MRC National Institute for Medical Research, London

Supervisors: Sanjeev Krishna (St Georges, University of London, UK, .(JavaScript must be enabled to view this email address)) and Christian Doerig (University of Glasgow, UK, .(JavaScript must be enabled to view this email address)).

Since the first suggestion that artemisinins kill P. falciparum by inhibiting the activity of PfATP61, more recent studies have produced results consistent with this hypothesis. In 2005 it was suggested that selectivity for malarial SERCA orthologues could be modulated by small changes in sequence2. Studies from field isolates in French Guiana and Senegal seem to corroborate this prediction. We now propose a genetic strategy to validate PfATP6 as a drug target as well as testing the hypothesis that artemisinins act through interaction with PfATP6. This work will draw upon extensive experience of knock out-complementation technologies, developed at the University of Glasgow using Plasmodium kinases as a model system. Initially knock out plasmids will be constructed to disrupt the genomic PfATP6 gene (which exists as a single copy with no paralogues). Co-transfection with native full-length PfATP6 is anticipated in order to rescue the otherwise presumably lethal transfected line. Once episomal rescue is verified using conventional molecular and cellular techniques, then the phenotype of amino acid-substitution mutants will be characterised, with appropriate controls, in detail. These studies, using the most current technologies to study drug targets in P. falciparum, are intended to provide both first class scientific training within a collaborative context as well as yielding high impact outputs.

Transfection vectors will be produced at SGUL, while generation of transgenic parasite lines will be performed at UGLA/INSERM. The phenotype of transgenic parasites will be examined by SGUL. This will ensure direct access to specific technical expertise at all steps of the project.

References:

1. Eckstein-Ludwig U, Webb RJ, Van Goethem ID, East JM, Lee AG, Kimura M, O'Neill PM, Bray PG, Ward SA, Krishna S (2003). Eckstein-Ludwig U, Webb RJ, Van Goethem ID, East JM, Lee AG, Kimura M, O'Neill PM, Bray PG, Ward SA, Krishna S (2003). Artemisinins target the SERCA ofP. falciparum. Nature 424, 957–961.
2. Jambou, R, Legrand, E, Niang, M, Khim, N, Lim, P, Volney, B, Ekala MT, Bouchier C, Esterre P, Fandeur T, Mercereau-Puijalon O (2005). Resistance of Plasmodium falciparum field isolates to in vitro artemether and point mutations of the SERCA- type PfATPase6. Lancet 366, 1960–1963.

Supervisors: Steven Ward (Liverpool School of Tropical Medicine, UK, .(JavaScript must be enabled to view this email address)) and Alexis Nzila (KEMRI/Wellcome Trust Centre for Geographic Medicine Research, Kenya, .(JavaScript must be enabled to view this email address)).

Methotrexate (MTX) is an analog of folic acid and is used at high dose (5,000 to 12,000 mg/week over several months) in the treatment of diverse malignancies. We have previously demonstrated that MTX is potent against P. falciparum malaria, including multi-drug resistant isolates, with IC50 values of around 30 nM. Two small clinical trials carried out in the 1970s indicate that a dose as low as 2.5 mg of MTX per day for 3 to 5 days can be used to treat malaria; however, this information was not exploited further because of concerns of toxicity1,2. However, since the 1980s, this drug has been used at low doses (less 25 mg/week over several years) in the treatment of rheumatoid arthritis, and the resulting data indicate at this dose MTX is safe and non toxic. In this project we will explore the potential of MTX as an antimalarial drug.

We propose to establish the efficacy, pharmacokinetics, pharmacodynamic and toxicology of properties of MTX in baboons infected with Plasmodium knowlesi. These studies will form the basis of the development of MTX in human. We also propose to identify a mouse malaria model for MTX and this model could be used to select MTX resistant strains which will be used to study the mechanism of MTX resistance. Finally, we will identify and characterize the mechanism of transport and the molecular targets of MTX in P. falciparum.

