Human red blood cell polymorphisms and malaria

Genetic factors are a major determinant of child survival in malaria endemic countries. Identifying which genes are involved and how they affect the malaria disease risk potentially offers a powerful mechanism through which to learn more about the host-parasite relationship. The past few years have seen significant progress towards achieving this goal for some of the best-known malaria resistance genes that determine the structure or function of red blood cells: Gerbich blood group antigen negativity; polymorphisms of the complement receptor genes (most notably CR1); Southeast Asian ovalocytosis; pyruvate kinase deficiency; haemoglobin E; the sickle cell trait; and alpha-thalassaemia are all examples. The challenge for the future must be to translate such advances into fresh approaches to the prevention and treatment of malaria.     

Transport mechanisms at the malaria parasite-host cell interface

Obligate intracellular malaria parasites reside within a vacuolar compartment generated during invasion which is the principal interface between pathogen and host. To subvert their host cell and support their metabolism, these parasites coordinate a range of transport activities at this membrane interface that are critically important to parasite survival and virulence, including nutrient import, waste efflux, effector protein export, and uptake of host cell cytosol. Here, we review our current understanding of the transport mechanisms acting at the malaria parasite vacuole during the blood and liver-stages of development with a particular focus on recent advances in our understanding of effector protein translocation into the host cell by the Plasmodium Translocon of EXported proteins (PTEX) and small molecule transport by the PTEX membrane-spanning pore EXP2. Comparison to Toxoplasma gondii and other related apicomplexans is provided to highlight how similar and divergent mechanisms are employed to fulfill analogous transport activities.     

Home improvements: malaria and the red blood cell

In real-estate agent's terms, the red blood cell is a renovator's dream. The mature human erythrocyte has no internal organelles, no protein synthesis machinery and no infrastructure for protein trafficking. The malaria parasite invades this empty shell and effectively converts the erythrocyte back into a fully functional eukaryotic cell. In this article, Michael Foley and Leann Tilley examine the Plasmodium falciparum proteins that interact with the membrane skeleton at different stages of the infection and speculate on the roles of these proteins in the remodelling process.     

Abnormal blood flow and red blood cell deformability in severe malaria

Obstruction of the microcirculation plays a central role in the pathophysiology of severe malaria. Here, Arjen Dondorp and colleagues describe the various contributors to impaired microcirculatory flow in falciparum malaria: sequestration, rosetting and recent findings regarding impaired red blood cell deformability. The correlation with clinical findings and possible therapeutic consequences are discussed.     

Multiscale modeling of red blood cell mechanics and blood flow in malaria

Red blood cells (RBCs) infected by a Plasmodium parasite in malaria may lose their membrane deformability with a relative membrane stiffening more than ten-fold in comparison with healthy RBCs leading to potential capillary occlusions. Moreover, infected RBCs are able to adhere to other healthy and parasitized cells and to the vascular endothelium resulting in a substantial disruption of normal blood circulation. In the present work, we simulate infected RBCs in malaria using a multiscale RBC model based on the dissipative particle dynamics method, coupling scales at the sub-cellular level with scales at the vessel size. Our objective is to conduct a full validation of the RBC model with a diverse set of experimental data, including temperature dependence, and to identify the limitations of this purely mechanistic model. The simulated elastic deformations of parasitized RBCs match those obtained in optical-tweezers experiments for different stages of intra-erythrocytic parasite development. The rheological properties of RBCs in malaria are compared with those obtained by optical magnetic twisting cytometry and by monitoring membrane fluctuations at room, physiological, and febrile temperatures. We also study the dynamics of infected RBCs in Poiseuille flow in comparison with healthy cells and present validated bulk viscosity predictions of malaria-infected blood for a wide range of parasitemia levels (percentage of infected RBCs with respect to the total number of cells in a unit volume).     

Hemodynamic effects of red blood cell aggregation

The influence of red blood cell (RBC) aggregation on blood flow in vivo has been under debate since early 1900's, yet a full understanding has still has not been reached. Enhanced RBC aggregation is well known to increase blood viscosity measured in rotational viscometers. However, it has been demonstrated that RBC aggregation may decrease flow resistance in cylindrical tubes, due to the formation of a cell-poor zone near the tube wall which results from the enhanced central accumulation of RBC. There is also extensive discussion regarding the effects of RBC aggregation on in vivo blood flow resistance. Several groups have reported increased microcirculatory flow resistance with enhanced RBC aggregation in experiments that utilized intravital microscopy. Alternatively, whole organ studies revealed that flow resistance may be significantly decreased if RBC aggregation is enhanced. Recently, new techniques have been developed to achieve well-controlled, graded alterations in RBC aggregation without influencing suspending phase properties. Studies using this technique revealed that the effects of RBC aggregation are determined by the degree of aggregation changes, and that this relationship can be explained by different hemodynamic mechanisms.     

