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A method for visualizing surface-exposed and internal PfEMP1 adhesion antigens in Plasmodium falciparum infected
Dominique Bengtsson1, Kordai M Sowa, Ali Salanti
1Centre for Medical Parasitology, Department of International Health, Immunology & Microbiology, Faculty of Health Sciences, University of Copenhagen, Copenhagen K, Denmark. dominique@cmp.dk
Background:
The insertion of parasite antigens into the host erythrocyte membrane and the structure and distribution of Plasmodium falciparum adhesion receptors on that membrane are poorly understood. Laser scanning confocal microscopy (LSCM) and a novel labelling and fixation method have been used to obtain high resolution immuno-fluorescent images of erythrocyte surface PfEMP1 and internal antigens which allow analysis of the accumulation of PfEMP1 on the erythrocyte membrane during asexual development.
Methods:
A novel staining technique has been developed which permits distinction between erythrocyte surface PfEMP1 and intracellular PfEMP1, in parasites whose nuclear material is exceptionally well resolved. Primary antibody detection by fluorescence is carried out on the live parasitized erythrocyte. The surface labelled cells are then fixed using paraformaldehyde and permeabilized with a non-ionic detergent to permit access of antibodies to internal parasite antigens. Differentiation between surface and internal antigens is achieved using antibodies labelled with different fluorochromes and confocal microscopy
Results:
Surface exposed PfEMP1 is first detectable by antibodies at the trophozoite stage of intracellular parasite development although the improved detection method indicates that there are differences between different laboratory isolates in the kinetics of accumulation of surface-exposed PfEMP1.
Conclusion:
A sensitive method for labelling surface and internal PfEMP1 with up to three different fluorochromes has been developed for laser scanning confocal optical microscopy and the analysis of the developmental expression of malaria adhesion antigens.
Insights
A new method visualizes Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) on infected red blood cells. This technique reveals how PfEMP1 accumulates during parasite development, aiding malaria research.
Area of Science:
- Malariology
- Cell Biology
- Immunology
Background:
- The precise mechanisms of parasite antigen insertion into host erythrocytes and the structure of Plasmodium falciparum adhesion receptors remain unclear.
- Understanding these processes is crucial for developing effective malaria interventions.
Purpose of the Study:
- To develop and validate a high-resolution imaging technique for analyzing erythrocyte surface and internal antigens.
- To investigate the accumulation of PfEMP1 on the erythrocyte membrane during asexual parasite development.
Main Methods:
- A novel staining and fixation method was developed for live, infected erythrocytes.
- Laser scanning confocal microscopy (LSCM) combined with differential fluorochrome labeling was used.
- Paraformaldehyde fixation and detergent permeabilization allowed simultaneous detection of surface and internal antigens.
Main Results:
- The method successfully distinguished between surface-exposed and intracellular PfEMP1 with high resolution.
- Surface-exposed PfEMP1 was detectable from the trophozoite stage onwards.
- Variations in the kinetics of PfEMP1 accumulation were observed among different laboratory isolates.
Conclusions:
- A sensitive, multi-fluorochrome labeling method using LSCM was established for analyzing malaria adhesion antigens.
- This technique provides valuable insights into the developmental expression of PfEMP1 and its role in malaria pathogenesis.
