Virulence gene dynamics in the human malaria parasite.

Lead Research Organisation: Keele University
Department Name: Inst for Science and Tech in Medicine

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 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.

Technical Summary

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.

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. In the case of malaria, the affected communities include human populations across the tropics and sub-tropics, with almost 250 million cases of disease and 1 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. 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. 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.

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. My continuing career as a malaria 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 benefit the communities affected by the parasite by bringing 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 work includes undergraduate and postgraduate teaching as well as research, and 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. To reach students at an earlier age, I am raising the profile of malaria research by taking part in regular education days for school teachers.

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.

Publications

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Description ERC Consolidator Grants
Amount € 1,998,696 (EUR)
Funding ID PlasmoCycle 
Organisation European Research Council (ERC) 
Sector Public
Country European Union (EU)
Start 06/2017 
End 05/2022
 
Description Faculty Research Support Fund
Amount £707 (GBP)
Organisation Keele University 
Sector Academic/University
Country United Kingdom
Start 01/2016 
End 01/2016
 
Description Jean Shanks Foundation Intercalated Grants
Amount £6,532 (GBP)
Organisation The Jean Shanks Foundation 
Sector Charity/Non Profit
Country United Kingdom
Start 08/2014 
End 07/2015
 
Description MRC Standard Responsive Mode
Amount £481,203 (GBP)
Funding ID MR/P010873/1 
Organisation Medical Research Council (MRC) 
Sector Academic/University
Country United Kingdom
Start 05/2017 
End 05/2020
 
Description Royal Society Kan Tong Po fellowship
Amount £3,000 (GBP)
Organisation The Royal Society 
Sector Academic/University
Country United Kingdom
Start 03/2018 
End 07/2018
 
Description Wellcome Trust Biomedical Vacation Scholarship
Amount £2,000 (GBP)
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 06/2014 
End 07/2014
 
Title BrdU labelling of DNA replication in Plasmodium parasites 
Description The labelling of DNA replication with bromo-/iodo-/chloro-deoxyuridine (BrdU/IdU/CldU), has long been a workhorse assay in human cell-cycle studies, identifying cells that are in S-phase swiftly and sensitively. However, this was not previously possible in Plasmodium - despite conflicting reports in the literature going back 25 years - because the parasite relies only on de novo synthesis of pyrimidines and does not salvage thymidine analogues like BrdU for conversion into nucleotides. I have recently showed that expressing a TK enzyme in transgenic parasites supplies the pyrimidine salvage pathway and allow parasites to be labelled with BrdU (see paper by Merrick, Malar J. 2015). Therefore, this can now be exploited to investigate how DNA replication is organised, from the whole-cell to the single-molecule level, during both schizogony and gametogenesis. 
Type Of Material Technology assay or reagent 
Year Produced 2015 
Provided To Others? Yes  
Impact I am actively exploiting this method to study replication dynamics in Plasmodium. I have two major grants under assessment (BBSRC and ERC) based on the technique, and I have shared it with other groups, at least one of whom is actively using it to answer related questions in Plasmodium biology. 
URL https://malariajournal.biomedcentral.com/articles/10.1186/s12936-015-1014-7
 
Title DNA combing in P. falciparum 
Description Part of an ongoing project to develop DNA replication labelling in Plasmodium parasites, which was developed by Dr Merrick as a side-project from the referenced grants. See also the methodology paper Merrick, Malar J. 2015. This collaborative work with the Sterkers lab at Montpellier University has, for the first time, developed DNA combing and DNA fibre analysis for Plasmodium. See paper currently under review at Scientific Reports: 'Single-molecule Analysis Reveals that DNA Replication Dynamics Vary Across the Course of Schizogony in the Malaria Parasite Plasmodium falciparum.' Slavica Stanojcic, Nada Kuk, Imran Ullah, Yvon Sterkers, Catherine J. Merrick. 
Type Of Material Technology assay or reagent 
Year Produced 2017 
Provided To Others? Yes  
Impact Paper on this technique is still under review. Once published, we anticipate wider uptake, as has already occurred with the preceding methods paper (Merrick, Malar J. 2015.) which has prompted several requests for collaborative work. This methodology was a key contributor to a successful ERC grant awarded to Dr Merrick in 2016. 
 
