Malaria Programme: Rayner group

The Malaria Programme uses genomic and genetic approaches to discover molecular mechanisms of host-parasite interactions that may lead to new biological insights and improved strategies for disease prevention.

Plasmodium falciparum is the parasite responsible for the overwhelming majority of malaria mortality.

The Rayner group is investigating the molecular details of the interactions between P. falciparum and human red blood cells (erythrocytes), using both genomic and proteomic approaches.

The Rayner lab has a particular focus on erythrocyte invasion, the process by which P. falciparum parasites recognise and invade human red blood cells. This is a critical step in the parasite's life cycle, because once inside erythrocytes, P. falciparum parasites are protected from the antibody-mediated immune system, and use the hemoglobin within the blood cell to grow and multiply. By understanding erythrocyte invasion in depth, the Rayner group aims to identify molecular steps in the process that could be targeted by new drugs or vaccines.

More information on the Malaria Programme.

[David Goulding, Genome Research Limited]

Background

Figure 1. Erythrocyte invasion.

Figure 1. Erythrocyte invasion.

Erythrocyte invasion: a complex dance between two genomes

All the symptoms and pathology of malaria are initiated when the invasive stage of P. falciparum, called merozoites, infect erythrocytes, leading to the colonisation and eventual destruction of these erythrocytes and the release of a new wave of invasive merozoites 48 hours later. Erythrocyte invasion is critical for parasite survival because P. falciparum is an intracellular parasite - the merozoite can not exist outside of an erythrocyte for any length of time. It is also a complex point of interaction between two genomes and the proteins that they encode.

Erythrocyte invasion consists of several phases. Recognition and attachment of merozoites to erythrocytes is driven by multiple interactions between proteins present on the surface of the merozoite and it's target erythrocyte. Although many P. falciparum ligands thought to play a role in this process have been identified, in most cases it is not known what erythrocyte receptors they bind to, or what their precise function is. This initial interaction step is further complicated by genetic variation in both the host and parasite genomes, which can dictate which erythrocytes the P. falciparum merozoites can recognise. Understanding the receptor-ligand interactions that mediate merozoite-erythrocyte attachment, and how those interactions are influenced by natural genetic variation, is one of the main goals of the Rayner group.

Once attachment has occurred, the P. falciparum merozoite literally drives its way into the erythrocyte, a process that is entirely driven by an actin-myosin motor within the parasite, similar to the actin myosin motors that drive muscle contraction. This invasion motor is in turn regulated by a complex web of post-translational modifications, and understanding these regulatory networks is another key goal of the Rayner group.

Research

Figure 2. By labeling a sub-population of erythrocytes with a fluorescent dye (green), and parasites with a fluorescent dye of a different colour (blue), we can use flow cytometry to count how many parasites have invaded those erythrocytes.

Figure 2. By labeling a sub-population of erythrocytes with a fluorescent dye (green), and parasites with a fluorescent dye of a different colour (blue), we can use flow cytometry to count how many parasites have invaded those erythrocytes.

zoom

The Rayner group aims to develop a better understanding of the molecular details of host-parasite interactions during the P. falciparum blood stages, with a particular focus on erythrocyte invasion. To achieve our aims we use a variety of approaches, many of which make use of the unique strengths of the Sanger Institute. Tiny changes in the DNA sequence called single nucleotide polymorphisms (SNPs) are known to be responsible for a lot of the variation seen in the DNA sequences of organisms. Genome-wide studies in the Sanger Institute's Malaria Programme are now identifying numerous SNPs within both human erythrocyte receptors and P. falciparum invasion ligands (Malaria Programme: Kwiatkowski group), but their effect on receptor-ligand binding or invasion is not yet known. We are particularly interested in developing new high-throughput phenotyping platforms to assess the effect that these SNPs have on specific steps during erythrocyte invasion, and have recently developed a two-colour flow cytometry based assay to quantitate erythrocyte invasion.

We are also using experimental genetic approaches to dissect the molecular roles of specific P. falciparum proteins in the process of erythrocyte invasion. We work with Gavin Wright's team to identify new receptor-ligand interactions involved in invasion, and to measure the impact of natural genetic variation on these interactions. In collaboration with Oliver Billker and the Sanger Institute Proteomics Mass Spectrometry core facility led by Jyoti Choudhary we are using mass spectrometry to study two posttranslational modifications that play key roles in regulating erythrocyte invasion - phosphorylation and palmitoylation.

Finally, we play a leading role in a global collaboration aimed at understanding the origins of P. falciparum parasites and what restricts certain parasites to certain hosts. In a recent comprehensive genetic study of parasites infecting wild apes in central Africa, we established that Plasmodium infection is widespread among apes, with at least six apparently independent P. falciparum related species present. All human P. falciparum parasites fall within a single clade of parasites infecting western lowland gorillas, arguing that Plasmodium falciparum passed from gorillas to humans at some time during our evolutionary history. Further studies are aimed at understanding whether such cross-species transmissions still occur and what the molecular requirements are that control which host species a Plasmodium parasite can infect.

Resources

Standard operating procedure for Plasmodium falciparum erythrocyte invasion assay - This document contains a detailed SOP for the two-colour flow cytometric erythrocyte invasion assay recently developed by the Rayner lab (Theron et al., Cytometry Part A, in press). It contains step by step methods for labeling erythrocytes using CFDA SE or DDAO SE, labeling parasite DNA with Hoechst 33342 or SYBR Green I, and how to combine erythrocyte and DNA dyes to measure P. falciparum invasion.

For further information, contact Leyla Bustamante.

Selected Publications

  • Basigin is a receptor essential for erythrocyte invasion by Plasmodium falciparum.

    Crosnier C, Bustamante LY, Bartholdson SJ, Bei AK, Theron M, Uchikawa M, Mboup S, Ndir O, Kwiatkowski DP, Duraisingh MT, Rayner JC and Wright GJ

    Nature 2011;480;7378;534-7

  • A scalable pipeline for highly effective genetic modification of a malaria parasite.

    Pfander C, Anar B, Schwach F, Otto TD, Brochet M, Volkmann K, Quail MA, Pain A, Rosen B, Skarnes W, Rayner JC and Billker O

    Nature methods 2011;8;12;1078-82

  • A plethora of Plasmodium species in wild apes: a source of human infection?

    Rayner JC, Liu W, Peeters M, Sharp PM and Hahn BH

    Trends in parasitology 2011;27;5;222-9

  • Malaria immunoepidemiology in low transmission: correlation of infecting genotype and immune response to domains of Plasmodium falciparum merozoite surface protein 3.

    Jordan SJ, Oliveira AL, Hernandez JN, Oster RA, Chattopadhyay D, Branch OH and Rayner JC

    Infection and immunity 2011;79;5;2070-8

  • An adaptable two-color flow cytometric assay to quantitate the invasion of erythrocytes by Plasmodium falciparum parasites.

    Theron M, Hesketh RL, Subramanian S and Rayner JC

    Cytometry. Part A : the journal of the International Society for Analytical Cytology 2010;77;11;1067-74

  • Origin of the human malaria parasite Plasmodium falciparum in gorillas.

    Liu W, Li Y, Learn GH, Rudicell RS, Robertson JD, Keele BF, Ndjango JB, Sanz CM, Morgan DB, Locatelli S, Gonder MK, Kranzusch PJ, Walsh PD, Delaporte E, Mpoudi-Ngole E, Georgiev AV, Muller MN, Shaw GM, Peeters M, Sharp PM, Rayner JC and Hahn BH

    Nature 2010;467;7314;420-5

  • Plasmodium falciparum erythrocyte invasion: a conserved myosin associated complex.

    Jones ML, Kitson EL and Rayner JC

    Molecular and biochemical parasitology 2006;147;1;74-84

The Team

Team members

Paula Cawkill
Advanced Research Assistant
Nadia Cross
Advanced Research Assistant
Nicola Hodson
Postdoctoral Fellow
Jessica Hostetler
PhD student
Gathoni Kamuyu
Visiting Scientist
Alena Pance
Staff Scientist
Liam Prestwood
Laboratory Manager
Will Proto
Postdoctoral Fellow
Theo Sanderson
ts10@sanger.ac.ukPhD Student

Paula Cawkill

- Advanced Research Assistant

Paula is a medical microbiologist. Previous to joining the Sanger Paula worked in industry for ten years following a Post Doc at Imperial College on Bordetella pertussis and a PhD at Leeds University on invasion of respiratory epithelial cells by Burkholderia cepacia.

