G-quadruplex biology in the human malaria parasite Plasmodium falciparum

The proposed research concerns the most important human malaria parasite, Plasmodium falciparum. Malaria is one of the world's most debilitating infectious diseases, killing over half a million people every year and affecting several hundred 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 new biological mechanism that the parasite may use for switching between var genes and for generating new variants. Our recent work has showed that an unusual DNA structure called a G-quadruplex that is concentrated around var genes seems to affect both these processes. To investigate this further, we now propose to map the G-quadruplexes throughout the Plasmodium genome, both in DNA and also in the messenger molecule, RNA. We will use a range of cutting-edge genome-wide technologies to do this, and will then check whether the distribution of the structures changes when the enzymes that unwind them are removed.

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 adhesive proteins 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 the distribution and metabolism of G-quadruplex (G4) motifs in both the DNA and RNA of the malaria parasite Plasmodium falciparum. G4-forming sequences are strikingly rare in the AT-rich P. falciparum genome, and are concentrated around the major family of virulence genes, called var genes. Our recent work has showed that G4 motifs may affect the expression and recombination of var genes, thus contributing to antigenic variation and diversification. Special classes of helicases are required to unwind G4s and we have knocked out two of these helicases in P. falciparum, revealing phenotypes in var gene expression, rates of genomic recombination, and telomere maintenance.

We now propose to use genome-wide chromatin immunoprecipitation to establish the distribution of G4 motifs and their cognate helicases throughout the P. falciparum genome. At the RNA level, we will examine the G4 content of the transcriptome via RNA base modification and structure-specific sequencing. In a second arm of experiments, we will follow up our work on the two 'RecQ' G4 helicases by making and characterising a double RecQ knockout, and also a knockout of the second class of putative G4 helicase termed FANCJ. Finally, we will characterise these knockouts not only in terms of G4-specific phenotypes, but also in their effects on DNA replication dynamics throughout the genome, using DNA fibre analysis.

Together, these experiments will provide multiple lines of evidence for the presence and roles of G4s and their cognate helicases in P. falciparum, thus improving our understanding of a novel aspect of malaria biology that is highly relevant to virulence.

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, since apicomplexan parasites such as the malaria parasite Plasmodium have a severe impact on both human and animal health within the UK and globally. In the case of malaria, affected communities include human populations across the tropics and sub-tropics, with several hundred million cases of disease and more than 0.5 million deaths per year in endemic areas. Also affected are travellers, from tourists to military personnel, who visit endemic regions. 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.

This project could provide at least two potential routes to new interventions: through 'anti-virulence' drugs or through drugs that interfere with G-quadruplex (G4) metabolism more generally and thus cause cell death. 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 main subject of this proposal - subsequently moving into field or clinical studies. Examples include the identification of the PfEMP1 adhesin involved in pregnancy malaria, which is now under investigation for a pregnancy malaria vaccine, and the identification of var genes encoding adhesins that facilitate rosetting on infected cells. Inhibitors of rosetting (a major factor in malaria pathology) are also now under investigation as anti-pathology drugs. The second potential route, of killing cells by interfering with their G4 metabolism, is under very active study in the cancer field. We are working with a small biotech company that specialises in G4-binding drugs to repurpose some of their agents as potential antimalarials, since some of them appear highly potent against Plasmodium in vitro.

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 I hope that my future work will 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 citizens on public transport about the importance of completing courses of malaria treatment, treating young children promptly, and using bednets, amongst other issues. The value of such contact in educating local communities and raising the profile of MRC-funded researchers should not be underestimated.

Turning to impacts within the UK, my work includes undergraduate and postgraduate teaching as well as research. It is particularly incumbent upon scientists studying neglected parasites to raise their profile and enthuse students about working on important topics that can remain 'invisible' in the developed world. I hope to inspire a new generation of students for careers in biological research, international development, or medicine - all of which will benefit both the students themselves and the communities affected by parasites. To reach students at an earlier age, I take part in regular education days for school children, teachers and the local community. Finally, I am employed at Keele University in North Staffordshire, an area of high unemployment where this project will immediately provide skilled academic work in a region with few such opportunities.