Project Descriptions

Project 1. Antigenic variation in Malaria: Molecular factors involved in differential expression of surface antigens.
Supervisors:
Artur Scherf, Institut Pasteur, Paris, France
Michael Lanzer, University of Heidelberg, Germany

Epigenetic factors control mutually exclusive transcription of members of the var gene family. Several nuclear factors have been identified, that interfere with var gene silencing. Among those are the proteins called Silent Information Regulators (Sir), which accumulate in the nuclear periphery. The aim of this PhD project would be to investigate these potential factors that establish gene silencing of telomeric and other genes located at the nuclear periphery.

The following tools will be used: Gene knock out, candidate proteins fused to different fluorescent proteins (live studies), chromatin analysis (ChIP) and phenotype analysis of var gene expression and location. The candidate will learn state of the art molecular and cell biology technologies of malaria parasites and will integrate into two malaria teams who are leading groups in the field of antigenic variation of malaria parasites.

Project 2. Functional analysis of invasion-related proteins shared between multiple stages of Plasmodium
Supervisors:
R. Menard, Institut Pasteur, Paris, France
M. Blackman, MRC-NIMR, London, UK
K. Matuschewski, University of Heidelberg, Germany

The three invasive stages of the malaria parasite share many of the critical elements that are directly involved in host cell invasion. These include cell surface proteins involved in host cell recognition, as well as components of the molecular motor that propels the parasite into its host cell. A current limitation of addressing the function of such molecules through genetic strategies is our inability to disrupt or modify the corresponding genes, as only the haploid erythrocytic stage can be transfected. Recently a conditional mutagenesis technique has been developed that allows modification of the gene in the erythrocytic stages such that it can be subsequently deleted in insect stages. The system is based on site-specific recombination using the FLP recombinase. This project aims to apply this technology to studying the function of two proteins that are important in invasion across stages. AMA-1 is a transmembrane surface protein long recognised as being directly implicated in red cell invasion, and recently identified on the sporozoite surface. MCP-1 is a cytoplasmic protein that localises to the moving junction during merozoite invasion of red cells and is also expressed in sporozoites. Our approach will establish the function of two key proteins in the malaria life cycle. This project will involve a collaboration between four partner institutes (University of Heidelberg, Institut Pasteur, National Institute for medical Research, and Imperial College).

Project 3. The role of phosphoinositide signaling in life cycle progression of malaria parasites.
Supervisors:
H. Vial, University of Montpellier-CNRS, France
O. Billker, Imperial College, London, UK
D. Soldati, Imperial College London, UK

Essential events in the malarial life cycle, such as microneme secretion, invasion and sexual reproduction, rely on common principles of signal transduction. Pharmacological and biochemical evidence suggest a key function phosphoinositide metabolites generated by an unusual phospholipase C. This results in the release of calcium from intracellular stores that plays a key role in these divers events. Upstream pathways regulating phospholipase C are most likely unique to the Apicomplexa, making them interesting targets for pharmacological intervention. This project is proposed to combine biochemical and genetic approaches to study the role of parasite phospholipase C, its regulation by upstream signaling pathways, and the phosphoinositide metabolites generated. The highly synchronized activation of P. berghei and P. falciparum gametocytes by xanthurenic acid will be exploited to analyze phosphoinositides in the Vial lab. PLC gene knock out parasites and epitope tagging will be carried out and transgenic parasites analyzed in the Billker lab. In the likely event of PLC being an essential enzyme conditional gene knock-outs will be developed in collaboration with the Soldati lab. This will serve as a starting point for a structural and functional analysis of unique domains in PLC and its potential as a pharmacological target will be evaluated.

