Creative interior design by Plasmodium falciparum: Lipid metabolism and the parasite's secret chamber.

Malaria parasites conceal themselves within host erythrocytes and establish a necessary logistics system through the three-membrane layered structures of these cells. To establish this system, lipid metabolism is needed for the de novo synthesis of lipids and the recycling of extracellular lipids and erythrocyte lipid components. Cholesterol supply depends on its uptake from the extracellular environment and erythrocyte cytoplasm, but phospholipids can be synthesized on their own. This differential production of lipid species creates unique modifications in the lipid profile of parasitized erythrocytes, which in turn may influence the biophysical and/or mechanical properties of organelles and vesicles and communication among them. Variations in local membrane properties possibly influence the transportation of various molecules such as parasite-derived proteins, because efficiencies in secretion, vesicle fusion and budding are partly determined by the lipid profiles. Comprehensive understanding of the parasite's lipid metabolism and the biophysics of lipid membranes provides fundamental knowledge about these pathogenic organisms and could lead to new anti-malarials.

A prospective mechanism and source of cholesterol uptake by Plasmodium falciparum-infected erythrocytes co-cultured with HepG2 cells.

Plasmodium falciparum (P. falciparum) parasites still cause lethal infections worldwide, especially in Africa (https://www.who.int/publications/i/item/world-malaria-report-2019). During P. falciparum blood-stage infections in humans, low-density lipoprotein, high-density lipoprotein and cholesterol levels in the blood become low. Because P. falciparum lacks a de novo cholesterol synthesis pathway, it must import cholesterol from the surrounding environment. However, the origin of the cholesterol and how it is taken up by the parasite across the multiple membranes that surround it is not fully understood. To answer this, we used a cholesterol synthesis inhibiter (simvastatin), a cholesterol transport inhibitor (ezetimibe), and an activating ligand of the peroxisome proliferator-activated receptor α, called ciprofibrate, to investigate the effects of these agents on the intraerythrocytic growth of P. falciparum, both with and without HepG2 cells as the lipoprotein feeders. P. falciparum growth was inhibited in the presence of ezetimibe, but ezetimibe was not very effective at inhibiting P. falciparum growth when used in the co-culture system, unlike simvastatin, which strongly promoted parasite growth in this system. Ezetimibe is known to inhibit cholesterol absorption by blocking the activity of Niemann-Pick C1 like 1 (NPC1L1) protein, and simvastatin is known to enhance NPC1L1 expression in the human body's small intestine. Collectively, our results support the possibility that cholesterol import by P. falciparum involves hepatocytes, and cholesterol uptake into the parasite occurs via NPC1L1 protein or an NPC1L1 homolog during the erythrocytic stages of the P. falciparum lifecycle.

Real-time cholesterol sorting in Plasmodium falciparum-erythrocytes as revealed by 3D label-free imaging.

Cholesterol, a necessary component of animal cell membranes, is also needed by the lethal human malaria parasite Plasmodium falciparum. Because P. falciparum lacks a cholesterol synthesis pathway and malaria patients have low blood cholesterol, we speculated that it scavenges cholesterol from them in some way. We used time-lapse holotomographic microscopy to observe cholesterol transport in live P. falciparum parasites and structurally investigate erythrocyte membranes, both during and after P. falciparum invasion of human erythrocytes. After P. falciparum initially acquired free cholesterol or inner erythrocytic membrane-derived cholesterol, we observed budding lipid membranes elongating into the cytosol and/or membrane segments migrating there and eventually fusing with the parasite membranes, presumably at the parasitophorous vacuole membrane (PVM). Finally, the cholesterol-containing segments were seen to surround the parasite nucleus. Our imaging data suggest that a novel membrane transport system operates in the cytosol of P. falciparum-infected erythrocytes as a cholesterol import system, likely between the PVM and the erythrocyte membrane, and that this transportation process occurs during the live erythrocyte stages of P. falciparum.

Upper gastrointestinal pathophysiology due to mouse malaria Plasmodium berghei ANKA infection.

