Abstract
Plasmodium falciparum (Pf) malaria parasites remodel host erythrocytes by placing membranous structures in the host cell cytoplasm and inserting proteins into the surrounding erythrocyte membranes. Dynamic imaging techniques with high spatial and temporal resolutions are required to study the trafficking pathways of proteins and the time courses of their delivery to the host erythrocyte membrane.Methodology and FindingsUsing a tetracysteine (TC) motif tag and TC-binding biarsenical fluorophores (BAFs) including fluorescein arsenical hairpin (FlAsH) and resorufin arsenical hairpin (ReAsH), we detected knob-associated histidine-rich protein (KAHRP) constructs in Pf-parasitized erythrocytes and compared their fluorescence signals to those of GFP (green fluorescent protein)-tagged KAHRP. Rigorous treatment with BAL (2, 3 dimercaptopropanol; British anti-Lewisite) was required to reduce high background due to nonspecific BAF interactions with endogenous cysteine-rich proteins. After this background reduction, similar patterns of fluorescence were obtained from the TC- and GFP-tagged proteins. The fluorescence from FlAsH and ReAsH-labeled protein bleached at faster rates than the fluorescence from GFP-labeled protein.ConclusionWhile TC/BAF labeling to Pf-infected erythrocytes is presently limited by high background signals, it may offer a useful complement or alternative to GFP labeling methods. Our observations are in agreement with the currently-accepted model of KAHRP movement through the cytoplasm, including transient association of KAHRP with Maurer's clefts before its incorporation into knobs in the host erythrocyte membrane.
Highlights
Upon invading erythrocytes, Plasmodium falciparum (Pf) parasites extensively remodel their host cells [1]
While TC/biarsenical fluorophores (BAFs) labeling to Pf-infected erythrocytes is presently limited by high background signals, it may offer a useful complement or alternative to GFP labeling methods
Our observations are in agreement with the currently-accepted model of knob-associated histidine-rich protein (KAHRP) movement through the cytoplasm, including transient association of KAHRP with Maurer’s clefts before its incorporation into knobs in the host erythrocyte membrane
Summary
Plasmodium falciparum (Pf) parasites extensively remodel their host cells [1]. Some Pf-produced proteins are localized in electron-dense protrusions (‘‘knobs’’) at the parasitized erythrocyte (PE) surface [2,3]. These proteins provide points of cytoadherence to endothelium of microvenules, enabling the parasitized erythrocytes to sequester and avoid elimination by the spleen [4,5,6,7,8,9]. KAHRP serves as an essential structural element of knobs, binds to the membrane skeleton of the host erythrocyte, and helps to anchor the acid terminal segment (ATS) of PfEMP1 [10,11], which is responsible for cytoadherence through antigenically variant regions that recognize receptors such as CD36, TSP (thrombospondin), and ICAM-1 (Inter-Cellular Adhesion Molecule 1) [12,13,14]. A current model of intraerythrocytic protein delivery to knobs invokes parasite export of proteins across the PVM to the erythrocyte cytoplasm, transient association with Maurer’s clefts and, docking at the erythrocyte membrane [15,16]
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