Multiple stiffening effects of nanoscale knobs on human red blood cells infected with Plasmodium falciparum malaria parasite

During its asexual development within the red blood cell (RBC), Plasmodium falciparum (Pf), the most virulent human malaria parasite, exports proteins that modify the host RBC membrane. The attendant increase in cell stiffness and cytoadherence leads to sequestration of infected RBCs in microvasculature, which enables the parasite to evade the spleen, and leads to organ dysfunction in severe cases of malaria. Despite progress in understanding malaria pathogenesis, the molecular mechanisms responsible for the dramatic loss of deformability of Pf-infected RBCs have remained elusive. By recourse to a coarse-grained (CG) model that captures the molecular structures of Pf-infected RBC membrane, here we show that nanoscale surface protrusions, known as “knobs,” introduce multiple stiffening mechanisms through composite strengthening, strain hardening, and knob density-dependent vertical coupling. On one hand, the knobs act as structural strengtheners for the spectrin network; on the other, the presence of knobs results in strain inhomogeneity in the spectrin network with elevated shear strain in the knob-free regions, which, given its strain-hardening property, effectively stiffens the network. From the trophozoite to the schizont stage that ensues within 24–48 h of parasite invasion into the RBC, the rise in the knob density results in the increased number of vertical constraints between the spectrin network and the lipid bilayer, which further stiffens the membrane. The shear moduli of Pf-infected RBCs predicted by the CG model at different stages of parasite maturation are in agreement with experimental results. In addition to providing a fundamental understanding of the stiffening mechanisms of Pf-infected RBCs, our simulation results suggest potential targets for antimalarial therapies.The most virulent human malaria parasite, Plasmodium falciparum (Pf), causes ∼700,000 deaths each year (1, 2). Following entry into red blood cells (RBCs), the parasite matures through the ring (0–24 h), trophozoite (24–36 h), and schizont stages (40–48 h). This intraerythrocyte maturation is accompanied by striking changes in the surface topography and membrane architecture of the infected RBC (3–5). A notable modification is the formation of nanoscale protrusions, commonly known as knobs, at the RBC surface during the second half (24–48 h) of the asexual cycle. These protrusions mainly comprise the knob-associated histidine-rich protein (KAHRP) and the membrane-embedded cytoadherence protein, Pf-erythrocyte membrane protein 1 (PfEMP1). KAHRP binds to the fourth repeat unit of the spectrin α-chain, to ankyrin, to spectrin–actin–protein 4.1 complexes, and to the cytoplasmic domain of PfEMP1 (6–9). These attachments enhance the vertical coupling between the lipid bilayer and the spectrin network. Another striking modification in the Pf-infected RBC membrane is the reorganization of the cytoskeletal network caused by parasite-induced actin remodeling (10). As a result of these molecular-level modifications, the Pf-infected RBC exhibits markedly increased stiffness [the shear modulus increases on average from ∼4−10 µN/m in normal/uninfected RBCs, to ∼40 µN/m at the trophozoite stage, and to as high as 90 µN/m at the schizont stage (11–13)] and cytoadherence to the vascular endothelium, which enable sequestration from circulation in vasculature, and evasion from the surveillance mechanisms of the spleen. Although in vitro experimental studies have revealed roles of particular parasite-encoded proteins in remodeling the host RBC (14–22), the mechanism by which Pf-infected RBCs gain dramatically increased stiffness has remained unclear. Indeed, uncertainty remains as to whether the loss of deformability arises from the structural reorganization of the host membrane components or from the deposition of parasite proteins. That is, it is not clear whether the stiffening is due to remodeling of the spectrin network, or to the formation of the knobs, or both. As experimental studies alone have heretofore not been able to determine the molecular details, numerical modeling, combined with a variety of experimental observations and measurements, offers an alternative approach to reveal the underlying mechanisms.We present here a coarse-grained (CG) molecular dynamics (MD) RBC membrane model to correlate structural modifications at the molecular ultrastructure level with the shear responses of the Pf-infected RBC membrane, focusing on the second half of the parasite’s intra-RBC asexual cycle (24–48 h), i.e., the trophozoite and schizont stages. The CG model is computationally efficient, and able to capture the molecular structures of the RBC membrane in both normal and infected states. CGMD simulations reveal that spectrin network remodeling accounts for a relatively small change in shear modulus. Instead, the knobs stiffen the membrane by multiple mechanisms, including composite strengthening, strain hardening, and knob density-dependent vertical coupling. Our findings provide molecular-level understanding of the stiffening mechanisms operating in Pf-infected RBCs and shed light on the pathogenesis of falciparum malaria.     