Description DNA combing, Montpellier University 
Organisation University of Wurzburg
Department Parasitology
Country Germany 
Sector Academic/University 
PI Contribution I conducted method-development in preparing Plasmodium for DNA combing, and provided parasite material to collaborators in Montpellier.
Collaborator Contribution The collaborators provide access to the DNA combing facility, and data analysis skills for the resultant DNA fibre images.
Impact Preliminary data in support of two major grant applications (BBSRC and ERC); the ERC application being successful in 2017. Publication in the journal Scientific Reports, 2017.
Start Year 2016
 
Description Development of BrdU-incorporation methodology in Plasmodium berghei & Plasmodium knowlesi 
Organisation Imperial College London
Department VIGILab
Country United Kingdom 
Sector Academic/University 
PI Contribution Technical advice & discussion concerning BrdU-labelling methodology for development of TK-expressing P. berghei at ICL; technical advice & plasmid provided for development of TK-expressing P. knowlesi at Sanger.
Collaborator Contribution A researcher in the Vigilab developed a TK-expressing P. berghei which they are now characterising. At WT Sanger, existing collaborators in the Rayner lab have developed a TK P. knowlesi line. My lab now has both these lines to be taken forward in a compartive study of replication across 3 species of Plasmodium.
Impact Prelim data showing the BrdU incorporation is feasible in TK-expressing P. berghei (presented at MAM 2015 conference) & also in TK-expressing P. knowlesi.
Start Year 2015
 
Description Development of BrdU-incorporation methodology in Plasmodium berghei & Plasmodium knowlesi 
Organisation The Wellcome Trust Sanger Institute
Country United Kingdom 
Sector Charity/Non Profit 
PI Contribution Technical advice & discussion concerning BrdU-labelling methodology for development of TK-expressing P. berghei at ICL; technical advice & plasmid provided for development of TK-expressing P. knowlesi at Sanger.
Collaborator Contribution A researcher in the Vigilab developed a TK-expressing P. berghei which they are now characterising. At WT Sanger, existing collaborators in the Rayner lab have developed a TK P. knowlesi line. My lab now has both these lines to be taken forward in a compartive study of replication across 3 species of Plasmodium.
Impact Prelim data showing the BrdU incorporation is feasible in TK-expressing P. berghei (presented at MAM 2015 conference) & also in TK-expressing P. knowlesi.
Start Year 2015
 
Description Drug mode-of-action analysis via BrdU-incorporation into P. falciparum parasites 
Organisation Heidelberg University
Department Centre for Infectious Diseases, Parasitology
Country Germany 
Sector Academic/University 
PI Contribution TK-expressing parasite lines that incorporate BrdU to monitor DNA replication; reagents, methodology & hosting of visiting researchers
Collaborator Contribution Since publication of this new method (Merrick, Malar J 2015) several research groups have approached me wishing to use the technique. The Lanzer and Birkholtz group have provided/will provide antimalarial drugs to test, plus visiting researchers.
Impact Prelim data on the mode of action of 1 candidate antimalarial in blocking DNA replication (data generated by visiting researcher from Heidelberg to Keele).
Start Year 2016
 
Description Drug mode-of-action analysis via BrdU-incorporation into P. falciparum parasites 
Organisation University of Pretoria
Country South Africa 
Sector Academic/University 
PI Contribution TK-expressing parasite lines that incorporate BrdU to monitor DNA replication; reagents, methodology & hosting of visiting researchers
Collaborator Contribution Since publication of this new method (Merrick, Malar J 2015) several research groups have approached me wishing to use the technique. The Lanzer and Birkholtz group have provided/will provide antimalarial drugs to test, plus visiting researchers.
Impact Prelim data on the mode of action of 1 candidate antimalarial in blocking DNA replication (data generated by visiting researcher from Heidelberg to Keele).
Start Year 2016
 
Description Investigation of basic parasite biology via BrdU-incorporation into P. falciparum parasites 
Organisation Boston Children's Hospital
Country United States 
Sector Hospitals 
PI Contribution TK-expressing parasite lines that incorporate BrdU to monitor DNA replication; reagents & methodology for this work.
Collaborator Contribution Since publication of this new method (Merrick, Malar J 2015) several research groups have approached me wishing to use the technique. The Dvorin and Mota groups have expressed interest in sharing the technology to investigate their various phenotypes of interest in Plasmodium biology (genetic mutants etc.): work is ongoing or in development.
Impact None yet
Start Year 2018
 
