The cellular and molecular rheology of malaria.

Biorheology 51 (2014) 99–119 DOI 10.3233/BIR-140654 IOS Press

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Review article

The cellular and molecular rheology of malaria Brian M. Cooke a,∗ , John Stuart b and Gerard B. Nash b a

Department of Microbiology, Monash University, Victoria, Australia Centre for Cardiovascular Sciences, School of Clinical and Experimental Medicine, College of Medical and Dental Science, University of Birmingham, UK

b

Received 21 February 2014 Accepted in revised form 1 April 2014 Abstract. During development inside red blood cells (RBCs), Plasmodium falciparum malaria parasites export a number of proteins beyond the confines of their own plasma membrane where they associate with the RBC membrane skeleton. Here they participate in protein–protein interactions with both RBC proteins and other parasite proteins and assemble into complex multi-component structures known as knobs. These interactions cause profound changes to the rheological properties of RBCs, particularly increased cell resistance to deformation and increased adhesiveness, which underpin the severe and often fatal clinical manifestations of falciparum malaria. Here, we bring together recent insights that have been made into understanding the molecular mechanisms that underlie these parasite-induced alterations to RBCs. We describe some of the well-established methods that have been used to quantify the altered rheological properties of parasitized RBCs (PRBCs) and discuss emerging techniques that have already begun to advance our knowledge of the molecular basis of this important human disease. Finally, we suggest potential new avenues for rheological anti-malaria therapy. Keywords: Red blood cell, plasmodium, cell mechanics, adhesion

1. Introduction The evolving story of the importance of red blood cell (RBC) rheology in malaria is a lesson in the complexity and clinical significance of rheological processes. Although a number of rapid diagnostic tests are available and frequently used, microscopy remains the gold standard for the diagnosis of malaria, and classification of the species of malaria parasites, but the process of examining dried RBCs in a thin blood film on a glass slide has never allowed any understanding of the dynamics of blood flow. This is of particular relevance in malaria caused by Plasmodium falciparum (the most lethal of the five species of malaria parasites currently known to infect humans) in which impaired blood flow is an undisputed contributor to the pathogenesis of this severe and frequently fatal human parasitic disease. For this reason, the focus of this review centres on the molecular rheology of falciparum malaria. *

Address for correspondence: Professor Brian M. Cooke, Department of Microbiology, Monash University, Victoria 3800, Australia. Tel.: +61 3 9902 9146; Fax: +61 3 9902 9222; E-mail: [email protected]. 0006-355X/14/$27.50 © 2014 – IOS Press and the authors. All rights reserved

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B.M. Cooke et al. / Cellular and molecular rheology of malaria

An acutely ill individual with malaria will show a series of rheological changes including an acutephase rise in plasma fibrinogen (with a consequential rise in plasma viscosity and rouleaux formation), a rise in white cell count (with increased adhesion to endothelium) and a decrease in the number of circulating platelets, but, arguably, impaired rheology of the parasite-infected RBC (altered morphology, decreased deformability and increased adhesiveness) is more important in pathogenesis. P. falciparum parasites are transmitted in many tropical and sub-tropical regions of the world [96] by the bite of the female Anophelese mosquito. Symptoms arise as the parasite invades and replicates within circulating RBCs causing haemolysis and impaired blood flow. Individuals with falciparum malaria, principally young children, experience particularly severe clinical symptoms and may become comatose and die from cerebral malaria, in part due to impaired blood supply to the brain [71]. Similarly, pregnant women (and their developing foetus) are particularly susceptible to severe gestational (pregnancy-associated) malaria as a result of altered blood flow and sequestration of parasite-infected RBCs (PRBCs) in the placenta. The rheological significance of RBCs containing the abnormal beta chains of sickle haemoglobin (HbS) or other haemoglobins such as HbC, HbE or HbF, is also well known [98]. HbS heterozygotes or HbC homozygotes for example show dramatic protection against the more severe forms of malaria in African children [134]. This is due, in part, to the entry of P. falciparum into RBCs, that compromises deformability further than is caused by the presence of the abnormal haemoglobin itself, as well as inhibition of other parasite-induced modifications to RBCs that are known to mediate their adhesive properties [34,133]; these RBCs being selectively removed from the circulation and thus lowering the total PRBC count. As multi-drug resistant forms of malaria continue to emerge and spread [148], a better understanding of the mechanisms by which malaria parasites alter RBC rheology and cause impaired blood flow has potentially important therapeutic consequences that, unfortunately, continue to be under-recognised and consequently under-exploited [90]. Here, we review current knowledge of the structure-function relationships that govern the altered rheological properties of PRBCs, the molecular mechanisms that underpin them and some methods, old and new, useful for quantifying these changes. Finally, we suggest that understanding the molecular rheology of PRBCs opens up new avenues for future rheological anti-malaria therapy.

2. The red blood cell – Structure and function Far too often, RBCs are considered as no more than simple biological containers of aqueous haemoglobin that transport and exchange oxygen and carbon dioxide throughout the body. They are of course far more sophisticated; arguably the best understood eukaryotic cell, particularly in the composition and physical nature of the membrane skeleton over which the plasma membrane is draped and its relationship to the cells’ unique rheological properties [14,48,93–95]. At the molecular level, the membrane skeleton comprises at least 12 ‘classical’, extensively characterised proteins [49]; and in light of more recent proteomic studies, possibly many more [109]. The ordered arrangement of tetramers of α- and β-spectrin, their interconnection at the ternary complex, with actin and protein 4.1, and the connectivity with the overlying RBC membrane via band 3 and glycophorin C (GPC) provide the structural resilience of the RBC and the basis for its ability to repeatedly deform during passage through the microcirculation. Our understanding of the relationship of this protein network to the rheological properties of the whole cell has been advanced in the main by studies of pathological inherited disorders