Research

Paula joined the Sanger Institute Malaria Programme (Rayner group) in January 2014 and is involved in applying new phenotyping methods to study Plasmodium falciparum intraerythrocytic development and erythrocyte invasion. The research also supports other groups of the Malaria Programme.

Nadia Cross

- Advanced Research Assistant

Nadia is an advanced research assistant working under the supervision of Julian Rayner. She holds a BSc (HONS) in Medical Biology from Brunel University. She has worked at The Walter and Eliza Hall Institute of Medical Research in their Infection and Immunity Division and also at The Macfarlane Burnet Institute both in Melbourne, Australia. Nadia’s previous work focused on leading candidate antigens of malaria such as MSP2 and AMA-1. Specifically she was involved in measuring immune responses in clinical trials and in population studies from malaria endemic areas.

Research

Nadia joined the Sanger Institute Malaria Programme in 2014. Her current work utilises phenotyping assay platforms developed in the lab to help further our understanding of erythrocyte invasion.

References

  • New insights into acquisition, boosting, and longevity of immunity to malaria in pregnant women.

    Fowkes FJ, McGready R, Cross NJ, Hommel M, Simpson JA, Elliott SR, Richards JS, Lackovic K, Viladpai-Nguen J, Narum D, Tsuboi T, Anders RF, Nosten F and Beeson JG

    Macfarlane Burnet Institute of Medical Research, Melbourne, Australia. fowkes@burnet.edu.au

    Background: How antimalarial antibodies are acquired and maintained during pregnancy and boosted after reinfection with Plasmodium falciparum and Plasmodium vivax is unknown.

    Methods: A nested case-control study of 467 pregnant women (136 Plasmodium-infected cases and 331 uninfected control subjects) in northwestern Thailand was conducted. Antibody levels to P. falciparum and P. vivax merozoite antigens and the pregnancy-specific PfVAR2CSA antigen were determined at enrollment (median 10 weeks gestation) and throughout pregnancy until delivery.

    Results: Antibodies to P. falciparum and P. vivax were highly variable over time, and maintenance of high levels of antimalarial antibodies involved highly dynamic responses resulting from intermittent exposure to infection. There was evidence of boosting with each successive infection for P. falciparum responses, suggesting the presence of immunological memory. However, the half-lives of Plasmodium antibody responses were relatively short, compared with measles (457 years), and much shorter for merozoite responses (0.8-7.6 years), compared with PfVAR2CSA responses (36-157 years). The longer half-life of antibodies to PfVAR2CSA suggests that antibodies acquired in one pregnancy may be maintained to protect subsequent pregnancies.

    Conclusions: These findings may have important practical implications for predicting the duration of vaccine-induced responses by candidate antigens and supports the development of malaria vaccines to protect pregnant women.

    Funded by: Wellcome Trust

    The Journal of infectious diseases 2012;206;10;1612-21

  • A phase 1 trial of MSP2-C1, a blood-stage malaria vaccine containing 2 isoforms of MSP2 formulated with Montanide® ISA 720.

    McCarthy JS, Marjason J, Elliott S, Fahey P, Bang G, Malkin E, Tierney E, Aked-Hurditch H, Adda C, Cross N, Richards JS, Fowkes FJ, Boyle MJ, Long C, Druilhe P, Beeson JG and Anders RF

    Queensland Institute of Medical Research, University of Queensland, Brisbane, Australia. J.McCarthy@uq.edu.au

    Background: In a previous Phase 1/2b malaria vaccine trial testing the 3D7 isoform of the malaria vaccine candidate Merozoite surface protein 2 (MSP2), parasite densities in children were reduced by 62%. However, breakthrough parasitemias were disproportionately of the alternate dimorphic form of MSP2, the FC27 genotype. We therefore undertook a dose-escalating, double-blinded, placebo-controlled Phase 1 trial in healthy, malaria-naïve adults of MSP2-C1, a vaccine containing recombinant forms of the two families of msp2 alleles, 3D7 and FC27 (EcMSP2-3D7 and EcMSP2-FC27), formulated in equal amounts with Montanide® ISA 720 as a water-in-oil emulsion.

    The trial was designed to include three dose cohorts (10, 40, and 80 µg), each with twelve subjects receiving the vaccine and three control subjects receiving Montanide® ISA 720 adjuvant emulsion alone, in a schedule of three doses at 12-week intervals. Due to unexpected local reactogenicity and concern regarding vaccine stability, the trial was terminated after the second immunisation of the cohort receiving the 40 µg dose; no subjects received the 80 µg dose. Immunization induced significant IgG responses to both isoforms of MSP2 in the 10 µg and 40 µg dose cohorts, with antibody levels by ELISA higher in the 40 µg cohort. Vaccine-induced antibodies recognised native protein by Western blots of parasite protein extracts and by immunofluorescence microscopy. Although the induced anti-MSP2 antibodies did not directly inhibit parasite growth in vitro, IgG from the majority of individuals tested caused significant antibody-dependent cellular inhibition (ADCI) of parasite growth.

    As the majority of subjects vaccinated with MSP2-C1 developed an antibody responses to both forms of MSP2, and that these antibodies mediated ADCI provide further support for MSP2 as a malaria vaccine candidate. However, in view of the reactogenicity of this formulation, further clinical development of MSP2-C1 will require formulation of MSP2 in an alternative adjuvant.

    Australian New Zealand Clinical Trials Registry 12607000552482.

    PloS one 2011;6;9;e24413

Nicola Hodson

- Postdoctoral Fellow

Nicola graduated from the University of York in 2009 with a degree in Molecular Cell Biology, which specialised in Immunology and Parasitology. During her time at the University of York she undertook a final year research project at the Centre for Immunology and Infection (CII) where she studied palmitoyl transferase enzymes in kinetoplastid parasites. Subsequently, she undertook a PhD at the Cambridge Institute for Medical Research (CIMR) where she undertook high throughput screening approaches to study novel protein trafficking machinery in human cell lines.

Research

The aim of her project is to use new experimental genetic and microscopy technologies to study erythrocyte invasion by P.falciparum. Invasion is challenging to study genetically because if the parasite genes involved are essential for invasion, they will also be essential for growth. This means that standard gene knockout techniques are impossible, as any parasites that have the essential genes deleted will never grow. Therefore her aim is to use a recently developed inducible knockout system to study the role of both invasion ligands and palmitoyl transferase enzymes in the invasion of the red blood cell by the parasite.

References

  • A human genome-wide screen for regulators of clathrin-coated vesicle formation reveals an unexpected role for the V-ATPase.

    Kozik P, Hodson NA, Sahlender DA, Simecek N, Soromani C, Wu J, Collinson LM and Robinson MS

    Cambridge Institute for Medical Research, Wellcome Trust/MRC Building, University of Cambridge, Hills Road, Cambridge CB2 0XY, UK. pk275@cam.ac.uk

    Clathrin-mediated endocytosis is essential for a wide range of cellular functions. We used a multi-step siRNA-based screening strategy to identify regulators of the first step in clathrin-mediated endocytosis, formation of clathrin-coated vesicles (CCVs) at the plasma membrane. A primary genome-wide screen identified 334 hits that caused accumulation of CCV cargo on the cell surface. A secondary screen identified 92 hits that inhibited cargo uptake and/or altered the morphology of clathrin-coated structures. The hits include components of four functional complexes: coat proteins, V-ATPase subunits, spliceosome-associated proteins and acetyltransferase subunits. Electron microscopy revealed that V-ATPase depletion caused the cell to form aberrant non-constricted clathrin-coated structures at the plasma membrane. The V-ATPase-knockdown phenotype was rescued by addition of exogenous cholesterol, indicating that the knockdown blocks clathrin-mediated endocytosis by preventing cholesterol from recycling from endosomes back to the plasma membrane.

    Funded by: Wellcome Trust: 079919, 086598, 100140

    Nature cell biology 2013;15;1;50-60

  • Distinct and overlapping roles for AP-1 and GGAs revealed by the "knocksideways" system.

    Hirst J, Borner GH, Antrobus R, Peden AA, Hodson NA, Sahlender DA and Robinson MS

    University of Cambridge, Cambridge Institute for Medical Research, Cambridge CB2 0XY, UK. jh228@cam.ac.uk

    Although adaptor protein complex 1 (AP-1) and Golgi-localized, γ ear-containing, ADP-ribosylation factor-binding proteins (GGAs) are both adaptors for clathrin-mediated intracellular trafficking, the pathways they mediate and their relationship to each other remain open questions. To tease apart the functions of AP-1 and GGAs, we rapidly inactivated each adaptor using the "knocksideways" system and then compared the protein composition of clathrin-coated vesicle (CCV) fractions from control and knocksideways cells. The AP-1 knocksideways resulted in a dramatic and unexpected loss of GGA2 from CCVs. Over 30 other peripheral membrane proteins and over 30 transmembrane proteins were also depleted, including several mutated in genetic disorders, indicating that AP-1 acts as a linchpin for intracellular CCV formation. In contrast, the GGA2 knocksideways affected only lysosomal hydrolases and their receptors. We propose that there are at least two populations of intracellular CCVs: one containing both GGAs and AP-1 for anterograde trafficking and another containing AP-1 for retrograde trafficking. Our study shows that knocksideways and proteomics are a powerful combination for investigating protein function, which can potentially be used on many different types of proteins.