References:

1. Sheehy, T.W. and H. Dempsey, Methotrexate therapy for Plasmodium vivax malaria. Jama, 1970. 214(1): p. 109-14.
2. Wildbolz, A., Methotrexate in the therapy of malaria. Ther Umsch, 1973. 30(3): p. 218-22.

Supervisors: Michael Lanzer (University of Heidelberg, Germany, .(JavaScript must be enabled to view this email address)) and Patrick Bray (Liverpool School of Tropical Medicine, UK, .(JavaScript must be enabled to view this email address)).

Prior to the development and spread of parasite resistance, chloroquine (CQ) was one of the most successful antimicrobial agents ever developed. CQ binds to haematin, (a reactive iron-bearing hemoglobin metabolite), preventing its sequestration and detoxification in the parasite digestive vacuole (DV). In CQ-resistant parasites mutations in PfCRT (an integral DV membrane protein) are responsible for removing CQ from the DV and reducing its binding to haematin. The exact means by which this is achieved is controversial and proposed mechanisms include an active carrier-mediated process1 and a channel-like leak pathway2. This project will address these and other key issues using contemporary molecular techniques. Studies at UKHD will employ Xenopus laevis oocytes engineered to express both mutant and wild-type PfCRT, whilst at LSTM complementary studies will take advantage of a recently developed proteoliposome system. A variety of structural analogues are available “in house” to characterize the structural features necessary for transport.

The two partners are among the most active in this area and their combined expertise is considerable. This project will provide a highly stimulating and fruitful environment for the prospective student with added value coming from the opportunity to take advantage of additional expertise in DV physiology and medicinal chemistry in the two centres.

References:

1. Sanchez, C.P., Stein, W., and Lanzer, M. (2003) Trans stimulation provides evidence for a drug efflux carrier as the mechanism of chloroquine resistance in Plasmodium falciparum. Biochemistry 42: 9383-9394.
2. Johnson, D.J., Fidock, D.A., Mungthin, M., Lakshmanan, V., Sidhu, A.B.S., Bray, P.G., and Ward, S.A. (2004) Evidence for a central role for PfCRT in conferring Plasmodium falciparum resistance to diverse antimalarial agents. Mol Cell 15: 867-877.

Supervisors: Henri Vial (University of Montpellier 2, France, .(JavaScript must be enabled to view this email address) ) and Clemens Kocken (Biomedical Primate Research Centre, The Netherlands, .(JavaScript must be enabled to view this email address) ).

Phospholipid metabolism is crucial to development of the malaria parasite. Many of the enzymes in this pathway have features that distinguish them from their mammalian counterparts, making them excellent putative targets for drug development. Several structural classes of biscationic antiphospholipid antimalarial compounds, developed in this lab and currently under clinical development, have been shown to inhibit parasite phospholipid metabolism1. The intricacies of the interactions with specific enzyme targets remain to be clarified.

This project aims to perform a biochemical and structural characterization of these enzymes and to elucidate their interactions with biscationic drugs. Some classes of biscationic compounds interact with heme (ferriprotoporphyrin IX), indicating additional specificities that contribute to their antimalarial activities2. For these reasons we will also probe the interaction of our molecules with heme. We envisage that there will be differences among the structural classes in their affinities for both the enzymes and for heme. These studies will therefore provide important information on the mode of action of biscationic compounds to feed into the drug development program, aiding refinement and optimization of lead molecules against multiple targets in this pathway.

Recombinant proteins will be produced for extensive biochemical and structural studies. We have already successfully over-expressed some of the recombinant enzymes (choline kinase and CDP-phosphocholine cytidylyltransferase). Recombinant proteins will be designed to probe the function of the different domains of the enzymes. The effects of the drugs will be quantified. We will use x-ray crystallography for atomic structural studies. Using NMR, we will be able to study smaller constructs as well as the molecular dynamic behaviour of complexes between protein domains and the drugs. Interactions of the drugs with heme will be studied using UV spectroscopy and the effect on the formation of hemozoin crystals will be studied using standard techniques.

