基因改造和相关生命阶段的培养和纯化的寄生虫。此外，我们已经拥有了一系列表达转基因LAPS的转基因寄生虫系，这为拟议的研究提供了独特的工具。具体目标是：(1)测试寄生虫发育过程中晶体的功能需求。我们将使用囊泡运输的抑制剂来化学破坏寄生虫的晶体组装。这对下游寄生虫发育的影响将使我们能够测试LAPS是否在晶体形成的下游发挥作用，以及下游寄生虫的发育是否需要晶体组装；(2)定义LAP‘相互作用体’。我们拥有转基因寄生虫株系，表达带有绿色荧光蛋白(GFP)的LAP。我们将使用可以与GFP结合的磁珠从这些寄生虫中拉出LAP复合体，然后通过质谱仪(MS)分析这些复合体以确定它们的蛋白质组成。LAP复合体(LAP相互作用组)中其他蛋白质的鉴定将为其作用模式提供新的线索；(3)定义晶体蛋白质组。我们拥有没有晶体的转基因寄生虫株系。利用MS，我们将比较晶体阳性(野生型)和晶体阴性寄生虫的总蛋白质含量，以确定其他晶体成分。作为一种平行的方法，我们将使用MS分析纯化的晶体组分。对晶体(晶体蛋白质组)中其他蛋白质的鉴定将对其作用模式和LAPS的作用模式提供新的线索；(4)研究新的LAP相互作用组和晶体蛋白质组的亚细胞定位、功能和作用模式。从目标2和3中鉴定的新鉴定的LAP相互作用体和晶体蛋白质组的蛋白质将在转基因寄生虫中使用荧光蛋白标签和基因敲除方法进行功能表征。这些结果将为LAP的功能和作用模式提供新的机制见解。拟议的研究将建立LAP复合体和晶体细胞器一部分的分子的全面图景，这将导致对LAP的功能和作用模式的更好理解。这将形成一个平台，用于开发新的和合理的干预战略，以对抗由顶丝复合体寄生虫引起的人类和家畜疾病。

项目成果

Biogenesis of the crystalloid organelle in Plasmodium involves microtubule-dependent vesicle transport and assembly.

• DOI: 10.1016/j.ijpara.2015.03.002
• 发表时间: 2015-07
• 期刊: INTERNATIONAL JOURNAL FOR PARASITOLOGY
• 影响因子: 4
• 作者: Saeed, Sadia;Tremp, Annie Z.;Dessens, Johannes T.
• 通讯作者: Dessens, Johannes T.

A potent series targeting the malarial cGMP-dependent protein kinase clears infection and blocks transmission.

• DOI: 10.1038/s41467-017-00572-x
• 发表时间: 2017-09-05
• 期刊: Nature communications
• 影响因子: 16.6
• 作者: Baker DA;Stewart LB;Large JM;Bowyer PW;Ansell KH;Jiménez-Díaz MB;El Bakkouri M;Birchall K;Dechering KJ;Bouloc NS;Coombs PJ;Whalley D;Harding DJ;Smiljanic-Hurley E;Wheldon MC;Walker EM;Dessens JT;Lafuente MJ;Sanz LM;Gamo FJ;Ferrer SB;Hui R;Bousema T;Angulo-Barturén I;Merritt AT;Croft SL;Gutteridge WE;Kettleborough CA;Osborne SA
• 通讯作者: Osborne SA

The Plasmodium alveolin IMC1a is stabilised by its terminal cysteine motifs and facilitates sporozoite morphogenesis and infectivity in a dose-dependent manner.

• DOI: 10.1016/j.molbiopara.2016.09.004
• 发表时间: 2017-01
• 期刊: Molecular and biochemical parasitology
• 影响因子: 1.5
• 作者: Al-Khattaf FS;Tremp AZ;El-Houderi A;Dessens JT
• 通讯作者: Dessens JT

Detection of malaria sporozoites expelled during mosquito sugar feeding.

• DOI: 10.1038/s41598-018-26010-6
• 发表时间: 2018-05-15
• 期刊: Scientific reports
• 影响因子: 4.6
• 作者: Brugman VA;Kristan M;Gibbins MP;Angrisano F;Sala KA;Dessens JT;Blagborough AM;Walker T
• 通讯作者: Walker T