    Funded by: Wellcome Trust

    Current biology : CB 2012;22;18;1711-6

Jessica Hostetler

- PhD student

Jessica graduated cum laude from Duke University with a B.S. degree in Biology in 2003. Her undergraduate research experience covered cancer drug research in the oncology lab of Dr. Susan Ludeman and molecular evolution work in the labs of Professors John Mercer and V. Louise Roth. Jessica expanded into the field of genomics in the Genome Finishing and Analysis group at the J. Craig Venter Institute (formerly TIGR) in 2003. She managed the group from 2007-2011 before entering her PhD program.

Research

Jessica joined the Rayner lab as part of the National Institutes of Health Oxford-Cambridge Scholars Program. Under her mentors, Julian Rayner at WTSI and Rick Fairhurst at the NIH, she studies how Plasmodium vivax invades red blood cells. Jessica is building a library of parasite surface proteins to use for functional and immuno-epidemiological studies. As part of this, she will use AVEXIS (Avidity-based extracellular interaction screen) developed by Gavin Wright’s group to search for receptor-ligand interactions between P. vivax surface proteins and red blood cell surface proteins.

Gathoni Kamuyu

- Visiting Scientist

Gathoni holds a MSc. in Molecular Biology of Infectious Diseases from the London School of Hygiene and Tropical Medicine and is currently a PhD student registered with the Open University, UK and primarily based at the KEMRI-Wellcome Trust Research Programme, Kilifi. Kenya.

Gathoni has previously worked at the KEMRI-Wellcome Trust Research Programme, Kilifi Kenya on identifying parasitic infections as risk factors for active-convulsive epilepsy in sub-Saharan Africa.

Research

Her research work is focused on identifying the merozoite targets of protective immunity to Plasmodium falciparum malaria using sera from malaria immune adults. An immuno-proteomic approach will be used to identify immunogenic merozoite antigens and thereafter, selected antigens will be validated using sera from a longitudinally monitored cohort of malaria immune adults.

References

  • New antigens for a multicomponent blood-stage malaria vaccine.

    Osier FH, Mackinnon MJ, Crosnier C, Fegan G, Kamuyu G, Wanaguru M, Ogada E, McDade B, Rayner JC, Wright GJ and Marsh K

    Pathogen Vector and Human Biology Department, Kenya Medical Research Institute Centre for Geographical Medicine Research, Coast, P. O. Box 230, 80108 Kilifi, Kenya.

    An effective blood-stage vaccine against Plasmodium falciparum remains a research priority, but the number of antigens that have been translated into multicomponent vaccines for testing in clinical trials remains limited. Investigating the large number of potential targets found in the parasite proteome has been constrained by an inability to produce natively folded recombinant antigens for immunological studies. We overcame these constraints by generating a large library of biochemically active merozoite surface and secreted full-length ectodomain proteins. We then systematically examined the antibody reactivity against these proteins in a cohort of Kenyan children (n = 286) who were sampled at the start of a malaria transmission season and prospectively monitored for clinical episodes of malaria over the ensuing 6 months. We found that antibodies to previously untested or little-studied proteins had superior or equivalent potential protective efficacy to the handful of current leading malaria vaccine candidates. Moreover, cumulative responses to combinations comprising 5 of the 10 top-ranked antigens, including PF3D7_1136200, MSP2, RhopH3, P41, MSP11, MSP3, PF3D7_0606800, AMA1, Pf113, and MSRP1, were associated with 100% protection against clinical episodes of malaria. These data suggest not only that there are many more potential antigen candidates for the malaria vaccine development pipeline but also that effective vaccination may be achieved by combining a selection of these antigens.

    Funded by: Medical Research Council: MR/J002283/1; Wellcome Trust: 077092, 077176, 088634, 089833, 092654, 092741, 098051

    Science translational medicine 2014;6;247;247ra102

  • Prevalence and risk factors for active convulsive epilepsy in rural northeast South Africa.

    Wagner RG, Ngugi AK, Twine R, Bottomley C, Kamuyu G, Gómez-Olivé FX, Connor MD, Collinson MA, Kahn K, Tollman S and Newton CR

    Studies of Epidemiology of Epilepsy in Demographic Surveillance Systems (SEEDS)-INDEPTH Network, Accra, Ghana; MRC/Wits Rural Public Health & Health Transitions Research Unit (Agincourt), School of Public Health, Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, South Africa; Division of Epidemiology and Global Health, Department of Public Health and Clinical Medicine, Umeå University, Umeå, Sweden. Electronic address: Ryan.wagner@wits.ac.za.

    Rationale: Epilepsy is among the most common neurological disorders worldwide. However, there are few large, population-based studies of the prevalence and risk factors for epilepsy in southern Africa.

    Methods: From August 2008 to February 2009, as part of a multi-site study, we undertook a three-stage, population-based study, embedded within the Agincourt health and socio-demographic surveillance system, to estimate the prevalence and identify risk factors of active convulsive epilepsy (ACE) in a rural South African population.

    Results: The crude prevalence of ACE, after adjusting for non-response and the sensitivity of the screening method, was 7.0/1,000 individuals (95% CI 6.4-7.6) with significant geographic heterogeneity across the study area. Being male (OR=2.3; 95% CI 1.6-3.2), family history of seizures (OR=4.0; 95% CI 2.0-8.1), a sibling with seizures (OR=7.0; 95% CI 1.6-31.7), problems after delivery (OR=5.9; 95% CI 1.2-24.6), and history of snoring (OR=6.5; 95% CI 4.5-9.5) were significantly associated with ACE. For children, their mother's exposure to some formal schooling was protective (OR=0.30; 95% CI 0.11-0.84) after controlling for age and sex. Human immunodeficiency virus was not found to be associated with ACE.

    Conclusions: ACE is less frequent in this part of rural South Africa than other parts of sub-Saharan Africa. Improving obstetric services could prevent epilepsy. The relationship between snoring and ACE requires further investigation, as does the relative contribution of genetic and environmental factors to examine the increased risk in those with a family history of epilepsy.

    Funded by: Wellcome Trust: 058893/Z/99/A, 069683/Z/02/Z, 085477/Z/08/Z

    Epilepsy research 2014;108;4;782-91

  • Clinical features, proximate causes, and consequences of active convulsive epilepsy in Africa.

    Kariuki SM, Matuja W, Akpalu A, Kakooza-Mwesige A, Chabi M, Wagner RG, Connor M, Chengo E, Ngugi AK, Odhiambo R, Bottomley C, White S, Sander JW, Neville BG, Newton CR, SEEDS writing group, Twine R, Gómez Olivé FX, Collinson M, Kahn K, Tollman S, Masanja H, Mathew A, Pariyo G, Peterson S, Ndyomughenyi D, Bauni E, Kamuyu G, Odera VM, Mageto JO, Ae-Ngibise K, Akpalu B, Agbokey F, Adjei P, Owusu-Agyei S, Kleinschmidt I, Doku VC, Odermatt P, Nutman T, Wilkins P and Noh J

    Centre for Geographic Medicine Research Coast, Kenya Medical Research Institute, Kilifi, Kenya; Studies of Epidemiology of Epilepsy in Demographic Surveillance Systems (SEEDS)-INDEPTH Network, Accra, Ghana; Department of Psychiatry, University of Oxford, Oxford, United Kingdom.

    Purpose: Epilepsy is common in sub-Saharan Africa (SSA), but the clinical features and consequences are poorly characterized. Most studies are hospital-based, and few studies have compared different ecological sites in SSA. We described active convulsive epilepsy (ACE) identified in cross-sectional community-based surveys in SSA, to understand the proximate causes, features, and consequences.

    Methods: We performed a detailed clinical and neurophysiologic description of ACE cases identified from a community survey of 584,586 people using medical history, neurologic examination, and electroencephalography (EEG) data from five sites in Africa: South Africa; Tanzania; Uganda; Kenya; and Ghana. The cases were examined by clinicians to discover risk factors, clinical features, and consequences of epilepsy. We used logistic regression to determine the epilepsy factors associated with medical comorbidities.