Project 4. Trafficking of parasite proteins to the Maurer’s clefts
Supervisors:
C. Braun-Breton, University of. Montpellier, France
M. Lanzer, University of. Heidelberg, Germany

P. falciparum exports proteins beyond its own confines to the host erythrocyte cytoplasm and plasma membrane. These proteins are important virulence factors mediating cytoadhesive interactions, nutrient uptake and merozoite release. Maurer’s clefts are part of the intra-erythrocytic secretory machinery. Recent studies have identified several signals necessary for the targeting of parasite proteins to the Maurer’s clefts and beyond. Other work has identified several resident proteins of the Maurer’s clefts, that may be essential for the biological function of this secretory compartment and may interact with the trafficking signalling. The goal of the study is to characterise interactions between resident clefts’ proteins and passenger proteins using state of the art bio-imaging combined with biochemical studies. This will involve the generation of GFP chimera, transfection of P. falciparum, FRET, confocal microscopy, affinity chromatography and two-hybrid assays. This is a joint project between the Universities of Montpellier and Heidelberg.

Project 5. Protective versus pathogenic effects of leucocyte stimulation in malaria
Supervisors:
David Roberts, Oxford University, UK
Pierre Druilhe, Institut Pasteur, Paris, France

The activation of cells of the macrophage lineage during malaria has been one of the first to be recognised. Infected RBC, released schizonts, free merozoites, are taken up by tissue macrophages, particularly in the spleen and liver, and by blood monocytes and polymorphonuclears.

This leads to the activation of these cells and the release of numerous mediators among which, oxygen and nitrogen radicals, TNF, Interferon, are abundant and known to be pharmacologically potent. The remarkable size of malarial, as compared to eg.bacterial, septicemia, implies that this activation is extremely strong. These cells and mediators are known to play important roles in the clinical expression of the disease, although the precise role of each of these and potentially others is not well defined and now requires to be better investigated.

In terms of defence, these cells play several roles, some being already identified. The most obvious is parasite's uptake which is increased by activated cells and when specific antibodies agglutinate parasites or SIRBC, or when they bridge a parasite and a macrophage. This obviously contributes to parasite clearance. In addition, mediators particularly TNF-a induce fever that has been reported to reduce parasite growth. Finally Monocytes, though not macrophages, activation leads to the release of monokines that can block the division of surrounding intra-erythrocytic parasites. Indeed, in vitro studies connected with in vivo clinical experiments have shown that monocyte mediators triggered by protective IgG and merozoites play a major role in defence in promoting an Antibody Dependent Cellular Inhibition (ADCI). This mechanism may potentially also induce pathogenic effects, that remain to be investigated. The identification of the critical mediators affecting Plasmodium will be instrumental in this part of the work.

The very wide range of phenotypes that can be expressed by cells of the myeloid lineage require to better characterize them in malaria patients at different stages of their interaction with the parasite. Further this state will be influenced by the host –parasite interaction through direct and indirect (lymphocytes, platelets) mechanisms triggered by the parasite, resulting in distinct profiles of cytokines' - monokines - expression. Thus, cytokines have at the same time, an effect on the parasite but also on the host cells, with an often dual role (pro or anti disease) according to their concentrations and time-frame of expression.

It thus appears valuable to determine the respective contribution to the host pathology and defence of blood monocytes, polymorphonuclears and as much as possible tissue macrophages both in models and in individuals being at various stages of the infection (first-attack, high parasitaemia with chronic enlarged spleen, immune asymptomatic adults, severe or chronic anaemia, cerebral malaria) and in a kinetic manner .

Project 6. Erythrocyte surface molecule expression and severe malaria
Supervisors:
Mats Wahlgren, Karolinska Institute, Stockholm, Sweden
Fred Kironde, Makerere University, Kampala, Uganda

The aim of this research project is to identify parasite-derived surface-located adhesion molecules on Plasmodium falciparum-infected erythrocytes. We will usesurface-proteomics and RNA-profiling of virulent Plasmodium falciparum

Project 7. Genetic basis of ethnic differences in susceptibility to malaria
Supervisors:
Ogobara Doumbo, University of Bamako, Mali
Dominic Kwiatkowski, University of Oxford, UK
David Modiano, University of Rome, Italy

