Membrane and host cytoskeleton reorganization during malaria parasite egress from erythrocytes

Malaria is a major global killer, most deadly for children in the developing world. Of the five species that infect humans, Plasmodium falciparum is the most lethal. Although there are currently effective anti malarial drugs, the appearance and spread of resistant strains of P falciparum pose an increasing threat. The parasite has a complex life cycle with several different stages in its mosquito and human hosts, but the clinical symptoms in humans arise from waves of parasite release during the asexual blood stages, in which parasites invade red blood cells and multiply within an internal membrane compartment called a vacuole. The growing parasites hijack their host blood cells, consume their haemoglobin, and redirect the cell activity for the benefit of the parasite. Of particular importance, the parasite exports some of its own proteins to build new structures on the surface of the blood cell. A uniquely lethal aspect of P. falciparum is that it creates surface protrusions (called knobs) on the blood cell that make it adhere to the lining of blood vasculature. This prevents the infected cells from being captured and destroyed by the spleen, but can also block brain blood capillaries, the main cause of death in malaria infection. Once they have matured, 16-24 daughter parasites break through their surrounding vacuole membrane as well as the red blood cell membrane (a process collectively called egress) to enter the bloodstream, where they immediately invade a fresh round of blood cells. The processes of invasion, adhesion and egress are regulated through a cascade of enzyme reactions, as well as expression and transport of structural components. Some of these components are unique to malaria, making them potential targets for future drug development. Currently, most of them are poorly characterised.
In this project, we focus on the steps by which the mature parasites break through the two bounding membranes to undergo egress. A highly regulated series of steps leads to the explosive release of parasites from the blood cell. To examine these membrane breakage events in detail, we use electron microscopy to image at nanoscale resolution the three-dimensional structures of the infected cells during the late stages of parasite development. This imaging is combined with use of parasite mutants and drug-like molecules and enzyme inhibitors to trap the parasites at different steps of egress. This approach has already led us to discover a new, initial step in egress that had not been previously detected. We now know that the process begins with the parasites causing the membrane surrounding their vacuole to become leaky. Subsequently, this membrane is completely disrupted, allowing the parasites to move freely inside the blood cell. Shortly after that, the blood cell membrane itself becomes leaky and then finally the cell membrane and its underlying cytoskeleton rupture to allow the parasites to escape and invade new host blood cells.
With recent advances in gene editing technology, it has now become possible to conditionally modify gene expression in P. falciparum, and we will use this powerful technology to probe the molecular nature, functions and subcellular localisations of key malaria components involved in the sequence of steps during egress. The results of these studies could form the basis for future development of novel therapeutics.

Intracellular pathogens such as the malaria parasite hijack host cells and redirect their activity into facilitating replication and dispersal of the pathogen. The major clinically relevant phase of malaria is the asexual blood stage, during which parasites invade erythrocytes, multiply inside a vacuole and then escape for a new round of invasion. Egress of mature parasites from the erythrocyte requires the parasites to break through the two membranes enclosing them - the parasitophorous vacuole membrane and the erythrocyte cell membrane. These events are highly regulated, involving a cascade of activation of at least one parasite kinase, at least 2 proteases and likely other membrane modifying proteins such as phospholipases and/or pore-forming proteins. We have used electron tomography, video microscopy of live cells and soft X-ray tomography to uncover new details of the changes to the vacuolar and erythrocyte membranes and cytoskeleton during egress. These studies led to the discovery of a previously undescribed, initial step in egress, in which permeabilisation of the vacuole membrane precedes its complete disruption. This is rapidly followed by permeabilisation and breakage of the erythrocyte cell membrane and collapse of the cytoskeleton to allow the explosive release of parasites. With the methods we have developed, along with powerful new conditional gene modification tools in P. falciparum, we now wish to pursue the molecular and cellular basis of membrane and cytoskeleton reorganization during P. falciparum egress. Understanding the details of the egress process will lead to improved means of malaria control.

Malaria is one of the biggest burdens of infectious disease in the developing world, causing around 600,000 deaths annually, mainly of children. Our work seeks to decipher the fundamental mechanisms by which this pathogen causes disease, yielding new insights into the molecular and cellular events underlying parasite proliferation during the asexual blood stage of malaria infection, the sole source of clinical symptoms and mortality. Our research has the potential to reveal new targets for therapeutics and vaccines. It is a matter of the most serious global concern that P. falciparum is developing resistance to the drug artemisinin, the treatment of last resort when all other malaria drugs fail due to resistant strains.

This application specifically addresses the process in which the mature parasites modify their host erythrocyte to facilitate their own proliferation. P. falciparum-infected cells express parasite-encoded adhesive protrusions (known as knobs) on their surface, enabling them to escape capture by the spleen and causing the most dangerous form of malaria, leading to death by obstructing blood capillaries in the brain. We previously discovered a remarkable spiral scaffold underlying the membrane protrusions. The major focus of this proposal is on the highly regulated process of egress, by which the mature parasites escape their host cell and immediately invade many others. This process is common to all malaria species. In our recent work we have discovered a new, initial stage in egress, which is now leading to the search for the triggering event that initiates egress. If this process can be blocked, clinical symptoms and progression of malaria would be greatly inhibited.

A better understanding of how the malaria parasite hijacks cellular processes in its erythrocyte host has the potential to benefit all researchers and clinicians working on malaria, and it is hoped that development of new therapeutic or vaccine strategies would ultimately benefit the millions of malaria patients world wide. The proposed research will also contribute to training in state of the art electron microscopy methods, a field in which there is on ongoing explosion of interest both in academia and industry. Researchers trained in these methods at Birkbeck are extremely highly sought after in the top research and industrial laboratories internationally. The proposed work will combine this training with expertise in studying a major human pathogen. Seeding well-trained scientists into academic and industry is essential to ensure the competitiveness of the UK science base in the future. This yields not only academic but also substantial economic and social benefits.