    Half (51%) of the 2,170 people with ACE were children and 69% of seizures began in childhood. Focal features (EEG, seizure types, and neurologic deficits) were present in 58% of ACE cases, and these varied significantly with site. Status epilepticus occurred in 25% of people with ACE. Only 36% received antiepileptic drugs (phenobarbital was the most common drug [95%]), and the proportion varied significantly with the site. Proximate causes of ACE were adverse perinatal events (11%) for onset of seizures before 18 years; and acute encephalopathy (10%) and head injury prior to seizure onset (3%). Important comorbidities were malnutrition (15%), cognitive impairment (23%), and neurologic deficits (15%). The consequences of ACE were burns (16%), head injuries (postseizure) (1%), lack of education (43%), and being unmarried (67%) or unemployed (57%) in adults, all significantly more common than in those without epilepsy.

    Significance: There were significant differences in the comorbidities across sites. Focal features are common in ACE, suggesting identifiable and preventable causes. Malnutrition and cognitive and neurologic deficits are common in people with ACE and should be integrated into the management of epilepsy in this region. Consequences of epilepsy such as burns, lack of education, poor marriage prospects, and unemployment need to be addressed.

    Funded by: NIAID NIH HHS: Z01 AI000197-28; Wellcome Trust: 083744, 092654, 099782

    Epilepsia 2014;55;1;76-85

  • Exposure to multiple parasites is associated with the prevalence of active convulsive epilepsy in sub-Saharan Africa.

    Kamuyu G, Bottomley C, Mageto J, Lowe B, Wilkins PP, Noh JC, Nutman TB, Ngugi AK, Odhiambo R, Wagner RG, Kakooza-Mwesige A, Owusu-Agyei S, Ae-Ngibise K, Masanja H, Osier FH, Odermatt P, Newton CR and Study of Epidemiology of Epilepsy in Demographic Sites (SEEDS) group

    KEMRI/Wellcome Trust Research Programme, The Centre of Geographical Medicine Research - Coast, Kilifi, Kenya; Studies of the Epidemiology of Epilepsy in Demographic Surveillance Systems (SEEDS)-INDEPTH Network, Accra, Ghana.

    Background: Epilepsy is common in developing countries, and it is often associated with parasitic infections. We investigated the relationship between exposure to parasitic infections, particularly multiple infections and active convulsive epilepsy (ACE), in five sites across sub-Saharan Africa.

    A case-control design that matched on age and location was used. Blood samples were collected from 986 prevalent cases and 1,313 age-matched community controls and tested for presence of antibodies to Onchocerca volvulus, Toxocara canis, Toxoplasma gondii, Plasmodium falciparum, Taenia solium and HIV. Exposure (seropositivity) to Onchocerca volvulus (OR = 1.98; 95%CI: 1.52-2.58, p<0.001), Toxocara canis (OR = 1.52; 95%CI: 1.23-1.87, p<0.001), Toxoplasma gondii (OR = 1.28; 95%CI: 1.04-1.56, p = 0.018) and higher antibody levels (top tertile) to Toxocara canis (OR = 1.70; 95%CI: 1.30-2.24, p<0.001) were associated with an increased prevalence of ACE. Exposure to multiple infections was common (73.8% of cases and 65.5% of controls had been exposed to two or more infections), and for T. gondii and O. volvulus co-infection, their combined effect on the prevalence of ACE, as determined by the relative excess risk due to interaction (RERI), was more than additive (T. gondii and O. volvulus, RERI = 1.19). The prevalence of T. solium antibodies was low (2.8% of cases and 2.2% of controls) and was not associated with ACE in the study areas.

    Conclusion: This study investigates how the degree of exposure to parasites and multiple parasitic infections are associated with ACE and may explain conflicting results obtained when only seropositivity is considered. The findings from this study should be further validated.

    Funded by: Wellcome Trust: 083744, 084538, 092654, 096392

    PLoS neglected tropical diseases 2014;8;5;e2908

  • Analysis of antibodies to newly described Plasmodium falciparum merozoite antigens supports MSPDBL2 as a predicted target of naturally acquired immunity.

    Tetteh KK, Osier FH, Salanti A, Kamuyu G, Drought L, Failly M, Martin C, Marsh K and Conway DJ

    Department of Pathogen Molecular Biology, London School of Hygiene and Tropical Medicine, London, United Kingdom.

    Prospective studies continue to identify malaria parasite genes with particular patterns of polymorphism which indicate they may be under immune selection, and the encoded proteins require investigation. Sixteen new recombinant protein reagents were designed to characterize three such polymorphic proteins expressed in Plasmodium falciparum schizonts and merozoites: MSPDBL1 (also termed MSP3.4) and MSPDBL2 (MSP3.8), which possess Duffy binding-like (DBL) domains, and SURFIN4.2, encoded by a member of the surface-associated interspersed (surf) multigene family. After testing the antigenicities of these reagents by murine immunization and parasite immunofluorescence, we analyzed naturally acquired antibody responses to the antigens in two cohorts in coastal Kenya in which the parasite was endemic (Chonyi [n = 497] and Ngerenya [n = 461]). As expected, the prevalence and levels of serum antibodies increased with age. We then investigated correlations with subsequent risk of clinical malaria among children <11 years of age during 6 months follow-up surveillance. Antibodies to the polymorphic central region of MSPDBL2 were associated with reduced risk of malaria in both cohorts, with statistical significance remaining for the 3D7 allelic type after adjustment for individuals' ages in years and antibody reactivity to whole-schizont extract (Chonyi, risk ratio, 0.51, and 95% confidence interval [CI], 0.28 to 0.93; Ngerenya, risk ratio, 0.38, and 95% CI, 0.18 to 0.82). For the MSPDBL1 Palo Alto allelic-type antigen, there was a protective association in one cohort (Ngerenya, risk ratio, 0.53, and 95% CI, 0.32 to 0.89), whereas the other antigens showed no protective associations after adjustment. These findings support the prediction that antibodies to the polymorphic region of MSPDBL2 contribute to protective immunity.

    Funded by: Wellcome Trust: 074695/Z/04/B, 092654

    Infection and immunity 2013;81;10;3835-42

  • A threshold concentration of anti-merozoite antibodies is required for protection from clinical episodes of malaria.

    Murungi LM, Kamuyu G, Lowe B, Bejon P, Theisen M, Kinyanjui SM, Marsh K and Osier FH

    KEMRI Centre for Geographic Medicine Research, Coast, P.O. Box 230-80108, Kilifi, Kenya. lmurungi@kemri-wellcome.org

    Antibodies to selected Plasmodium falciparum merozoite antigens are often reported to be associated with protection from malaria in one epidemiological cohort, but not in another. Here, we sought to understand this paradox by exploring the hypothesis that a threshold concentration of antibodies is necessary for protection. We analyzed data from two independent cohorts along the Kenyan coast, one in which antibodies to AMA1, MSP-2 and MSP-3 were associated with protection from malaria (Chonyi) and another in which this association was not observed (Junju). We used a malaria reference reagent to standardize antibody measurements across both cohorts, and applied statistical methods to derive the threshold concentration of antibodies against each antigen that best correlated with a reduced risk of malaria (the protective threshold), in the Chonyi cohort. We then tested whether antibodies in Junju reached the protective threshold concentrations observed in the Chonyi cohort. Except for children under 3 years, the age-matched proportions of children achieving protective threshold concentrations of antibodies against AMA1 and MSP-2 were significantly lower in Junju compared to Chonyi (Fishers exact test, P<0.01). For MSP-3, this difference was significant only among 4-5 year olds. We conclude that although antibodies are commonly detected in malaria endemic populations, they may be present in concentrations that are insufficient for protection. Our results have implications for the analysis and interpretation of similar data from immuno-epidemiological studies.

    Funded by: Medical Research Council: G1002624; Wellcome Trust: 085880/Z/08/Z, 089833/Z/09/Z, 092654

    Vaccine 2013;31;37;3936-42

  • Prevalence of active convulsive epilepsy in sub-Saharan Africa and associated risk factors: cross-sectional and case-control studies.

    Ngugi AK, Bottomley C, Kleinschmidt I, Wagner RG, Kakooza-Mwesige A, Ae-Ngibise K, Owusu-Agyei S, Masanja H, Kamuyu G, Odhiambo R, Chengo E, Sander JW, Newton CR and SEEDS group

    Studies of Epidemiology of Epilepsy in Demographic Surveillance Systems, International Network for the Demographic Evaluation of Populations and Their Health (INDEPTH), Accra, Ghana. kngugi26@gmail.com

    Background: The prevalence of epilepsy in sub-Saharan Africa seems to be higher than in other parts of the world, but estimates vary substantially for unknown reasons. We assessed the prevalence and risk factors of active convulsive epilepsy across five centres in this region.

