Hemoglobins S and C Interfere with Actin Remodeling in Plasmodium falciparum–Infected Erythrocytes

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Science  02 Dec 2011:
Vol. 334, Issue 6060, pp. 1283-1286
DOI: 10.1126/science.1213775


The hemoglobins S and C protect carriers from severe Plasmodium falciparum malaria. Here, we found that these hemoglobinopathies affected the trafficking system that directs parasite-encoded proteins to the surface of infected erythrocytes. Cryoelectron tomography revealed that the parasite generated a host-derived actin cytoskeleton within the cytoplasm of wild-type red blood cells that connected the Maurer’s clefts with the host cell membrane and to which transport vesicles were attached. The actin cytoskeleton and the Maurer’s clefts were aberrant in erythrocytes containing hemoglobin S or C. Hemoglobin oxidation products, enriched in hemoglobin S and C erythrocytes, inhibited actin polymerization in vitro and may account for the protective role in malaria.

The malaria parasite Plasmodium falciparum has exerted a selective pressure on the human population that has led to the emergence of several polymorphisms within the human genome that protect carriers from severe malaria-related disease and death (1). The best-known examples are the structural hemoglobinopathies S (sickle cell trait; HbS) and C (HbC), in which glutamate at the sixth position within the β-globin chain is replaced by valine and lysine, respectively (2, 3). Protection against severe malaria correlates with a distorted display of parasite-encoded adhesins on the surface of infected erythrocytes (4, 5). By reducing the cytoadhesive capacity of parasitized erythrocytes, HbS and HbC seem to mitigate the life-threatening complications resulting from the sequestration of infected erythrocytes in postcapillary microvessels of the brain and other organs. How HbS and HbC bring about this effect is unclear. We tested the hypothesis that HbS and HbC interfere with the machinery that directs parasite-encoded proteins to the erythrocyte surface.

Human erythrocytes lack a secretory system and are rapidly cleared from circulation by the spleen when damaged or infected. To develop within human erythrocytes and to avoid passage through the spleen, P. falciparum extensively modifies its host cell (6), for example, by placing the disease-mediating immunovariant adhesin PfEMP1 in knob-like protrusions in the erythrocyte plasma membrane (7). To direct PfEMP1 and other determinants of virulence and pathology to the erythrocyte’s plasma membrane, the parasite establishes a trafficking system within the cytoplasm of its host cell, of which a prominent feature are Maurer’s clefts, unilamellar membrane profiles that serve as intermediary compartments for proteins en route to the erythrocyte surface (810). To better define the elements of this machinery and how they are altered when P. falciparum develops in HbS and HbC erythrocytes, we applied electron tomography to parasitized erythrocytes preserved by rapid freezing (fig. S1).

We initially investigated P. falciparum–infected erythrocytes (at the trophozoite stage, 20 to 26 hours after invasion) containing the wild-type hemoglobin HbA (homozygous). The tomograms revealed the erythrocyte plasma membrane, the knobs, and the Maurer’s clefts (Fig. 1A). In addition, we observed an extended network of long, sometimes branched filaments that connected the Maurer’s clefts with the knobs. Of the 20 knobs identified in 12 tomograms, all were connected to Maurer’s clefts by filaments. The filaments were between 40 and 950 nm long (Fig. 1B) and 6.8 ± 0.5 nm in diameter (Fig. 1C). Some of the filaments branched at main angles of 70° ± 5° and 110° ± 5° (Fig. 1D).

Fig. 1

Structural features in the cytoplasm of P. falciparum–infected erythrocytes. (A) Section through a cryoelectron tomogram (left) and corresponding surface-rendered view (right) of a trophozoite-infected HbAA erythrocyte. Labeling and color code in all figures: PM, erythrocyte plasma membrane (dark blue); K, knobs (red); V, vesicles (cyan); MC, Maurer’s clefts (cyan); filaments (yellow) indicated by arrowheads. (B) Frequency histogram of filament lengths in infected (filled circles) and uninfected (open circles) erythrocytes containing HbAA, HbSC, and HbCC. T, trophozoite; R, ring. MC, close to (+), or far from (–), Maurer’s clefts. n > 150 filaments in each case. **P < 0.001 compared with uninfected HbAA erythrocytes (Student’s t test). (C) Mean densitogram across 30 filaments. Dotted lines indicate width of filament. (D) Distribution of branching angles in filaments in P. falciparum–infected erythrocytes containing HbAA (blue, close to; brown, far from Maurer’s clefts), HbSC (gray), HbCC (green). (E) Filament-vesicle contacts, indicated by arrowheads. (F) Different planes of a tomogram of a “Tokuyasu” section and surface rendered view, showing immunolabeling of vesicles with PfEMP1 antibodies (red, 10-nm gold). Scale bars: (A) and (F), 100 nm; (E), 50 nm.

The tomograms further revealed vesicles of various sizes, ranging from 20 nm to more than 200 nm in diameter (fig. S2). About 70% of the observed vesicles (47 out of 68) were attached to filaments (Fig. 1E), independent of their size. The remaining vesicles were associated with Maurer’s clefts or appeared free in the erythrocyte cytosol. Some vesicles carried PfEMP1 (Fig. 1F) (9, 11).