One of the possible approaches in the study of human variation in the susceptibility to malaria consists in comparing malariologic indicators between populations differing in their genetic background and living in the same epidemiological context, i.e. exposed to the same transmission level and to the same parasite strains. Actually, the possible observation of interethnic differences of susceptibility in such conditions may provide new opportunities to detect factors associated to protection. This inter-ethnic comparative approach was applied in extensive studies performed in hyperendemic rural areas of Burkina Faso and Mali, West Africa. These studies showed striking interethnic heterogeneities in the susceptibility to Plasmodium falciparum malaria among the sympatric ethnic groups, Fulani, Mossi, Rimaibé and Dogon (Modiano D. et al. 1995, 1996; Dolo A. et al. unpublished). Despite similar entomological inoculation rates and comparable use of protective measures, the Fulani were less parasitized and less affected by the disease. This resistance was not associated to classic malaria resistance genes (Modiano D. et al. 2001) and the analysis of the humoral immune response to P. falciparum sporozoite and blood stage antigens revealed higher immune reactivity in Fulani. (Modiano D. et al. 1995, 1996, 1998, 1999). This group is genetically distinct from the neighbouring groups (Modiano et al. 2001) and the presence of unknown immunogenetic protective factor/s is the most likely explanation for their higher response to malaria.

The general objective of the present PhD project is to explore the biological basis of the ethnic group specific resistance to malaria shown in the Fulani. Polymorphic MHC and non-MHC genes, related to the immune system will be investigated. Among the most promising region to investigate is the 5q31-q33 region. This region, 31Mb long, codes for several cytokines involved in the regulation of immunity (i.e. Th2 cytokine cluster, IRF1, CSF2, CD14 and IL12B) and it has been recently linked to some common diseases among which malaria (Marquet S. et al. 1996; Rihet P. et al. 1998; Walley AJ et al. 2001). Furthermore, Interleukin-4, which lies, in the 5q31-q33 region was studied in the three ethnic groups of Burkina Faso and a promoter polymorphism (-589 C/T) was found to be associated with elevated antibody levels against malaria antigens in the Fulani (Luoni G. et al. 2001).

Exploiting the advances of the human genome project, a map of nucleotide and haplotype diversity, specific for the West African populations under study, will be generated. Haplotype tagging Single Nucleotide Polymorphisms (htSNPs) specific for the ethnic groups involved will be identified from SNP Consortium and will be analysed either as single functional factors (direct association analysis) or will help to map an unknown gene using the different Linkage Disequilibrium patterns of the relevant chromosomic region (indirect association analysis). Intra- and inter-ethnic association analysis between genotypes and humoral and cellular immune phenotypes will be performed for each SNP in the Fulani and in sympatric ethnic groups from Burkina Faso, Mali and Sudan.

The PhD project will be performed in the frame of a collaboration among the following institutions: Department of Public Health, Section of Parasitology, University of Rome “La Sapienza” (Prof. David Modiano); Centre National de Recherche et de Formation sur le Paludisme, Ministère de la Santé, Burkina Faso (Dr. Bienvenu Sodiomon Sirima); University of Oxford (Prof. Dominic Kwiatkowski); Malaria Research and Training Center, University of Bamako, Mali, (Prof. Ogobara Doumbo); Institute of Endemic Diseases, University of Khartoum, Sudan (Prof. Ibrahim).

Project 8. Transcriptional analysis of the biology of P. berghei parasites in the mosquito midgut
Supervisors:
E. Levashina, IBMC-CNRS , Strasbourg, France
A. P. Waters, Leiden, Netherlands
 

The development of malaria parasites in the mosquito vector represents a critical step in the transmission of the disease. Recently, we have demonstrated that An. gambiae is not just a passive malaria transmitter but is able to detect parasite presence and to limit its development in the midgut tissues. We identified a mosquito complement-like protein, TEP1, which is implicated in parasite surveillance (Blandin et al., 2004, Cell 116, 661-670). We have next developed two mosquito transgenic lines which exhibit loss- and gain-of-function phenotypes for TEP1. Interestingly, the loss-of-function line is highly susceptible to infections with Plasmodium berghei and two P. yoelii species. In contrast, development of these parasite in the gain-of-function line is very restricted and the parasite numbers are dramatically reduced.