Biochemical and structural studies of the enzymes in complex with drugs will be done at UM2, whilst BPRC has extensive expertise with Pichia pastoris expression. Work on drug-heme interactions will be in collaboration with Pat Bray, an Associated Partner (LSTM). The three centres involved in this project are at the forefront of research in their own areas. Their combined knowledge and technical expertise are highly complementary in the context of this project and will provide a stimulating environment for the prospective student.

References:

1. Wengelnik K., Vidal V., Ancelin ML, Cathiard AM, Morgat JL, Kocken C., Calas M., Herrera, S. Thomas, A. and Vial, H. Science, 2002, 295, 1311-1314.
2. Biagini GA, Pasini E, Hughes R, De Koning HP, Vial H, O'Neill P, Ward S and Bray P. Blood, 2004, 104, 3372-3377.

Supervisors: Giuseppe Campiani (University of Siena, Italy, .(JavaScript must be enabled to view this email address)) and Alister Craig (Liverpool School of Tropical Medicine, UK, .(JavaScript must be enabled to view this email address)).

Cytoadherence of P. falciparum infected erythrocytes has been implicated in the pathogenesis of severe malaria. Adhesion can take place through a number of host receptors but only a subset of these are used frequently by isolates from children with disease, including intercellular adhesion molecule 1 (ICAM-1). We have identified a lead compound for inhibition of cytoadherence through in silico screening of mimeotopes modelled on the region of ICAM-1 implicated in binding to P. falciparum combined with ex-vivo binding assays1 developed at LSTM. The inhibitory compound is (+)-epigalloyl-catechin-gallate (EGCG), a polyphenol compound found at high levels in green tea. EGCG blocks ICAM-1-meditaed adhesion but is unable to reverse established binding. In the proposed project we will first chemically modify the EGCG lead molecule to improve its binding inhibition, based on structural comparisons with the L43-loop of human ICAM-1 and molecular modelling, an area of expertise at NatSyn. Next, we will examine the ability to inhibit ICAM-1-mediated adhesion in a range of parasite isolates. Finally we will investigate interference by EGCG with associated binding phenotypes (e.g. CD36) previously observed in ICAM-1-binding strains1. Our aim is to provide adjunct therapies to standard anti-parasite drugs that will support the sick child during the first 24 hours after admission to hospital, when much of the mortality of severe malaria is seen.

References:

1. Dormeyer et al, (2006) Rational design of anti-cytoadherence inhibitors for P. falciparum based on the crystal structure of human ICAM-1 Antimicrob. Agents Chemother. 50: 724-730.

Supervisors: Pietro Alano (Istituto Superiore di Sanità, Italy, .(JavaScript must be enabled to view this email address)) and Robert Sauerwein (Radboud University, The Netherlands, .(JavaScript must be enabled to view this email address)).

Gametocytes ensure transmission of Plasmodium parasites from the human host to the mosquito vector, playing a key role in the spread of the disease. A unique feature of P. falciparum gametocyte development is their initial sequestration, primarily in the bone marrow, before their release in the peripheral circulation at maturity1. High selectivity for bone marrow, and the absence of ‘knob’ structures on the surface of gametocyte-infected erythrocytes (PfGiE) indicate that mechanisms of PfGiE adhesion are different from those used by asexual parasites. In particular, the mechanisms of homing of immature PfGiE to bone marrow and for release of mature PfGiE are completely unknown1. The proposed collaborative project, which will be in close collaboration with a third Associated Partner, BPRC, will address fundamental questions about this unexplored aspect of P.  falciparum biology using a multidisciplinary approach, as follows:

First, PfGiE adhesion assays with bone marrow epithelial cell lines and purified host ligands will be established, relying on gametocyte and cell culture expertises at RU/ISS. Transgenic fluorescent PfGiE (ISS) will allow quantification of adherence, and stage-specific antibodies will be used to identify different gametocyte maturation stages. Next, identification of parasite molecules exported to the surface of PfGiE will be sought by surface labelling of staged gametocytes and mass-spectrometric identification. Gene products identified in this approach will be functionally characterised through construction and transfection of fusion proteins and generation of specific KO parasite lines. The transmission potential of KO lines will be assessed in mosquito transmission experiments at RU. Third, antibodies against selected PfGiE candidate gene products will be produced for biochemical, cellular and ultrastructural characterisation of the respective proteins and their localisation in gametocytes, and to be used in inhibition assays of PfGiE adhesion.