    Methods: We did large population-based cross-sectional and case-control studies in five Health and Demographic Surveillance System centres: Kilifi, Kenya (Dec 3, 2007-July 31, 2008); Agincourt, South Africa (Aug 4, 2008-Feb 27, 2009); Iganga-Mayuge, Uganda (Feb 2, 2009-Oct 30, 2009); Ifakara, Tanzania (May 4, 2009-Dec 31, 2009); and Kintampo, Ghana (Aug 2, 2010-April 29, 2011). We used a three-stage screening process to identify people with active convulsive epilepsy. Prevalence was estimated as the ratio of confirmed cases to the population screened and was adjusted for sensitivity and attrition between stages. For each case, an age-matched control individual was randomly selected from the relevant centre's census database. Fieldworkers masked to the status of the person they were interviewing administered questionnaires to individuals with active convulsive epilepsy and control individuals to assess sociodemographic variables and historical risk factors (perinatal events, head injuries, and diet). Blood samples were taken from a randomly selected subgroup of 300 participants with epilepsy and 300 control individuals from each centre and were screened for antibodies to Toxocara canis, Toxoplasma gondii, Onchocerca volvulus, Plasmodium falciparum, Taenia solium, and HIV. We estimated odds ratios (ORs) with logistic regression, adjusted for age, sex, education, employment, and marital status.

    Results: 586,607 residents in the study areas were screened in stage one, of whom 1711 were diagnosed as having active convulsive epilepsy. Prevalence adjusted for attrition and sensitivity varied between sites: 7·8 per 1000 people (95% CI 7·5-8·2) in Kilifi, 7·0 (6·2-7·4) in Agincourt, 10·3 (9·5-11·1) in Iganga-Mayuge, 14·8 (13·8-15·4) in Ifakara, and 10·1 (9·5-10·7) in Kintampo. The 1711 individuals with the disorder and 2032 control individuals were given questionnaires. In children (aged <18 years), the greatest relative increases in prevalence were associated with difficulties feeding, crying, or breathing after birth (OR 10·23, 95% CI 5·85-17·88; p<0·0001); abnormal antenatal periods (2·15, 1·53-3·02; p<0·0001); and head injury (1·97, 1·28-3·03; p=0·002). In adults (aged ≥18 years), the disorder was significantly associated with admission to hospital with malaria or fever (2·28, 1·06-4·92; p=0·036), exposure to T canis (1·74, 1·27-2·40; p=0·0006), exposure to T gondii (1·39, 1·05-1·84; p=0·021), and exposure to O volvulus (2·23, 1·56-3·19; p<0·0001). Hypertension (2·13, 1·08-4·20; p=0·029) and exposure to T solium (7·03, 2·06-24·00; p=0·002) were risk factors for adult-onset disease.

    Interpretation: The prevalence of active convulsive epilepsy varies in sub-Saharan Africa and that the variation is probably a result of differences in risk factors. Programmes to control parasitic diseases and interventions to improve antenatal and perinatal care could substantially reduce the prevalence of epilepsy in this region.

    Funded by: Wellcome Trust: 083744, 084538, 092654

    The Lancet. Neurology 2013;12;3;253-63

  • The ratio of monocytes to lymphocytes in peripheral blood correlates with increased susceptibility to clinical malaria in Kenyan children.

    Warimwe GM, Murungi LM, Kamuyu G, Nyangweso GM, Wambua J, Naranbhai V, Fletcher HA, Hill AV, Bejon P, Osier FH and Marsh K

    The Jenner Institute, University of Oxford, Oxford, United Kingdom. george.warimwe@ndm.ox.ac.uk

    Background: Plasmodium falciparum malaria remains a major cause of illness and death in sub-Saharan Africa. Young children bear the brunt of the disease and though older children and adults suffer relatively fewer clinical attacks, they remain susceptible to asymptomatic P. falciparum infection. A better understanding of the host factors associated with immunity to clinical malaria and the ability to sustain asymptomatic P. falciparum infection will aid the development of improved strategies for disease prevention.

    Here we investigate whether full differential blood counts can predict susceptibility to clinical malaria among Kenyan children sampled at five annual cross-sectional surveys. We find that the ratio of monocytes to lymphocytes, measured in peripheral blood at the time of survey, directly correlates with risk of clinical malaria during follow-up. This association is evident among children with asymptomatic P. falciparum infection at the time the cell counts are measured (Hazard ratio (HR)  =  2.7 (95% CI 1.42, 5.01, P  =  0.002) but not in those without detectable parasitaemia (HR  =  1.0 (95% CI 0.74, 1.42, P  =  0.9).

    Conclusions: We propose that the monocyte to lymphocyte ratio, which is easily derived from routine full differential blood counts, reflects an individual's capacity to mount an effective immune response to P. falciparum infection.

    Funded by: Medical Research Council: G1002624; Wellcome Trust: 090532, 092654, 092741/Z/10/Z

    PloS one 2013;8;2;e57320

  • The blood-stage malaria antigen PfRH5 is susceptible to vaccine-inducible cross-strain neutralizing antibody.

    Douglas AD, Williams AR, Illingworth JJ, Kamuyu G, Biswas S, Goodman AL, Wyllie DH, Crosnier C, Miura K, Wright GJ, Long CA, Osier FH, Marsh K, Turner AV, Hill AV and Draper SJ

    Jenner Institute, University of Oxford, Old Road Campus Research Building, Roosevelt Drive, Oxford OX3 7DQ, UK. sandy.douglas@ndm.ox.ac.uk

    Current vaccine strategies against the asexual blood stage of Plasmodium falciparum are mostly focused on well-studied merozoite antigens that induce immune responses after natural exposure, but have yet to induce robust protection in any clinical trial. Here we compare human-compatible viral-vectored vaccines targeting ten different blood-stage antigens. We show that the full-length P. falciparum reticulocyte-binding protein homologue 5 (PfRH5) is highly susceptible to cross-strain neutralizing vaccine-induced antibodies, out-performing all other antigens delivered by the same vaccine platform. We find that, despite being susceptible to antibody, PfRH5 is unlikely to be under substantial immune selection pressure; there is minimal acquisition of anti-PfRH5 IgG antibodies in malaria-exposed Kenyans. These data challenge the widespread beliefs that any merozoite antigen that is highly susceptible to immune attack would be subject to significant levels of antigenic polymorphism, and that erythrocyte invasion by P. falciparum is a degenerate process involving a series of parallel redundant pathways.

    Funded by: Medical Research Council: G0600424, G1000527, G1000527(95710); Wellcome Trust: 077108, 084113/z/07/z, 089455, 089455/2/09/Z, 089833/Z/09/Z, 090532, 095540

    Nature communications 2011;2;601

Alena Pance

- Staff Scientist

Alena Pance completed a BSc in biology and an MSc in cell biology and tropical medicine in Venezuela, where she worked on Chagas as well as Malaria. She then did a PhD at the University of Cambridge, UK, during which she started working on transcriptional control of gene expression and differentiation pathways. The first post-doctoral position was held in the Faculty of Medicine at the Univeristy of Burgundy in Dijon France, where she strengthened her expertise in transcriptional regulation. Alena returned to Cambridge as an Individual Marie Curie Research Fellow, to study the transcriptional regulation of the neuroendocrine phenotype.

Research

Alena returned to parasitology joining the Sanger Institute malaria programme. She is using her knowledge of cell and molecular biology to develop a stem cell-based strategy to study the host components of malaria. Genes associated with the disease are manipulated in stem cells that are differentiated into erythrocytes to analyse their role in infection. We also study the transcriptional regulation of haematopoietic differentiation and we generate IPS cells from a range of samples with polymorphisms associated to malaria. Alena coordinates a collaborative effort to develop genetically engineered cell lines for forward programming for erythrocytic differentiation with various groups in Cambridge.

References

  • SDF-1 chemokine signalling modulates the apoptotic responses to iron deprivation of clathrin-depleted DT40 cells.

    Pance A, Morrissey-Wettey FR, Craig H, Downing A, Talbot R and Jackson AP

    The Wellcome Trust Sanger Institute, Hinxton, Cambridge, United Kingdom.