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of RBCs including the haemoglobinopathies, thalassaemias and hereditary sphero- and ovalo-cytoses [129]. However, more recently, elucidation of the structural changes in RBCs induced by infection with malaria parasites, particularly P. falciparum, and an understanding of the corresponding functional alterations to the RBC has considerably extended our knowledge of both structure/function relationships in RBCs and the mechanisms of the pathogenesis of malaria. 3. Malaria infection and the red blood cell After entering the blood stream following the bite from an infected mosquito, all five species of Plasmodium currently known to cause malaria in humans (P. falciparum, P. vivax, P. malariae, P. ovale and the most recently recognised, P. knowlesi) first undergo a brief but obligatory stage of replication in the liver. After a period of approximately 2 weeks, parasites burst from the hepatocytes and rapidly invade RBCs in which they undergo repeating cycles of asexual replication. The duration of the parasites’ asexual life cycle in RBCs depends on the species, but for P. falciparum is approximately 48 hours (Fig. 1). Over the first half of the cycle, young, newly-invaded ‘ring’ forms of the parasite develop into mature, pigmented ‘trophozoites’. During the second 24 hours of the cycle, trophozoites mature further into schizont stages that eventually lyse the RBC releasing 8–32 daughter ‘merozoites’ into the circulation which rapidly invade new RBCs. RBCs containing ring-stage parasites show only mild and subtle rheological impairment and continue to circulate in peripheral blood. In contrast, RBCs containing trophozoites or schizonts are notably absent from the peripheral circulation because these mature forms accumulate in the microvasculature of most, if not all organs of the body [86,114,124]. These sequestered PRBCs can perturb or completely obstruct blood flow in small diameter vessels of the microcirculation and contribute, in part, to severe and frequently fatal clinical sequelae associated with malaria infection, such as cerebral malaria [71,116]. Interestingly, in stark contrast to P. falciparum, RBCs infected with all other species of human malaria parasites that cause less severe and fatal disease are generally believed not to sequester to any significant degree. All asexual forms of the parasite circulate in the peripheral blood, suggesting that the magnitude of the rheological alterations to RBCs caused by all other human malaria parasites are likely to be less than those seen with P. falciparum. The modifications to RBCs that occur following infection with P. falciparum are particularly interesting. The RBC acquires a series of new properties and is converted from a relatively simple ‘bag of haemoglobin’ into a more complex cell by the appearance of new structures in the RBC cytoplasm, and new proteins at the RBC membrane skeleton and on the surface. As mentioned above, these modifications are most pronounced after infection with P. falciparum; the species that causes the most deadly form of malaria in humans [96]. The structural, morphological and functional changes that occur in infected RBCs are striking and include loss of the normal discoid shape and perturbations in the mechanical and adhesive properties of the cell. Infected RBCs become spherocytic, with their surface punctuated by 5,000–10,000 distinct electron-dense elevations known as knobs [61]; intriguing structures that are associated with alteration of the cell’s mechanical and adhesive properties. Knobs are located over some junctional complexes of the RBC membrane skeleton (through direct interaction with both spectrin and actin) [108] and vary in diameter (60–160 nm), height (10–20 nm) and density (7–70 µm2 ), depending on the infecting parasite strain, becoming smaller and more numerous as the parasite matures [61, 82,97]. Knobs appear to be essential for PRBCs to sequester in the microvasculature in vivo [30,116] and are invariably found on PRBCs from malaria infected individuals (after a brief period of in vitro culture to mature them to the trophozoite stage) [138]. Further, ultra-structural studies suggest that the knob structure is the intimate point of contact between the PRBC and the surface of the cell to which

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Fig. 1. Schematic representation of the life-cycle of P. falciparum in human red blood cells. Following invasion of red blood cells (RBCs) by merozoites, parasites form fine ring-forms (Rings). Over a period of 48 hours, rings mature through a pigmented trophozoite stage (Trophozoites) and then further into the multi-nucleated schizont stage (Schizonts). Schizonts then rupture, releasing newly formed merozoites back into the circulation. These merozoites rapidly invade new RBCs and the cycle continues. Note that only uninfected and ring-stage-infected RBCs circulate freely in the peripheral blood of malaria-infected individuals. Mature trophozoite- and schizont-infected RBCs are grossly rheologically-impaired and sequester in the microvasculature (predominantly post-capillary venules) of a variety of organs by adhering to vascular endothelial cells (or syncytiotrophoblasts in the placenta). A small proportion of parasites develop into terminally-differentiated, male (|) and female (~) sexual forms (Gametocytes) that are responsible for transmitting malaria from infected humans to mosquitoes. In stark contrast to asexual-stage parasites, RBCs infected with immature gametocytes sequester (predominantly in the spleen and bone marrow) but are released into the peripheral circulation once they are fully mature to avail themselves to blood-feeding female mosquitoes. (Colors are visible in the online version of the article; http://dx.doi.org/10.3233/BIR-140654.)

it adheres [99] and that the parasite proteins that mediate the interaction are clustered over the surface of the knob [6,80]. Surface potential spectroscopy performed using an atomic force microscope has also revealed that knobs are positively charged, with a membrane potential of +20 mV, when compared with the remainder of the RBC membrane, which may further enhance PRBC-host cell interactions [1]. Within the cytoplasm of the PRBC, novel membranous structures called Maurer’s clefts (MCs) are elaborated by the parasite and appear to play a role in transporting new proteins to the RBC membrane and possibly their assembly into knobs [115]. The loss of normal RBC deformability associated with increased membrane rigidity, and unusual adhesiveness of infected RBCs for a variety of other cells (including other RBCs, vascular endothelial cells, platelets, dendritic cells and placental syncytiotrophoblasts (see [46,105] for reviews) are arguably the two most pathophysiologically-important rheological consequences of falciparum malaria infection.


