G-quadruplex biology in the human malaria parasite Plasmodium falciparum

Lead Research Organisation: Keele University
Department Name: Faculty of Natural Sciences


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.

Technical Summary

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.

Planned Impact

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.
Description Work on this grant is ongoing at Cambridge University: the 3-year duration was split after year-1 due to a move of the research group from Keele University to Cambridge University. For outcomes of the whole project, please therefore refer to the outcomes from the work on this project at Cambridge, which will be completed in Sept 2020.
Exploitation Route The grant is still ongoing, see above. For outcomes of the whole project, please therefore refer to the outcomes from the work on this project at Cambridge, which will be completed in Sept 2020.
Sectors Healthcare

Description Royal Society Kan Tong Po fellowship
Amount £3,000 (GBP)
Organisation The Royal Society 
Sector Charity/Non Profit
Country United Kingdom
Start 03/2018 
End 07/2018
Title RNA Structure-seq and in-vivo Structurome mapping in P. falciparum malaria parasites 
Description This grant has included the development and optimisation - in collaboration with Dr Kwok at City University Hong Kong - of methodology for RNA structure-mapping in P. falciparum parasites. This has never been conducted before in an intracellular parasite, and was published in 2021 in two manuscripts in Nucleic Acids Research and BioRxiv (still under peer review). The rG4 seq dataset has also been submitted to PlasmoDB.org. 
Type Of Material Technology assay or reagent 
Year Produced 2019 
Provided To Others? No  
Impact Impact will be broader once the methodology is published and the data appears on PlasmoDB. 
Description ChIP for PfGBP2 in P. falciparum parasites, Radboud University 
Organisation Radboud University Nijmegen
Country Netherlands 
Sector Academic/University 
PI Contribution A candidate P. falciparum telosome protein, PfGBP2, identified in this project, was epitope-tagged, subjected to chromatin immunoprecipitation, and sent to collaborators Dr Bartfai and coworkers at Radboud University.
Collaborator Contribution Dr Bartfai's group conducted and the GBP2 ChIP-seq and analysed resultant data.
Impact After considerable optimisation and repeats, we concluded that GBP2 is not amenable to ChIP. This conflicts with a publication that appeared in Dec 2020 from a competitor lab, reporting ChIP of GBP2 with the same methodology. However, poor reporting standards in this paper make it impossible to verify or reanalyse their results. Our data are still destined for a paper on PfGBP2 (which in late 2019 was already complete except for the ChIP data, and was scheduled for submission in early 2020 - considerably delayed by the pandemic shutdown, and then by the competing publication, which now necessitates some re-writing). Our work will be submitted within 2021.
Start Year 2019
Description Plasmodium RNA structurome 
Organisation City University of Hong Kong
Country Hong Kong 
Sector Academic/University 
PI Contribution Hosted Dr Chun Kit Kwok from Hong Kong City University at Cambridge to commence collaboration on a Plasmodium structurome. PDRA on this grant then produced the requisite RNA, which was processed by Dr Kwok in Hong Kong and then data were analysed at Cambridge in collaboration with Dr Anton Enright.
Collaborator Contribution Dr Kwok provided expertise and handled the processing of our structurome-probed RNA. Dr Enright provided informatics expertise in processing resultant data.
Impact A structurome was generated, fully analysed, and a paper is now nearly written (considerably delayed by the pandemic shutdown), aiming for submission in early 2021.
Start Year 2018
Description RNA Shape-Sequencing, Hong Kong City University 
Organisation City University of Hong Kong
Country Hong Kong 
Sector Academic/University 
PI Contribution Provision of P. falciparum RNA and data analysis. The G4 content of the P. falciparum transcriptome is being examined by RNA shape sequencing in collaboration with Dr Chun Kit Kwok - previously of Cambridge University, now at Hong Kong City University - and his bioinformatics collaborators in the Chan group.
Collaborator Contribution Dr Kwok has generated a dataset from P. falciparum RNA, providing labour, reagents and technical expertise. Dr Chan is supporting in silico data analysis.
Impact Duplicate dataset of RNA G4s found in the P. falciparum transcriptome. Follow-on RoyalSoc. Kang Ton Po fellowship for Dr Kwok to visit the Merrick lab and pursue this work. Manuscript describing this dataset about to be submitted in early 2021 (delayed by pandemic).
Start Year 2017
Description RNA structurome in Toxoplasma with Bill Sullivan 
Organisation Indiana University
Country United States 
Sector Academic/University 
PI Contribution Arising from our publication on the RNA structurome of Plasmodium, Prof Sullivan requested support for his RO1 application to carry out a similar technique in Toxoplasma.
Collaborator Contribution None yet
Impact None yet (RO1 outcome pending)
Start Year 2021
Description 2 Bugbitten blogs, PDRA F. Noulin 2017 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Media (as a channel to the public)
Results and Impact These blog entries highlight important papers in the parasitology/vector biology literature, and regularly receive over 1000 hits.
Year(s) Of Engagement Activity 2017
URL http://blogs.biomedcentral.com/bugbitten/2017/12/15/plasmespins-ix-and-x-as-drug-targets-putting-a-s...
Description Mock Oxbridge interviews, Stoke on Trent college 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact PI Dr Merrick was asked by a local sixth form college (which has traditionally low participation in elite higher education) to conduct mock interviews for students applying to Oxbridge.
Year(s) Of Engagement Activity 2017