1st year PhD student, University of Edinburgh.
Supervisors: Joanne Thompson and J.Alexandra Rowe
BSc.: University of Ghana, Legon
Internship: Noguchi Memorial Institute for Medical Research
Analysis of red cell binding proteins and their effect on rosetting
Background:
Interaction of the malaria parasite Plasmodium falciparum with human red blood cells (RBC) plays a crucial role in the biology of the parasite. Merozoites bind and invade human RBC to establish blood stage infection, a process involving many different merozoite proteins with RBC-binding capability. Other parasite proteins that bind RBC are expressed during the pigmented trophozoite and the schizont stages of asexual blood stage infection, including some members of the PfEMP1 variant antigen family, expressed on the surface of infected RBC. PfEMP1 variants that bind RBC bring about rosetting, the spontaneous binding of infected to uninfected RBC, a phenotype that is linked to the development of life-threatening malaria.
A further family of Plasmodium proteins that may meditate host-parasite interactions are the Plasmodium Cysteine Repeat Modular Proteins (PCRMPs). Two members of this family, PfCRMP1 and 2 co-localize with PfEMP1 within structures (the Maurer’s Clefts) that traffic exported parasite proteins to the surface of mature blood-stage parasites, and so may ultimately be exposed at the infected RBC surface. In addition, the NH2-terminal of PCRMP1 uniquely contains two motifs implicated in intercellular adhesion; a Kringle domain and a microneme adhesive repeat (MAR) domain. MAR domains found in micronemal proteins of the coccidian parasites Toxoplasma and Eimeria recognize sialic acid, present on the surface of many host cells, including RBCs. It is not currently known, however, whether interactions between the MAR domain of PCRMP1 and sialylated glycoconjugates on RBCs play a role in rosette formation.
Hypothesis and Aims
The aim of this project is to compare and contrast a known P. falciparum RBC binding protein (a PfEMP1 variant involved in rosetting) with a protein of unknown function that is hypothesised to have red cell binding properties based on bioinformatic analysis. The specific aims are:
- to express potential RBC binding domains as recombinant proteins in E. coli
- to raise antibodies (polyclonal and monoclonal) to putative RBC binding domains
- to develop RBC binding assays to examine function of putative RBC binding proteins
- to determine whether the MAR domain of PfCRMP1 and PvCRMP1 binds to RBCs
Methods:
- Molecular biology: DNA extraction, primer design, PCR, subcloning, sequencing
- Protein chemistry: recombinant protein expression and purification
- Immunology: antibody experiments eg. IFA/FACS
- Cell biology: parasite culture, adhesion assays