The project's objective will be to identify novel gametocyte-specific molecules that may be targets for intervention (chemotherapeutic or vaccination) designed to interfere with transmission.

References:

1. Smalley, M.E., Abdalla, S., and Brown, J., (1980). The distribution of Plasmodium falciparum in the peripheral blood and bone marrow of Gambian children. Trans R Soc Trop Med Hyg. 75: 103-105.
2. Alano,P., Billker,O. (2005) Gametocytes and gametes. In Molecular Approaches to Malaria. Washington DC: ASM Press

Supervisors: Michael Blackman (National Institute for Medical Research, UK, .(JavaScript must be enabled to view this email address)) and Oliver Billker (Imperial College London, UK, .(JavaScript must be enabled to view this email address)).

Following the bite of an infected mosquito, sporozoites enter the circulation and invade hepatocytes. The intracellular parasite replicates within a membrane-bound parasitophorous vacuole (PV) to yield several thousand liver-stage merozoites per infected cell1. Schizont rupture (egress) releases the merozoites into the bloodstream to initiate the erythrocytic cycle, which is responsible for the clinical manifestations of the disease. At each round of intraerythrocytic growth further mitotic division takes place, also inside a PV, producing blood-stage merozoites which are released upon egress to repeat the cycle.

Recent work in P. falciparum has shown that egress of blood-stage merozoites is preceded by discharge into the PV of a serine protease called SUB12. There, SUB1 mediates the proteolytic maturation of the SERA family of papain-like proteases as well as a number of merozoite surface proteins. Studies with SUB1 inhibitors have shown that these events may be required for schizont rupture and may additionally 'prime' the merozoite surface for invasion, suggesting that SUB1 plays a key regulatory role during the final stages of blood-stage merozoite maturation We aim to investigate the role of SUB1 in liver-stage zoite development and egress. The project comprises two elements that will very effectively combine the interests and expertise of the two PIs involved. First, we will epitope-tag and produce antibodies to the SUB1 orthologue of the rodent malaria parasite P. berghei (PbSUB1) to allow analysis of its expression and sub-cellular localisation throughout the entire parasite lifecycle. In the second part of the project we will use a conditional expression system to examine the function of PbSUB1 in liver stages and the signals that trigger its activity. The results will shed light on how the parasite manipulates and exits its host cell, and will characterize the function of an enzyme with potential as a new malarial drug target. The project will provide the PhD student with training in a wide range of biological techniques, including recombinant DNA and transfection technology, protein biochemistry and cell biology, in a stimulating research environment.

References:

1. Sturm, A. & Heussler, V. (2007). Live and let die: manipulation of host hepatocytes by exoerythrocytic Plasmodium parasites. Med Microbiol Immunol. 196:127-133.
2. Yeoh, S., O'Donnell, R. A., Koussis, K., Dluzewski, A. R., Ansell, K. H., Osborne, S. A., Hackett, F., Withers-Martinez, C., Mitchell, G. H., Bannister, L. H., et al. (2007). Subcellular discharge of a serine protease mediates release of invasive malaria parasites from host erythrocytes. Cell 131, 1072-1083.

Supervisors: Dominique Soldati (University of Geneva, Switzerland, .(JavaScript must be enabled to view this email address)) and Catherine Braun-Breton (University of Montpellier 2, France, .(JavaScript must be enabled to view this email address)).