BACKGROUND: Epigastric pain, vomiting, and other gastrointestinal problems are among the most important symptoms of malaria infection as they suggest the possibility that the condition is serious. Pathophysiologies such as gastric mucosal changes and delayed gastric emptying have been reported in serious cases of malaria infection. However, it is unclear whether or not pathophysiological involvement of the upper gastrointestinal tract occurs in Plasmodium berghei ANKA (PbA)-infected mice. METHODS: PbA-infective Anopheles mosquitoes were used to infect mice via the natural route of infection. Fifteen PbA-C57BL/6 mice were used as a cerebral malaria model and the same numbers of PbA-BALB/c mice were used as a cerebral malaria-resistant model, and then we investigated the pathophysiological involvement of the stomach and small intestine. RESULTS: On day 8 post infection, six PbA-C57BL/6 mice showed cerebral malaria and nine others had uncomplicated infection. All the PbA-C57BL/6 mice on that same day showed severe weight loss with multiple, red gastric patches and changes to the course of the small intestine with villus goblet cell enlargement. In addition, cerebral malaria cases showed gastric gas retention with submucosal edema and small intestinal shortening. In PbA-BALB/c mice, overextension of the stomach and gas retention were evident from week 2 after PbA infection, as well as changes to the course of the small intestine and mesenteric thinning with fragility. CONCLUSIONS: We described the upper gastrointestinal pathophysiology representing new findings directly linked to malarial severity and subsequent death in PbA-infected mice as a mouse model of malaria infection.

The localization of α-synuclein in the process of differentiation of human erythroid cells.

Although the neuronal protein α-synuclein (α-syn) is thought to play a central role in the pathogenesis of Parkinson's disease (PD), its physiological function remains unknown. It is known that α-syn is also abundantly expressed in erythrocytes. However, its role in erythrocytes is also unknown. In the present study, we investigated the localization of α-syn in human erythroblasts and erythrocytes. Protein expression of α-syn increased during terminal differentiation of erythroblasts (from day 7 to day 13), whereas its mRNA level peaked at day 11. α-syn was detected in the nucleus, and was also seen in the cytoplasm and at the plasma membrane after day 11. In erythroblasts undergoing nucleus extrusion (day 13), α-syn was detected at the periphery of the nucleus. Interestingly, we found that recombinant α-syn binds to trypsinized inside-out vesicles of erythrocytes and phosphatidylserine (PS) liposomes. The dissociation constants for binding to PS/phosphatidylcholine (PC) liposomes of N-terminally acetylated (NAc) α-syn was lower than that of non NAc α-syn. This suggests that N-terminal acetylation plays a significant functional role. The results of the present study collectively suggest that α-syn is involved in the enucleation of erythroblasts and the stabilization of erythroid membranes.

Detailed methodology for high resolution scanning electron microscopy (SEM) of murine malaria parasitized-erythrocytes.

Scanning electron microscopy (SEM) is a powerful tool used to investigate object surfaces and has been widely applied in both material science and biology. With respect to the study of malaria, SEM revealed that erythrocytes infected with Plasmodium falciparum, a human parasite, display 'knob-like' structures on their surface comprising parasitized proteins. However, detailed methodology for SEM studies of malaria parasites is lacking in the literature making such studies challenging. Here, we provide a step-by-step guide to preparing Plasmodium-infected erythrocytes from two mouse strains for SEM analysis with minimal structural deterioration. We tested three species of murine malaria parasites, P. berghei, P. yoelii, and P. chabaudi, as well as non-parasitized human erythrocytes and P. falciparum-infected erythrocytes for comparisons. Our data demonstrated that the surface structures of parasitized erythrocytes between the three species of murine parasites in the two different strains of mice were indistinguishable and no surface alterations were observed in P. falciparum-erythrocytes. Our SEM observations contribute towards an understanding of the molecular mechanisms of parasite maturation in the erythrocyte cytoplasm and, along with future studies using our detailed methodology, may help to gain insight into the clinical phenomena of human malaria.

Physicochemical Aspects of the Plasmodium chabaudi-Infected Erythrocyte.

Membrane electrochemical potential is a feature of the molecular profile of the cell membrane and the two-dimensional arrangement of its charge-bearing molecules. Plasmodium species, the causative agents of malaria, are intracellular parasites that remodel host erythrocytes by expressing their own proteins on erythrocyte membranes. Although various aspects of the modifications made to the host erythrocyte membrane have been extensively studied in some human Plasmodium species (such as Plasmodium falciparum), details of the structural and molecular biological modifications made to host erythrocytes by nonhuman Plasmodium parasites have not been studied. We employed zeta potential analysis of erythrocytes parasitized by P. chabaudi, a nonhuman Plasmodium parasite. From these measurements, we found that the surface potential shift was more negative for P. chabaudi-infected erythrocytes than for P. falciparum-infected erythrocytes. However, electron microscopic analysis of the surface of P. chabaudi-infected erythrocytes did not reveal any modifications as compared with nonparasitized erythrocytes. These results suggest that differences in the membrane modifications found herein represent unique attributes related to the pathogenesis profiles of the two different malaria parasite species in different host animals and that these features have been acquired through parasite adaptations acquired over long evolutionary time periods.

