Project

Back to overview

Sequence and expression analysis of Plasmodium falciparum var genes and immunological and functional analysis of PfEMP1 in naturally infected human blood samples

English title Sequence and expression analysis of Plasmodium falciparum var genes and immunological and functional analysis of PfEMP1 in naturally infected human blood samples
Applicant Beck Hans-Peter
Number 118456
Funding scheme Project funding (Div. I-III)
Research institution Swiss Tropical and Public Health Institute
Institution of higher education University of Basel - BS
Main discipline Molecular Biology
Start/End 01.10.2007 - 30.09.2010
Approved amount 335'000.00
Show all

All Disciplines (3)

Discipline
Molecular Biology
Immunology, Immunopathology
Cellular Biology, Cytology

Keywords (10)

MALARIA; PLASMODIUM FALCIPARUM; Microarray; PFEMP1; ANTIGENIC VARIATION; VAR GENES; TAR cloning; sequence analysis; trafficking; transfection

Lay Summary (English)

Lead
Lay summary
Adherence of infected red blood cells to blood capillaries are responsible for the majority of the observed pathology in malaria. This is mediated by a parasite protein on the surface of the infected cell, called PfEMP1. PfEMP1 is variable and encoded by a large number of genes in a given population. In this project we will study the diversity of expressed genes coding for PfEMP1 (var genes) to test our hypothesis that only a limited number of these genes are responsible for pathology. We will extent our studies to sequence larger fragments of the molecule which until now was elusive due to its size. Using a targeting cloning and sequencing strategy, we plan to specifically sequence only var genes but from a large number of patients.We will use this sequence information to select synthetic peptides using an bioinformatics approach to test their immunological recognition and binding capacity to selected molecules.Finally, we will study the fate of PfEMP1 from the parasite’s cytosol through the parasitophorous vacuole into the red blood cell membrane. In this subproject we are interested in potential interaction partners enabling the parasite to modify the host cell to such extend.We hope that the information gained during this project will identify potential virulence factors (selected PfEMP1 molecules) with the option to use these as vaccine candidates. Furthermore, by studying the transport and trafficking of this clinically important molecule through the cell we hope to identify structures and transporters which might become targets for interventions. Understanding the limits of diversity and understanding the molecular events which are necessary to transport PfEMP1 to the surface is of utmost importance. Because PfEMP1 is essential for parasite survival in vivo, understanding this process in detail may open completely new strategies for intervening with this devastating disease.
Direct link to Lay Summary Last update: 21.02.2013

Responsible applicant and co-applicants

Employees

Associated projects

Number Title Start Funding scheme
104043 Analysis of Plasmodium falciparum var genes: genomic distribution, regulation of gene expression and binding properties in naturally infected human blood samples 01.06.2004 Project funding (Div. I-III)
132709 Proteomic analysis and studies of interactions of exported proteins in Plasmodium falciparum 01.10.2010 Project funding (Div. I-III)

Abstract

Rationale and hypothesis: Cytoadherence of Plasmodium falciparum is a major contributor to the pathology of malaria. Adherence to endothelial receptors is mediated by a parasite derived protein termed PfEMP1 which shows antigenic variation and is encoded by var family comprising 50 to 60 genes per haploid genome. var genes are grouped into 3 major groups according to their up stream region which also coincides with chromosomal location. This genetic structuring lead to the question whether certain var genes are associated with different disease presentations. We have conducted longitudinal and case control studies isolating parasite mRNA to study expression of var genes in naturally occurring infections. In this project we like to carry our ongoing work further and address new open questions. We want: I) to generate data on the degree of variation in naturally occurring infections to test the hypothesis that the expressed var gene repertoire is limited and that identification of virulent var genes is feasible. This would help to understand how P. falciparum both escapes immunological attacks but still remain as chronic infection in the human host.II) to generate more sequence information 3’ of the var DBLa domain for further binding and immunological assays. Information on diversity in these parts of the molecule is crucial to understand functional and immunological properties of the whole PfEMP1 molecule. III) to generate a limited peptide and recombinant protein microarray based on amino acid sequences derived from I) and II) for immunological and functional studies. IV) to analyse PfEMP1 interaction partners responsible for transport of PfEMP1 through the erythrocyte cytosol. In particular to analyse the interaction between the two Maurer’s clefts histidine rich proteins MAHRP1 and MAHRP2.Approach: We will use various bioinformatic approaches to test whether groups of var genes expressed associate with clinical disease. We also will estimate the diversity of var genes in two geographically separated populations using species richness estimates.To generate a more comprehensive sequence data base on var genes, we will use either long range PCR and cloning, or transformation associated recombination (TAR) cloning to generate large sequences 3’ of the DBLa domain of var genes. Sequences obtained with this approach will be analysed as described under objective I). Deriving from these analyses, we will generate a microarray containing peptide features representing conserved, unstructured and coiled-coil domains for serological and functional binding assays. We will also generate a microarray using recombinantly produced PfEMP1 fragments also for functional and immunological assays. With this approach we hope to identify those var genes which confer solid binding to endothelial receptors. Finally, we will study protein-protein interaction between PfEMP1 and two Maurer’s clefts resident proteins (MAHRP1 and MAHRP2) in an attempt to study the translocation of PfEMP1 to the membrane of the infected erythrocyte. This is because we were able to show that knock down of PfEMP1 blocks transport from the parasite or the parasitophorous vacuole to the Maurer’s clefts and beyond. Using transgenic parasites, affinity purification and co-precipitation we expect to identify the interaction partners of the two MAHRP proteins and probably of PfEMP1. Since transport of PfEMP1 is crucial for parasite survival in vivo, understanding this process in detail may open completely new strategies for intervening with this devastating disease.Expected value: Through the combination of basic laboratory research with molecular-epidemiological studies, this project will significantly contribute to our understanding of var gene expression, diversity, and its association with disease severity and chronicity. The immunological and functional analyses will enable us to identify the most important ligands for endothelial adhesion and thus will provide the opportunity to develop new strategies against malaria (e.g. development of an anti-disease vaccine). Understanding the molecular events which are necessary to transport PfEMP1 to the surface is also of utmost importance. Because transport of PfEMP1 is essential for parasite survival in vivo, understanding this process in detail may open completely new strategies for intervening with this devastating disease.
-