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

拟议的PHD项目结合了尖端方法，以提供关于疟原虫入侵红细胞的机制的更多信息。由于每年有40多万人死于疟疾，东南亚出现了对一线抗疟疾药物的抗药性，而目前的疫苗只有部分疗效(对5-17个月的儿童最多为36%)，随着时间的推移而逐渐减弱，因此更好地了解疟疾的发病机制，以便确定和优先考虑新的疫苗靶点，是至关重要的。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，因为被称为裂殖子的入侵形式是细胞外的，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，因此阻止红细胞入侵将阻止寄生虫的繁殖，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裂殖子的重新定向，使顶端与红细胞接触，从而允许紧密连接的形成，从而承诺入侵。拟议的PHD项目旨在研究使用基因工程CRISPR/Cas9荧光诺氏疟原虫菌株的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。这将涉及对顶端和膜蛋白的荧光标记，以用作方向性的标记。这些菌株将被用作工具来研究抑制剂的影响和不同的红细胞生物物理性质。一旦建立了重定位分析，它将通过标记更多的蛋白质来扩展，以允许对与入侵相关的不同细胞器进行成像，并允许在整个过程中跟踪它们。整个项目的结构如下：1.收集关于诺氏疟原虫附着和入侵的数据，以便与现有的恶性疟原虫数据进行比较；2.开发一种定量分析方法，以表征疟原虫裂殖子在入侵过程的早期阶段的重新定位和膜包裹。这将涉及：a)在单行中产生带有多个标记标记的荧光菌株b)使用现场视频显微镜对这些菌株成像，并开发结合成像和跟踪算法的定位分析。应用定位分析测试抑制物、抗体和红细胞生物物理特性对重定向、膜包裹和侵袭的影响；4.跟踪其他侵袭细胞器在入侵过程后期的动态运动/部署；更好地了解裂殖体入侵所涉及的蛋白质和过程将有助于确定和优先考虑预防裂殖体入侵的疫苗和小分子靶点，从而减少疾病的健康和经济和社会负担。

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.