Levamisole inhibits sequestration of infected red blood cells in patients with falciparum malaria

BACKGROUND: Sequestration of infected red blood cells (iRBCs) in the microcirculation is central to the pathophysiology of falciparum malaria. It is caused by cytoadhesion of iRBCs to vascular endothelium, mediated through the binding of Plasmodium falciparum erythrocyte membrane protein-1 to several endothelial receptors. Binding to CD36, the major vascular receptor, is stabilized through dephosphorylation of CD36 by an alkaline phosphatase. This is inhibited by the alkaline phosphatase-inhibitor levamisole, resulting in decreased cytoadhesion. METHODS: Patients with uncomplicated falciparum malaria were randomized to receive either quinine treatment alone or treatment with a single 150-mg dose of levamisole as an adjunct to quinine. Peripheral blood parasitemia and parasite stage distribution were monitored closely over time. RESULTS: Compared with those in control subjects, peripheral blood parasitemias of mature P. falciparum parasites increased during the 24 h after levamisole administration (n=21; P=.006). The sequestration ratio (between observed and expected peripheral blood parasitemia) of early trophozoite and midtrophozoite parasites increased after levamisole treatment, with near complete prevention of early trophozoite sequestration and >65% prevention of midtrophozoite sequestration. CONCLUSION: These findings strongly suggest that levamisole decreases iRBC sequestration in falciparum malaria in vivo and should be considered as a potential adjunctive treatment for severe falciparum malaria. TRIAL REGISTRATION: Current Controlled Trials identifier: 15314870.     

Transport of the essential nutrient isoleucine in human erythrocytes infected with the malaria parasite Plasmodium falciparum

The intraerythrocytic malaria parasite derives much of its requirement for amino acids from the digestion of the hemoglobin of its host cell. However, one amino acid, isoleucine, is absent from adult human hemoglobin and must therefore be obtained from the extracellular medium. In this study we have characterized the mechanisms involved in the uptake of isoleucine by the intraerythrocytic parasite. Under physiologic conditions the rate of transport of isoleucine into human erythrocytes infected with mature trophozoite-stage Plasmodium falciparum parasites is increased to approximately 5-fold that in uninfected cells, with the increased flux being via the new permeability pathways (NPPs) induced by the parasite in the host cell membrane. Transport via the NPPs ensures that protein synthesis is not rate limited by the flux of isoleucine across the erythrocyte membrane. On entering the infected erythrocyte, isoleucine is taken up into the parasite via a saturable, ATP-, Na+-, and H+-independent system which has the capacity to mediate the influx of isoleucine in exchange for leucine (liberated from hemoglobin). The accumulation of radiolabeled isoleucine within the parasite is mediated by a second (high-affinity, ATP-dependent) mechanism, perhaps involving metabolism and/or the concentration of isoleucine within an intracellular organelle.     

Characterization and localization of Plasmodium falciparum surface antigens on infected erythrocytes from west African patients

The malaria-induced surface antigens on Plasmodium falciparum-infected erythrocytes from West African patients were characterized by agglutination of infected cells by human sera, surface immunofluorescence of live infected cells, inhibition of cytoadherence to C32 melanoma cells by human sera, immunoelectron microscopy (immunoEM), and immunoprecipitation. In a nonimmune individual, serum antibody reactivity to surface antigens of infected cells was acquired during convalescence, as tested by all five methods, and was generally parasite isolate-specific. By contrast, adult hyperimmune West African sera reacted with many isolates, including isolates from geographically distinct regions. A quantitative correlation was established between agglutination and surface immunofluorescence assay titers, and between surface immunofluorescence assay and immunoEM reactivity, suggesting that a single antigen or a set of coexpressed antigens is being detected. Surface iodination of infected cells identified trypsin-sensitive high M, antigens in the sodium dodecyl sulfate extract. All sera tested that agglutinated infected cells also immunoprecipitated these antigens. The same surface antigens were immunoprecipitated by the homologous convalescent serum as by adult sera. By immunoEM these antigens were localized exclusively at the knob-like protrusions of infected cells, where they may participate in adherence to vascular endothelium.     

