Virulence gene dynamics in the human malaria parasite

The proposed research concerns the most important human malaria parasite, Plasmodium falciparum. Malaria is one of the world's most debilitating infectious diseases, killing almost a million people every year and affecting up to 300 million. Most of the deaths occur in young children in sub-Saharan Africa, but adults can also suffer from malaria throughout their lives, reducing quality of life and retarding economic development in endemic countries. The lack of an effective vaccine and the emergence of drug-resistant parasites mean that there is now an urgent need for research leading to a better understanding of the malaria parasite, and hence to new vaccine targets and treatment strategies for this disease.

The malaria parasite causes illness via the infection of red blood cells. It multiplies inside these cells and modifies their surfaces with proteins called PfEMP1s that bind to the walls of blood vessels. This is crucial for parasite survival as it removes infected cells from the circulating blood and protects them from passing through the spleen, which might recognize and destroy them. It also contributes to disease, with severe malaria being particularly associated with the accumulation of infected cells in vessels of the brain and placenta. It is therefore of great interest to malaria biologists to understand the mechanisms that control the expression of these adhesive PfEMP1 proteins.

PfEMP1s are not expressed uniformly by all malaria parasites: instead, individual parasites regularly switch between different variants. This allows them to stay ahead of the immune system and sustain a chronic infection for months or even years. The parasites have a large, variable family of 'var' genes for different PfEMP1 proteins and they vary the expression of these genes by so-called 'epigenetic switching'. Furthermore, var genes recombine very readily to generate new variants, so each parasite strain - of which there are many hundreds circulating in endemic areas - has a unique repertoire of possible surface proteins. This is one reason why immunity to repeated malaria infections is slow to develop in humans: every new parasite strain looks different to the immune system, so people can be re-infected repeatedly throughout their lives.

Understanding, and ultimately interfering with, the expression, switching and recombination of var genes - and thus the variant expression of PfEMP1 proteins - could be a key to more effective immune control of malaria. Therefore, this research focuses on a possible biological mechanism for switching between var genes and for generating new variants. An unusual DNA structure called a G-quadruplex that is concentrated around var genes may affect both of these processes.

Experiments to investigate this idea will include stabilizing the G-quadruplexes with specific chemicals and testing the effect on var gene expression. G-quadruplexes will also be isolated to test their effect on 'reporter' genes (genes coding for an easily-measurable product, such as a fluorescent protein). Finally, we will also test the effects of mutating some proteins called helicases that unwind the G-quadruplex DNA structures.

These studies will lead to a better understanding of the mechanisms underlying var gene dynamics, and may ultimately inform new strategies to combat malaria, since var genes - and the PfEMP1s that they encode - are central to malarial disease. 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 summaries on appropriate websites and via science-writing in magazines and/or online. Work such as this remains vital as long as the malaria parasite continues to cause an immense burden of human disease.

The proposed work will investigate a novel mechanism for silencing sub-telomeric var genes and promoting their recombination in Plasmodium falciparum. The var gene family is known to undergo epigenetic silencing and switching, involving sirtuin histone deacetylases. Var genes also recombine readily to generate new variants. However, the mechanisms underlying these virulence processes are not fully understood.
G-quadruplex (G4) DNA structures may influence both transcription and recombination of var genes. G4-forming sequences are strikingly rare in the AT-rich P. falciparum genome, and are concentrated around var genes. In model systems, these structures can affect both transcription and recombination by stalling RNA or DNA polymerases. A special class of 'RecQ' helicases unwind G4s and a human RecQ helicase was recently shown to operate in concert with a sirtuin: cells lacking either of these proteins developed genome instability and chromosome fusions at G-rich telomeres. In P. falciparum, I have shown that similar phenotypes are seen in a sirtuin knockout, suggesting that G4s, RecQ helicases and sirtuins may also affect DNA metabolism in the parasite.
Several complementary molecular-genetic approaches will address this hypothesis. Parasites will be treated with G4-stablizing drugs and tested for altered var gene transcription, telomere maintenance and karyotypic changes. G4 sequences will be cloned upstream of reporter genes, as integrated transgenes, and their effect on transcription and recombination will be examined. Putative RecQ helicases will be knocked out and the mutant parasites phenotyped, and ChIP studies of the helicases will be carried out in strains with or without a sirtuin. Together, these experiments will provide multiple lines of evidence for the role of G4s, RecQ helicases and sirtuins in controlling transcription and recombination of P. falciparum virulence genes, thus improving our understanding of a fundamental aspect of malaria biology.

Most research on infectious pathogens ultimately aims to inform or develop new therapeutics or control strategies, and thus to benefit the communities affected by the disease. The project proposed here is no exception. In the case of malaria, the affected communities include human populations across the tropics and sub-tropics, with hundreds of millions of cases of disease and almost a million deaths per year. The burden of disease is clearly huge and the need for a better understanding of the malaria parasite to inform new control strategies is urgent. Alleviating the burden of malaria will not only reduce the global death toll; it will also reduce the social and economic impact of the disease, which includes the loss of economic activity through illness, as well as neurological sequelae such as epilepsy and attention disorders in children, and retarded growth and development due to anaemia.

Basic research on virulence mechanisms usually takes some years to translate to the clinic, but there are many examples of research on var genes and PfEMP1s - the subject of this proposal - subsequently moving into field or clinical studies. One example is the identification of the PfEMP1 adhesin involved in pregnancy malaria, which is now under investigation for a pregnancy malaria vaccine. Another is the identification of var genes encoding adhesins that facilitate rosetting on infected cells. Inhibitors of rosetting, a major factor in malaria pathology, are now under investigation.

The impact of this project will extend beyond the possibility of discovering new routes towards malaria therapy and control. Supporting more malaria researchers will expand the community working on this important disease, thus raising awareness and facilitating more research funding. The grant will support a dedicated postdoctoral researcher who will gain specialized skills in malaria genetics; while my own continuing career as a malaria biologist depends upon early-career grants such as this one. I hope that this career will ultimately include both basic research and field studies, as my postdoctoral work did. Field studies can, in turn, benefit the communities affected by the parasite by bringing local people into contact with educated researchers, as well as by providing employment and direct scientific education. While working in the Gambia as a postdoc, 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 MRC researchers should not be underestimated.

Turning to impacts within the UK, my career will include undergraduate and postgraduate teaching as well as research. It is particularly incumbent upon scientists studying neglected and tropical diseases to raise the profile of these diseases 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 research on neglected diseases, in international development, or in medicine - all of which will benefit both the students themselves and the communities affected by malaria.

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 bring skilled academic work to a region with few such opportunities in which the university is a significant and valuable employer.