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Finally, in this review, it would be remiss of us not to comment on the rheology of another intraerythrocytic life-cycle stage of the malaria parasite, the gametocyte. Gametocytes represent a small but significant proportion of blood-stage parasites, which by complex and incompletely understood stimuli and mechanisms, develop after the ring-stage into terminally-differentiated, male and female sexual forms (rather than into trophozoites) that are responsible for transmitting malaria from humans to mosquitoes. In P. falciparum, mature gametocytes are easily recognisable in Giemsa-stained blood smears and are morphologically distinct from mature asexual forms; the male resembling a sausage shape and the female, somewhat more curved, resembling a banana, sickle, or falciform shape; from which P. falciparum derives its name (Fig. 1). Within RBCs, gametocytes develop through 5 distinct stages of maturation (I–V) [64] over a period of approximately two weeks. Similar to RBCs infected with mature trophozoitestage parasites, RBCs harbouring immature (stage I–IV) gametocytes sequester in the host, but unlike asexual stages, sequestration seems to occur in the spleen and bone marrow rather than in the microvasculature of most other organs [72,117,127]. Interestingly, once the parasites mature to stage V however, the PRBCs become liberated and circulate freely in the in peripheral blood, availing themselves for transfer to biting mosquitoes via the blood meal. While the precise mechanisms underlying this process critical to parasite transmission and survival remain uncertain, RBCs infected with stage V gametocytes are more deformable (measured by ektacytometry or microsphiltration [135] or micropipette aspiration [2]) than RBCs infected with more immature gametocyte stages. The rheological basis for this fascinating late-stage-specific increase in the deformability of PRBCs has only recently become a subject of investigation. Studies so far suggest that it is more likely due to increased deformability (reduced rigidity) of the parasite itself rather than to changes in the geometry (surface area:volume) or mechanical properties of the PRBC membrane skeleton [2,38,42,63]. Furthermore, sequestering RBCs infected with immature gametocytes do not appear to express membrane knobs or PfEMP-1 on their surface or cytoadhere to the same adhesion molecules used by asexual stage parasites to bind to the vascular endothelium (see Section 5.3) [5,136] indicating that they sequester by different, yet unknown mechanisms to those that mediate sequestration of asexual RBC stages of the parasite. Given the potential impact that blocking the release of mature gametocyte-infected RBCs into the circulation would have on the transmission of human malaria, further work in this particular area of malaria rheology is clearly warranted. 4. Techniques for quantifying the altered rheological properties of infected red blood cells Quantifying the rheological abnormalities of RBCs that follow malaria parasite invasion has required the application of a number of biophysical techniques, some of which have only recently become available. Measurements have been made on bulk populations of RBCs (either dilute suspensions or in whole blood) or on individual cells. Recent advances have led to the development of techniques such as optical traps or laser tweezers, atomic force microscopy and optical stretchers, that, together with advances in microfluidics, permit direct, real-time manipulation and analysis of single RBCs at the micro- and nano-meter level and with nano- and pico-Newton force resolution. 4.1. Filtration, viscometry and rheoscopy Assessment of the bulk mechanical properties of RBCs by measuring their transit time through pores in polycarbonate filters has been a popular method to quantify the deformability or resistance to flow of RBCs due to its relative simplicity and ease of performance. Filtration methods, however, have been of limited utility for studying malaria-infected RBCs due to their relatively insensitivity to the low number

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of infected RBCs present in clinical blood samples or cultures of laboratory-adapted parasites. Similarly, rotational viscometers (such as cone-and-plate or bob-and-cup) to derive a viscosity profile of a suspension of RBCs under defined shear formed the basis of very early pioneering studies to quantify the altered mechanical properties of monkey RBCs infected with malaria parasites [91]. However, for similar reasons of relative insensitivity, this method too failed to be widely adopted. Direct or indirect visualization of individual infected RBCs under defined rotational shear stress using a rheoscope allows the derivation of a shear-induced elongation index for individual cells in a suspension and overcomes some of the disadvantages of filtration and viscometry by allowing more precise quantitation at the single cell level. Direct visualization of individual malaria-infected RBCs in sheared bulk suspension in a rheoscope allowed Cranston et al. [33] to extend the earlier viscometric studies of Miller [91]. The former was the first study to describe the worsening in the RBC mechanical properties during advance of the life cycle stages of the parasite. Rheoscopy has not been utilized to any great extent in malaria rheology since those early studies. A related technique for measuring RBC deformability (for mixed infected and uninfected RBCs), is ektacytometry using a laser-assisted optical rotational cell analyzer (LORCA) [43]. This device measures the averaged elongation of a cell population from the pattern of light diffraction. Again, data derived from these measurements needs to be carefully interpreted as the abnormal pattern seems to arise from changes in the uninfected RBCs as well as the small sub-population of PRBCs present in the bulk population. Micropipette analysis of single RBCs overcomes the inherent disadvantages associated with bulk population methods (see below) although it is much more laborious and technically-challenging, especially for clinical samples where the proportion of PRBCs is frequently low. 4.2. Laminar shear flow systems The use of parallel-plate flow chambers allows visualization and quantitation of the interactions of flowing blood cells with endothelial (or other) cells or purified adhesion proteins, under conditions of flow that mimic those in the circulation. Unlike in vivo models, laminar flow systems allow fine control over the shear stress, shear rate and viscosity as well as the biochemical composition of the haemodynamic environment. Although such flow-based methods have been used most extensively for studies of leucocytes and platelets, they have been (and continue to be) developed and used for studies of the altered adhesive properties of malaria-infected RBCs ([19,27,101] for examples). Direct microscopic observation of adherent cells under defined and well-controlled flow conditions allows the number binding and their behaviour to be monitored. Interestingly, studies of malaria-infected RBCs in flow chambers have revealed that, like leucocytes, they show different adhesive behaviour (rolling or stationary) depending on the identity of the endothelial cell receptor with which they interact [17,19]; PRBCs interacting with CD36 for example remain stationary while those interacting with ICAM-1 constantly roll [17]. Adhesive forces between PRBCs and different receptors have also been quantified by applying increasing shear in these flow-based assays, providing a useful measure of the likely physiological relevance of different PRBC-receptor interactions. Technically simplified flow-based assays, more suitable for use in resource-poor settings, have also been developed and used to obtain important quantitative information about the adhesive properties of PRBCs taken directly from infected individuals with uncomplicated or severe disease in malaria endemic areas [24,25]. Flow chambers have also been used to determine the mechanical properties of PRBCs, deriving estimates of membrane shear elastic modulus from shear-induced elongation indices for pointattached RBCs [103,132]. Such measurements are also influenced, however, by cellular factors such


