Project
Descriptions
International
PhD Programme on Biology and Pathology of the Malaria
Parasite (July 8th - August 31st, 2004)
BioMalPar is a collaborative
research Network of Excellence, funded by the European
Commission under the sixth frame
work programme and coordinated by the Pasteur Institute.
It is currently engaging 44 Principal Investigators
from 20 institutions in Europe and Africa [17 European
and 3 African institutions]. It conducts research
on malaria parasites and their interactions with
both
mammalian hosts and mosquito vectors. It also aims
to train talented young scientists from both North
and South in malaria research, with emphasis on
interdisciplinary approaches.
Training is conducted through the BioMalPar International
PhD Programme, which is organized and operated
in collaboration with the European Molecular
Biology Laboratory (EMBL,
also a partner of BioMalPar) in Heidelberg. It
is independent of but modelled on the successful
EMBL
International PhD Programme.
Applications are invited for studentships linked
to collaborative research projects, each supervised
by
two or more BioMalPar investigators, as listed
below:
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 and posttranscriptional analysis
of the biology of the P.berghei parasites
in the mosquito midgut using TEP-1 transgenic mosquito
lines
Supervisors:
E.
Levashina, IBMC-CNRS , Strasbourg,
France
A.
P. Waters, Leiden,
Netherlands
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, TEP!, 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 parasites
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. The proposed project
will extend this characterization
to the simultaneous analysis of mosquito and P.berghei proteomes.
Several important questions will be addressed. (i) Identification of genes
that cooperate with
TEP1 in parasite killing in the mosquito. Genes and proteins representing
differential patterns (assessed by microarrays and proteomic analysis) in
tow 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 microarrays and 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 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).
Applications are to
be submitted online
and are due by 31 August 2004.
Shortlisted candidates will be invited to EMBL in 26-27
October 2004, for interviews with the BioMalPar Admissions
Committee.
Offers of admission to the BioMalPar International
PhD programme will be made on the last day of interviews.
Students can start depending on availability
as early as November
2004 but no later than 19 February 2005. Admitted candidates
will be expected to arrive in Heidelberg, Germany on
19/20 February 2005 for the BioMalPar Core PhD Course
organized
by Prof. Michael Lanzer (University of Heidelberg).
The
course will include the BioMalPar Annual meeting on
3-4 March 2005 and will end on 12
March 2005. Subsequent
workshops and practical courses will provide additional
training
opportunities.
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