Description Investigation of basic parasite biology via BrdU-incorporation into P. falciparum parasites 
Organisation University of Lisbon
Country Portugal 
Sector Academic/University 
PI Contribution TK-expressing parasite lines that incorporate BrdU to monitor DNA replication; reagents & methodology for this work.
Collaborator Contribution Since publication of this new method (Merrick, Malar J 2015) several research groups have approached me wishing to use the technique. The Dvorin and Mota groups have expressed interest in sharing the technology to investigate their various phenotypes of interest in Plasmodium biology (genetic mutants etc.): work is ongoing or in development.
Impact None yet
Start Year 2018
 
Description RNA Shape-Sequencing, Cambridge University 
Organisation University of Cambridge
Department Department of Chemistry
Country United Kingdom 
Sector Academic/University 
PI Contribution The G4 content of the P. falciparum transcriptome is being examined by RNA shape sequencing in collaboration with Dr Chun Kit Kwok in the Balasubramanian lab at Cambridge University.
Collaborator Contribution Dr Kwok is providing reagents and technical expertise in the process, as well as access to bioinformatic support for data analysis.
Impact RNA shape-seq data was produced within 2016. This informed a second, successful, grant application to the MRC which is currently underway (2017-2020), including followup work on this dataset. A publication is also in preparation for submission within 2019.
Start Year 2015
 
Description RNA-seq at Sanger Institute 
Organisation The Wellcome Trust Sanger Institute
Country United Kingdom 
Sector Charity/Non Profit 
PI Contribution Genetic mutants generated in this project were examined for genome-wide transcriptional perturbations via RNA-Seq in collaboration with the lab of Dr Julian Rayner (a named collaborator on this grant). PI Catherine Merrick and PDRA Lynne Harris visited the Sanger Institute, discussed and analysed resultant data.
Collaborator Contribution Dr Rayner's lab supplied both the knowledge and the materials to analyse these mutant parasites by RNA-seq
Impact A publication appeared in 2018 in Plos Genetics, including the outcomes of this collaboration. Dr Harris gained valuable skills to improve her employability in the future, and the project will gain 'omic level data which we could not generate at Keele without the input of our collaborators.
Start Year 2013
 
Description Tetragene collaboration 
Organisation TetraGene
Country United States 
Sector Private 
PI Contribution We are actively investigating the activity in malaria parasites of new G-quadruplex binding drugs to which Tetragene holds exclusive rights.
Collaborator Contribution Tetragene have freely shared (subject to MTA and confidentiality agreements) their G-quadruplex binding molecule(s).
Impact An iCASE PhD studentship application to further this collaborative work was submitted in summer 2014, but not funded. A publication appeared in ACC in 2018 detailing the outcomes of this study.
Start Year 2014
 
Description Whole Genome Sequencing by Malariagen, Sanger Institute 
Organisation The Wellcome Trust Sanger Institute
Country United Kingdom 
Sector Charity/Non Profit 
PI Contribution Genetic mutants generated in this project were examined by Whole Genome Sequencing in collaboration with the MalariaGen consortium, Dr Julian Rayner (a named collaborator on this grant) and Dr Antoine Claessens. Almost 100 genomes were sequenced in a large-scale 'clone tree' approach to track mutation rates over time.
Collaborator Contribution MalariaGen has provided the whole-genome sequencing facility; Dr Claessens has provided bioinformatic skills in data analysis.
Impact A publication including the results of this study appeared in Plos Genetics in 2018.
Start Year 2013
 
Description 'Women in Science' schools visit to Keele, 2014 and 2015 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? Yes
Geographic Reach Local
Primary Audience Schools
Results and Impact Highly positive feedback from students and school teachers involved

'It has been an absolutely brilliant day, with a really good blend of theory and practical activity. It was an excellent opportunity for our young people to engage with academics and so many Keele students.' - Paul Winterburn, Biddulph High School teacher.
'I am 100% coming to Uni now, man...' - Year-9 pupil.
Year(s) Of Engagement Activity 2014,2015
URL http://www.keele.ac.uk/lifesci/newsandevents/2014/womeninscience.php
 