    We have previously deleted both endogenous copies of the clathrin heavy-chain gene in the chicken pre B-cell-line DT40 and replaced them with clathrin under the control of a tetracycline-regulatable promoter (Tet-Off). The originally derived cell-line DKO-S underwent apoptosis when clathrin expression was repressed. We have also described a cell-line DKO-R derived from DKO-S cells that was less sensitive to clathrin-depletion. Here we show that the restriction of transferrin uptake, resulting in iron deprivation, is responsible for the lethal consequence of clathrin-depletion. We further show that the DKO-R cells have up-regulated an anti-apoptotic survival pathway based on the chemokine SDF-1 and its receptor CXCR4. Our work clarifies several puzzling features of clathrin-depleted DT40 cells and reveals an example of how SDF-1/CXCR4 signalling can abrogate pro-apoptotic pathways and increase cell survival. We propose that the phenomenon described here has implications for the therapeutic approach to a variety of cancers.

    Funded by: Biotechnology and Biological Sciences Research Council: BB/D001242/1

    PloS one 2014;9;8;e106278

  • Atypical mitogen-activated protein kinase phosphatase implicated in regulating transition from pre-S-Phase asexual intraerythrocytic development of Plasmodium falciparum.

    Balu B, Campbell C, Sedillo J, Maher S, Singh N, Thomas P, Zhang M, Pance A, Otto TD, Rayner JC and Adams JH

    Department of Global Health, College of Public Health, University of South Florida, Tampa, Florida, USA.

    Intraerythrocytic development of the human malaria parasite Plasmodium falciparum appears as a continuous flow through growth and proliferation. To develop a greater understanding of the critical regulatory events, we utilized piggyBac insertional mutagenesis to randomly disrupt genes. Screening a collection of piggyBac mutants for slow growth, we isolated the attenuated parasite C9, which carried a single insertion disrupting the open reading frame (ORF) of PF3D7_1305500. This gene encodes a protein structurally similar to a mitogen-activated protein kinase (MAPK) phosphatase, except for two notable characteristics that alter the signature motif of the dual-specificity phosphatase domain, suggesting that it may be a low-activity phosphatase or pseudophosphatase. C9 parasites demonstrated a significantly lower growth rate with delayed entry into the S/M phase of the cell cycle, which follows the stage of maximum PF3D7_1305500 expression in intact parasites. Genetic complementation with the full-length PF3D7_1305500 rescued the wild-type phenotype of C9, validating the importance of the putative protein phosphatase PF3D7_1305500 as a regulator of pre-S-phase cell cycle progression in P. falciparum.

    Funded by: NIAID NIH HHS: F31AI083053, R01 AI094973, R01AI033656, R01AI094973; Wellcome Trust

    Eukaryotic cell 2013;12;9;1171-8

  • Tailoring the models of transcription.

    Pance A

    The Welcome Trust Sanger Institute, Genome Campus Hinxton, Cambridge CB10 1SA, UK. ap9@sanger.ac.uk.

    Molecular biology is a rapidly evolving field that has led to the development of increasingly sophisticated technologies to improve our capacity to study cellular processes in much finer detail. Transcription is the first step in protein expression and the major point of regulation of the components that determine the characteristics, fate and functions of cells. The study of transcriptional regulation has been greatly facilitated by the development of reporter genes and transcription factor expression vectors, which have become versatile tools for manipulating promoters, as well as transcription factors in order to examine their function. The understanding of promoter complexity and transcription factor structure offers an insight into the mechanisms of transcriptional control and their impact on cell behaviour. This review focuses on some of the many applications of molecular cut-and-paste tools for the manipulation of promoters and transcription factors leading to the understanding of crucial aspects of transcriptional regulation.

    International journal of molecular sciences 2013;14;4;7583-97

  • Oct-1 cooperates with the TATA binding initiation complex to control rapid transcription of human iNOS.

    Reveneau S, Petrakis TG, Goldring CE, Chantôme A, Jeannin JF and Pance A

    EPHE Laboratory, Faculty of Medicine, University of Bourgogne, Dijon, France.

    Expression of the human inducible nitric oxide synthase (hiNOS) is generally undetectable in resting cells, but stimulation by a variety of signals including cytokines induces transcription in most cell types. The tight transcriptional regulation of the enzyme is a complex mechanism many aspects of which remain unknown. Here, we describe an octamer (Oct) element in hiNOS proximal promoter, located close to the TATA box. This site constitutively binds Oct-1 and its deletion abrogates cytokine-induced transcription, showing that it is indispensable though not sufficient for transcription. Increasing the distance between Oct and the TATA box by inserting inert DNA sequence inhibits transcription, and footprinting of this region shows no other protein binding in resting cells, suggesting an interaction between the two complexes. Chromatin immunoprecipitation assays detect the presence of Oct-1, RNA polymerase II and trimethyl K4 histone H3 on the proximal promoter in resting cells, confirming that the gene is primed for transcription before stimulation. RT-PCR of various fragments along the hiNOS gene shows that transcription is initiated in resting cells and this is inhibited by interference with Oct-1 binding to the proximal site of the promoter. We propose that, through interaction with the initiation complex, Oct-1 regulates hiNOS transcription by priming the gene for the rapid response required in an immune response.

    Cellular and molecular life sciences : CMLS 2012;69;15;2609-19

  • CCR4-associated factor 1 coordinates the expression of Plasmodium falciparum egress and invasion proteins.

    Balu B, Maher SP, Pance A, Chauhan C, Naumov AV, Andrews RM, Ellis PD, Khan SM, Lin JW, Janse CJ, Rayner JC and Adams JH

    Department of Global Health, College of Public Health, University of South Florida, College of Public Health, 3720 Spectrum Blvd., Suite 304, Tampa, FL, USA.

    Coordinated regulation of gene expression is a hallmark of the Plasmodium falciparum asexual blood-stage development cycle. We report that carbon catabolite repressor protein 4 (CCR4)-associated factor 1 (CAF1) is critical in regulating more than 1,000 genes during malaria parasites' intraerythrocytic stages, especially egress and invasion proteins. CAF1 knockout results in mistimed expression, aberrant accumulation and localization of proteins involved in parasite egress, and invasion of new host cells, leading to premature release of predominantly half-finished merozoites, drastically reducing the intraerythrocytic growth rate of the parasite. This study demonstrates that CAF1 of the CCR4-Not complex is a significant gene regulatory mechanism needed for Plasmodium development within the human host.

    Funded by: NIAID NIH HHS: R01 AI094973, R01 AI094973-01, R01AI033656, R01AI094973; Wellcome Trust

    Eukaryotic cell 2011;10;9;1257-63

  • A role for the transcriptional repressor REST in maintaining the phenotype of neurosecretory-deficient PC12 cells.

    Pance A, Livesey FJ and Jackson AP

    Department of Biochemistry, University of Cambridge, Cambridge, UK.

    The rat PC12 variant cell line, A35C, lacks regulated secretory organelles due to a selective transcriptional block. Hence, A35C may provide clues about the mechanisms that underlie control of neurosecretion. We used mRNA microarray profiling to examine gene expression in A35C. Genes for regulated secretory proteins were down-regulated, while other membrane trafficking pathways were unaffected. A subset of genes repressed in A35C contain binding sites for the neuronal transcriptional repressor, RE1-silencing transcription factor (REST), and REST is expressed in A35C but not normal PC12 cells. Blocking the activity of REST in A35C using a dominant-negative construct induced the reappearance of mRNAs for synaptophysin, chromogranin A, synaptotagmin IV and the beta3 subunit of the voltage-gated sodium channel (Scn3b), all of which contain RE1 sites in their genes. In the case of Scn3b, the corresponding protein was also re-expressed. Granule and synaptic vesicle proteins were not re-expressed at the protein level, despite reactivation of their mRNA, suggesting the existence of additional post-transcriptional control for these proteins. Our work identifies one of the mechanisms underlying the phenotype of neurosecretory-deficient neuroendocrine cells, and begins to define the critical components that determine a key aspect of the neuroendocrine phenotype.

    Journal of neurochemistry 2006;99;5;1435-44

  • Nitric oxide and hormones in breast cancer: allies or enemies?