Tomograms taken in areas more than 5 μm distant from Maurer’s clefts also revealed vesicles and a filamentous network (Fig. 2A). However, these filaments were shorter than those observed in the vicinity of Maurer’s clefts (Fig. 1B). Moreover, the two main branching angles of the filaments were equally distributed, whereas close to Maurer’s clefts the filaments preferentially branched at an angle of 70° ± 5° in the direction of the Maurer’s clefts (Fig. 1D).

Fig. 2

Organization of host actin in infected and uninfected HbAA erythrocytes. Tomograms of trophozoite-infected erythrocytes: (A) distant from Maurer’s clefts; (B) after treatment with cytochalasin D (1 μM). (C) Immuno-EM of infected erythrocytes, using a gold-coupled monoclonal antibody to β-actin (see also fig. S3A). Tomograms of uninfected (D) and ring-stage–infected (E) HbAA erythrocytes. Cyan, vesicles and early-stage Maurer’s clefts. Scale bars, 100 nm.

The filaments had features reminiscent of actin filaments, including diameter and branching pattern (Fig. 1, C and D) (12). Indeed, treating P. falciparum–infected erythrocytes (trophozoites) for 10 min with the actin depolymerizing agent cytochalasin D (1 μM) destroyed the filaments and altered the morphology of the Maurer’s clefts (Fig. 2B). Vesicles were also not observed under these conditions. Immunolabeling of high-pressure frozen electron microscopy (EM) sections, using a gold-coupled monoclonal antibody specific for β-actin (13), provided further evidence that the long filaments contained actin (Fig. 2C and fig. S3). We noted a higher density of actin labeling in the area between the Maurer’s clefts and the erythrocyte plasma membrane compared with areas elsewhere in the erythrocyte cytoplasm (fig. S3), supporting the conclusion that actin filaments connect the Maurer’s clefts with the erythrocyte plasma membrane (13). Ankyrin may anchor the actin filaments to the Maurer’s clefts (14).

The erythrocyte owes its shape and physical properties to a membrane skeleton that is primarily composed of spectrin tetramers joined by a junctional complex that is mainly composed of actin protofilaments (15). The length of the actin protofilaments is tightly regulated and is restricted to 14 to 16 monomers (15). Tomograms of plunge-frozen uninfected human erythrocytes revealed short filaments of 30 to 40 nm underneath the erythrocyte plasma membrane (Figs. 2D and 1B)—which in terms of length, thickness (6.8 ± 0.5 nm), and location are consistent with actin protofilaments. The spectrin filaments are significantly thinner than actin filaments (16) and were not detected in our tomograms but were visible by classical EM in negatively stained preparations of the membrane skeleton (fig. S4). The hexagonal arrangements characterizing the membrane skeleton of uninfected red blood cells (17) were not observed in membrane skeletal preparations from parasitized erythrocytes (fig. S4). These data suggest that the parasite remodels the membrane skeleton of its host cell during intra-erythrocytic development to build an actin network of its own design. Consistent with this hypothesis, the parasite-generated actin cytoskeleton in ring-stage-infected erythrocytes (10 to 14 hours after invasion) was less pronounced in areas close to Maurer’s clefts (Fig. 2E), with short filaments of 20 to 60 nm dominating (Fig. 1B) (P < 0.001), suggesting that actin remodeling occurs as the parasite develops.

We next analyzed tomograms of P. falciparum–infected erythrocytes (at the trophozoite stage) containing homozygous HbCC and heterozygous HbSC. The tomograms revealed the erythrocyte plasma membrane with knobs, vesicles of different sizes (ranging from 20 to 100 nm in diameter) (fig. S2), and large amorphous membrane conglomerates, but no membrane profiles characteristic of Maurer’s clefts (Fig. 3, A and B). However, an antiserum against the Maurer’s clefts marker PfSBP1 identified the amorphous membrane conglomerates as aberrant Maurer’s clefts (Fig. 3C). Classical EM of chemically fixed or high-pressure frozen cells confirmed that in HbSC- and HbCC-containing erythrocytes, Maurer’s clefts did not form the elaborated, multilayered membrane stacks seen in HbAA erythrocytes (fig. S5). Despite the altered morphology of Maurer’s clefts, parasites appeared to develop normally in HbSC and HbCC erythrocytes (figs. S6 and S7).

Fig. 3

Aberrant actin cytoskeleton and Maurer’s clefts in trophozoite-infected HbSC and HbCC erythrocytes. Tomograms of infected HbSC (A) and HbCC (B) erythrocytes. (C) Immunolabeling of infected HbCC erythrocyte, using an antiserum against the Maurer’s clefts marker PfSBP1. (D) Tomogram of an uninfected HbCC erythrocyte. Scale bars, 100 nm.