Immunoelectron microscopic localization of vivax malaria antigens to the clefts and caveola-vesicle complexes of infected erythrocytes

Erythrocytes infected with Plasmodium vivax show unique ultrastructural changes which include membranous structures in the host cell cytosol, called clefts, and caveola-vesicle complexes (CVC) in the infected erythrocyte membrane. It has been suggested that the latter structures correspond with the Schuffner's dots observed on Giemsastained thin films. The subcellular localization of a 28 kDa and a 95 kDa antigen of the erythrocytic stages of P. vivax was determined by post-embedding immunoelectron microscopy. Four monoclonal antibodies (MAbs) (2H12.B4,2H8.E10, 1H4.B6, and 4C12.G4) against the 95 kDa protein reacted with the vesicles of CVC and vesicles scattered in the cytoplasm of the infected erythrocytes. Two other MAbs (4C12.B10 and 4D7.B1) against a 28 kDa protein reacted with the cytoplasmic clefts and were also reactive with the vesicles and electron dense materials in parasitophorous vacuole. These parasite-induced structures make a contribution to the movement of some malaria proteins from the parasite to the erythrocyte surface.     

Serum protein concentrations in Plasmodium falciparum malaria.

In patients with uncomplicated Plasmodium falciparum infection cytokine-mediated serum protein levels of C-reactive protein (CRP), coeruloplasmin (COE), beta 2-microglobulin (B2M), alpha 1-acid glycoprotein (AAG), alpha 1-antitrypsin (AAT), haptoglobin (HPT), prealbumin (PRE), retinol binding protein (RBP), albumin (ALB) and transferrin (TRF) were measured in an endemic area of the Amazonian rain forest. Semi-immune (SI) and nonimmune (NI) patients were investigated. In both patient groups the serum concentrations of CRP, COE and B2M were elevated on admission. In addition AAG and AAT concentrations were increased in NI patients compared to control subjects. Significantly lower serum concentrations of HPT, PRE, RBP, ALB and TRF were seen in both patient groups during the acute phase of the disease, and were more pronounced in NI patients. After a 28-day follow-up, AAT and B2M were normal in SI patients but HPT, AAT and B2M were still significantly altered in NI patients.     

Transfection of Plasmodium falciparum within human red blood cells.

Plasmodium falciparum malaria parasites within human red blood cells (RBCs) have been successfully transfected to produce chloramphenicol acetyltransferase (CAT). Electroporation of parasitized RBCs was used to introduce plasmids that have CAT-encoding DNA flanked by 5' and 3' untranslated sequences of the P. falciparum hsp86, hrp3, and hrp2 genes. These flanking sequences were required for expression as their excision abolished CAT activity in transfected parasites. Transfection signals from native CAT-encoding DNA compared well with those from a synthetic DNA sequence adapted to the P. falciparum major codon bias, demonstrating effective expression of the bacterial sequence despite its use of rare P. falciparum codons. Transfected ring-stage parasites produced CAT signals at least as strong as transfected schizont-stage parasites even though ring stages are surrounded by more RBC cytoplasm than schizonts. The transfection of erythrocyte-stage P. falciparum parasites advances our ability to pursue genetic analysis of this major pathogen.     

The enzymes of the glycolytic pathway in erythrocytes infected with Plasmodium falciparum malaria parasites

Enzymes of the glycolytic pathway as well as some ancillary enzymes were studied in normal red cells parasitized with Plasmodium falciparum in culture at varying parasitemias as well as in isolated parasites. The levels of all enzymes except diphosphoglycerate mutase, glucose-6- phosphate dehydrogenase, and adenylate kinase were elevated. Extreme elevations of hexokinase, aldolase, enolase, pyruvate kinase, and adenosine deaminase concentrations were noted. In most cases, electrophoretically distinct bands of enzyme activity were also seen. These findings partly explain the previously noted 50- to 100-fold increase in glucose consumption of infected red cells and suggest that further knowledge of these parasite enzymes and their genetic basis may aid both in designing new chemotherapy and in understanding the evolution of these parasites.     