    Pance A

    Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QW, UK. a.pance@bioc.cam.ac.uk

    Unlike other types of cancer, tumors of the breast are greatly influenced by steroid hormones. The effect of estrogen and progesterone depends on the presence of their specific receptors and these constitute important parameters in determining the aggressiveness of the tumor, the feasibility of certain therapies and the prediction of relapse. The molecular mechanisms of steroid hormone action have not been fully elucidated but recent findings implicate the nitric oxide (NO) pathway in some of these effects. Both hormones can regulate the nitric oxide synthases (NOS) and, in turn, the NO produced has profound consequences on tumor cell homeostasis. On one hand, estrogen increases the activity of endothelial NOS (eNOS or NOSIII), while progesterone activates inducible NOS (iNOS or NOSII) expression. The data presented suggest that the low levels of NO produced by NOSIII mediate the proliferative effect of estrogen. On the other hand, the increase in apoptosis in response to progesterone could implicate the high levels of NO produced by induction of NOSII expression. Understanding of the mechanisms and interactions of steroid hormones with the NO pathway could lead to the development of new approaches and strategies for the effective treatment of breast cancer.

    Future oncology (London, England) 2006;2;2;275-88

  • Progesterone enhances cytokine-stimulated nitric oxide synthase II expression and cell death in human breast cancer cells.

    Bentrari F, Arnould L, Jackson AP, Jeannin JF and Pance A

    EPHE INSERM 517, Faculty of Medicine, University of Bourgogne, Dijon, France.

    The presence of hormone receptors is related to survival outcome in breast cancer. Previous results from our laboratory established a correlation between the presence of nitric oxide synthase II (NOSII) and nitric oxide (NO) production with progesterone receptors in a series of human breast tumours. Furthermore, this was directly related to a lower tumour grade and a lower proliferation rate of the tumour cells. To examine these results in further detail, the effect of progesterone (Pg) and 17beta-oestradiol (E2) on NOSII expression was analysed in the human breast cancer cell line MCF-7. By Northern blot and promoter activity, we show that a cytokine mix (TNF-alpha, IL-beta, and IFN-gamma) induces NOSII transcription after 6 h stimulation. In the absence of cytokines, neither hormone affects NOSII expression. However, Pg but not E2, enhances cytokine-induced NOSII transcription as well as NO synthesis, mainly by cooperation with gamma-interferon. The increase in NO accumulation in the media induced by addition of Pg to the cytokine treatment significantly increases cell death, mainly accounted for by apoptosis, as compared to the effect of cytokines alone. Our findings help clarify the role of steroid hormones in NOSII expression as well as the effect on cell viability and may suggest novel approaches towards hormonotherapy and the treatment of cancer.

    Laboratory investigation; a journal of technical methods and pathology 2005;85;5;624-32

  • Chemosensitization by a non-apoptogenic heat shock protein 70-binding apoptosis-inducing factor mutant.

    Schmitt E, Parcellier A, Gurbuxani S, Cande C, Hammann A, Morales MC, Hunt CR, Dix DJ, Kroemer RT, Giordanetto F, Jäättelä M, Penninger JM, Pance A, Kroemer G and Garrido C

    Institut National de la Santé et de la Recherché Médicale U-517, Faculty of Medicine and Pharmacy, Dijon, France.

    Heat shock protein 70 (HSP70) inhibits apoptosis and thereby increases the survival of cells exposed to a wide range of lethal stimuli. HSP70 has also been shown to increase the tumorigenicity of cancer cells in rodent models. The protective function of this chaperone involves interaction and neutralization of the caspase activator apoptotic protease activation factor-1 and the mitochondrial flavoprotein apoptosis-inducing factor (AIF). In this work, we determined by deletional mutagenesis that a domain of AIF comprised between amino acids 150 and 228 is engaged in a molecular interaction with the substrate-binding domain of HSP70. Computer calculations favored this conclusion. On the basis of this information, we constructed an AIF-derived protein, which is cytosolic, noncytotoxic, yet maintains its capacity to interact with HSP70. This protein, designated ADD70, sensitized different human cancer cells to apoptosis induced by a variety of death stimuli by its capacity to interact with HSP70 and therefore to sequester HSP70. Thus, its chemosensitizing effect was lost in cells in which inducible HSP70 genes had been deleted. These data delineate a novel strategy for the selective neutralization of HSP70.

    Cancer research 2003;63;23;8233-40

  • Heat shock protein 70 binding inhibits the nuclear import of apoptosis-inducing factor.

    Gurbuxani S, Schmitt E, Cande C, Parcellier A, Hammann A, Daugas E, Kouranti I, Spahr C, Pance A, Kroemer G and Garrido C

    INSERM U-517, Faculty of Medicine and Pharmacy, 7 Boulevard Jeanne d'Arc, 21033 Dijon, France.

    Heat shock protein 70 (HSP70) can inhibit apoptosis by neutralizing and interacting with apoptosis-inducing factor (AIF), a mitochondrial flavoprotein that translocates upon apoptosis induction to the nucleus, via the cytosol. Here, we show that only members of the HSP70 family interact with AIF. Systematic deletion mapping revealed the existence of three distinct functional regions in the AIF protein: (1) a region between amino acids 150 and 228 that binds HSP70, (2) a domain between residues 367 and 459 that includes a nuclear localization sequence (NLS) and (3) a C-terminal domain beyond residue 567 required for its chromatin-condensing activity. Deletion of the 150-268 domain completely abolished HSP70 binding and facilitated the nuclear import of AIF, resulting in a gain-of-function phenotype with enhanced AIF-mediated chromatin condensation as compared to wild-type AIF. This gain-of-function phenotype was observed in wild-type control cells (which express low but significant levels of HSP70), yet was lost when AIFDelta150-268 was introduced into HSP70 knockout cells, underscoring the functional importance of the AIF-HSP70 interaction. Altogether, our data demonstrate that AIF inhibition by HSP70 involves cytosolic retention of AIF. Moreover, it appears that endogenous HSP70 protein levels are sufficiently elevated to modulate the lethal action of AIF.

    Oncogene 2003;22;43;6669-78

Liam Prestwood

- Laboratory Manager

I am the Laboratory Manager for multiple research groups within the Malaria Programme, including Oliver Billker and Julian Rayner’s teams. I completed my degree in Natural Sciences at the University of Bath, including research into the transdifferentiation of pancreatic cells into hepatocytes. I previously worked at the company GlaxoSmithKline on drug discovery for Rheumatoid Arthritis, focusing on assay development. Until 2014, I worked at the Department of Medicine, University of Cambridge within the Division of Infectious Diseases, investigating retroviruses (HIV-1, HIV-2 and FIV), including research on viral packaging, RNA structure and RNA / protein interactions.

Research

I joined the Sanger Institute Malaria Programme in 2014, managing health and safety for a number of research laboratories, including Containment Facilites and work with Plasmodium parasites. I oversee office and laboratory spaces on behalf of the teams, including equipment maintenance, purchasing and day to day running of the laboratories. I also manage the development of new office and lab facilities for the Malaria Programme.

References

  • Current perspectives on RNA secondary structure probing.

    Kenyon J, Prestwood L and Lever A

    *University of Cambridge Department of Medicine, Box 157 Level 5 Addenbrooke's Hospital, Hills Road, Cambridge, Cambridgeshire CB2 0QQ, U.K.

    The range of roles played by structured RNAs in biological systems is vast. At the same time as we are learning more about the importance of RNA structure, recent advances in reagents, methods and technology mean that RNA secondary structural probing has become faster and more accurate. As a result, the capabilities of laboratories that already perform this type of structural analysis have increased greatly, and it has also become more widely accessible. The present review summarizes established and recently developed techniques. The information we can derive from secondary structural analysis is assessed, together with the areas in which we are likely to see exciting developments in the near future.

    Funded by: Medical Research Council: G0800142; Wellcome Trust: 078007/Z/05/Z

    Biochemical Society transactions 2014;42;4;1251-5

  • In-gel probing of individual RNA conformers within a mixed population reveals a dimerization structural switch in the HIV-1 leader.

    Kenyon JC, Prestwood LJ, Le Grice SF and Lever AM

    Department of Medicine, University of Cambridge, Addenbrooke's Hospital, Hills Road, Cambridge, Cambridgeshire, CB2 0QQ, UK and HIV-Drug Resistance Program, Centre for Cancer Research, National Cancer Institute, P.O. Box B, Building 535, Frederick, MD 21702-1201, USA.

    Definitive secondary structural mapping of RNAs in vitro can be complicated by the presence of more than one structural conformer or multimerization of some of the molecules. Until now, probing a single structure of conformationally flexible RNA molecules has typically relied on introducing stabilizing mutations or adjusting buffer conditions or RNA concentration. Here, we present an in-gel SHAPE (selective 2'OH acylation analysed by primer extension) approach, where a mixed structural population of RNA molecules is separated by non-denaturing gel electrophoresis and the conformers are individually probed within the gel matrix. Validation of the technique using a well-characterized RNA stem-loop structure, the HIV-1 trans-activation response element, showed that authentic structure was maintained and that the method was accurate and highly reproducible. To further demonstrate the utility of in-gel SHAPE, we separated and examined monomeric and dimeric species of the HIV-1 packaging signal RNA. Extensive differences in acylation sensitivity were seen between monomer and dimer. The results support a recently proposed structural switch model of RNA genomic dimerization and packaging, and demonstrate the discriminatory power of in-gel SHAPE.