The role of malaria merozoite proteases in red blood cell invasion

Invasion of red blood cells by the malaria merozoite is an essential step in the life cycle of this obligate intracellular pathogen. The molecular details of invasion are only recently becoming understood, largely through studies in related apicomplexan parasites such as Toxoplasma. Protease activity is required for successful invasion to disengage interactions between parasite adhesins and host cell receptors. Shedding of at least two essential surface proteins from the merozoite is thought to occur continuously during invasion as the parasite moves into the nascent parasitophorous vacuole. This shedding is performed by way of juxtamembrane cleavage and is mediated by a sheddase, which probably belongs to the subtilisin-like superfamily. Recent revelations have shown that transmembrane adhesins that are secreted onto the Toxoplasma tachyzoite surface and capped to its posterior pole are shed by way of cleavage within their transmembrane domains. A family of intramembrane serine proteases called rhomboids have now been identified within Apicomplexa, and one Toxoplasma rhomboid has been localized to the posterior end of the parasite. This supports their role in capping proteolysis. Proteases involved in invasion constitute potential targets for the development of new protease inhibitor-based drugs.     

Pulmonary vascular effects of red blood cells containing S-nitrosated hemoglobin

The role of S-nitrosated hemoglobin (SNO-Hb) in the regulation of blood flow is a central and controversial question in cardiopulmonary physiology. In the present study, we investigate whether intact human red blood cells (RBCs) synthesized to contain high SNO-Hb levels are able to export nitric oxide bioactivity and vasodilate the pulmonary circulation, and whether SNO-Hb dependent vasodilation occurs secondary to an intrinsic oxygen-linked, allosteric function of Hb. RBCs containing supraphysiological concentrations (100-1,000x normal) of SNO-Hb (SNO-RBCs) were synthesized and added to isolated, perfused rat lungs during anoxic or normoxic ventilation, and during normoxic ventilation with pulmonary hypertension induced by the thromboxane mimetic U-46619. SNO-RBCs produced dose-dependent pulmonary vasodilation compared with control RBCs during conditions of both normoxic (U-46619) and hypoxic pulmonary vasoconstriction. These effects were associated with a simultaneous, rapid, and temperature-dependent loss of SNO from Hb. Both vasodilatory effects and the rate of SNO-Hb degradation were independent of oxygen tension and Hb oxygen saturation. Furthermore, these effects were not affected by inhibition of the RBC membrane band 3 protein (anion exchanger-1), a putative membrane facilitator of NO export from RBCs. Whereas these data support observations by multiple groups that synthesized SNO-Hb can vasodilate, this effect is not under intrinsic oxygen-dependent allosteric control, nor likely to be relevant in the pulmonary circulation at normal physiological concentrations.     

A multifunctional serine protease primes the malaria parasite for red blood cell invasion

The malaria parasite Plasmodium falciparum replicates within an intraerythrocytic parasitophorous vacuole (PV). Rupture of the host cell allows release (egress) of daughter merozoites, which invade fresh erythrocytes. We previously showed that a subtilisin-like protease called PfSUB1 regulates egress by being discharged into the PV in the final stages of merozoite development to proteolytically modify the SERA family of papain-like proteins. Here, we report that PfSUB1 has a further role in ‘priming' the merozoite prior to invasion. The major protein complex on the merozoite surface comprises three proteins called merozoite surface protein 1 (MSP1), MSP6 and MSP7. We show that just before egress, all undergo proteolytic maturation by PfSUB1. Inhibition of PfSUB1 activity results in the accumulation of unprocessed MSPs on the merozoite surface, and erythrocyte invasion is significantly reduced. We propose that PfSUB1 is a multifunctional processing protease with an essential role in both egress of the malaria merozoite and remodelling of its surface in preparation for erythrocyte invasion.     