We are interested in mechanisms of TEP1-dependent parasite killing and to address this question, we initiated the transcriptional analysis of the genes that are co-regulated with TEP1 after parasite infection. The proposed project will extend this characterization to the simultaneous analysis of mosquito and P. berghei genomes. Several important questions will be addressed. (i) Identification of genes that cooperate with TEP1 in parasite killing in the mosquito. Genes representing differential patterns (assessed by microarray analysis) in two transgenic lines will be further functionally characterized using dsRNA knockdown and the biology of parasite killing will be explored by cell biology approaches. (ii) Characterization by microarray analysis of parasite responses to distinct selective pressures exhorted by the mosquito host: strong immune response in the TEP1 GOF lines; and limited immune responses in the TEP1 LOF mosquito line. Identified parasite genes will be subjected to KO analysis and their role in parasite invasion will be characterized.


Project 9. Genome wide expression profiling of Anopheles gambiae infection with Plasmodium falciparum and comparative analysis with P.berghei infections
Supervisors:
F. C. Kafatos, EMBL, Heidelberg, Germany
M. Lanzer, University of Heidelberg, Germany
R. Sinden, Imperial College, London, UK

The journey of Plasmodium through the mosquito vector is accompanied by complex interactions with the vector, particularly at the level of midgut and salivary gland epithelia. Utilization of the model system of Anopheles gambiae and the rodent malaria, Plasmodium berghei let to the identification of a robust and efficient innate immune responses in the vector against parasite infection and has shaped our understanding of the interactions between these two species, However, the expression of distinct surface proteins by the human malaria parasite raises the question whether distantly related Plasmodium species, like P.berghei and P.falciparum , interact similarly with the mosquito vector. This project will investigate the similarities and differences of A. gambiae immune responses against these to Plasmodium species using functional genomics approaches.

The successful candidate will learn and perform experimental infections of A.gambiae mosquitoes with the human malaria parasite P.falciparum in the laboratories of Prof. M. Lanzer at the University of Heidelberg and Prof. R. Sinden at the Imperial College, London. The candidate will then focus on large-scale expression profiling of the genome of A.gambiae infected with P.falciparum using the A.gambiae 20,000 EST microarray platform in the Kafatos laboratory. The expression profiles will be compared with ones previously obtained from P.berghei infected A.gambiae mosquitoes, with specila emphasis on identifying innate immunity genes expressed differentially by A.gambiae during the interaction with these two distantly related Plasmodium species. In a later stage, differentially expressed genes will be functionally analysed by RNAi knockout in A. gambiae by observing the effect of the knockout genes on the survival of both parasite species in the mosquito vector.

Project 10. Accessory cell regulation of B-cell activation and development in human and murine malaria
Supervisors:
J. Langhorne, MRC-NIMR, Lodon, UK
B. Urban, Oxford University, UK
K. Marsh, Kilifi, Kenya

In malaria-endemic areas, almost all children are infected with the parasite Plasmodium falciparum. About 1% of children suffer from severe disease and death whereas others show mild clinical symptoms or are asymptomatically infected. The bloodstages of the parasite * which cause malarial disease - are extremely variant. It is now believed that children living in endemic areas have to be infected repeatedly with different parasite variants so that with increasing exposure they develop a repertoire of protective antibody responses to parasite variants. Therefore, clinical immunity to P. falciparum malaria is never sterile but allows infected individuals to control parasitaemia without succumb to disease. Recent evidence suggests that antibody responses to P. falciparum antigens are short-lived and dependent on the presence of bloodstage parasites. However, it is not known whether short-lived antibody responses are due to a defect in B-cell differentiation, T-helper cell differentiation or inadequate support by accessory cells such as dendritic cells, monocytes or stromal cells.

During the course of the project the candidate will establish in vitro assays for B-cell differentiation, T- helper cell and accessory cell function. These assays will be used to analyse the ability of different accessory cells to support B-cell differentiation in human and rodent malaria. The project is a collaboration between the University of Oxford (Britta Urban), NIMR (Jean Langhorne) and the Wellcome Trust Research Laboratories/KEMRI (Kevin Marsh) in Kilifi, Kenya and the candidate is expected to spend time in all three locations.