Human natural killer cells control Plasmodium falciparum infection by eliminating infected red blood cells

Immunodeficient mouse–human chimeras provide a powerful approach to study host-specific pathogens, such as Plasmodium falciparum that causes human malaria. Supplementation of immunodeficient mice with human RBCs supports infection by human Plasmodium parasites, but these mice lack the human immune system. By combining human RBC supplementation and humanized mice that are optimized for human immune cell reconstitution, we have developed RBC-supplemented, immune cell-optimized humanized (RICH) mice that support multiple cycles of P. falciparum infection. Depletion of human natural killer (NK) cells, but not macrophages, in RICH mice results in a significant increase in parasitemia. Further studies in vitro show that NK cells preferentially interact with infected RBCs (iRBCs), resulting in the activation of NK cells and the elimination of iRBCs in a contact-dependent manner. We show that the adhesion molecule lymphocyte-associated antigen 1 is required for NK cell interaction with and elimination of iRBCs. Development of RICH mice and validation of P. falciparum infection should facilitate the dissection of human immune responses to malaria parasite infection and the evaluation of therapeutics and vaccines.Malaria is caused by infection with parasites of the Plasmodium species which are transmitted by bites of infected Anopheles mosquitoes. Plasmodium species are highly host specific. making it difficult to model human parasite infection in laboratory animals. So far, most in vivo experimental studies of malaria have been carried out with mouse and rat Plasmodium strains in rodents. Differences in invasion and disease pathology between human and rodent parasite species have impeded the translation of findings from rodents into human. The lack of appropriate small animal models also has hampered the evaluation of new drugs and vaccines before clinical trials (1).To overcome this challenge, one approach is to supplement SCID mice with human RBCs. The resulting mice support a limited blood-stage P. falciparum infection (2–4). The need to inject large volumes of human RBCs repeatedly and to treat mice with anti-neutrophil antibody and highly toxic clodronate liposomes to suppress the rapid clearance of the injected human RBCs by macrophages in the recipient mice makes working with this system difficult. More recently NOD-SCID Il2rg^−/− (NSG) mice have been shown to support a more efficient P. falciparum infection without the treatment of clodronate liposomes or anti-neutrophil antibody (5). Furthermore, a recent report shows the development of liver-stage P. falciparum infection in immunocompromised and fumarylacetoacetate hydrolase-deficient (Fah^−/−, Rag2^−/−, Il2rg^−/−) (FRG) mice. Backcrossing of FRG mice to the NOD background and supplementing the resulting mice with human RBCs led to reproducible transition from liver-stage infection to blood-stage infection (6). Despite such progress, none of the existing mouse models of human parasite infection has a human immune system.The immune system plays a critical role in the control of parasite infection. Studies in mice using mouse Plasmodium strains have shown that mouse immune cells such as natural killer (NK) cells, T cells, dendritic cells, and B cells all contribute to antiparasitic immunity (7–10). Notably, depletion of NK cells in a mouse model of Plasmodium chabaudi infection results in more severe disease associated with higher parasitemia and mortality (11). In vitro, P. falciparum-infected human RBCs are shown to interact with human NK cells, leading to the induction of IFN-γ (12). Compared with P. falciparum schizonts, live infected RBCs (iRBCs) induce more rapid activation and more production of IFN-γ by NK cells (13). More recently, it has been shown that, in addition to IFN-γ, activated human NK cells also produce perforin and granzyme against P. falciparum-infected RBCs (14). However, because of the lack of appropriate models, little is known about the role of human NK cells in the control of P. falciparum infection in vivo. NK cells are cytolytic and can lyse virus-infected cells and tumor cells (15). However, whether NK cells also can eliminate parasite-infected RBCs directly has not been demonstrated comprehensively.In our study of humanized mice, we previously had developed a simple and effective method of enhancing human cell reconstitution by hydrodynamic expression of human cytokines. Expression of human IL-15 and Flt-3/Flk-2 ligand (Flt-3L) enhances the reconstitution of human NK cells, monocytes, and macrophages (16). In this study, we have constructed humanized mice that have an optimized human immune cell reconstitution as well as high levels of human RBCs through supplementation. We show that such humanized mice support an efficient infection by P. falciparum. Depletion of human NK cells, but not macrophages, in these mice results in a significant increase in parasitemia. Our additional studies in vitro show that NK cells interact preferentially with iRBCs and become activated, resulting in the elimination of iRBCs in a contact-dependent manner. We further show that the cell adhesion molecules lymphocyte-associated antigen 1 (LFA-1) and to some extent DNAX accessory molecule 1 (DNAM-1) mediate NK cell interaction with and elimination of iRBCs. Development of humanized mice with robust reconstitution of human immune cells and human RBCs and validation of the model for P. falciparum infection should facilitate the dissection of human immune responses to malaria parasite infection and the evaluation of therapeutics and vaccines.     