Rapid isolation of single malaria parasite-infected red blood cells by cell sorting

Malaria research often requires isolation of individually infected red blood cells (RBCs) or of a homogenous parasite population derived from a single parasite (clone). Traditionally, isolation of individual, parasitized RBCs or parasite cloning is achieved by limiting dilution or micromanipulation. This protocol describes a method for more efficient cloning of the malaria parasite; the method uses a cell sorter to rapidly isolate Plasmodium falciparum-infected RBCs singly. By gating the parameters of forward-angle light scatter and side-angle light scatter in a cell sorter, singly infected RBCs can be isolated and automatically deposited into a 96-well culture plate within 1 min. Including a Percoll purification step; the entire procedure to seed a 96-well plate with singly infected RBCs can take <40 min. This highly efficient single-cell sorting protocol should be useful for cloning of both laboratory parasite populations from genetic manipulation experiments and clinical samples.     

Analysis of short RNAs in the malaria parasite and its red blood cell host

RNA interference (RNAi) is an RNA degradation process that involves short, double-stranded RNAs (dsRNA) as sequence specificity factors. The natural function of the RNAi machinery is to generate endogenous short double-stranded RNAs to regulate gene expression. It has been shown that treatment of Plasmodium falciparum, the etiologic agent of malaria, with dsRNA induces degradation of the corresponding microRNA (miRNA), yet typical RNAi-associated genes have not been identifiable in the parasite genome. To clarify this discrepancy we set out to clone short RNAs from P. falciparum-infected red blood cells and from purified parasites. We did not find any short RNA that was not a rRNA or tRNA fragment. Indeed, only known human miRNAs were isolated in parasite preparations indicating that very few if any short RNAs exist in P. falciparum. This suggests a different mechanism than classical RNAi in observations of dsRNA-mediated degradation. Of the human miRNAs identified, the human miRNA mir-451 accumulates at a very high level in both infected and healthy red blood cells. Interestingly, mir-451 was not detectable in a series of immortalised cell lines representing progenitor stages of all major blood lineages, suggesting that mir-451 may play a role in the differentiation of erythroid cells.     

Classification and localization of hemoglobin binding sites on the red blood cell membrane.

The binding of hemoglobin to the red cell membrane was characterized over a wide range of free hemoglobin concentrations by measurement of membrane bound and supernatant hemoglobin. Scatchard analysis of the binding data revealed two classes of sites: high affinity sites with a binding constant of 1 X 10(8) M-1 and 1.2 X 10(6) sites per cell, and a second, low affinity class of sites with a binding constant of 6 X 10(6)M-1 and 6 X 10(6) sites per cell. The low affinity sites are shown to be nonspecific and appear to be a result of the ghost preparation. The high affinity sites are shown to be specific to the inner surface of the red cell membrane. The competition of hemoglobin and glyceraldehyde-3-phosphate dehydrogenase suggests band III proteins as a potential binding site for hemoglobin.     

Plasmodium yoelii: effects of red blood cell modification and antibodies on the binding characteristics of the 235-kDa rhoptry protein

The 235-kDa rhoptry protein of the rodent malaria parasite Plasmodium yoelii yoelii was shown to bind to the surface of mouse red blood cells in a calcium-independent process, using a erythrocyte-binding assay. This binding is affected by modification of the surface of the red blood cells by enzymatic treatment. Chymotrypsin and trypsin but not neuraminidase treatment of the erythrocytes significantly reduced the binding of the 235-kDa proteins. The binding of an unrelated 135-kDa protein was abolished by treatment with chymotrypsin. Although the 235-kDa proteins bind to both reticulocytes and mature red blood cells, the binding to mature cells was more pronounced. In the presence of hyperimmune infection serum or specific polyclonal antibodies to the 235-kDa protein its binding to erythrocytes was reduced, further demonstrating the specificity of this ligand-receptor interaction.     

Organization of ETRAMPs and EXP-1 at the parasite-host cell interface of malaria parasites

The parasite-host cell interface is a key compartment of vacuolated intracellular pathogens but little is known about its molecular composition and architecture. We used in vivo cross-linking to analyse the parasite-host cell interface of asexual stages of the most virulent human malaria parasite Plasmodium falciparum. We show that the integral membrane protein members of the early transcribed membrane protein (ETRAMP) family and exported protein 1 (EXP-1), which are components of the parasite-host cell interface, form complexes of oligomeric arrays in this compartment. The most notable feature is that each ETRAMP member and EXP-1 define separate arrays, demonstrating that the protein distribution in this membrane is non-random. Each of three recombinant ETRAMPs readily oligomerized in bacterial membranes, confirming that these arrays can form independently of other Plasmodium proteins. We propose that the malaria parasite-host cell interface contains patches of integral membrane proteins forming a mosaic of different microdomains in this membrane.     

