Proteases in host cell invasion by the malaria parasite

Lead Research Organisation: MRC National Inst for Medical Research

Abstract

Each year, over 2 million people die as a result of infection with the malaria parasite. Development of an effective vaccine has proved difficult, and in many parts of the world there is widespread resistance to available antimalarial drugs. In an infected person, the malaria parasite grows within red blood cells. To do this, the invasive merozoite form of the parasite must exit from its host cell at regular intervals of about 48 hours then bind to the outside of a new red cell and invade it. Exit and invasion require the activity of a number of parasite enzymes, called proteases. Work in this laboratory aims to understand how these proteases work, and to devise compounds which prevent their action, thereby blocking the parasites life cycle and the progress of the disease. Our work is intended to aid the development of a new class of antimalarial drugs based on protease inhibitors that will combat and control this devastating disease.

Technical Summary

Malaria is directly responsible for an estimated 2-3 million deaths per annum, imposing an immense economic burden on much of the developing world. There is no malaria vaccine, and resistance against mainstay antimalarial drugs is widespread, resulting in an increasing threat to travellers.||Malaria is caused by an obligate intracellular parasite, which invades and replicates within red blood cells. We are interested in the molecular mechanisms by which the invasive blood-stage form of the parasite, the merozoite, modifies, exits and enters its host red cell.||Blood stages of the most virulent species of the malaria parasite, Plasmodium falciparum, can be cultured in vitro, and our work uses classical biochemical approaches, transgenesis and microscopy to study invasion in this system. A major element of our work makes use of heterologous expression systems for structural and enzymological studies.||At or around invasion, several essential parasite proteins are dramatically restructured as a result of proteolytic activity. A subtilisin-like parasite protease called PfSUB1 plays a role in release of the parasite from its host cell. At invasion, several merozoite surface proteins known to play a key role in invasion are precisely cleaved by another parasite subtilisin-like protease called PfSUB2 and shed from the merozoite surface. Our investigations of these proteases are focused on determining their structure, their fine substrate specificity and their precise function in the parasite as well as the identification of selective low molecular weight inhibitors of the enzymes that have potential for development as antimalarial drugs. In addition we have found that members of a different family of serine proteases called rhomboids, first discovered in Drosophila, also play an important role in invasion. We are engaged in active collaborations with other groups interested in the role of serine proteases in host cell invasion by related Apicomplexan parasites, including the pathogen Toxoplasma gondii. Our research focuses on the physiological significance of these proteolytic processing events, and the structural and functional characterization of the enzymes involved in these and other important protease-mediated processes during invasion. A better understanding of these will help to lay the groundwork for development of new, much needed drugs or vaccines which target processes and molecules involved in this critical step in the parasites life cycle.

Publications

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Blackman MJ (2013) Recent insights into apicomplexan parasite egress provide new views to a kill. in Current opinion in microbiology

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Blackman MJ (2008) Malarial proteases and host cell egress: an 'emerging' cascade. in Cellular microbiology

 
Description EU- Network of Excellence-NOE-(Evimalar)
Amount £102,888 (GBP)
Funding ID 242095 
Organisation European Commission 
Sector Public
Country European Union (EU)
Start 10/2009 
End 09/2014
 
Description EU-Networks for Initial Training-(InterMalTraining)
Amount £807,654 (GBP)
Funding ID PITN-GA-2008-215281 
Organisation European Commission 
Sector Public
Country European Union (EU)
Start 10/2008 
End 09/2012
 
Description EU-funded Marie Curie ITN (MalParTraining)
Amount £111,924 (GBP)
Organisation Marie Sklodowska-Curie Actions 
Sector Academic/University
Country Global
Start 10/2006 
End 10/2010
 
Description EU-funded Marie Curie ITN (STARS)
Amount £184,907 (GBP)
Funding ID PITN-GA-2009-238490 
Organisation Marie Sklodowska-Curie Actions 
Sector Academic/University
Country Global
Start 09/2010 
End 09/2013
 
Description EU-funded Network of Excellence (BioMalPar)
Amount £280,000 (GBP)
Organisation European Commission 
Sector Public
Country European Union (EU)
Start 10/2006 
End 10/2009
 
Description EviMalAR PhD Fellowship
Amount £61,617 (GBP)
Organisation European Commission 
Sector Public
Country European Union (EU)
Start 12/2010 
End 11/2013
 
Description FP7-SP1-Cooperation-2009 ("MALSIG")
Amount £159,360 (GBP)
Organisation European Commission 
Department Seventh Framework Programme (FP7)
Sector Public
Country European Union (EU)
Start 02/2009 
End 01/2012
 
Description MRC Project Grant
Amount £10,000 (GBP)
Funding ID G1100013 
Organisation Medical Research Council (MRC) 
Sector Academic/University
Country United Kingdom
Start 10/2011 
End 09/2014
 
