Combining Video Microscopy and Experimental Genetics to Explore Plasmodium Merozoite Re-Orientation and Erythrocyte Invasion

结合视频显微镜和实验遗传学探索疟原虫裂殖子重新定向和红细胞侵袭

• 批准号: 2119714
• 金额: --
• 依托单位: University of Cambridge
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2018
• 资助国家: 英国
• 起止时间: 2018 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2119714
• 关键词: Combining; Video; Microscopy; Experimental; Genetics

The proposed PhD project combines cutting-edge methodologies to provide more information about the mechanism of erythrocytes invasion by Plasmodium parasites. With over 400,000 deaths per year, resistance to the front-line antimalarials emerging in Southeast Asia, and the current vaccine having only a partial efficacy (at best 36% in children aged 5-17 months) that wanes over time, it is crucial that malaria disease mechanisms are better understood in order to identify and prioritise new vaccine targets. The blood stage of the parasite life cycle provides a unique window of opportunity for vaccines, as the invasive form known as the merozoite is extracellular, and therefore vulnerable to the host immune system. In addition, Plasmodium parasites cannot replicate outside a host cell, so blocking erythrocyte invasion would prevent parasite multiplication, and therefore disease. Invasion is a complex process, with one critical element being reorientation of the asymmetric merozoite to bring the apex into contact with the erythrocyte to allow tight junction formation to occur, and thus a commitment to invasion to be made. The proposed PhD project aims to study reorientation and the mechanism of membrane wrapping using genetically engineered CRISPR/Cas9 fluorescent Plasmodium knowlesi strains, applying live video microscopy techniques to capture reorientation and invasion events in real-time. This will involve the fluorescent tagging of apex and membrane proteins to use as markers for directionality. These lines will be used as tools to investigate the effects of inhibitors and varying erythrocyte biophysical properties. Once the reorientation assay is established, it will be expanded by labelling additional proteins to allow imaging of different organelles associated with invasion, and allowing their tracking through the process. The full project will be structured as follows:1. Collecting data about Plasmodium knowlesi attachment and invasion to allow comparison with existing Plasmodium falciparum data;2. Developing a quantitative assay to characterise Plasmodium merozoite re-orientation and membrane wrapping during the early stages of the invasion process. This will involve: a) Generating fluorescent strains with multiple markers tagged in a single line b) Imaging these strains using live video microscopy and developing an orientation assay combining imaging with a tracking algorithm.3. Applying the orientation assay to test the impact of inhibitors, antibodies and erythrocyte biophysical features on reorientation, membrane wrapping and invasion;4. Tracking dynamic movement / deployment of other invasive organelles during later stages of the invasion process;A greater understanding of the proteins and processes involved in merozoite invasion will aid in the identification and prioritisation of vaccine and small molecule targets to prevent invasion and therefore reduce the health, but also economic and social burden of the disease.

建议的博士项目结合前沿的方法，提供更多关于疟原虫入侵红细胞机制的信息。由于每年有40多万人死亡，东南亚出现了对一线抗疟疾药物的耐药性，而且目前的疫苗只有部分效力（在5-17个月的儿童中最多为36%），并且随着时间的推移而减弱，因此必须更好地了解疟疾发病机制，以便确定新的疫苗目标并确定其优先次序。寄生虫生命周期的血液阶段为疫苗提供了一个独特的机会窗口，因为称为merozoite的侵入形式是细胞外的，因此容易受到宿主免疫系统的攻击。此外，疟原虫不能在宿主细胞外复制，因此阻断红细胞入侵可以防止寄生虫繁殖，从而防止疾病。入侵是一个复杂的过程，其中一个关键因素是不对称的裂殖子重新定向，使其顶点与红细胞接触，从而形成紧密的连接，从而承诺入侵。本博士项目旨在利用基因工程CRISPR/Cas9荧光诺氏疟原虫菌株研究重定向和膜包裹机制，应用实时视频显微镜技术实时捕捉重定向和入侵事件。这将涉及到尖端和膜蛋白的荧光标记，作为定向标记。这些细胞系将被用作研究抑制剂的作用和红细胞生物物理特性变化的工具。一旦重新定位试验建立，它将通过标记额外的蛋白质来扩展，以允许成像与入侵相关的不同细胞器，并允许在整个过程中跟踪它们。整个项目的结构如下：1。1 .收集诺氏疟原虫的附着和侵袭数据，与现有恶性疟原虫数据进行比较；在入侵过程的早期阶段，发展一种定量分析，以表征疟原虫的分裂子体重新定向和膜包裹。这将涉及：a)产生在单线上标记多个标记的荧光菌株b)使用实时视频显微镜对这些菌株进行成像，并开发将成像与跟踪算法相结合的定向试验。3 .应用定向实验检测抑制剂、抗体和红细胞生物物理特征对重定向、膜包裹和侵袭的影响；在入侵过程后期跟踪其他入侵细胞器的动态运动/部署；更好地了解参与裂殖子侵入的蛋白质和过程将有助于确定疫苗和小分子靶点并确定其优先次序，以防止侵入，从而减少疾病的健康负担，以及经济和社会负担。