Project 11. The innate immune response to malaria- correlates of susceptibility
Supervisors:
R. Sauerwein, Nijmegen, Netherlands
M. Troye-Blomberg, Stockholm University, Sweden
O. Doumbo, University of Bamako, Mali

In the course of malaria infection there is a complex interaction between the host immune system and the Plasmodium parasite, which is uniquely adapted to survival in the human host.
The mechanisms of malaria immunity are poorly understood but play a pivotal role in the control of the parasite lifecycle progression in the human host.

The innate immune system forms the first line of defence against malaria and it is thought that interactions at this “front” determine not only the level of (pathological) inflammatory reactions, but also the fine-tuning (and therefore effectiveness) and the subsequent (acquired) immune response. Better understanding of this complex situation could lead not only to new therapeutical interventions in clinical malaria, but also to more effective vaccination strategies.

Toll-like receptors (TLRs) are likely to play a major role in this interaction. This family of pathogen associated molecular pattern (PAMP) receptors recognises various conserved microbial products and host-derived signals of tissue damage. The role of TLRs in the protection against bacterial, viral and other parasitical infections is currently under intense research. New knowledge is also emerging about the functions of TLR’s in the fine-tuning of the subsequent immune-response.
In this project in vitro studies and experimental human P.falciparum infections will be used to study 1) parasite-TLR interactions and 2) dissect early innate immune responses. In vitro correlates of malaria susceptibility will be linked with in vivo findings.

Studies in vitro will include (i) Analysis of malaria-antigen recognition by human TLR’s using anti-TLR blocking antibodies, cell lines transfected with specific TLR’s and TLR-KO cells harvested from KO-mice; (ii) FACS analysis of TLR expression on various cell populations (including monocytes, macrophages and dendritic cells) following stimulation with malaria antigens, (iii) Downstream signalling pathways; and (iv) Analysis of cytokine pathways and cell populations involved in TLR-mediated inflammatory responses to malaria antigens.

Studies in human experimental malaria will include (i) In vitro PBMC responses to malaria antigens versus in vivo parasite suppression; (ii) Gene-expression profiling; (iii) FACS analysis of TLR expression ; (iv) Regulatory T cells.

Project 12. Development and adaptation of computational approaches to sequence analysis in the study of Plasmodium spp. genomes.
Supervisors
Elisabetta Pizzi, Istituto Superiore di Sanità, Roma, Italy.
Matthew Berriman, Wellcome Trust Sanger Institute, Hinxton, United Kingdom.

In spite of the large amount of data obtained through genome sequencing and gene expression experiments, little is still known on sequence elements involved in gene expression in Plasmodium. The automatic location of promoters in a eukaryotic genome is hampered by their complex combinatorial nature and the weak conservation in their sequence elements. The proposed project concerns the development of a novel procedure for analysis of intergenic regions and identification of structural and regulatory elements in Plasmodium genomes based on their compositional/statistical properties, integrated with structural information (bendability; flexibility scale based on direct measurements on DNA oligos). Results will be compared with those obtained by applying other methods (pattern matching; phylogenetic footprinting).

Computational work will rely on genomic sequence data produced by sequencing projects and on results obtained by microarray and proteomic data. Particular attention will be paid to the analysis of sequences neighbouring genes involved in sexual differentiation or specifically expressed in sexual stages. Predicted structural and regulatory elements will be functionally characterised in transfection experiments with reporter genes in the rodent and the human malaria parasites Plasmodium berghei and Plasmodium falciparum.

Sequence analysis and, in particular, comparison of protein sequences between Plasmodium and other organisms is hampered by specific base composition of the malaria parasites. A second area of activity of this project will be the adaptation of existing algorithms that are used in protein sequence comparison to such properties (e.g. construction of a specific amino acid substitution matrix).

Project 13.  Anti-parasitic activities of antibodies to surface antigens of asexual blood stages of Plasmodium falciparum

Supervisors: Klavs Berzins, Stockholm University, Sweden; Jürg Gysin, Institut Pasteur, France

It is well established that antibody dependent mechanisms are important in protective immunity against the asexual blood stages of P. falciparum. These parasite neutralizing mechanisms include inhibition of parasite growth or invasion, phagocytosis of infected erythrocytes or merozoites, prevention of the dispersal of merozoites and blocking of the cytoadherence of infected erythrocytes. Several potential target antigens in this context have been identified, including merozoite surface associated antigens, antigens present in the apical organelles of merozoites and antigens expressed on the surface of infected erythrocytes.