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as haemoglobin concentration and the presence of inclusion bodies and may therefore differ quite substantially from the value obtained for the modulus determined by micropipette aspiration techniques (see below). Although information on the deformability of whole, intact PRBCs is clearly of physiological relevance, assays that quantify whole cell deformability have limited use for more detailed dissection of the multiple individual parameters that contribute collectively to the overall altered mechanical properties of whole PRBCs. 4.3. Micropipette aspiration In this technique, a negative hydrostatic pressure is used to partially or completely aspirate a single RBC into a glass micropipette with diameter ranging from less than 1 µm to 10 µm. The time taken for a whole RBC to enter a micropipette with a diameter smaller than the diameter of the cell being aspirated is related to the overall deformability of the whole cell. Aspiration of a membrane tongue and measurement of tongue length as a function of aspiration pressure allows derivation of the shear elastic modulus of the membrane skeleton itself and is not influenced by other cellular properties that contribute to whole cell deformability. In addition, point-attached cells can be elongated by aspiration and retraction of the pipette and the rate of their recovery measured after they come free. The time constant for shape recovery is proportional to the membrane shear viscosity divided by the shear elastic modulus [68]. Micropipette techniques have been used extensively, albeit by a small number of specialised laboratories to quantify mechanical alterations in the membrane skeleton of RBCs infected with a variety of different malaria parasites [59,73,84,102,104,110]. Such studies, for example, have revealed that there is little change in membrane viscoelasticity following the early stages of parasite invasion and maturation but there is progressive rigidification of the RBC membrane skeleton once the more mature, pigmented trophozoite has developed. The elastic modulus of uninfected RBCs increases more than 3 fold (from 5 µN/m to greater than 15 µN/m) as parasites mature inside the RBC and correlates with the export of parasite proteins that interact with the RBC membrane skeleton and the formation of RBC membrane knobs [59,110]. Micropipettes have also been used to quantify the forces involved in the adhesive interactions between PRBCs and the endothelium or other RBCs. For example, the force of interaction between normal RBCs and a malaria-infected RBC in a rosette (a single PRBC surrounded by multiple non-infected RBCs) is at least 5 times higher than those between PRBCs and endothelial cells, indicating that these abnormal aggregates of RBCs could easily exist in the microcirculation of humans infected with malaria [100, 101]. 4.4. Atomic force microscopy More recently, the atomic force microscope (AFM) has been used as a powerful imaging tool with nanometer and sub-nanometer scale as well as a force sensor with pico-Newton resolution [83]. AFM tips coated with purified proteins allow forces of interaction to be accurately quantified between specific ligands and their cell-expressed receptors, often down to the level of a single molecular interaction. The technique has been used to study adhesive interactions at the molecular level for both leucocytes and RBCs [62,122]. For PRBCs, it has provided an excellent tool to image and quantify knobs on the PRBC surface in much greater detail than was previously possible by electron microscopy as well as to obtain a better understanding of the proteins responsible for their formation [70,82,118]. It has also revealed significant morphological differences in knobs on RBCs from individuals with genetically-abnormal

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RBCs (such as those containing abnormal haemoglobins) [3] that are well known to be protective against the most severe complications, such as cerebral malaria, that often accompany severe malaria in humans. 4.5. Optical traps or laser tweezers Optical traps (or laser tweezers) use high intensity laser light to trap, control and manipulate minute particles in a suspending medium and have become an important research tool in cell biology. RBCs can be stretched over a large range using optical tweezers by attaching dielectric silica microbeads of 1–4 µm diameter, non-specifically to the surface of the cell which then form ‘handles’ by which the RBC can be dragged through a viscous suspending medium, allowing quantitation of its overall elasticity [10]. Alternatively, two beads, attached diametrically opposite each other on the RBC can form a trap in which the cell can be uniaxially stretched [37,84,92]. The shear elastic modulus of the RBC can be inferred from force/length measurements and has been estimated to be between 3.0 µN/m to 8.0 µN/m for normal RBCs, which is within the range previously determined by others using micropipette aspiration [41,65,67,126]. Interestingly, when RBCs infected with different life-cycle stages of P. falciparum parasites were examined using laser tweezers [131], the increase in RBC rigidity caused by malaria infection was much greater (up to 10-fold) than that measured in earlier studies of individual PRBCs using micropipette aspiration techniques (up to 3-fold) [59,110]; the difference presumably being explained by the fact that optical tweezers measure the overall deformability of the whole RBC (including the poorly deformable intracellular parasite itself) whereas micropipettes measure only the altered elastic modulus of the RBC membrane skeleton. 4.6. Microfluidics Over the last 2 decades, there has been a tremendous increase in the interest and use of microfluidic devices in all areas of biomedicine [140,149]. They offer numerous advantages to studying the rheological properties of RBC. They can be easily manufactured with a high degree of accuracy, sophistication and reproducibility using micro-fabrication and lithography techniques to mimic the architecture and flow dynamics of the microcirculation in vivo. Materials such as silicone elastomer can be used to fabricate silicone channels with varying dimensions that approximate those of the human microvasculature. Furthermore, the devices use extremely small sample volumes and are highly suited to high-throughput screening and analysis. Elastomeric microfluidic channels with a height of 2 µm and width of 2–8 µm have been used to study the flow properties of both normal [58] and malaria-infected RBCs [123]. Providing further functionality to microfluidic channels by coating with proteins expressed on the surface of endothelial cells, together with more accurate quantitation, will further increase the utility of such devices for more advanced studies the mechanical and adhesive properties of RBCs. 4.7. In vivo and ex vivo models Since the pathophysiological endpoint of the altered mechanical and adhesive properties of P. falciparum-infected RBCs is their highly efficient sequestration within vital organs, this presents obvious challenges for studying these rheological phenomena in humans. Direct observation of the microcirculation in the human eye is relatively straightforward and has been used to visualise blood flow in individual vessels in malaria-infected individuals [8]. Such observations however, remain largely qualitative since high resolution imaging required to accurately quantify either the flow or rheological properties