P. falciparum has 10 putative aspartic proteases (PfPMI to PfPMX). Four of these (PfPMI, PfPMII, HAP and PfPMIV) are involved in hemoglobin degradation and have been extensively studied. PfPMIX and PfPMX are highly similar proteins expressed in late schizont and in sporozoite stages. We have localized PfPMIX to the Maurer’s Clefts of infected red blood cells while PfPMX is reported to interact with PfAARP2, a protein exported to the infected red blood cell, most likely to the Maurer’s Clefts1,2. The localization and expression patterns of PfPMIX and PfPMX are consistent with a role in parasite egress from host cells, either by direct action on host-cell components or by maturation of parasite-derived proteins. The aim of this project is the functional characterization of PfPMIX and PfPMX throughout the cell cycle. The results from this project will, together with the current studies on serine and cysteine proteases in egress, ultimately be integrated to give us a model of how parasites escape from host cells, and how this essential process can be targeted for chemotherapy.

The aims of the project are as follows; i) confirm the sub-cellular localization of PMIX and PMX in P. falciparum and P. berghei; and ii) the functional analysis of PfPMIX and PfPMX during the erythrocytic cycle will be assessed by generation of knock-out parasites. Should these genes prove to be essential, a conditional knock-out strategy will be adopted. Similarly, a functional analysis of P. berghei homologues PbPMIX and PbPMX in sporozoites will be performed either by using the tet-inducible system (currently under development by O. Billker and D. Soldati) or by using a stage specific promoter; iii) characterization of protease activity: recombinant PfPMIX and PfPMX will be expressed in a bacterial or eukaryotic expression system, purified, and enzyme kinetics and substrate preferences will be determined; iv) If the expression of the enzymes is successful, their sensitivity against a range of aspartyl protease inhibitors will be determined.

References:

1. Blisnick T, Vincensini L, Fall G, Braun-Breton C. (2006) Protein phosphatase 1, a Plasmodium falciparum essential enzyme, is exported to the host cell and implicated in the release of infectious merozoites. Cell Microbiol. 4:591-601.
2. Michael Shea, Ursula Jäkle, Qing Liu, Colin Berry, Keith A. Joiner, and Dominique Soldati-Favre (2007). Family of aspartic proteases and a novel, dynamic, and cell-cycle dependent protease localization in the secretory pathway of Toxoplasma gondii. Traffic 8:1018-34.

Supervisors: Artur Scherf (Institut Pasteur, France, .(JavaScript must be enabled to view this email address) ) and Christian Doerig (University of Glasgow, UK, .(JavaScript must be enabled to view this email address) ).

P. falciparum uses differential gene expression to establish phenotypic variation at different stages of the erythrocytic life cycle, a mechanism believed to allow immune clearance and to adapt to different host cell phenotypes. A number of distinct variant parasite proteins are expressed at the erythrocyte surface such as PfEMP1, rifin and surfin. A common feature among these proteins is that they are encoded by multi-gene families. A precise counting mechanism appears to exist for each type of gene family, controlling the switching of expression of surface molecules. For example, var genes are expressed in a mono allelic fashion, whereas rifin appear to express a small subset of members in a single cell. Epigenetic factors appear to orchestrate gene activation and the switching process1. This project aims to investigate factors that control the expression of the var, rif and surfin gene families. The role of reversible chromatin marks in antigenic variation and parasite proliferation will be investigated using specific inhibitors of histone deacetylation and methylation. In other systems, links have been established between regulatory protein kinases (e.g the yeast BUR1 cyclin-dependent kinase) and histone methylation, and a function for the BUR1 kinase in transcriptional regulation through the selective control of histone modifications has been proposed2. Chromatin marks will be compared in wild-type parasites, and in mutant clones lacking specific kinases. State-of-the-art technologies in chromatin research will be used to investigate the molecular mechanism of phenotypic variation. We will use a new generation of microarrays for ‘ChIP on chip’ studies to analyze critical chromatin factors such as histone marks or variant histones. The participant laboratories have ample experience in parasite adhesion, invasion inhibition and kinase assays to monitor the phenotype of parasites with deficiencies in chromatin components.