The tomograms further revealed actin filaments underneath the erythrocyte plasma membrane in both HbSC and HbCC erythrocytes. However, the filaments were significantly shorter than those seen in infected HbAA erythrocytes (P < 0.001) (Fig. 1B), and the two main branching angles were equally distributed (Fig. 1D). Only a few actin filaments (8 of 150 in nine tomograms) were attached to the degenerated Maurer’s clefts, and they did not interconnect them with the knobs. Only 15% (6 out of 40) of the vesicles observed were attached to filaments; 85% were free in the cytoplasm.

Erythrocytes containing HbS and HbC have a dysfunctional cytoskeleton among other abnormalities (18), which we confirmed in uninfected HbCC erythrocytes by cryoelectron tomography (compare Figs. 2D and 3D). The actin filaments appeared less confined to the volume underneath the erythrocyte plasma membrane (Fig. 3D) and were significantly longer (P < 0.001) than those in uninfected HbAA erythrocytes (Fig. 1B).

These changes in actin organization result from endogenous factors and may involve oxidized forms of hemoglobin (19). Both HbS and HbC are unstable and readily oxidize to methemoglobin and finally to hemichromes, which accumulate more than 10-fold over normal levels (18, 20). Hemoglobin and methemoglobin have a stabilizing effect on the membrane skeleton by promoting the self-association of spectrin dimers to tetramers (19, 21), whereas hemichromes can destabilize the erythrocyte membrane skeleton by decreasing the spectrin-protein 4.1-actin interaction (19). Thus, oxidized hemoglobins might interfere with the actin reorganization brought about by the parasite in HbS and HbC erythrocytes. Consistent with this hypothesis, shorter actin filaments were formed in in vitro actin polymerization assays in the presence of total protein extracts (5 μM) from HbSC and HbCC erythrocytes compared with extracts from HbAA erythrocytes (P < 0.001) (Fig. 4A). This inhibitory effect could be reproduced by ferryl hemoglobin, but not by hemoglobin or methemoglobin, and was dependent on the percentage of ferryl hemoglobin of total hemoglobin (Fig. 4, B and C, and fig. S8). Ferryl hemoglobin is readily formed in human erythrocytes under conditions of oxidative stress (22, 23), as occurs in HbS and HbC erythrocytes (18). Ferryl hemoglobin is also known to oxidize actin and to alter actin filament dynamics (24).

Fig. 4

Inhibition of actin polymerization in vitro. (A) Actin filament length in the presence of total protein extracts from erythrocytes containing HbAA, HbSC, and HbCC. (B) Effect of 5 μM of hemoglobin (Hb), methemoglobin (met-Hb), and ferryl hemoglobin (ferryl-Hb) on actin filament length. (C) Concentration-dependent decrease of actin filament length by ferryl hemoglobin. The percentage of ferryl hemoglobin of total hemoglobin (5 μM) is indicated. The means ± SEM of at least four independent experiments are shown. **P < 0.01 compared with HbAA extracts and hemoglobin.

Here, we have shown that P. falciparum establishes an actin cytoskeleton of its own design within the host cell cytoplasm. The parasite’s own actin seems not to play a role in this process because Plasmodium actin is not known to be exported into the erythrocyte cytoplasm (25). Instead, the parasite seems to establish this actin cytoskeleton by mining the actin and, possibly, other components from the erythrocyte membrane skeleton. The parasite-organized actin cytoskeleton connects the Maurer’s clefts with the knobs, defines the morphology of the Maurer’s clefts, and may facilitate the movement of cargo vesicles from the Maurer’s clefts to the erythrocyte plasma membrane.

The predicted secretome of P. falciparum (26, 27) is expected to contain actin regulatory factors that help to generate and maintain the parasite-generated actin cytoskeleton. Other proteins of parasite origin may fix the actin-deprived membrane skeleton of the host cell, thereby redefining the mechanical and morphological properties of the infected erythrocyte (28, 29). Oxidized forms of hemoglobins, in particular ferryl hemoglobin, might interfere with actin remodeling, thereby preventing the parasite from creating its own actin cytoskeleton within the host cell cytoplasm. As a result, Maurer’s clefts do not properly form, vesicular transport is impaired, and the export of parasite-encoded disease-mediating adhesins to the erythrocyte surface is distorted. It seems to be through this mechanism that HbS and HbC confer their protective role against malaria.

Supporting Online Material

Materials and Methods

Figs. S1 to S8

References (3044)

References and Notes

  1. Acknowledgments: We thank S. Prior, L. Lemgruber, and D. Ouermi for technical assistance and help. We thank the Max Planck Institutes (Martinsried and Frankfurt), the European Molecular Biology Laboratory, Centre for Organismal Studies, and Bioquant (Heidelberg) for access to EM facilities. Supported by the Deutsche Forschungsgemeinschaft (research focus Host-Parasite Coevolution); the Bundesministerium für Bildung, Wissenschaft, Forschung und Technologie, and the Chica and Heinz Schaller Foundation. M.L. and F.F. are members of the Heidelberg Excellence Cluster CellNetworks and the European Network of Excellence EVIMalaR.
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