This project will analyse the parasite neutralizing activities of antibodies with regard to mechanisms, antibody specificity and quality (e.g. isotype, affinity). In the initial phase of the project the work will focus on antigens expressed on the surface of infected erythrocytes, in particular the conserved antigens Pf332, RSP2 and variant antigens PfEMP1CSA encoded by var1 /var2CSA, but work on other selected antigens will be phased in along the run of the project. For these analyses polyclonal antibodies against epitopes in different parts of these antigens will be generated, including mouse, human monoclonal antibodies (Stockholm) and single chain antibodies (Marseille), but also polyclonal affinity purified human antibodies (Stockholm and Marseille).

Antibodies to Pf332, RSP2 PfEMP1CSA and some new and not yet characterized surface antigens have been shown to inhibit parasite growth and adherence to host cells in vitro and act for some of them in synergy with monocytes in this context. The mechanisms involved in these inhibitions with or without monocytes, apart from phagocytosis, are still unknown. Growth of parasites in vitro for extended time periods in the presence of suboptimal inhibitory concentrations of antibodies, renders the parasites specifically less sensitive to inhibition by those antibodies by as yet unknown mechanisms. Using already existing antibodies and newly developed antibodies, the mechanisms for antibody dependent parasite inhibition will be analysed in detail  and evasion mechanisms that the parasite may use to escape immune pressure will be defined (Stockholm). Furthermore, the basis for the cytoadherence blocking activity of certain antibodies to Pf332, RSP2 and PfEMP1CSA, while other antibodies reactive with these antigens lack this activity, will be analysed under static and in flow conditions on various cell types or tissue cryosections over the entire blood stage cycle (Marseille).

Project 14.  Posttranscriptional analysis of the biology of the P. beghei parasites in the mosquito midgut

Supervisors: E. Levashina, IBMC-CNRS , Strasbourg, France;  H. Stunnenberg, NCMLS, Nijmegen, Netherlands

The development of malaria parasites in the mosquito vector represents a critical step in the transmission of the disease. Recently, we have demonstrated that An. gambiae is not just a passive malaria transmitter but is able to detect parasite presence and to limit its development in the midgut tissues. We identified a mosquito complement-like protein, TEP1, which is implicated in parasite surveillance (Blandin et al., 2004, Cell 116, 661-670). We have next developed two mosquito transgenic lines which exhibit loss- and gain-of-function phenotypes for TEP1. Interestingly, the loss-of-function line is highly susceptible to infections with Plasmodium berghei and two P. yoelii species. In contrast, development of these parasite in the gain-of-function line is very restricted and the parasite numbers are dramatically reduced.

We are interested in mechanisms of TEP1-dependent parasite killing and to address this question, we initiated the transcriptional analysis of the genes that are co-regulated with TEP1 after parasite infection. Although informative, this method does not allow detecting differences between two strains at the post-translational level. To complement our transcriptional profiling, we propose to a successful candidate to perform a comparative proteomic analysis of the mosquito midgut and blood cell extracts from loss-of-function (hypersusceptible) and gain-of-function (resistant) transgenic lines. Recently, a proteomic analysis provided the first characterization of the P. falciparum proteome throughout the parasite life cycle.However, this method has not yet been applied to study mosquiot proteome. The proposed project will provide simultaneous analysis of mosquito and P. berghei proteomes. Several important questions will be addressed. (i) Identification of proteins that cooperate with TEP1 in parasite killing in the mosquito.Candidate genes will be further functionally characterized using dsRNA knockdown and the biology of parasite killing will be explored by cell biology approaches. (ii) Characterization by proteomic analysis of parasite responses to distinct selective pressures exhorted by the mosquito host: strong immune response in the TEP1 GOF lines; and limited immune responses in the TEP1 LOF mosquito line. Identified parasite genes will be subjected to KO analysis and their role in parasite invasion will be elucidated.

Project 15. Looking for the functional role of Plasmodium proteases in gametocyte to oocyst transition.

