Functional dissection of Condensin and Cohesin in atypical mitosis and meiosis in Plasmodium

Plasmodium is the causative agent of malaria and is responsible for about 584,000 human deaths annually, mostly children under the age of 5. The increasing resistance of the parasite to existing drugs and the lack of an efficient vaccine represent the main obstacles to combat this burden. Therefore, there is an urgent need for the development of innovative therapeutics. Plasmodium has a complex life cycle with diverse host environments, changes in cell shape, size and motility and an atypical mode of cell proliferation. The symptoms of the disease are manifest when malaria parasites invade host red blood cells, wherein the parasite divides and multiplies many times (endomitosis), eventually leading to destruction of the host cell. Some of the parasite cells may cease to divide and they become precursor 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 precursor sex cells within the mosquito gut. The male gametocytes undergo rapid cell division (endoreduplication) 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 when the mosquito feeds.

The process of Plasmodium cell division in host red blood cells, and during sexual development in the mosquito vector is very different, but both are essential for parasite growth and transmission. If any of these stages are blocked then both proliferation and transmission are curtailed. Therefore, it is critically important to understand how the parasite multiplies and divides at these stages so that we can devise ways to interfere with them by developing appropriate drugs.

The molecules that control these types of atypical cell division in the parasite are very poorly understood. The project proposed here is to study the role of two important protein complexes: condensin and cohesin. Although these complexes are known to be involved in cell division in many model systems, there is no knowledge of how they function during malaria parasite cell division and proliferation. We can start by using the knowledge gathered in model systems and applying this to the malaria parasite. For example, we have recently identified one such protein (CDC20) in the malaria parasite and showed that it has a key role in regulating male gamete formation. In preliminary work showing that our approach is feasible, we have obtained evidence for the presence of condensin and cohesin in the parasite multiplying within the red blood cell, in the male sex cell and in the meiotic cell (an ookinete). Recent advances in analysing genes in malaria allow us to study the function of these molecules. For example, by taking away the gene, we can see what happens when the proteins are no longer made, and if they are tagged experimentally with a fluorescent marker we can see under the microscope where they are located in the parasite. We will also study how these molecules interact with other proteins during various processes such as chromosome segregation and cytokinesis, to name two. We can also purify these protein complexes and study their interactions using mass spectroscopy-based approaches. Therefore, we are now in a position where we can explore how cell division in malaria is controlled by these complexes and we can study how these molecules regulate different stages of cell division.

Our study may identify molecular targets important in parasite cell division in host red blood cells and in the cells dividing within the mosquito vector. As a result, we may identify potential targets for drugs or vaccines that could in the future be used to block parasite replication in the blood and transmission from one individual to another through the mosquito.

Plasmodium is a unicellular parasite and the causative agent of malaria. These parasites have an atypical cell division that differs from that of the host they invade. They divide within red blood cells (schizogony) in a process resembling endomitosis where the nuclear envelope remains intact and a single cell becomes multinucleated. Moreover, during this stage their chromosomes do not condense. The parasite also shows endo-reduplication during male gametocyte differentiation, as well as a short meiotic stage within the mosquito vector. The molecular mechanisms that regulate these atypical cell divisions in Plasmodium are not well studied.

The main aim of this project is to dissect out the molecular mechanisms controlling two important components, Condensin and Cohesin in these types of atypical cell division and establish how they differ from the classical cell division of host cells, ultimately to identify intervention targets.

Condensin and cohesin belong to an important family of proteins that have crucial roles in chromosome dynamics and gene regulation, both in mitosis and meiosis in most eukaryotes. This has been widely studied in some cells, yet nothing is known of their localisation, function, regulation or the complexes associated with them during atypical cell division in Plasmodium.

Complementary approaches will be used to achieve three main objectives, using Plasmodium berghei as a model system: (1) To identify the subcellular location of condensin and cohesin at various stages of parasite cell division. 2) To study the function and transcriptional regulation of cohesin and condensin during parasite cell division and (3) To characterise the cohesion and condensin complexes using proteomic approaches.

We will use reverse genetics, state of the art cell biology methods with fluorescence, confocal, and electron microscopy, and protein biochemistry with proteomics to achieve our goals.

This proposal fits well within the strategic aim of the MRC towards Global health, Research to people and picking research that delivers. We plan to address the role of Condensin and Cohesin in the core stages of atypical cell division during the malaria parasite life cycle, in both mammalian host and mosquito vector. To achieve this we have chosen an experimentally tractable rodent malaria parasite, Plasmodium berghei, to enable us to examine these atypical stages during both the sexual and asexual stages of the life cycle.

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 studies in yeast, human and plant cells, very little is known about such processes and their regulation in unicellular parasitic protozoans like Plasmodium. 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.

Understanding the molecular mechanisms controlling multiplication of the parasite following atypical cell division 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 processes of chromosome biology and nuclear division; thereby controlling cell division and hence the identification of probable targets for intervention both to treat the disease state and to prevent transmission.

This research will provide further fundamental knowledge on the role of these conserved condensin and cohesin complexes whilst the parasite develops and proliferates using a very different mode of cell division and closed mitosis, compared with the classical processes observed in mammalian cells. It will be informative for both pure research in cell and developmental biology, and for understanding the evolution of these molecules and their function.

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 and wider community studying other parasites. Most of the resources generated during the course of this project will be deposited in research databases such as PlasmoDB or the RMgm database. The research on malaria parasites will hopefully also impact on our understanding of cell division in other parasites such as Trypanosoma, Giardia and others.