Transmission of the malaria parasite requires ferlin for gamete egress from the red blood cell

Ferlins mediate calcium‐dependent vesicular fusion. Although conserved throughout eukaryotic evolution, their function in unicellular organisms including apicomplexan parasites is largely unknown. Here, we define a crucial role for a ferlin‐like protein (FLP) in host‐to‐vector transmission of the rodent malaria parasite Plasmodium berghei. Infection of the mosquito vectors requires the formation of free gametes and their fertilisation in the mosquito midgut. Mature gametes will only emerge upon secretion of factors that stimulate the disruption of the red blood cell membrane and the parasitophorous vacuole membrane. Genetic depletion of FLP in sexual stages leads to a complete life cycle arrest in the mosquito. Although mature gametes form normally, mutants lacking FLP remain trapped in the red blood cell. The egress defect is rescued by detergent‐mediated membrane lysis. In agreement with ferlin vesicular localisation, HA‐tagged FLP labels intracellular speckles, which relocalise to the cell periphery during gamete maturation. Our data define FLP as a novel critical factor for Plasmodium fertilisation and transmission and suggest an evolutionarily conserved example of ferlin‐mediated exocytosis.     

Interaction of the exported malaria protein Pf332 with the red blood cell membrane skeleton

Intra-erythrocytic Plasmodium falciparum malaria parasites synthesize and export numerous proteins into the red blood cell (RBC) cytosol, where some bind to the RBC membrane skeleton. These interactions are responsible for the altered antigenic, morphological and functional properties of parasite-infected red blood cells (IRBCs). Plasmodium falciparum protein 332 (Pf332) is a large parasite protein that associates with the membrane skeleton and who's function has recently been elucidated. Using recombinant fragments of Pf332 in in vitro interaction assays, we have localised the specific domain within Pf332 that binds to the RBC membrane skeleton to an 86 residue sequence proximal to the C-terminus of Pf332. We have shown that this region partakes in a specific and saturable interaction with actin (K_d = 0.60 µM) but has no detectable affinity for spectrin. The only exported malaria protein previously known to bind to actin is PfEMP3 but here we demonstrate that there is no competition for actin-binding between PfEMP3 and Pf332, suggesting that they bind to different target sequences in actin.     

蜂毒溶血肽对鸡红细胞及膜的生化作用

本文采用荧光分光光度、薄层层析、原子吸收、荧光显微图像等多种生化技术,系统研究了蜂毒肽作用于鸡红细胞及膜的生化机理。结果表明：蜂毒肽影响红细胞膜上及胞内两种酶的功能。它抑制膜Na⁺-K⁺-ATPase活性,导致胞内外离子转运异常,K⁺浓度失衡;它也抑制细胞内葡萄糖-6-磷酸脱氢酶活性,其正电区域干扰胞内带负电小分子的作用,影响红细胞正常代谢。蜂毒肽干扰膜中阴离子通道的转运功能,使细胞渗透压改变,引起膨胀而溶血。蜂毒肽对有核红细胞核内DNA没有作用,与其他抗微生物多肽作用的靶向不同。据此认为,抗菌蛋白类抗生素对细菌作用的生化机理与传统抗生素不同,这是细菌对其不易产生耐药性的重要原因。     

Side-dependent effects of internal versus external Na and K on ouabain binding to reconstituted human red blood cell ghosts

The side-dependent effects of internal and external Na and K on the ouabain binding rate, as promoted by inside MgATP, has been evaluated utilizing reconstituted human red blood cell ghosts. Such ghost systems provide the situation where [Na]i, [K]i, [Na]o, and [K]o can each be varied under conditions in which the others are either absent or fixed at constant concentrations. It was found that, in the presence of Ko, increasing either [Na]i or [K]i resulted in decreasing the rate at which ouabain was bound. Changes in [Na]i or [K]i in the absence of Ko were without effect on the ouabain binding rate. Thus, the ouabain binding rate was found to vary inversely with the rate of Na:K and K:K exchange but was independent of the rate of Na:Na exchange. The effect of Ko in antagonizing ouabain binding, as well as the influence of Nao on this interaction, were found to require the presence of either Nai or Ki. The results are interpreted in terms of a model relating the availability of the ouabain binding site to different conformational states of the pump complex. Differences were observed in the ouabain binding properties of red cell ghosts compared to microsomal preparations but it is not known whether the basis for the differences resides in the different preparations studied or in the lack of control of sidedness in the microsomal systems.