Thanasis G. Loukeris, University of Crete and A. P. Waters, Leiden, Netherlands

It has been recognized for over two decades that proteases are critical to the blood-stage life cycle of the malaria parasite and therefore they have been selected as targets for drug development. Lately there is increasing evidence that Plasmodium proteases may also play significant roles during the development of their mosquito stages and therefore may serve as targets for an alternative transmission blocking strategy. We are planning to combine strategies and tools in order to investigate the functional role of two plasmodial proteases,  berghepain 1 and P. berghei SUB2 that belong to two major groups, papain family cysteine proteases and subtilases respectively, in gametocyte to oocyst transition. Combination of strategies and tools is high desirable  because of the probable  vital and multi-stage role of these proteases in invasion processes and/or parasite developmental transition. The collaboration encompasses three interrelated projects:

A) SUB2: gene specific knock out has proven unsuccessful in the functional study of SUB2, since sub2 transgene KO causes lethality in the asexual stages. Therefore for the purpose of our studies we are going to generate transgenic parasites in which the expression and/or function of SUB2 will be affected only in gametocytes and ookinete. This will be approached in several ways: a) by restricting SUB2 expression to the asexual stages. This approach should produce viable parasites since SUB2 expression in the asexual stages will be unaffected and will be only abolished in gametocytes and ookinetes, b) functional inhibition of SUB2 through stage specific expression of its inhibitory prodomain and c) functional inhibition of SUB2 through the stage specific expression of molecularly engineered serpins aiming to inhibit specifically SUB2.

B)  The role of bergepain 1 in the mosquito/parasite interactions will be investigated by the generation of a bergepain KO strain

C) Silencing proteases in gametocytes: A stimulating approach has been recently developed in Waters/Janse lab.  By providing a specific 3’ UTR region to an open reading frame, silencing of the corresponding gene can be achieved in female gametocytes. Application of this approach in the case of bergepain 1 and PbSUB2 may provide additional information as regards the functional role of these proteases in gametocyte development and fusion.

Although the PhD thesis will be based mainly on B and C projects, participation of the student in project A is not a priori excluded.

Project 16.  The role of C-type lectins in A. gambiae innate immunity

Supervisors: F. C. Kafatos, EMBL, Heidelberg, Germany; R. Sinden, Imperial College, London, UK

The development of Plasmodium ookinetes in the midguts of wildtype susceptible mosquitoes is regulated, at least in part, by a complex interplay of mosquito immune-related factors (agonists and antagonists). Parasite agonists include two C-type lectins (CTLs), CTL4 and CTLMA2, which protect the parasite from the potent vector immune reaction, melanization. The knockdown (KD) of these CTLs in A. gambiae susceptible mosquitoes induced massive melanization of P. berghei ookinetes egressing from the vector midgut epithelium. Preliminary studies suggest that both proteins are secreted into the mosquito hemolymph, potentially forming a protein complex that may involve other immune-related proteins belonging to the clip-domain serine protease homologue subfamily (CLIPA). The KD of specific CLIPA genes induced ookinete melanization, similar to what was observed in CTL4 and CTLMA2 KD mosquitoes.

The successful candidate will investigate the potential interactions between CTL4 and CTLMA2 and candidate CLIPA proteins in hemolymph extracts from wildtype adult mosquitoes. To investigate further the mode of action of these CTLs, the candidate will utilize the A. gambiae 20,000 EST microarray platform established in the Kafatos laboratory to compare the expression profiles of CTL4 and CTLMA2 KD mosquitoes to that of GFP KD control mosquitoes. Genes whose transcriptional profiles are strongly altered by the CTLs KD will be functionally analyzed by RNAi in adult mosquitoes to assay their role in ookinete melanization. The candidate will also assay the effect of CTL4 and CTLMA2 KD on the survival of P. falciparum ookinetes, in collaboration with Prof R.E. Sinden at Imperial College, London. The role of CTL4 in microbial defence will also be investigated. In the frame of this project, the candidate will perform experimental infections of A. gambiae with P. berghei and P. falciparum. The candidate will also receive training in gene KD by reverse genetics, transcriptomics and various molecular biology techniques and fluorescence microscopy.