Description Article in Slightly Foxed Magazine 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Article 'A Scientist for all Seasons' (A review of E.O Wilson's 'Biophilia') Merrick, C.J. Slightly Foxed, Summer 2014 issue. Published as part of an occasional series reviewing classic books in the scientific literature, for a literary magazine with a circulation of ~7000.
Year(s) Of Engagement Activity 2014
URL https://foxedquarterly.com/Slightly-Foxed-Issue-42-Catherine-Merrick-on-Biophilia/
 
Description BSP YouTube channel 
Form Of Engagement Activity Engagement focused website, blog or social media channel
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact PI Catherine Merrick became Hon. Communications Secretary for the British Society of Parasitology in 2015. As part of this job she has set up a YouTube channel to host videos about the society, as well as curating the society's webpage (receiving ~200 hits per day) and Facebook and Twitter groups (almost 2000 members). Collectively, this BSP activity provides up-to-date and accessible news and information on parasitology to a very broad sector of the public.
Year(s) Of Engagement Activity 2015
URL https://www.youtube.com/channel/UCo46EvqlMbWmgABiM1cBfdA
 
Description BSP social media 
Form Of Engagement Activity Engagement focused website, blog or social media channel
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact PI Catherine Merrick became Hon. Communications Secretary for the British Society of Parasitology in 2015. As part of this job she curates the society's fast-growing Facebook and Twitter groups (which have approximately doubled in membership during her tenure and now have almost 3000 members). https://www.facebook.com/groups/8573726067/ and https://twitter.com/BSPparasitology. They provide up-to-date and accessible news and information on parasitology to a broad sector of the public: in the case of the Twitter group, primarily parasitology researchers; in the Facebook group, a large cohort of interested parties and practitioners in developing countries.
Year(s) Of Engagement Activity 2015,2016,2017
URL https://twitter.com/BSPparasitology
 
Description Bugbitten blog, C Merrick 2014 
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, highlighting important papers in the parasitology/vector biology literature, regularly receive over 1000 hits.
Year(s) Of Engagement Activity 2014
URL http://blogs.biomedcentral.com/bugbitten/2014/04/01/throwing-the-switch-gametocytogenesis-in-malaria...
 
Description Bugbitten blog, C Merrick 2015 
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, highlighting important papers in the parasitology/vector biology literature, regularly receive over 1000 hits.
Year(s) Of Engagement Activity 2015
URL http://blogs.biomedcentral.com/bugbitten/2015/08/21/rinse-and-repeat-can-mosquito-passage-reset-mala...
 
Description New BSP website 
Form Of Engagement Activity Engagement focused website, blog or social media channel
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact In her role as Hon. Communications Secretary for the British Society for Parasitology, PI Dr Catherine Merrick has had a new website for the British Society of Parasitology commissioned, built and gone-live in autumn 2016. The website includes a much-expanded public engagement section.
Year(s) Of Engagement Activity 2016
URL http://bsp.uk.net/
 
Description School visit, Madeley High School 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Keele staff including PI Catherine Merrick visited a local school to talk about their careers as female scientists and to run hands-on workshops for GCSE-level pupils.
Year(s) Of Engagement Activity 2015
URL http://www.keele.ac.uk/lifesci/newsandevents/2015/intwomensday.php
 
Description School visit, Malbank School 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact An evening talk on malaria for students and their parents as part of an extension programme at a local school
Year(s) Of Engagement Activity 2015
 
Description School visit, South Cheshire College 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Visit to give a talk on malaria and on university entry at a local sixth form college
Year(s) Of Engagement Activity 2015
URL http://www.keele.ac.uk/lifesci/newsandevents/2015/scheshirecollege.php
 
Description School visit, Walsall College 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact Visit to give a talk on malaria and on university entry at a regional sixth form college
Year(s) Of Engagement Activity 2014
 
Description Talk at Stoke on Trent college 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact A talk by PI Dr Merrick to 6th formers in the Stoke on Trent college university extension programme, to encourage interest in biology & applications to university
Year(s) Of Engagement Activity 2016