Title: Fluorescence Reporter Proteins for Studying the Protein Trafficking of Plasmodium falciparum Infected Human Red Blood Cells

Georgeta Crivat1,2 , Juliana Martha Sa2, Fuyuki Tokumasu2 , Thomas Wellems2 , Jeeseong Hwang1

Affiliation: 1.Optical Technology Division (844), NIST Gaithersburg, MD 20899.

                      2. NIH/NIAID, Twinbrook III, Rockville, MD 20852

               Plasmodium falciparum (Pf), a protozoan parasite, is the cause of most deadly human malaria. In the intraerythrocytic phase of (Pf), the parasite exports proteins to the erythrocyte cytoplasm and to the plasma membrane of the erythrocyte. During this process, the plasma membrane of erythrocyte distorts due to knobby protrusions or “knobs” which are known to induce cytoadherence of Pfs infected erythrocytes to endothelial cell receptors in the wall of capillary blood vessels and subsequently avoid elimination by spleen. These “knobs”, contain knob-associated histidine-rich protein (KAHRP) that interacts with the cytoskeleton of the host erythrocyte and with Pf-produced erythrocyte membrane proteins (PfEMPs) and therefore they are involved in the pathogenesis of malaria disease [1]. Common strategies to visualize the details of the trafficking process involve the expression of green fluorescent protein (GFP) fused to trafficking proteins and measurements of an ensemble of proteins, such as fluorescence recovery after photo-bleaching (FRAP) technique. However, this technique is diffusion-limited and insufficient to reveal the fast dynamical process of trafficking involving proteins localized in specific cellular sites at different stages.   Furthermore, the exact mechanism of the transport may be obscured by the failure of delivering GFP- proteins to the target sites , due to the large size of GFP (25 to 27 kD) moieties and the perturbation of the conformation of the protein of interest [2]. To improve the study of protein trafficking in P. falciparum, we employed a previously described approach in other systems of protein tagging “antibody-free” engineering fusion proteins to express tetracysteine motifs (TC) [2]. These fusion proteins can be further labelled with red and green biarsenical fluorophores, FlAsH-EDT2 and ReAsH-EDT2, respectively, that are more photo-stable and less perturbing than GFP tags. They also provide excellent image contrast in electron microscopy without additional immuno-labeling. Here we report on our success in engineering KAHRP-GFP-TC (control) and KAHRP-TC constructs and describes details of experimental strategies involving gene cloning techniques. Results of our work can provide a better understanding of the interactions between KAHRP and PfEMPs essential to the investigation of malaria pathogenesis.

 [1] E. Knuepfer, M. Rug, N. Klonis, L.Tilley, A.F. Cowman, Blood, 105(10), 4078-4087

 [2]  S.R. Adams, R.E. Campbell, L.A. Gross, B.R. Martin, G.K.Walkup, Y.Yao, J. Llopis, R.Y. Tsien, J. Am.Chem.Soc. 124, 6063-6076 


Mentors Name: Jeeseong Hwang

Optical Technology Division (844), 100 Bureau Drive, Stop 8440

Gaithersburg, MD 20899-8440

 Tel: 301-975-4580

Fax: 301-869-5700

Email: jeeseong.hwang@nist.gov

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