Identification and characterization of telomere proteins in Plasmodium falciparum.

The proposed research concerns the human malaria parasite Plasmodium. Malaria is one of the world's most debilitating infectious diseases and Plasmodium is related to several other major parasites of humans and livestock, so a better understanding of it may help us to understand and control other human and animal diseases as well. The lack of effective vaccines against most of these parasites and the emergence of drug-resistant parasites mean that there is an urgent need for research leading to new treatment strategies for malaria and related diseases.

Single-celled parasites can replicate very rapidly inside infected people or animals. One basic function that all rapidly-replicating cells must perform is the maintenance of their telomeres: the ends of the chromosomes that make up the cell's genome. Telomeres become slightly shorter every time a cell divides and will eventually become too short for survival if they are not regularly extended. Cancer cells, for example, usually develop a method for replenishing their telomeres that allows them to replicate endlessly and grow out of control, whereas most normal cells in an adult body cannot do this.

The malaria parasite, like any other single-celled organism, replenishes its telomeres every time it divides, giving it an infinite capacity for replication. However, Plasmodium is a rather unusual microbe and we know very little about the machinery that controls its telomeres. Our understanding of telomeres in human cells is quite good, but the machinery that maintains telomeres in malaria parasites does not resemble the machinery in human cells. This project therefore focuses on the maintenance of telomeres in the malaria parasite. A better understanding of this could point us in the direction of new drug targets, since disrupting the structure and maintenance of telomeres is often lethal.

To find this machinery will require a biological fishing expedition. The whole genome of the malaria parasite will be broken up into pieces and the telomeric DNA will be fished out along with the proteins bound to it. These will then be identified and the experiment will be followed up by knocking out some of these proteins in parasites grown in the lab, to test whether they actually do control telomeres.

Overall, the study will give us a better understanding of the mechanisms that maintain the telomeres of these unusual parasites. It may inform new drug strategies to combat malaria and related parasitic diseases affecting both humans and animals. The outcomes of the research will be published in open-access scientific journals and presented at international conferences. They will be communicated to the general public via lay summaries on appropriate websites and via science-writing in magazines and/or online.

The proposed work will identify and characterise novel telomere-associated proteins in the protozoan parasite Plasmodium falciparum. This parasite (along with other apicomplexans of medical and veterinary importance: Toxoplasma, Cryptosporidium, Eimeria, Babesia and Theileria) belongs to an early-branching, highly-divergent lineage and few of the factors that cap, regulate and maintain its telomeres have been identified by homology with model organisms. Telomere maintenance is, however, a vital basic function, and may also represent a novel drug target.

This project will use Proteomics of Isolated Chromatin Segments (PICh) to identify the protein complement of telomeric DNA from P. falciparum. The PICh technique, in which cross-linked protein-DNA complexes are isolated via a specific DNA probe before identifying the proteins by mass spectrometry, was recently very successful in identifying known and novel components of the human telomere-binding complex.
After identifying the telomeric proteome in wildtype P. falciparum, telomeric proteins in sirtuin-knockout parasites will be identified for comparison. The two 'sirtuin' histone deacetylases play key roles in regulating expression of sub-telomeric virulence genes. I have shown that they also affect telomere length and genome stability, so these enzymes may be central components of a telomere-regulating complex.
Telomeric proteins of particular interest will be validated by immunofluorescence with fluorescence in situ hybridisation, to verify their specificity for the telomere. Genetic knockouts will then be generated and phenotyped for an initial characterisation of the roles of these proteins: telomere length and telomere stability in knockout lines will be analysed by Southern blotting and karyotyping.

The project will improve our understanding of a fundamental aspect of Plasmodium biology, as well as opening up new avenues for research on Plasmodium and other important apicomplexan parasites.

Most research on infectious pathogens ultimately aims to inform or develop new therapeutics or control strategies, and thus to benefit affected communities. The project proposed here is no exception, since apicomplexan parasites have a severe impact on both human and animal health within the UK and across the world. In the case of malaria, caused by Plasmodium, the affected communities include human populations in the tropics and sub-tropics (almost 250 million cases of disease and 1 million deaths per year) as well as travellers, from tourists to military personnel, who visit malaria-endemic regions. The burden of disease and the socio-economic impact are clearly huge and there is an urgent need for a better understanding of the parasite to inform new control strategies. Besides Plasmodium, the related parasites Toxoplasma and Cryptosporidium cause significant human disease in immuno-compromised individuals, while Babesia, Theileria and Eimeria are major parasites of cattle and poultry.

Basic biological studies, leading through to applied research, have the potential to reveal new drug targets for these pathogens. Telomere-associated proteins represent a particularly promising target because a rapidly-replicating protozoan parasite must maintain its telomeres to survive, and the proposed project aims to identify exactly such targets. Newly discovered proteins usually take some years to translate to the clinic as drug targets, but there are many examples of basic research generating targets that soon move into drug screening studies. New drugs to combat apicomplexan parasites could ultimately benefit diverse groups of people in the UK and the wider world, including residents of malaria-endemic countries, travellers, military personnel and those in the agricultural industry.

The impact of this project will extend beyond the long-term possibility of discovering new anti-parasitic drugs. Supporting more malaria researchers will expand the community working on this important disease, thus raising awareness and facilitating more research funding. My continuing career as a parasite biologist depends upon early-career grants such as this one and I hope that my career will ultimately include both basic research and field studies, as my postdoctoral work did. Field studies can bring many particular benefits to the communities affected by the malaria parasite - by bringing people into contact with educated researchers as well as by providing employment and direct scientific education. For example, while working in the Gambia, I had many conversations with Gambian citizens on public transport about the importance of completing courses of malaria treatment, treating young children promptly before severe disease develops and using bednets, amongst other issues. The value of such contact in educating local communities and raising the profile of UK-funded researchers should not be underestimated.

Turning to impacts within the UK, my career will include undergraduate and postgraduate teaching as well as research, and it is particularly incumbent upon scientists studying neglected and tropical parasites to raise their profile and enthuse students about working on important public health topics that can remain 'invisible' in the developed world. I hope to inspire a new generation of students to consider careers in biological research, in international development, or in human or veterinary medicine - all of which will benefit both the students themselves and the communities affected by parasitic diseases.

Finally, I am employed at Keele University in North Staffordshire, an area of high unemployment that was recently designated by the government for a Local Enterprise Partnership to promote economic redevelopment. The proposed project will immediately bring skilled academic work to a region with few such opportunities in which the university is a significant and valuable employer.