Description MRCT Development Gap Funding (Inhibitors of a malarial serine protease)
Amount £166,000 (GBP)
Organisation MRC-Technology 
Sector Academic/University
Country United Kingdom
Start 01/2006 
End 01/2007
 
Description Project Grant
Amount £10,000 (GBP)
Funding ID 094752 
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 05/2011 
End 04/2014
 
Description Project Grant-2009 "Proteases in egress of the malaria parasite from its host cell"
Amount £177,006 (GBP)
Funding ID 086550/Z/08/Z 
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 05/2009 
End 04/2012
 
Description Swiss National Science Fund Fellowship
Amount £35,000 (GBP)
Organisation Swiss National Science Foundation 
Sector Public
Country Switzerland
Start 11/2011 
End 10/2012
 
Description Training Day-2007 (MalParTraining")
Amount £20,941 (GBP)
Organisation European Commission 
Sector Public
Country European Union (EU)
Start 03/2007 
End 04/2007
 
Description Travel Award
Amount £200 (GBP)
Organisation British Society for Parasitology (BSP) 
Sector Learned Society
Country United Kingdom
Start 09/2009 
End 10/2009
 
Description Wellcome Trust/NIH PhD Studentship
Amount £80,000 (GBP)
Funding ID 103459/Z/14/Z 
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 10/2014 
End 09/2018
 
Title Anti-PfSUB1 mAbs 
Description Monoclonal antibodies (4 in total) specific for a malarial processing protease called PfSUB1. 
Type Of Material Antibody 
Year Produced 2013 
Provided To Others? Yes  
Impact These have been used to follow the trafficking, subcellular sites of storage, and secretion of the protease, as well as to produce Fab fragments that have been used to solve the x-ray crystal structure of the enzyme. Initial data were recently published (Collins CR, Hackett F, Strath M, Penzo M, Withers-Martinez C, Baker DA, Blackman MJ [2013] Malaria parasite cGMP-dependent protein kinase regulates blood stage merozoite secretory organelle discharge and egress. PLoS Pathog. 9:e1003344). 
URL http://www.ncbi.nlm.nih.gov/pubmed/24785947
 
Title DiCre in the malaria parasite 
Description We have adapted the DiCre gene disruption system to the humana malarial parasite P. falciparum. This for the first time provides a robust means of conditionally disrupting essential genes in order to study their function. This will revolutionise the malaria research field. 
Type Of Material Technology assay or reagent 
Year Produced 2014 
Provided To Others? Yes  
Impact This system has already allowed the genetic study of a number of essential malarial genes, and will continue to generate a high degree of interest across the malaria research field because of its enormous potential for the discovery of gene function. 
URL http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=2348932...
 
Title Human-adapted Plasmodium knowlesi 
Description Plasmodium knowlesi is an emerging human pathogen in large parts of SE Asia, but until recently could only be maintained by growth in vivo in primates. We have adapted a line of P. knowlesi to long-term continuous growth in vitro in human red blood cells, providing a new tool for researchers interested in this zoonotic pathogen. 
Type Of Material Cell line 
Year Produced 2013 
Provided To Others? Yes  
Impact This has enabled in-depth genomic and genetic studies on this important human pathogen, as well as a range of biological studies that have yet to reveal their full impact. 
URL http://www.ncbi.nlm.nih.gov/pubmed/23267069
 
Title PfSUB1 inhibitor screen 
Description Development of a high throughput screen to search for inhibitors of an important malarial protease involved in host cell egress. Development of this took around 3 years of work and involved both intramural and industrial collaborations. 
Type Of Material Technology assay or reagent 
Year Produced 2006 
Provided To Others? Yes  
Impact It has been used to identify physiological substrates and the function of an important malarial enzyme and drug target 
 
Description BioMalpar-funded studentship 
Organisation University of Geneva
Country Switzerland 
Sector Academic/University 
PI Contribution This work involved a study of cell surface receptors used by the pathogen Toxoplasma gondii to invade cells. The main work was performed by a PhD student funded jointly by my research group and that of a collaborator at the University of Geneva (Prof Dominique Soldati)
Collaborator Contribution The partnership used funding obtained from an EU source (the BioMalPar Network of excellence). The student was hosted jointly at the University of Geneva and NIMR.
Impact This has resulted in an improved understanding of host cell invasion by the causative agent of toxoplasmosis, and important and potentially serious opportunistic infection. Publications directly arising from it have PubMed ID numbers 19901027, 18818666 and 17491595.
 
