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

Department of Infectious Diseases, Parasitology, Heidelberg University, 69120 Heidelberg, Germany.
Science (Impact Factor: 33.61). 12/2011; 334(6060):1283-1286. DOI: 10.1126/science.1213775

ABSTRACT 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.

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Available from: Friedrich Frischknecht, Jun 01, 2014
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    • "Consistent with this, a cytosolic actin population has been visualized via immunogold labeling of sections of intact RBCs (Cyrklaff et al., 2011). Second, it has been shown that infection of human RBCs with the malaria-causing parasite Plasmodium falciparum results in dramatic remodeling of RBC actin filaments into an aberrantly branched network in the cytosol to facilitate export of virulence factors (Cyrklaff et al., 2011; Rug et al., 2014). Presumably, such reorganization requires dynamic disassembly/reassembly of actin filaments. "
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    ABSTRACT: Short, uniform-length actin filaments function as structural nodes in the spectrin-actin membrane skeleton to optimize the biomechanical properties of red blood cells (RBCs). Despite the widespread assumption that RBC actin filaments are not dynamic (i.e., do not exchange subunits with G-actin in the cytosol), this assumption has never been rigorously tested. Here, we show that a subpopulation of human RBC actin filaments is indeed dynamic based on rhodamine-actin incorporation into filaments in resealed ghosts, and by fluorescence recovery after photobleaching (FRAP) analysis of actin filament mobility in intact RBCs (∼25-30% of total filaments). Cytochalasin-D inhibition of barbed-end exchange reduces rhodamine-actin incorporation and partially attenuates FRAP recovery, indicating functional interaction between actin subunit turnover at the single-filament level and mobility at the membrane-skeleton level. Moreover, perturbation of RBC actin filament assembly/disassembly with latrunculin-A or jasplakinolide induces a ∼twofold increase or a ∼60% decrease, respectively, in soluble actin, resulting in altered membrane deformability, as determined by alterations in RBC transit time in a microfluidic channel assay, as well as abnormalities in spontaneous membrane oscillations (flickering). These experiments identify a heretofore-unrecognized but functionally important subpopulation of RBC actin filaments, whose properties and architecture directly control the biomechanical properties of the RBC membrane. © 2015 by The American Society for Cell Biology.
    Molecular Biology of the Cell 02/2015; 26(9). DOI:10.1091/mbc.E14-12-1583 · 4.47 Impact Factor
    • "This hypothesis of poor attachment is consistent with the low number and very abbreviated nature of filamentous extensions from the few MC that could be found in our images of the AlF4 − -treated parasitized erythrocytes. The remodeling evident in our images of unroofed P. falciparuminfected erythrocytes also agrees with the cryoelectrontomography findings of Cyrklaff et al. (2011), who described altered host actin filaments in parasitized erythrocytes and connections of the MC by cytochalasin D-sensitive actin tethers to the interior of the erythrocyte membrane, and with the image reconstructions of Hanssen et al. (2008b), which showed small vesicles in association with tethered MC and extensions to the erythrocyte membrane. The observed dimensions of 100–650 nm for MC in unroofed preparations are in good agreement with the reported range 200–500 nm for MC at the periphery of the P. falciparum-infected erythrocytes in those reconstructions (Hanssen et al., 2010). "
    Biophysical Journal 01/2015; 108(2):618a. DOI:10.1016/j.bpj.2014.11.3365 · 3.97 Impact Factor
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    • "In P. falciparum trophozoite-stage iRBCs, actin is remodeled to allow controlled trafficking of cargo vesicles important for functioning of Maurer's clefts and knobs [104], and can be oxidized [35] [101] [102]. Blood group O-derived hemoglobin variants somehow interfere with this remodeling and the establishment of a parasite-directed actin cytoskeleton in the infected cells [105]. In a rat model of oxidative stress, after use of x-irradiation to induce reactive oxygen species, actin was extensively oxidized, with partial oxidation of methionines including met-82 sulfone, oxidation of two of four tryptophans , and oxidation of several cysteines [106]. "
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    ABSTRACT: Unlabelled: Plasmodium vivax is the causative infectious agent of 80-300 million annual cases of malaria. Many aspects of this parasite's biology remain unknown. To further elucidate the interaction of P. vivax with its Saimiri boliviensis host, we obtained detailed proteomes of infected red blood cells, representing the trophozoite-enriched stage of development. Data from two of three biological replicate proteomes, emphasized here, were analyzed using five search engines, which enhanced identifications and resulted in the most comprehensive P. vivax proteomes to date, with 1375 P. vivax and 3209 S. boliviensis identified proteins. Ribosome subunit proteins were noted for both P. vivax and S. boliviensis, consistent with P. vivax's known reticulocyte host-cell specificity. A majority of the host and pathogen proteins identified belong to specific functional categories, and several parasite gene families, while 33% of the P. vivax proteins have no reported function. Hemoglobin was significantly oxidized in both proteomes, and additional protein oxidation and nitration was detected in one of the two proteomes. Detailed analyses of these post-translational modifications are presented. The proteins identified here significantly expand the known P. vivax proteome and complexity of available host protein functionality underlying the host-parasite interactive biology, and reveal unsuspected oxidative modifications that may impact protein function. Biological significance: Plasmodium vivax malaria is a serious neglected disease, causing an estimated 80 to 300 million cases annually in 95 countries. Infection can result in significant morbidity and possible death. P. vivax, unlike the much better-studied Plasmodium falciparum species, cannot be grown in long-term culture, has a dormant form in the liver called the hypnozoite stage, has a reticulocyte host-cell preference in the blood, and creates caveolae vesicle complexes at the surface of the infected reticulocyte membranes. Studies of stage-specific P. vivax expressed proteomes have been limited in scope and focused mainly on pathogen proteins, thus limiting understanding of the biology of this pathogen and its host interactions. Here three P. vivax proteomes are reported from biological replicates based on purified trophozoite-infected reticulocytes from different Saimiri boliviensis infections (the main non-human primate experimental model for P. vivax biology and pathogenesis). An in-depth analysis of two of the proteomes using 2D LC/MS/MS and multiple search engines identified 1375 pathogen proteins and 3209 host proteins. Numerous functional categories of both host and pathogen proteins were identified, including several known P. vivax protein family members (e.g., PHIST, eTRAMP and VIR), and 33% of protein identifications were classified as hypothetical. Ribosome subunit proteins were noted for both P. vivax and S. boliviensis, consistent with this parasite species' known reticulocyte host-cell specificity. In two biological replicates analyzed for post-translational modifications, hemoglobin was extensively oxidized, and various other proteins were also oxidized or nitrated in one of the two replicates. The cause of such protein modification remains to be determined but could include oxidized heme and oxygen radicals released from the infected red blood cell's parasite-induced acidic digestive vacuoles. In any case, the data suggests the presence of distinct infection-specific conditions whereby both the pathogen and host infected red blood cell proteins may be subject to significant oxidative stress.
    Journal of Proteomics 12/2014; 115. DOI:10.1016/j.jprot.2014.12.010 · 3.89 Impact Factor
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