Imaging of the subsurface structures of "unroofed" Plasmodium falciparum-infected erythrocytes.

Intraerythrocytic stages of Plasmodium falciparum parasites modify the membranes of their host erythrocytes with numerous expressed proteins. They also install new membranous structures in the erythrocyte cytoplasm, including Maurer's clefts (MC) and a tubulovesicular network. These structures support molecular trafficking processes that are necessary for the growth and multiplication of P. falciparum intraerythrocytic stages. To study the morphology and organization of these modifications, we prepared samples of P. falciparum-infected erythrocytes by 'unroofing' techniques and examined them by transmission electron microscopy. Images of the 'unroofed' parasitized erythrocytes feature cytoskeleton alterations and the presence of new membranous structures generated by P. falciparum, including small vesicles and MC connected by extensions to the inner erythrocyte membrane. Non-parasitized erythrocytes showed no evidence of these structures or extensions. In further experiments, we observed a relative absence of MC and extensions after treatment of parasitized erythrocytes with aluminum tetrafluoride (AlF4(-)), an inhibitor of vesicle trafficking. The morphology and physical location of MC, extensions and small vesicles in unroofed specimens are consistent with the role of these structures in the trafficking of P. falciparum proteins to the surface of parasitized erythrocytes.

The effect of hydrophilic ionic liquids 1-ethyl-3-methylimidazolium lactate and choline lactate on lipid vesicle fusion.

Ionic liquids (ILs) are room-temperature molten salts that have applications in both physical sciences and more recently in the purification of proteins and lipids, gene transfection and sample preparation for electron microscopy (EM) studies. Transfection of genes into cells requires membrane fusion between the cell membrane and the transfection reagent, thus, ILs may be induce a membrane fusion event. To clarify the behavior of ILs with cell membranes the effect of ILs on model membranes, i.e., liposomes, were investigated. We used two standard ILs, 1-ethyl-3-methylimidazolium lactate ([EMI][Lac]) and choline lactate ([Ch][Lac]), and focused on whether these ILs can induce lipid vesicle fusion. Fluorescence resonance energy transfer and dynamic light scattering were employed to determine whether the ILs induced vesicle fusion. Vesicle solutions at low IL concentrations showed negligible fusion when compared with the controls in the absence of ILs. At concentrations of 30% (v/v), both types of ILs induced vesicle fusion up to 1.3 and 1.6 times the fluorescence intensity of the control in the presence of [Ch][Lac] and [EMI][Lac], respectively. This is the first demonstration that [EMI][Lac] and [Ch][Lac] induce vesicle fusion at high IL concentrations and this observation should have a significant influence on basic biophysical studies. Conversely, the ability to avoid vesicle fusion at low IL concentrations is clearly advantageous for EM studies of lipid samples and cells. This new information describing IL-lipid membrane interactions should impact EM observations examining cell morphology.

Comparison of protein behavior between wild-type and G601S hERG in living cells by fluorescence correlation spectroscopy.

The human ether-a-go-go-related gene (hERG) protein is a cardiac potassium channel. Mutations in hERG can result in reductions in membrane channel current, cardiac repolarization, prolongation of QT intervals, and lethal arrhythmia. In the last decade, it has been found that some mutants of hERG involved in long QT syndrome exhibit intracellular protein trafficking defects, while other mutants sort to the membrane but cannot form functional channels. Due to the close relationship between intracellular trafficking and functional protein expression, we aimed to measure differences in protein behavior/motion between wild-type and mutant hERG by directly analyzing the fluorescence fluctuations of green fluorescent protein-labeled proteins using fluorescence correlation spectroscopy (FCS). Our data imply that FCS can be applied as a new diagnostic tool to assess whether the defect in a particular mutant channel protein involves aberrant intracellular trafficking.