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of individual PRBCs in this dynamic in vivo situation is technically challenging. Orthogonal polarization spectral (OPS) imaging of human microvessels in the mucosa of the mouth or rectum improves image resolution and allows at least some level of quantitation [44] but really contributes little else other than confirmation that microvascular obstruction plays a role in the pathophysiology of severe malaria. Animal models of malaria (predominantly non-human primates and mice) overcome some of the technical challenges presented by trying to image the microcirculation at high resolution in humans. However, the extent to which they accurately mimic the human microvasculature or the pathogenesis of human malaria infections is always a point of contention [32,81]. A mouse model in which the vasculature of the maesocaecum was exposed and perfused ex vivo with human RBCs infected with P. falciparum was the first direct observation that knobs on the PRBC surface were necessary and sufficient for their adhesion to vascular endothelial cells and consequent microvascular flow obstruction [116]. Further, direct observation and quantitation of RBCs flowing through the vasculature of the relatively accessible cremaster muscle, in mice infected with murine species of malaria parasites (e.g. P. yoelii), provided the first in vivo evidence of the molecular nature of the interaction between PRBCs and the endothelium [76]. Further advances in the development of humanised mouse models, specifically immune-deficient mice grafted with xenogenic transplants of human RBCs (and ideally human vascular endothelium) that support the replication of human malaria parasites ([4] for example) will significantly improve our ability to study the interactions of flowing PRBCs with the vasculature in mice in a situation much more physiologically relevant to human malaria infections. This combined with new and more sophisticated quantitative imaging modalities ([53] for example) can potentially make these and other rodent models an extremely useful resource in this area. This is perhaps particularly important since the future development and use of unquestionably more relevant models using non-human primates will continue to be hampered by ethical and cost considerations. One relatively recent development, particularly noteworthy here in the context of RBCs and malaria, is the use of human spleens (surgically removed during resection of some pancreatic tumours) which are then perfused ex vivo [11]. The spleen is a complex organ that plays a protective role in malariainfected individuals by trapping and clearing PRBCs from the circulation as well as providing a site for the sequestration of RBCs infected with immature gametocytes ([12,39] for reviews). Interestingly, this model provided the first direct evidence that, in addition to mature-parasite-infected RBCs, a proportion of both uninfected and ring-stage-parasite-infected RBCs are also retained and removed by the spleen [13,119]. The extent of the rheological changes (particularly rigidity and adhesiveness) in non-infected (but parasite affected) or ring-infected RBCs, either in clinical samples or cultured parasites, is subtle (sometimes undetectable) when compared to RBCs infected with mature, pigmented parasites. Thus, their retention in the spleen demonstrates the exquisite sensitivity of this organ as a mechanical sensing device [40] and offers an explanation for the exacerbated level of anaemia (higher than would be expected from haemolysis and destruction of mature-parasite-infected RBCs alone) that is a commonly observed phenomenon in malaria-infected individuals. 4.8. Molecular techniques, genetics and genomics for P. falciparum malaria parasites Advances in our understanding of the molecular rheology of malaria infection are, in large part, attributed to major advances over the last 2 decades in parasite genomics and the ability to transfect and genetically manipulate P. falciparum. The completion of the first, annotated genome sequence for P. falciparum in 2002 [57], and the development of a stable transfection system for the RBC stages of the

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parasite [31,150] allowed us to take this research to a new level of exploration in which particular cellular changes could be unequivocally ascribed to the function of individual genes or gene families and proteins. The generation of transgenic parasites over the past decade or so, that do not express some of the accessory RBC membrane parasite-encoded molecules such as KAHRP, PfEMP3 or Pf332, in combination with new and more sophisticated rheological methods, have been responsible for ascribing a precise function to individual parasite proteins and linking them to specific rheological alterations in the PRBC.

5. Exported malaria proteins – Function and rheological consequences 5.1. Exported parasite proteins Development of abnormal circulatory behaviour of RBCs coincides with the production of a number of stage-specific proteins that are exported from the parasite to the RBC membrane and subsequently interact directly with RBC cytoskeletal proteins. These proteins are associated with many morphological alterations to the RBC including the appearance of knobs. Over the past decade or so, at least 10 proteins that are exported by the parasite to the RBC membrane skeleton have been identified and characterised to a greater or lesser extent (Table 1). However, more recent bioinformatic analysis of the annotated Table 1 Examples of some characterized exported malaria proteins that interact with the RBC membrane skeleton and alter the rheological properties of RBCs Protein RESA

Salient features 155 kDa phosphoprotein; present in RBCs infected with ring-stage parasites; binds to β-spectrin