References:

1. Freitas-Junior, L. H., Hernandez-Rivas, R., Ralph, S. A., Montiel-Condado, D., Ruvalcaba-Salazar, O. K., Rojas-Meza, A. P., Mâncio-Silva, L., Leal-Silvestre, R. J., Shorte, S., and Scherf, A. (2005) Telomeric heterochromatin propagation and histone acetylation control mutually exclusive expression of antigenic variation genes in malaria parasites. Cell 121:25-36.
2. Chu Y, Sutton A, Sternglanz R, Prelich G. (2006) The BUR1 cyclin-dependent protein kinase is required for the normal pattern of histone methylation by SET2. Mol Cell Biol. 26:3029-38.

Supervisors: Jean Langhorne (National Institute for Medical Research, UK, .(JavaScript must be enabled to view this email address)) and Eleanor Riley (London School of Hygiene and Tropical Medicine, UK, .(JavaScript must be enabled to view this email address) ).

Innate immune responses magnify and direct the subsequent adaptive immune responses; indeed this is one mechanism underpinning the immunostimulatory role of adjuvants, particularly those containing microbial ligands for pattern recognition receptors. However innate responses differ between individuals as a result of polymorphism of key innate response gene families in different populations. It is very likely that these genetic differences may translate into inter-individual and inter-population differences in responses to vaccination and may explain the reported low immunogenicity of vaccines (including malaria vaccines) in some African populations. Using two mouse malaria models, Plasmodium chabaudi and P. yoelii, we will investigate the impact of genetic diversity in innate response genes on the response to malaria vaccines. We will use two vaccine approaches using the C terminus of the major merozoite surface protein MSP1; recombinant protein and different adjuvants, and a DNA vaccine/virus prime boost strategy. We will compare innate and adaptive responses to vaccination in C56BL/6 and BALB/c mice, which differ in their susceptibility to malaria, and for which there are abundant data on genomic diversity in host response genes. We will monitor innate responses (dendritic cells, NK, NK-T and gamma-delta T cell; numbers, activation markers, cytokine production) following primary and secondary vaccination and identify key differences that correlate with vaccine responses. Using mice with defined genetic defects or polymorphisms in different arms of the innate response, we will attempt to narrow down the key genetic differences underpinning these differences and subsequent vaccine induced immunity. These studies will define important biomarkers of innate responses able to support protective adaptive responses, and will have wide application for malaria vaccine design.

References:

1. Norman PJ, et al Unusual selection on the KIR3DL1/S1 natural killer cell receptor in Africans (2007). Nat Genet. 39:1092-9.
2. Gavin AL, Hoebe K, Duong B, Ota T, Martin C, Beutler B, Nemazee D (2006). Adjuvant-enhanced antibody responses in the absence of toll-like receptor signaling. Science. 314:1936-8.

Supervisors: Paolo Arese (University of Torino, Italy, .(JavaScript must be enabled to view this email address)a>) and Thomas Williams (University of Oxford, UK, .(JavaScript must be enabled to view this email address)).

Host genetic factors are important determinants of malaria survival. Some of the best-described examples include polymorphisms that affect the red blood cell (RBC). This project aims to investigate the mechanisms of malaria protection in a number of (RBC) polymorphisms through a collaboration between the UOXF and UT groups, and a third collaborator in Rome/Burkina Faso (Modiano). The focus of this project will be on the hypothesis that common pathways, notably those involved in oxidant stress, are central to the malaria-protective properties of many of these polymorphisms. In their uninfected state such RBCs are characterized either by higher production of ROS or by lower levels of ROS scavenging pathways. Previous work suggests that this oxidative damage is accelerated upon infection with P. falciparum and that this leads to their enhanced removal from circulation by phagocytosis. Further, two membrane parameters of parasitized RBCs appear to be modified by moderate oxidative stress typically present in infected RBCs of carriers of protective mutations: firstly, serine-phosphorylation of band 3, band 4.1/2 and adducin is enhanced; second, 4-hydroxynonenal adducts to RBC membrane are increased. 4-hydroxynonenal-modified infected RBCs are rigid and intensely removed by phagocytes. The above parameters can be assessed in RBC ghosts prepared ex vivo from malaria patients and provide new data on mechanism of protection in common RBC mutations. Further, more recent work has shown the enhanced and co-ordinated mRNA expression of anti-oxidant enzymes and heat-shock proteins in mildy oxidatively stressed, ring-stage parasitized RBCs, most remarkable being increases in expression of pfHSP-90, SOD1 and SOD1, G6PD and the thioredoxin system. Of particular interest are the essential role of pfHSP-90 for parasite development, and the availability of specific drugs (geldanamycin and similar compounds) that have potent antimalarial properties. In this project we aim to exploit the unique opportunities offered by the collaborating groups to study these pathways ex vivo in children of known genotype who present with malaria through natural exposure.

