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

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.