Cellular rheological consequences References Little or no measurable effect on [36,54–56,112,125] membrane rigidity; increases the thermal stability of infected RBCs; may protect RBCs from fragmentation during malaria fever MESA 250–300 kDa phosphoprotein; Competes with RBC p55 protein for [28,29,85,87,142] binds to the 30 kDa domain of RBC binding to protein 4.1 and is likely protein 4.1 to modulate p55 function; precise function in RBCs remains unknown KAHRP 80–109 kDa histidine-rich protein; Essential for the formation of RBC [20,30,59,69,77,111,118,141,143,147] binds to α-spectrin, actin and membrane knobs and adhesion of ankyrin; interacts with the infected RBCs to the vascular cytoplasmic tail of the parasites’ endothelium under flow; anchors cytoadhesive ligand, PfEMP1 PfEMP1 into the infected RBC membrane; may be involved in trafficking PfEMP1 to the infected RBC surface; contributes about 50% of the total increase in membrane rigidity in infected RBCs PfEMP3 315 kDa protein; binds to Contributes about 15% of the total [78,113,146] α-spectrin at junctional complexes increase in membrane rigidity in infected RBCs; not essential for cytoadherence; destabilises the RBC membrane skeleton; may be involved in infected RBC rupture and merozoite release


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Table 1 (Continued) Protein Pf332

Salient features Giant protein (c. 750 kDa); binds specifically to actin via an 86 residue sequence in the C-terminal end of Pf332

FIKK

A family of 21 unique kinases, most remain uncharacterised, some members associated with the RBC membrane skeleton; possibly responsible for phosphorylation of other parasite or RBC proteins 265–285 kDa, highly antigenically-variable protein, spans the RBC membrane and binds to the RBC skeleton via a conserved cytoplasmic domain that binds to spectrin, actin and KAHRP; concentrated at knobs

PfEMP1

SBP1

46 kDa integral membrane protein of Maurer’s clefts; associates with the RBC membrane skeleton in infected RBCs

Cellular rheological consequences Modulates the level of increased RBC rigidity; plays a significant role in cytoadhesion by assisting transport of PfEMP1 to the infected RBC surface Likely to have a wide variety of different functions affecting membrane mechanical and adhesive properties of infected RBCs

Extracellular domain exposed on the RBC surface mediates adherence of infected RBCs to vascular endothelial cells, other RBCs (normal and malaria-infected), platelets and placental syncytiotrophoblasts; different variants of PfEMP1 bind to different receptors and mediate different forms of adhesion (rolling or stationary) Critical for trafficking PfEMP1 to the RBC surface and cytoadhesion of infected RBCs

References [60,66,89]

[75,106,107]

[6,80,108,139]

[9,18,88]

Abbreviations: RESA, ring-infected erythrocyte surface antigen; MESA, mature parasite-infected erythrocyte surface antigen; KAHRP, knob-associated histidine-rich protein; PfEMP, Plasmodium falciparum erythrocyte membrane protein; Pf332, Plasmodium falciparum protein 332; FIKK, FIKK kinases; SBP, skeleton binding protein; kDa, kilo Dalton; RBC, red blood cell.

genome sequence of P. falciparum predicts that almost 400 proteins are likely to be exported into the RBC, and at least 70 of those are likely to interact with the RBC membrane skeleton and play a role in RBC remodelling [120]. Of the proteins characterised so far, some, including ring-infected erythrocyte surface antigen (RESA), mature-parasite-infected erythrocyte surface antigen (MESA), P. falciparum erythrocyte membrane 3 (PfEMP3) and Pf332 appear to be distributed evenly around the skeleton, while others such as the knob associated histidine rich protein (KAHRP) and P. falciparum erythrocyte membrane 1 (PfEMP1) tend to cluster together in higher density beneath membrane knobs (Fig. 2). Intriguingly, in early maturing parasites, a number of these proteins, and others such as P. falciparum skeleton-binding protein 1 (PfSBP1), MAHRP, Rex1 and a number of novel, yet uncharacterised putative kinases, collectively termed FIKK kinases [121,145], appear to be associated with MCs or other punctate structures that are scattered throughout the RBC cytoplasm [75,106,107]. Interestingly, despite the fact that MCs were first described by Georg Maurer more than 100 years ago, their precise function in PRBCs remains unknown, although it is likely that they represent the vehicles by which many of these exported malaria proteins are delivered to their final destination in the RBC. Characterisation of the precise binding domains that mediate the interactions between such parasite proteins and the RBC membrane skeleton is now being performed and reports are appearing on both the RBC membrane skeletal target protein and the sequences involved in binding. For example, MESA binds in a strong non-covalent manner to protein 4.1

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Fig. 2. Morphological and molecular alteration of the RBC membrane induced by P. falciparum malaria parasites. (A) Schematic representation of the interaction of exported malaria proteins with proteins of the RBC membrane skeleton. An area of the RBC membrane forming a single knob structure is represented. (B) The surface of a P. falciparum-infected RBC imaged by atomic force microscopy (AFM) showing the presence of knobs on the RBC membrane. (C) Higher magnification of a portion of the RBC membrane in panel B showing a single knob on the RBC membrane. Abbreviations: K, knob-associated histidine-rich protein; PfEMP1, P. falciparum erythrocyte membrane protein 1; EMP3, P. falciparum erythrocyte membrane protein 3; Pf332, P. falciparum protein 332; FIKK, FIKK kinases; MESA, mature parasite-infected erythrocyte surface antigen; Ank, ankyrin; Gp, glycophorin; 4.1, protein 4.1; RBC, red blood cell. Figure modified and reprinted from [21] with permission from IOS Press.

[87,142] and RESA has been shown to bind to spectrin via a 48-residue domain in the RESA protein [112]. KAHRP binds to spectrin, actin and ankyrin, and although the precise binding domains of the parasite protein remain to be determined, it appears, for spectrin at least, to be somewhere in the aminoterminal region [77,111]. KAHRP also binds to the cytoplasmic tail of PfEMP1 and defined regions in both of these proteins that mediate the interaction have been mapped [139,141,143].