References:

1. Williams TN. Red blood cell defects and malaria. (2006) Mol Biochem Parasitol. 149:121-7.
2. Ayi K, Turrini F, Piga A, Arese P. Enhanced phagocytosis of ring-parasitized mutant erythrocytes: a common mechanism that may explain protection against falciparum malaria in sickle trait and beta-thalassemia trait (2004). Blood 104:3364-3371.

Supervisors: David Roberts (University of Oxford, UK, .(JavaScript must be enabled to view this email address)), Chetan Chitnis (ICGEB, India, .(JavaScript must be enabled to view this email address)) and Peter Bull (KEMRI/Wellcome Trust Centre for Geographic Medicine Research, Kenya).

Virulent falciparum malaria has been associated with the auto-agglutination or clumping of infected cells and platelets1,2. Moreover, there is increasing evidence to link accumulation of platelet clumps with end-organ damage. We have defined two distinct mechanisms of platelet-dependent clumping by infected red blood cells, dependent on CD36 or C1qR. We propose to study the different clumping phenotypes in parasites from a large case control study of cases of severe and mild malaria cases in Kilifi, Kenya, led by Peter Bull and in collaboration with a third Associated Partner, KEMRI. In ongoing studies we are defining the expressed var genes in clumping and non-clumping isolates by sequencing 100 DBL-1 var gene tags from cDNA from each parasite isolate, defining the spectrum of var gene DBL-1 domains expressed by clumping clones and fully sequencing the dominant var genes. We will define the variation in the respective platelet binding domains of 20 clinical isolates with a clear clumping phenotype. We will establish as many as possible of these isolates into long term culture and determine their clumping phenotypes. In parallel studies, we will define the var genes associated with the two major clumping phenotypes in existing laboratory lines and the specific domains responsible for adhesion to platelets. These studies will determine the relationship between different clumping phenotypes and severe malaria and the potential to define candidate vaccine antigens to prevent disease caused by these parasite phenotypes.

References:

1. Pain A, et al., PNAS 2001 98:1805-10.
2. Chotivanich K, et al., J Infect Dis. 2004 189:1052-5.
3. Biswas A, et al. PLoS Pathog 2007 3:1271-80.

Supervisors: Maria Mota (Institute of Molecular Medicine, Portugal, .(JavaScript must be enabled to view this email address)) and Freddy Frischknecht (University of Heidelberg, Germany, .(JavaScript must be enabled to view this email address)).

Sporozoites enter hepatocytes and transform into thousands of merozoites1. Although there are no clinical symptoms during the liver phase of infection, this stage can be targeted using different vaccine strategies. However, we currently understand very little about the cell biology of liver cell infection. Studies of the interaction of viruses or bacteria with their host cells show that most interfere with host cell signalling events that lead to the remodeling of the cytoskeleton of the host cell2. These pathogen-induced changes usually result in an enhanced spread and/or replication of the invading microorganism.