    Funded by: Medical Research Council: G0800142

    Nucleic acids research 2013;41;18;e174

Will Proto

- Postdoctoral Fellow

As an undergraduate I studied Microbiology at the University of Liverpool. In 2006, I moved to the University of Glasgow to undertake my PhD working in Jeremy Mottram’s lab. My PhD focused on investigating cysteine peptidases and autophagy in Trypanosoma brucei. After my PhD, I commenced a postdoctoral position in the same lab and expanded my research into cell death mechanisms of parasitic protozoa.

Research

In 2013, I joined Julian Rayner’s group at the Sanger Institute where I study factors influencing the host specificity of Plasmodium species. I am particularly interested in understanding erythrocyte invasion by Plasmodium parasites, and whether species-specific variation in key parasite and/or host genes involved in this process might influence the successful infection of different host species.

References

  • Plasmodium falciparum ATG8 implicated in both autophagy and apicoplast formation.

    Tomlins AM, Ben-Rached F, Williams RA, Proto WR, Coppens I, Ruch U, Gilberger TW, Coombs GH, Mottram JC, Müller S and Langsley G

    Institute of Infection, Immunity and Inflammation; College of Medical, Veterinary and Life Sciences; University of Glasgow; Glasgow, UK.

    Amino acid utilization is important for the growth of the erythrocytic stages of the human malaria parasite Plasmodium falciparum, however the molecular mechanism that permits survival of the parasite during conditions of limiting amino acid supply is poorly understood. We provide data here suggesting that an autophagy pathway functions in P. falciparum despite the absence of a typical lysosome for digestion of the autophagosomes. It involves PfATG8, which has a C-terminal glycine which is absolutely required for association of the protein with autophagosomes. Amino acid starvation provoked increased colocalization between PfATG8- and PfRAB7-labeled vesicles and acidification of the colabeled structures consistent with PfRAB7-mediated maturation of PfATG8-positive autophagosomes; this is a rapid process facilitating parasite survival. Immuno-electron microscopic analyses detected PfRAB7 and PfATG8 on double-membrane-bound vesicles and also near or within the parasite's food vacuole, consistent with autophagosomes fusing with the endosomal system before being routed to the food vacuole for digestion. In nonstarved parasites, PfATG8, but not PfRAB7, was found on the intact apicoplast membrane and on apicoplast-targeted vesicles and apicoplast remnants when the formation of the organelle was disrupted; a localization also requiring the C-terminal glycine. These findings suggest that in addition to a classical role in autophagy, which involves the PfRAB7-endosomal system and food vacuole, PfATG8 is associated with apicoplast-targeted vesicles and the mature apicoplast, and as such contributes to apicoplast formation and maintenance. Thus, PfATG8 may be unique in having such a second role in addition to the formation of autophagosomes required for classical autophagy.

    Funded by: Biotechnology and Biological Sciences Research Council: BB/D526329/1; Medical Research Council: G0700127; Wellcome Trust: WT061173MA-SM, WT085349

    Autophagy 2013;9;10;1540-52

  • Cell death in parasitic protozoa: regulated or incidental?

    Proto WR, Coombs GH and Mottram JC

    Wellcome Trust Centre for Molecular Parasitology, Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, G12 8TA, UK.

    Apoptosis and other types of regulated cell death have been defined as fundamental processes in plant and animal development, but the occurrence of, and possible roles for, regulated cell death in parasitic protozoa remain controversial. A key problem has been the difficulty in reconciling the presence of apparent morphological markers of apoptosis and the notable absence of some of the key executioners functioning in higher eukaryotes. Here, we review the evidence for regulated cell death pathways in selected parasitic protozoa and propose that cell death in these organisms be classified into just two primary types: necrosis and incidental death. It is our opinion that dedicated molecular machinery required for the initiation and execution of regulated cell death has yet to be convincingly identified.

    Funded by: Medical Research Council: 0700127, G9722968; Wellcome Trust: 085349

    Nature reviews. Microbiology 2013;11;1;58-66

  • Crystal structure of a Trypanosoma brucei metacaspase.

    McLuskey K, Rudolf J, Proto WR, Isaacs NW, Coombs GH, Moss CX and Mottram JC

    Wellcome Trust Centre for Molecular Parasitology, Institute of Infection, Immunity, and Inflammation, College of Medical, Veterinary, and Life Sciences, University of Glasgow, Glasgow G12 8TA, United Kingdom. karen.mcluskey@glasgow.ac.uk

    Metacaspases are distantly related caspase-family cysteine peptidases implicated in programmed cell death in plants and lower eukaryotes. They differ significantly from caspases because they are calcium-activated, arginine-specific peptidases that do not require processing or dimerization for activity. To elucidate the basis of these differences and to determine the impact they might have on the control of cell death pathways in lower eukaryotes, the previously undescribed crystal structure of a metacaspase, an inactive mutant of metacaspase 2 (MCA2) from Trypanosoma brucei, has been determined to a resolution of 1.4 Å. The structure comprises a core caspase fold, but with an unusual eight-stranded β-sheet that stabilizes the protein as a monomer. Essential aspartic acid residues, in the predicted S1 binding pocket, delineate the arginine-specific substrate specificity. In addition, MCA2 possesses an unusual N terminus, which encircles the protein and traverses the catalytic dyad, with Y31 acting as a gatekeeper residue. The calcium-binding site is defined by samarium coordinated by four aspartic acid residues, whereas calcium binding itself induces an allosteric conformational change that could stabilize the active site in a fashion analogous to subunit processing in caspases. Collectively, these data give insights into the mechanistic basis of substrate specificity and mode of activation of MCA2 and provide a detailed framework for understanding the role of metacaspases in cell death pathways of lower eukaryotes.

    Funded by: Medical Research Council: 0700127, G0700127; Wellcome Trust: 085349, 091790

    Proceedings of the National Academy of Sciences of the United States of America 2012;109;19;7469-74

  • Trypanosoma brucei metacaspase 4 is a pseudopeptidase and a virulence factor.

    Proto WR, Castanys-Munoz E, Black A, Tetley L, Moss CX, Juliano L, Coombs GH and Mottram JC

    Wellcome Trust Centre for Molecular Parasitology, Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary, and Life Sciences, University of Glasgow, 120 University Place, Glasgow G12 8TA, United Kingdom.

    Metacaspases are caspase family cysteine peptidases found in plants, fungi, and protozoa but not mammals. Trypanosoma brucei is unusual in having five metacaspases (MCA1-MCA5), of which MCA1 and MCA4 have active site substitutions, making them possible non-enzymatic homologues. Here we demonstrate that recombinant MCA4 lacks detectable peptidase activity despite maintaining a functional peptidase structure. MCA4 is expressed primarily in the bloodstream form of the parasite and associates with the flagellar membrane via dual myristoylation/palmitoylation. Loss of function phenotyping revealed critical roles for MCA4; rapid depletion by RNAi caused lethal disruption to the parasite's cell cycle, yet the generation of MCA4 null mutant parasites (Δmca4) was possible. Δmca4 had normal growth in axenic culture but markedly reduced virulence in mice. Further analysis revealed that MCA4 is released from the parasite and is specifically processed by MCA3, the only metacaspase that is both palmitoylated and enzymatically active. Accordingly, we have identified that the multiple metacaspases in T. brucei form a membrane-associated proteolytic cascade to generate a pseudopeptidase virulence factor.

    Funded by: Medical Research Council: G0000508, G0700127, G9722968

    The Journal of biological chemistry 2011;286;46;39914-25

Theo Sanderson

ts10@sanger.ac.uk PhD Student

Theo studied Natural Sciences at the University of Cambridge, before joining the Sanger Institute’s PhD programme in 2011. After rotation projects working with iPS cells and comparative genomics of trypanosomatid parasites, he entered Julian Rayner’s lab in early 2012.

Research

Theo's project involves conducting large-scale screens of the invasion machinery of malaria parasites, leveraging the reverse genetic technologies developed by the Billker and Rayner groups in a number of species of malaria parasite. His initial work has focused on the rodent malaria parasite Plasmodium berghei, but his focus is on transferring these high throughput approaches to the human-simian parasite P. knowlesi.

* quick link - http://q.sanger.ac.uk/mal-ray