G-quadruplex biology in the human malaria parasite Plasmodium falciparum
Lead Research Organisation:
University of Cambridge
Department Name: Pathology
Abstract
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 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.
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.
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.
Publications
Harris LM
(2018)
G-Quadruplex DNA Motifs in the Malaria Parasite Plasmodium falciparum and Their Potential as Novel Antimalarial Drug Targets.
in Antimicrobial agents and chemotherapy
Merrick, C J
(2018)
RecQ helicases and quadruplex structures in var gene evolution and expression
Edwards-Smallbone, J
(2019)
A novel putative telomere-binding protein in Plasmodium falciparum (poster presentation, MPM 2019)
Dumetz F
(2019)
Parasitic Protozoa: Unusual Roles for G-Quadruplexes in Early-Diverging Eukaryotes.
in Molecules (Basel, Switzerland)
Description | This award produced or contributed to 6 peer-reviewed primary research papers and one peer-reviewed review. These are listed below as outcome URLs. To summarise the contents of these papers, we produced a new understanding of the roles of G-quadruplex nucleic acid structures in the biology of human malaria parasites - particularly of the G-quadruplexes (G4s) that occur specifically in RNA, which we measured and functionally investigated for the first time. We found that malaria parasites encode quite large numbers of RNA G4s and that these can influence the translation of genes that have important roles in parasite biology. Furthermore, some these structures are variably encodes in the different parasite strains that are found in human malaria patients. Alongside this, we produced the first complete RNA 'structurome' from this important human parasite, and identified the first parasite protein that can bind to both DNA and RNA G4s. All these findings have the potential for follow-up in further research. Finally, we seeded a project investigating G4-binding drugs as potential new anti-malarial agents, and pursued this through a further grant from the Rosetrees Trust. |
Exploitation Route | The current topic of further grant applications is our finding that a G-quadruplex is variably encoded, in field strains of P. falciparum, in an important DNA repair gene called Rad54. We wish to investigate whether this polymorphic G4 site could affect the efficiency of DNA repair in different parasite strains. This could be highly relevant to their response to DNA-damaging antimalarial drugs. The first RNA structurome, which we produced, will inform other RNA researchers, and lead to further refinements in this field. The G4-binding protein that we identified, GBP2, is now under investigation by a collaborating lab that studies Plasmodium RNA binding proteins. Finally, we recently published our pilot work on the prospects for repurposing G4-binding drugs as novel antimalarials. Although the first candidates we tested proved unsuitable in their current form, due to off-target toxicity in a mouse model, they provide the basis for investigating other G4-binding compounds in this regard. |
Sectors | Healthcare |
URL | https://academic.oup.com/nar/article/49/21/12486/6430841 |
Description | From Anticancer to Antimalarial Agent: Repositioning Quarfloxin |
Amount | £58,590 (GBP) |
Funding ID | M857 |
Organisation | Rosetrees Trust |
Sector | Charity/Non Profit |
Country | United Kingdom |
Start | 08/2019 |
End | 09/2022 |
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 | Cambridge Festival of Ideas, Debate Panel |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | Regional |
Primary Audience | Public/other audiences |
Results and Impact | Dr Merrick and PDRA Dr Franck Dumetz were panellists in a public debate on the morality and feasibility of parasite elimination, held at the Cambridge Law Faculty as part of the Cambridge Festival of Ideas in Oct 2018. More than 40 audience members attended this debate, engaging in Q&A and voting. |
Year(s) Of Engagement Activity | 2018 |
URL | https://www.festivalofideas.cam.ac.uk/events/do-we-have-right-exterminate-all-parasites |
Description | Cambridge Science Festival, March 2020 |
Form Of Engagement Activity | Participation in an open day or visit at my research institution |
Part Of Official Scheme? | No |
Geographic Reach | Regional |
Primary Audience | Public/other audiences |
Results and Impact | Cambridge Science Festival is a large pan-university event attracting 1000s of attendees from the region each year. The Pathology Department holds a Saturday open day offering a range of displays and hands-on activities. These include a game exploring vector-borne disease transmission, together displays of insect vectors and malaria parasites. In 2020, it was planned that Dr Merrick, together with PDRAs Holly Craven, Franck Dumetz, Francis Totanes and Jennifer McDonald, would run the activity. The aim was to engage and educate children and parents on the topic of neglected and parasitic diseases. Several hundred visitors would normally pass through the department each year and interact with these activities. However, within days of the 2020 Festival, and after the 2020 ResearchFish submission had been made, the university was suddenly closed due to coronavirus and hands-on festival activities were cancelled. |
Year(s) Of Engagement Activity | 2020 |
URL | https://www.sciencefestival.cam.ac.uk/events/hector-vector |
Description | Dr Dumetz work, and media reporting thereon, at Cambridge coronavirus testing centre |
Form Of Engagement Activity | A magazine, newsletter or online publication |
Part Of Official Scheme? | No |
Geographic Reach | Regional |
Primary Audience | Media (as a channel to the public) |
Results and Impact | During the pandemic shutdown, Dr Dumetz volunteered to work at the Cambridge coronavirus testing centre for several months and his activities, amongst those of others, were reported in university press releases & websites. |
Year(s) Of Engagement Activity | 2020 |
Description | Popular Science magazine |
Form Of Engagement Activity | A press release, press conference or response to a media enquiry/interview |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Media (as a channel to the public) |
Results and Impact | Advised a journalist from Popular Science magazine for the 2018 'Best of What's New' special. The journalist contected me concerning a new non-invasive malaria testing kit (which was not ultimately featured in the magazine). |
Year(s) Of Engagement Activity | 2018 |
Description | Science Articles in Sawston Scene village magazine |
Form Of Engagement Activity | A magazine, newsletter or online publication |
Part Of Official Scheme? | No |
Geographic Reach | Local |
Primary Audience | Public/other audiences |
Results and Impact | During the pandemic shutdown, Dr Merrick wrote 2 articles for spring and autumn editions of the Sawston scene magazine (published in a S.Cambs village of ~8000 residents). These updated residents on the science behind the pandemic. |
Year(s) Of Engagement Activity | 2020 |
URL | http://www.sawstonscene.org/ |
Description | TRAVELLING: AT WHOSE EXPENSE? Exhibition in Cambridge Festival of Ideas 2019 |
Form Of Engagement Activity | Participation in an open day or visit at my research institution |
Part Of Official Scheme? | No |
Geographic Reach | Regional |
Primary Audience | Public/other audiences |
Results and Impact | Dr Franck Dumetz, PDRA, participated in this exhibition as part of the annual Cambridge Festival of Ideas: a large pan-university event attracting 1000s of attendees from the region. The exhibition considered the impact of global travel on culture and health (superstitions, dietary habits, environmental changes, infectious diseases etc.) through anthropology, biomedical research and culture. |
Year(s) Of Engagement Activity | 2019 |
URL | https://www.festivalofideas.cam.ac.uk/events/travelling-whose-expense |