Effect of plasmodial RESA protein on deformability of human red blood cells harboring Plasmodium falciparum

During intraerythrocytic development, Plasmodium falciparum exports proteins that interact with the host cell plasma membrane and subplasma membrane-associated spectrin network. Parasite-exported proteins modify mechanical properties of host RBCs, resulting in altered cell circulation. In this work, optical tweezers experiments of cell mechanical properties at normal physiological and febrile temperatures are coupled, for the first time, with targeted gene disruption techniques to measure the effect of a single parasite-exported protein on host RBC deformability. We investigate Pf155/Ring-infected erythrocyte surface antigen (RESA), a parasite protein transported to the host spectrin network, on deformability of ring-stage parasite-harboring human RBCs. Using a set of parental, gene-disrupted, and revertant isogenic clones, we found that RESA plays a major role in reducing deformability of host cells at the early ring stage of parasite development, but not at more advanced stage. We also show that the effect of RESA on deformability is more pronounced at febrile temperature, which ring-stage parasite-harboring RBCs can be exposed to during a malaria attack, than at normal body temperature.     

Granulocyte chemotaxis in acute human Plasmodium falciparum malaria

The non-lymphoid elements of the peripheral leucocyte pool were examined in the present study to determine their response to chemotactic stimulation. Our results indicate that granulocytes are effectively mobilized during malaria infections and are not deactivated by complement-derived chemotactic factors. These findings provide further evidence for the restriction of immunosuppression to some specific T and B-cell related functions only.     

Lymphocyte response to purified Plasmodium falciparum antigens during and after malaria

The peripheral blood lymphocyte response to affinity purified soluble Plasmodium falciparum antigens from in vitro cultures was studied in seven patients with acute falciparum malaria, on eight occasions, and in 15 persons having had malaria, at various times post infection, on 24 occasions. During infection, the response was low or absent in most patients (median stimulating index = [SI] = 1.4). One week post infection, a specific antigen response rose (SI = 2.9), but not to the levels found two weeks to one year post infection (SI = 5.8). At two to four years post infection, it was still present. During a recrudescence of malaria in a single patient, it was lost temporarily. The response to optimal concentrations of lectin mitogens and to tuberculin antigen was not suppressed in acute malaria.     

Haemozoin detection in infected erythrocytes for Plasmodium falciparum malaria diagnosis-prospects and limitations

Several methods based on the detection of the parasite-specific pigment haemozoin (Hz) in blood are currently being investigated as alternative diagnostic methods for malaria. Although this approach may appear attractive, the fact that in Plasmodium falciparum (P. f.) malaria, the severity of which should give it the highest diagnostic priority, the fact that most circulating intra-erythrocytic P. f. parasites contain little or no Hz raises some concern. We used flow cytometry to investigate the possibilities and limitations of the detection of intra-erythrocytic Hz in malaria infected patient blood samples and in vitro cultures. However, reliable detection of ring-forms or young trophozoites of P. f. parasites could not be achieved, although one-quarter of mature parasites could be detected after 24-48 h in culture. Our results strongly suggest that, although it may be useful for monitoring maturation, detection of intra-erythrocytic Hz by flow cytometry will not provide an optimal method for diagnosis of P. falciparum malaria.     

Antigen-specific immunosuppression in human malaria due to Plasmodium falciparum

Proliferative responses of T lymphocytes to antigens specific and not specific for malaria were investigated in 32 adult patients in eastern Thailand during acute infection with Plasmodium falciparum malaria and during their convalescence. Immune unresponsiveness to malarial antigen, which persisted for more than four weeks in 37.5% of the individuals, was present in all patients, irrespective of parasitemia or severity of clinical illness. Suppression of responses to nonspecific antigens was less profound and observed only in patients with moderately severe or cerebral malaria. The depressed functional responses were associated with a loss of T lymphocytes--both helper and suppressor subsets--from the peripheral blood; these responses were recovered once parasites were cleared. These results indicate that blood-stage plasmodial infections may suppress responses important for immunity to malaria and so allow the parasite to survive. They further suggest that patients acutely or even recently infected with P. falciparum may not respond as well to a malaria vaccine as would uninfected individuals.