Characterisation of complexes formed by parasite proteins exported into the host cell compartment of Plasmodium falciparum infected red blood cells.

During its intraerythrocytic life cycle, the human malaria parasite Plasmodium falciparum supplements its nutritional requirements by scavenging substrates from the plasma through the new permeability pathways (NPPs) installed in the red blood cell (RBC) membrane. Parasite proteins of the RhopH complex: CLAG3, RhopH2, RhopH3, have been implicated in NPP activity. Here, we studied 13 exported proteins previously hypothesised to interact with RhopH2, to study their potential contribution to the function of NPPs. NPP activity assays revealed that the 13 proteins do not appear to be individually important for NPP function, as conditional knockdown of these proteins had no effect on sorbitol uptake. Intriguingly, reciprocal immunoprecipitation assays showed that five of the 13 proteins interact with all members of the RhopH complex, with PF3D7_1401200 showing the strongest association. Mass spectrometry-based proteomics further identified new protein complexes; a cytoskeletal complex and a Maurer's clefts/J-dot complex, which overall helps clarify protein-protein interactions within the infected RBC (iRBC) and is suggestive of the potential trafficking route of the RhopH complex itself to the RBC membrane.

Fluorescent Nitrogen-Doped Carbon Dots for Label Live Elder Blood-Stage Plasmodium falciparum through New Permeability Pathways.

To verify the size and emergence time of new permeability pathways (NPPs) in malaria parasites, the permeability of the Plasmodium falciparum-infected erythrocytes was tested with different particle sizes of nanomaterials by flow cytometry assay. The results confirmed the permeability of the host cell membrane increases with parasite maturation for the stage-development evolution of NPPs, and especially found that a particle size of about 50 nm had higher efficiency. As a kind of the novel nanomaterials, nitrogen-doped carbon dots (NCDs) showed no toxicity, specificity binding ability to the malaria parasites, and could label live elder blood-stage P. falciparum through NPPs, indicating the potential application in cell imaging. NPPs and some nanomaterials such as NCDs deserve more attention and exploration for the elimination and prevention of malaria.

Choline and PEG dually modified artemether nano delivery system targeting intra-erythrocytic Plasmodium and its pharmacodynamics in vivo.

OBJECTIVE: The choline derivative (CD) and polyethylene-glycol (PEG) dually modified artemether (ARM) nanostructured lipid carriers (CD-PEG-ARM-NLC) have been designed to prolong the circulation of ARM in blood, as well as to develop targeting for new permeability pathways (NPPs) and erythrocyte choline carriers (ECCs) that are expressed on the Plasmodium-infected erythrocyte membrane. SIGNIFICANCE: The CD-PEG-ARM-NLC constructed in this study was found to be able to target endoerythrocytic Plasmodium by increasing the drug concentration and residence time in the infected erythrocytic microenvironment and minimizing toxicity and side effects. METHODS: CD-PEG-ARM-NLC was prepared using high-pressure homogenization followed by physicochemical characterization. The targeting ability of CD-PEG-NLC to infected erythrocytes probed by coumarin-6 was investigated by using fluorescence microscopy imaging. The SYBR Green I assay for parasite nucleic acid was adapted in order to assess the efficacy of inhibition against parasite growth in vitro. The antimalarial activity of ARM-loaded NLCs was evaluated by a Pearson four-day suppressive test in Pyy265BY-bearing mice. RESULTS: In vitro imaging indicated that the intracellular delivery of CD-PEG-ARM-NLC was efficiently taken up by the infected erythrocytes via ECCs and NPPs, which could be inhibited by addition of furosemide (an inhibitor of NPPs) and excessive choline (native substrate of ECCs). Moreover, in vitro and in vivo studies that evaluated antimalarial activity suggested that CD-PEG-ARM-NLC exhibited higher antimalarial activity in comparison to ARM-NLC and PEG-ARM-NLC. CONCLUSION: These findings suggested that choline and PEG dually modified NLC could be promising preparations for the production of hydrophobic antimalarial drugs, particularly for ARM.

Novel insights into red blood cell physiology using parasites as tools.

The mammalian red blood cell is a terminally differentiated cell that lacks a genetic programme and that has only a very limited metabolic capacity. Nonetheless, it serves as habitat for two parasites belonging to the monophyletic group of Apicomplexa, namely Plasmodium and Babesia. Studies of the parasitized red blood cell have revealed several properties that are unknown in the non-infected cell and that are difficult to conceptualize based on our view of red blood cell function. Here we review the current knowledge on host cell invasion and nutrient acquisition by these parasites. We attempt to dissect the factors that are directly contributed by the parasites from those that exist but have remained undetected in the non-infected cell.

Effect of Solanum nudum steroids on uninfected and Plasmodium falciparum-infected erythrocytes

Steroids from Solanum nudum (SNs) have demonstrated antiplasmodial activity against erythrocytic stages of the Plasmodium falciparum strain FCB-2. It is well known that steroids can alter the membrane function of erythrocytes. Thus, we assessed alterations in the membranes of uninfected red blood cells, the parasite invasiveness and the solute-induced lysis of parasitised red blood cells (pRBCs). induced by SNs. We found that most merozoites were unable to invade SN-treated erythrocytes. However, transmission electron microscopy revealed no effect on the morphology of uninfected erythrocytes treated with either SN2 or diosgenone and neither SN induced haemolysis of uninfected erythrocytes. SN2 and SN4 inhibited isosmotic sorbitol and alanine-induced haemolysis of pRBCs. In contrast, diosgenone and SN1 did not inhibit solute-induced haemolysis. The inhibition of solute-induced lysis of parasitised erythrocytes by SN2 and SN4 suggest an action of these SNs on new permeability pathways of pRBCs.