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5.2. Altered RBC mechanical properties Given that RBCs are highly structurally adapted to be both pliable and resilient, it is hardly surprising the interactions of exported malaria parasite proteins have major effects on their overall mechanical properties. When compared with normal RBCs, the shear elastic modulus of RBCs infected with mature P. falciparum parasites is increased up to 3-fold [104]. Of the proteins characterised to date, the effect of KAHRP appears to be the greatest, contributing to approximately 50% of the increased membrane rigidification seen in PRBCs, followed by PfEMP3 which appears to contribute an additional 15% [59]. In contrast, another protein, Pf332, that binds specifically to actin [144], appears to moderate the level of increased rigidity of the infected RBC since parasites that lack this protein rigidify RBCs to a greater level than those containing it [60]. The proteins or mechanisms which contribute the remainder of the rigidification remain to be identified, however, the oxidative stress due to the development of the highly metabolically-active parasite in the RBC likely plays and important, albeit difficult to quantify, role [45]. Further, since some proteins exported by parasites structurally resemble kinases and are localised in the vicinity of the RBC membrane skeleton [75,106,107], alteration of the phosphorylation status of integral RBC membrane proteins, or other parasite proteins may also play an important, yet unidentified role. 5.3. Altered RBC adhesive properties Exported proteins, in particular those located at knobs, also play a critical role in the altered adhesive properties of infected RBCs. The knob structures at the membrane skeleton of PRBCs are intriguing. They are essential for adhesion of PRBCs to the vascular endothelium [30] and the process of sequestration that causes many of the frequently fatal syndromes associated with P. falciparum infections. The major structural element of the knobs appears to be the knob-associated histidine-rich protein (KAHRP). Also incorporated into this structure is one of a large family of highly polymorphic parasite exported proteins known P. falciparum erythrocyte membrane protein-1 (PfEMP1). PfEMP1 is anchored in the knob complex via interaction of its cytoplasmic domain with KAHRP and spectrin (see Fig. 2). Its external domain acts as a ligand for binding to a number of receptors expressed on the surface of vascular endothelial cells including CD36, ICAM-1, chondroitin sulphate A, hyaluronic acid and others of less clear pathophysiological significance in malaria infection such as VCAM-1, E-selectin, PECAM-1, and most recently, endothelial protein C receptor [7,23,137]. In addition to adhesion to vascular endothelial cells, PfEMP1 also mediates binding of infected RBCs to other PRBCs, a phenomenon known as autoagglutination, and to normal RBCs, termed rosetting [52]. Controversy still exists about the importance of these adhesive interactions in vivo; however, on balance, it appears that both rosetting and agglutinating strains of parasites are associated with more severe disease. A number of counter receptors on the surface of RBCs have been described to which PfEMP1 can bind including complement receptor 1 (CR1), heparan sulphate or heparan sulphate-like glycosaminoglycans, and the ABO blood group antigens, particularly blood group A. The physical forces binding RBCs into a rosette have been measured using both dual micropipetting techniques and viscometry and are estimated to be at least 5 times higher than those involved in the adherence of PRBCs to vascular endothelial cells [22]. Recent studies implicate a role for RBC actin in alteration of the adhesive properties of PRBCs ([34] for review). During its development in RBCs, P. falciparum appears to ‘hijack’ actin from the RBC membrane skeleton and use it to establish a novel structural network within the RBC cytoplasm that seems to facilitate transport of MCs (and their associated proteins, including PfEMP1) to the RBC

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membrane skeleton and surface. Interestingly, this actin remodelling process seems to be absent, or dramatically reduced in HbS and HbC RBCs [35], which also have morphologically-abnormal knobs at the RBC membrane, reduced expression or abnormal distribution of PfEMP1 on the PRBC surface and show a decreased capacity to cytoadhere [15,50,51,79]. Intriguingly, this additional cytoskeletal remodelling phenomenon in PRBCs offers the most plausible explanation yet for the protective effect of abnormal haemoglobins against the severe clinical complications of severe falciparum malaria. 6. Rheological therapeutics for malaria Theoretically, therapeutics that prevent or reverse the mechanical or adhesive abnormalities of PRBCs should significantly reduce the severity of malaria infection. The feasibility and clinical benefit of such an approach has probably been best demonstrated for leucocyte or platelet adhesion disorders using anti-adhesion therapies designed to antagonise integrin receptors [16]. Understanding the molecular interactions of PRBCs with blood vessel walls has identified a number of ligands (see Section 5.3) which may be potential targets for small molecule or antibody-based adhesion inhibitors. Potential reagents have been developed and tested in the laboratory including recombinant protein fragments of the malaria adhesin PfEMP1 [26], however, none of these have yet been developed further with a view to clinical trial. Whether the mechanical properties of PRBCs could be modified in vivo, and whether this would have a significant effect on progression or severity of malaria remains uncertain, given that the precise contribution of abnormal RBC mechanics in the pathogenesis of severe malaria has not been demonstrated. However, previous studies demonstrating that ion-channel blockers such as ICA-17043 (Clotrimazole), Pentoxifylline, Cetiedil or Bepridil can significantly improve the morphology and increase the deformability of mechanically-impaired sickle RBCs, which presumably would reduce their trapping in the microcirculation, offers some degree of support for such an approach [47,74,128,130]. Perhaps more excitingly, recent identification of the specific domains within a number of exported malaria proteins that mediate their interaction with specific proteins of the RBC membrane skeleton, together with knowledge of their mechanical consequences at the cellular level, do present possibilities to develop small molecule inhibitors which could interfere specifically with these interactions. For example, preventing the interaction of KAHRP with spectrin or PfEMP1, PfEMP3 with actin or PfSBP1 with the RBC membrane skeleton, using small peptides [18,111,141,143,144] could have major beneficial effects on reducing both the mechanical and the adhesive abnormalities of PRBCs. 7. Summary and outlook Abnormal interaction between RBCs and the human vasculature is a highly complex process that plays a central role in the pathogenesis of malaria, particularly that caused by P. falciparum. Deleterious rheological changes occur in RBCs soon after they have been invaded by the parasite including a progressive increase in the rigidity of the RBC membrane skeleton, decreased whole cell deformability and increased cell adhesiveness. Numerous abnormal parasite-induced protein–protein interactions in the RBC membrane skeleton are direct causes of these changes and are of immense pathophysiological significance. During the past 20 years, our understanding of the precise molecular mechanisms that underlie these specific rheological alterations to RBCs caused by infection with the malaria parasite has increased significantly. This is largely due to the use of new and improved techniques in cellular and molecular bi-