Description Development of a conditional gene disruption system for malaria 
Organisation University of Glasgow
Country United Kingdom 
Sector Academic/University 
PI Contribution This involved the development of a Cre-based conditional gene disruption system for use in the human malaria parasite Plasmodium falciparum. My team adapted this approach to P. falciparum and demonstrated that it can be used for rapid robust regulation of gene expression. In further work, a system based on U1-mediated RNA destabilisation has been shown tobe functional in the related pathogen Toxoplasma gondii but not to function in Plasmodium falciparum. This suggests differences in RNA splicing mechanisms between these related organisms.
Collaborator Contribution Our partners showed that this system functions in the related parasitic pathogen Toxoplasma gondii, encouraging us to attempt its establishment in P. falciparum. They also provided reagents and genetic tools.
Impact This has now resulted in two joint publications (Collins CR, Das S, Wong EH, Andenmatten N, Stallmach R, Hackett, F, Herman, JP, Mueller, S, Meissner, M and Blackman, MJ (2013) Robust inducible Cre recombinase activity in the human malaria parasite Plasmodium falciparum enables efficient gene deletion within a single asexual erythrocytic growth cycle. Mol Microbiol. 88:687-701. PMID: 23489321; and Pieperhoff MS, Pall GS, Jiménez-Ruiz E, Das S, Melatti C, Gow M, Wong EH, Heng J, Müller S, Blackman MJ*, Meissner M* (2015) Conditional U1 Gene Silencing in Toxoplasma gondii. PLoS One. 10:e0130356). Ongoing work may lead to further high-impact publications.
Start Year 2011
 
Description Human adaptation of a malaria pathogen 
Organisation National Institute for Biological Standards and Control (NIBSC)
Country United Kingdom 
Sector Public 
PI Contribution This collaboration was initiated in order to adapt a newly recognised human malaria pathogen, Plasmodium knowlesi, to growth in vitro in human red blood cells. Adaptation was performed my members of my group.
Collaborator Contribution Our partners provided primate (cynomolgus macaque) red blood cells for the early stages of adaptation.
Impact A publication describing the successful adaptation of the parasite to in vitro growth in human red cells has been published (Moon RW, Hall J, Rangkuti F, Ho YS, Almond N, Mitchell GH, Pain A, Holder AA, Blackman MJ [2013] Adaptation of the genetically tractable malaria pathogen Plasmodium knowlesi to continuous culture in human erythrocytes. Proc Natl Acad Sci U S A. 110:531-6. PMID: 23267069 ). This work has led to the culture adaptation of an important human pathogen, which will be immensly valuable for all future work in this area, including validation of drug targets and vaccine development.
Start Year 2009
 
Description Inhibitors of a malarial protease 
Organisation InhiBox
Country United Kingdom 
Sector Private 
PI Contribution This work was in many ways an extension of the above collaborations. Inhibox specialise in use of structural data to screen in silico for tight-binding small molecule inhibitors. Our team at NIMR provided a homology model of a malarial protease of interest (built in collaboration with the Division of Mathematical Biology at NIMR) and experimental substrate data.
Collaborator Contribution Purchase and provision of selected ('focused') compound libraries for screening as potential malarial protease inhibitors. Acted as a pilot study to promote further collaboration in this area.
Impact The collaboration allowed preliminary selection of compounds that inhibit a malarial protease. Acted as a pilot study to promote further collaboration in this area. This has directly led to the establishment of a firm collaboration in the form of an EU Marie Curie funded network called STARS (total budget approx 2.1 million Euro) which will enable continuationof this work on the malarial enzyme. A publication has recently emanated from this collaboration (Withers-Martinez, C., Suarez, C., Fulle, S., Kher, S., Penzo, M., Ebejerb, J.-P., Koussis, K., Hackett, F., Jirgensons, A., Finn, P., and Blackman, M.J. (2012). Plasmodium subtilisin-like protease 1 (SUB1): insights into the active-site structure, specificity and function of a pan-malaria drug target. Int J Parasitol 42, 597-612. PMID: 22796182)
Start Year 2007
 
Description MRCT 
Organisation MRC-Technology
Department MRCT Centre for Therapeutics Discovery (CTD)
Country United Kingdom 
Sector Charity/Non Profit 
PI Contribution This valuable collaboration involved multiple screens, used robotic facilities and expertise available at MRCT, of compound libraries from a variety of sources. Our team contributed purified recombinant malarial enzyme (protease) and substrate for the screen, as well as expertise. The funding was used to support a postdoctoral worker in my group, as well as to fund synthetic chemistry performed at MRCT.
Collaborator Contribution Provision of expert help, chemistry advice and screening capacity for high throughput screens of compound libraries in search for a malaria protease inhibitor.
Impact This work (along with the Molecular Nature collaboration) has led to substantial advances on our understanding of the function of the protease undeer examination, as well as how the malaria parasite regulates release from its host red blood cell. Relevant publications leading from this work are PubMed ID 18083098, 18503638 and 19214190
 
