Unravelling the molecular mechanisms regulating cell division in the malaria parasite

Malaria is the third largest global health problem caused by a single infectious agent after HIV and TB, affecting millions of people, and resulting in one million deaths annually (http://www.who.int/topics/malaria/). The emergence of resistance to antimalarial drugs and the lack of any effective vaccine highlight the great need to develop new tools to control the disease. The symptoms of the disease are caused when the malaria parasite invades human red blood cells and multiplies many times every two days eventually leading to destruction of red blood cells and followed by further invasion and cell division. Some of these parasite cells may cease to divide and they become sex cells (male and female gametocytes). When a female mosquito bites an infected person they ingest parasites along with the blood and this acts as a trigger to activate the parasite sex cells within the mosquito gut. The male gametocytes undergo rapid cell division to produce eight male gametes, which then fertilise the female gametes and the parasite life cycle continues in the mosquito gut. After further development and multiplication the parasite moves to the mosquito's salivary glands and is passed again to a new human host.

The divisions made by parasites in blood cells and during sexual development are very different, but both are essential for the parasite If the parasite was unable to multiply and divide in the blood stream then it would not cause disease and if the male gametocytes were unable to divide then parasite transmissionwould be blocked. Therefore it is critically important to understand how the parasite multiplies and divides at these two stages so that we can develop ways to interfere with them by developing appropriate drugs.

The molecules that control these two types of cell division in the parasite are very poorly understood. The project proposed here is to identify what they are and how they work. We can start by using the knowledge gathered in model systems and applying this in the parasite. For example in yeast and other well-studied systems, a complex of proteins called the anaphase promoting complex/cyclosome (APC/C), plays a key role in cell multiplication and division. It is activated by the action of other proteins for example one called the cell division cycle protein-20 (CDC20). These proteins are also further regulated by the addition or removal of small 'tags', for example phosphate groups that can turn on or turn off particular functions.

We have recently identified one of these proteins (CDC20) in the malaria parasite and shown that it has a key role in regulating male gamete formation, and also that it is itself regulated by addition of phosphate tags. In preliminary work showing that our approach is feasible, we have obtained evidence for the presence of components of the APC/C protein complex in both the parasite multiplying within the red blood cell and in the male sex cell. Recent advances in analysing genes in malaria allow us to study the function of these molecules. For example, we can see what happens if the proteins are no longer made, and if they are tagged experimentally with a fluorescent marker we can see where they are located in the parasite under the microscope. Therefore, we are now in a position where we can explore further to understand how cell division in malaria is controlled by these different molecules. We will also study how these molecules interact together.

This research will enable us to identify mechanisms essential for parasite growth and multiplication that might be targeted in the development of new anti-malarial treatments. Our study may identify molecular targets important in parasite cell division in red blood cells and in the formation of male gametes, and therefore effective against either the stage responsible for the disease in humans or the transmission from one individual to another through the mosquito.

Malaria is caused by unicellular parasites belonging to the genus Plasmodium. These parasites invade red blood cells and divide asexually in a developmental process termed as schizogony, causing the disease. In addition, some parasites within red blood cells arrest cell division and differentiate to form sexual gametocytes. Following ingestion by Anopheles mosquitoes, the male gametocyte undergoes rapid cell division to produce 8 male gametes in a process of microgametogenesis. Schizogony and microgametogenesis are distinct types of mitotic cell division, and both also differ from classical cell division as studied in yeast, mammalian or other cells. The molecular mechanisms that regulate cell division in the malaria parasite are not well elucidated. Our main aim in this project is to unravel the molecular pathways controlling these types of cell division and establish how they differ from classical cell division.

Major molecular components regulating cell division in yeast and other eukaryotic cells are the anaphase promoting complex/cyclosome (APC/C) protein complex, its activators such as cell division proteins CDC20 and CDH1, and protein kinases and phosphatases that control the reversible phosphorylation of the complex/activators. We recently identified and partially characterized the single homologue of CDC20/CDH1 in Plasmodium, and have shown that many of the structural protein components of APC/C are not present in these parasites.

In this project we aim to investigate the role of putative APC/C components and their interaction with CDC20 during malaria parasite cell division at the two stages of the life cycle. This project will primarily use the rodent malaria model P. berghei since the whole life cycle can be completed in the laboratory and it is highly amenable to experimental manipulation. We will use reverse genetics, cell biological methods such as microscopy, protein biochemistry, and proteomics approaches to achieve our goal.

Cell division is a prerequisite for any organism to proliferate and develop during its life cycle. To understand how chromosomes are duplicated and segregated and how cell division occurs in a proper manner are basic questions in cell biology. Although various systems have been used to provide our understanding of these processes, including yeast, human and plant cells, very little is known about such processes and their regulation in parasitic protozoa, especially malaria parasites belonging to the genus Plasmodium.

Malaria is a major infectious disease of humans, with 300 million clinical cases and around a million deaths each year, mostly of children (http://www.who.int/topics/malaria/). Currently there is no effective vaccine, and the continual emergence of resistance to antimalarial drugs has resulted in human suffering, a strain on health care resources, and economic disadvantage. With a global agenda to substantially reduce malaria-related childhood mortality, to block transmission and eliminate malaria from large parts of the world, and eventually to eradicate the parasite, new tools are desperately needed both now and in the longer term.

Our research proposal is directed towards a better understanding of the molecular mechanisms involved in parasite multiplication and more specifically parasite cell division at two stages of its life cycle. One is the asexual multiplication (schizogony) that takes place within the red blood cell and results in the pathology of the disease, and the other is male gamete formation that commences within the host in the development of gametocytes, which further develop, divide and differentiate within the mosquito gut after being ingested by a mosquito. This stage of the parasite's life cycle is important in terms of transmission blocking interventions.

It is clear that important targets of intervention to stop parasite multiplication and the causation of disease are likely to be expressed within the red blood cell stage and targets of intervention to block transmission are likely to be expressed during development and differentiation within mosquito gut. Understanding the molecular mechanisms controlling multiplication in these stages is crucial to help develop intervention and control strategies. Our research will give us a better understanding of the molecules that are involved in these two processes of cell division and hence the identification of probable targets for intervention.

Our multifaceted approach will employ technologies that are at the cutting edge of research in the areas of parasite genetics, cell biology, protein chemistry and proteomics. This study will further strengthen the research potential of the malaria community. Most of the resources generated during the course of the project will be deposited in research data bases like PlasmoDB or the RGM data base. The research in malaria parasites may also impact on our understanding of the cell division in other parasites such as Trypanosoma, Giardia and others.

The research output from the proposed project could be used to identify strategic targets and these could be used by Nottingham Innovation scientists and companies involved in designing, making and testing antimalarial agents. Hence the project could identify new strategies towards drug development.
In addition another important impact will be the training of the named researchers in the project, allowing them to advance their own research careers and development. They will gain knowledge in a cutting edge area of research in the subject of malaria, a disease that is of great health and socio-economic importance.