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ology, genomics, proteomics and imaging in combination with well-established and new rheological and biophysical techniques and advances in micro- and nano-technology. This increased knowledge brings new opportunities for research and presents numerous exciting new challenges for the years to come. Importantly, further understanding of the precise molecular basis of this truly ‘rheological disease’ may in the future lead to the development of novel, next-generation therapeutic agents designed to prevent or reverse cellular rheological phenomena associated with this human infection that still claims the lives of almost a million humans every year [96]. Acknowledgements BMC is grateful to Harry Goldsmith who first suggested that I write a review on the molecular rheology of malaria. Here, I come together with two of my most respected mentors in haemorheology, to write this review that we now dedicate in memory of our friend and colleague, Oguz Baskurt. References [1] M. Aikawa, K. Kamanura, S. Shiraishi, Y. Matsumoto, H. Arwati, M. Torii et al., Membrane knobs of unfixed Plasmodium falciparum infected erythrocytes: new findings as revealed by atomic force microscopy and surface potential spectroscopy, Exp. Parasitol. 84 (1996), 339–343. [2] M. Aingaran, R. Zhang, S.K. Law, Z. Peng, A. Undisz, E. Meyer et al., Host cell deformability is linked to transmission in the human malaria parasite Plasmodium falciparum, Cell Microbiol. 14 (2012), 983–993. [3] T. Arie, R.M. Fairhurst, N.J. Brittain, T.E. Wellems and J.A. Dvorak, Hemoglobin C modulates the surface topography of Plasmodium falciparum-infected erythrocytes, J. Struct. Biol. 150 (2005), 163–169. [4] L. Arnold, R.K. Tyagi, P. Meija, C. Swetman, J. Gleeson, J.L. Perignon et al., Further improvements of the P. falciparum humanized mouse model, PLoS One 6 (2011), e18045. [5] A. Bachmann, C. Esser, M. Petter, S. Predehl, V. von Kalckreuth, S. Schmiedel et al., Absence of erythrocyte sequestration and lack of multicopy gene family expression in Plasmodium falciparum from a splenectomized malaria patient, PLoS One 4 (2009), e7459. [6] D.I. Baruch, B.L. Pasloske, H.B. Singh, X.H. Bi, X.C. Ma, M. Feldman et al., Cloning the P. falciparum gene encoding PfEMP-1, a malarial variant antigen and adherence receptor on the surface of parasitized human erythrocytes, Cell 82 (1995), 77–87. [7] D.I. Baruch, S.J. Rogerson and B.M. Cooke, Asexual blood stages of malaria antigens: cytoadherence, Chem. Immunol. 80 (2002), 144–162. [8] N.A. Beare, T.E. Taylor, S.P. Harding, S. Lewallen and M.E. Molyneux, Malarial retinopathy: a newly established diagnostic sign in severe malaria, Am. J. Trop. Med. Hyg. 75 (2006), 790–797. [9] T. Blisnick, M.E. Morales-Betoulle, J.-C. Barale, P. Uzureau, L. Berry, S. Desroses et al., Pfsbp1, a Maurer’s cleft Plasmodium falciparum protein, is associated with the erythrocyte skeleton, Mol. Biochem. Parasitol. 111 (2000), 107– 121. [10] M.M. Brandao, A. Fontes, M.L. Barjas-Castro, L.C. Barbosa, F.F. Costa, C.L. Cesar et al., Optical tweezers for measuring red blood cell elasticity: application to the study of drug response in sickle cell disease, Eur. J. Haematol. 70 (2003), 207–211. [11] P.A. Buffet, G. Milon, V. Brousse, J.M. Correas, B. Dousset, A. Couvelard et al., Ex vivo perfusion of human spleens maintains clearing and processing functions, Blood 107 (2006), 3745–3752. [12] P.A. Buffet, I. Safeukui, G. Deplaine, V. Brousse, V. Prendki, M. Thellier et al., The pathogenesis of Plasmodium falciparum malaria in humans: insights from splenic physiology, Blood 117 (2011), 381–392. [13] P.A. Buffet, I. Safeukui, G. Milon, O. Mercereau-Puijalon and P.H. David, Retention of erythrocytes in the spleen: a double-edged process in human malaria, Curr. Opin. Hematol. 16 (2009), 157–164. [14] J.A. Chasis and N. Mohandas, Erythrocyte membrane deformability and stability: two distinct membrane properties that are independently regulated by skeletal protein associations, J. Cell Biol. 103 (1986), 343–350. [15] R. Cholera, N.J. Brittain, M.R. Gillrie, T.M. Lopera-Mesa, S.A. Diakite, T. Arie et al., Impaired cytoadherence of Plasmodium falciparum-infected erythrocytes containing sickle hemoglobin, Proc. Natl. Acad. Sci. USA 105 (2008), 991– 996.

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