Description Malarial PKG in egress 
Organisation London School of Hygiene and Tropical Medicine (LSHTM)
Department Faculty of Infectious and Tropical Diseases
Country United Kingdom 
Sector Academic/University 
PI Contribution We have established the mechanism by which inhibitors of a malarial cyclic GMP-dependent protein kinase (PKG) prevent release (egress) of the malaria parasite from its host red blood cell. This work has now been extended into a detailed analysis of the role of cGMP-dependent signalling in egress.
Collaborator Contribution Our partners originally provided low molecular weight PKG inhibitors and expertise in their use for this work. This has now been expanded into a full-scale collaboration funded by a joint Wellcome Trust Senior Investigator award.
Impact A paper describing the first publication emanating from this collaboration (Collins CR, Hackett F, Strath M, Penzo M, Withers-Martinez C, Baker DA, Blackman MJ [2013] Malaria parasite cGMP-dependent protein kinase regulates blood stage merozoite secretory organelle discharge and egress. PLoS Pathog.9:e1003344. PMID: 23675297) was used as a springboard for a successful grant application to the Wellcome Trust's Senior Investigator Award Programme early in 2015.
Start Year 2011
 
Description Molecular Nature Ltd 
Organisation Molecular Nature Ltd
Country United Kingdom 
Sector Private 
PI Contribution Compounds provided by Molecular Nature were screened for activity against a malarial protease using an assay developed by my research team at NIMR.
Collaborator Contribution Provision of large amounts of a natural compound previously found (in a screen in collaboration with MRC Technology) to be a potent inhibitor of a malarial protease.
Impact Lead compounds selected from the screen were used to determine the function of the maalria protease under examination. This led to a breakthrough in our understanding of how the malaria parasite is released from its host red blood cell. See PubMed ID 18083098.
 
Description Structural analysis of malaria parasite egress. 
Organisation Birkbeck, University of London
Department Department of Biological Sciences
Country United Kingdom 
Sector Academic/University 
PI Contribution This project focuses on determining the changes to the malaria parasite-infected erythrocyte that take place prior to and during release (egress) of the parasite from its host cell. We provide stringently synchronised parasite material and advice on how to work with it.
Collaborator Contribution Our collaborators have special expertise in electron microscopy (EM), and they use EM and related technologies (e.g. tomography) to examine the structural changes that take place in the parasitised cell during egress.
Impact This work has resulted in a recent publication (Watermeyer JM, Hale VL, Hackett F, Clare DK, Cutts EE, Vakonakis I, Fleck RA, Blackman MJ, Saibil HR. A spiral scaffold underlies cytoadherent knobs in Plasmodium falciparum-infected erythrocytes. Blood. 2016 127:343-51. doi: 10.1182/blood-2015-10-674002). Further publications are under preparation.
Start Year 2011
 
Description University of Heidelberg 
Organisation Heidelberg University Hospital
Department Department of Infectious diseases, Virology
Country Germany 
Sector Academic/University 
PI Contribution Major contributions to the intellectual and research input.
Collaborator Contribution Basic research - collaborators provided expertise and reagents
Impact Two publications this year (Child et al and Woehlbier et al). Not multidisciplinary.
Start Year 2007
 
Title PfSUB1 malarial protease 
Description Development of an assay to screen for inhibitors of an essential malarial protease that is a potential drug target. 
Type Therapeutic Intervention - Drug
Current Stage Of Development Initial development
Year Development Stage Completed 2007
Development Status Under active development/distribution
Impact Identification of drug-like inhibitors tht may have potential for development as new antimalarial drugs. 
 
Description Malaria proteases as drug targets 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Type Of Presentation Paper Presentation
Geographic Reach International
Primary Audience Other academic audiences (collaborators, peers etc.)
Results and Impact I have given numerous presentations at a range of international scientific conferences (with audiences ranging from less than 100 to greater than 500).

Press release on a Cell paper describing the identification of a protease involved in red cell egress by the malaria parasite
Year(s) Of Engagement Activity Pre-2006,2006,2007,2008,
 
Description The impact of malaria 
Form Of Engagement Activity A broadcast e.g. TV/radio/film/podcast (other than news/press)
Part Of Official Scheme? No
Type Of Presentation Keynote/Invited Speaker
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Took part in a Michael Mosely documentary aired on nationwide BBC TV, called 'Infested' and dedicated to pathogenic parasites. The programme resulted in lots of interest in our work and that on parasitic diseases generally.

Wider dissemination of the results of our research on malaria.
Year(s) Of Engagement Activity 2014,2015
URL http://www.bbc.co.uk/programmes/b03vrwj8