We propose to investigate the dynamic interaction of the developing liver stage malaria parasite with the host cell cytoskeleton. To this end, IMM has already generated two stable hepatoma cell lines expressing either a fusion between mCherry and actin, or mCherry and beta-tubulin, which can be filmed at high magnification for approximately 10 hours. The PhD student will perform dynamic imaging of the cytoskeleton during the complete cycle of liver stage development (which lasts ~50 hours) in P. berghei after green fluorescent sporozoites successfully invade hepatocytes. As sporozoites migrate through several hepatocytes prior to invasion1, we will add a blue fluorescent nucleic acid stain (Sytox, Molecular Probes) to the medium to distinguish between 'visited' and 'non-visited' hepatocytes (Sytox is taken up by cells through which sporozoites have passed - i.e. ‘visited’ cells - as their plasma membrane is disrupted, but not non-visited ones, which will serve as controls). All imaging will be performed on available wide-field or spinning disc confocal microscopes at 37ºC under tissue culture conditions at UKHD or IMM. Movie sequences will be quantitatively analyzed2 at UKHD.

This approach will show whether P. berghei reorganizes host cell actin filaments and microtubules throughout the liver stage. In addition, the PhD student will make use of RNAi to manipulate the assembly and organization of the actin or microtubule cytoskeleton of hepatocytes and follow by live imaging any changes that arise in P. berghei sporozoite development. The IMM group has already observed that knockdown of cdc42 leads to a significant increase in infection. This preliminary observation paves the way for the PhD student’s project to elucidate the role of the host cytoskeleton in Plasmodium liver cell infection.

References:

1. Prudencio M, Rodriguez A, Mota MM. (2006). Nat Rev Microbiol. 4(11):849-56.
2. Munter S, Way M, Frischknecht F. (2006). Sci STKE. 2006(335):re5.

Supervisors: Inga Siden-Kiamos (IMBB FORTH, Crete, .(JavaScript must be enabled to view this email address) ) and Kai Matuschewski (University of Heidelberg, Germany, .(JavaScript must be enabled to view this email address) ).

Motility of the invasive stages of the malaria parasite is dependent upon actin filament formation. Actin filaments in Plasmodium are unusually short and labile compared to actins in other organisms. In other systems, a number of proteins are known to regulate actin filament formation and turnover. In Plasmodium some of these have clear orthologues, while the genes encoding others seem to be absent. It has become evident over the last few years that one layer of control of gliding motility is also achieved through the regulation of actin filament formation. This project will investigate this aspect of motility in depth, using as a model system the P. berghei ookinete. Ookinetes are easily cultured in large numbers and hence are the only Plasmodium life cycle stage where phenotypic analysis of in vitro motility and biochemistry can be combined. Importantly, even essential genes can be conditionally knocked out in the ookinete using a promoter swap approach. We have recently established this technology to study the role of two myosins in ookinete motility.

From the dozen potential Plasmodium actin regulatory proteins we will prioritize candidates after gene expression analysis in selected life stages, in addition to using publicly-available data. We will then continue by gene targeting of the actin-binding proteins that are most likely to exert a stage-specific function and are dispensable for asexual blood stage growth. In case of vital genes we will generate conditional knock-downs in ookinetes. We will build on our promoter swap approach or, once successful, we will use the tetracycline-induced repressor system. The resulting mutants will be analyzed phenotypically using a variety of established assays throughout the different life stages, in the mosquito as well as in the rodent host. Specific motility assays will be employed to determine the effects of the mutations in the ookinete and, when appropriate, in the sporozoite. Once we observe an important function in motility we will continue with a biochemical characterization of the direct role in microfilament turnover. UKHD has recognized expertise in this area and has established a range of relevant biochemical methods.

References:

1. Siden-Kiamos I., Pinder, J.C., Louis C. (2006) Involvement of actin and myosins in Plasmodium berghei ookinete motility Mol. Biochem. Parasitol. 150:308–317.
2. Schuler H., Mueller, A.A., Matuschewski, K. (2005) A Plasmodium actin-depolymerizing factor that binds exclusively to actin monomers. Mol. Biol. Cell 16: 4013-4023.