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• W2018122290 abstract "•Malaria antigens are enriched in microvesicles released from infected RBCs (RMVs)•RMV release peaks during schizogony but before parasite egress•RMVs derived from infected RBCs activate host monocytes and neutrophils•RMVs internalized by infected RBCs stimulate transmission stage parasite development Humans and mice infected with different Plasmodium strains are known to produce microvesicles derived from the infected red blood cells (RBCs), denoted RMVs. Studies in mice have shown that RMVs are elevated during infection and have proinflammatory activity. Here we present a detailed characterization of RMV composition and function in the human malaria parasite Plasmodium falciparum. Proteomics profiling revealed the enrichment of multiple host and parasite proteins, in particular of parasite antigens associated with host cell membranes and proteins involved in parasite invasion into RBCs. RMVs are quantitatively released during the asexual parasite cycle prior to parasite egress. RMVs demonstrate potent immunomodulatory properties on human primary macrophages and neutrophils. Additionally, RMVs are internalized by infected red blood cells and stimulate production of transmission stage parasites in a dose-dependent manner. Thus, RMVs mediate cellular communication within the parasite population and with the host innate immune system. Humans and mice infected with different Plasmodium strains are known to produce microvesicles derived from the infected red blood cells (RBCs), denoted RMVs. Studies in mice have shown that RMVs are elevated during infection and have proinflammatory activity. Here we present a detailed characterization of RMV composition and function in the human malaria parasite Plasmodium falciparum. Proteomics profiling revealed the enrichment of multiple host and parasite proteins, in particular of parasite antigens associated with host cell membranes and proteins involved in parasite invasion into RBCs. RMVs are quantitatively released during the asexual parasite cycle prior to parasite egress. RMVs demonstrate potent immunomodulatory properties on human primary macrophages and neutrophils. Additionally, RMVs are internalized by infected red blood cells and stimulate production of transmission stage parasites in a dose-dependent manner. Thus, RMVs mediate cellular communication within the parasite population and with the host innate immune system. Plasmodium falciparum causes more than 200 million cases of malaria and more than 1 million deaths each year (Snow et al., 2005Snow R.W. Guerra C.A. Noor A.M. Myint H.Y. Hay S.I. The global distribution of clinical episodes of Plasmodium falciparum malaria.Nature. 2005; 434: 214-217Crossref PubMed Scopus (2138) Google Scholar). Rapid asexual amplification of parasites in human red blood cells (RBCs) can result in severe and life-threatening disease, while development of sexual stages or gametocytes is required for successful parasite transmission to the mosquito vector. Here we show that microvesicles are quantitatively released by parasite-infected RBCs and transferred between parasites, regulating the production of malaria transmission stages. Microvesicles (MVs) are small vesicles (0.1–1 μm in size) that are produced by direct plasma membrane blebbing. MVs can contain proteins, RNA, and even organelles and act as messengers between cells (Skog et al., 2008Skog J. Würdinger T. van Rijn S. Meijer D.H. Gainche L. Sena-Esteves M. Curry Jr., W.T. Carter B.S. Krichevsky A.M. Breakefield X.O. Glioblastoma microvesicles transport RNA and proteins that promote tumour growth and provide diagnostic biomarkers.Nat. Cell Biol. 2008; 10: 1470-1476Crossref PubMed Scopus (3700) Google Scholar). In mammalian cells, the rate of MV release is usually low but can be increased by cell activation or apoptosis. Increased MV production by human cells has been observed in a variety of conditions, including cardiovascular disease, arthritis, and thalassemia, and tumor cells can constitutively shed a large number of MVs (Cocucci et al., 2009Cocucci E. Racchetti G. Meldolesi J. Shedding microvesicles: artefacts no more.Trends Cell Biol. 2009; 19: 43-51Abstract Full Text Full Text PDF PubMed Scopus (1409) Google Scholar). In recent studies, malaria patients infected with either P. falciparum or the related human parasite P. vivax showed elevated levels of MVs derived from platelets and RBCs (Campos et al., 2010Campos F.M. Franklin B.S. Teixeira-Carvalho A. Filho A.L. de Paula S.C. Fontes C.J. Brito C.F. Carvalho L.H. Augmented plasma microparticles during acute Plasmodium vivax infection.Malar. J. 2010; 9: 327Crossref PubMed Scopus (105) Google Scholar; Nantakomol et al., 2011Nantakomol D. Dondorp A.M. Krudsood S. Udomsangpetch R. Pattanapanyasat K. Combes V. Grau G.E. White N.J. Viriyavejakul P. Day N.P. Chotivanich K. Circulating red cell-derived microparticles in human malaria.J. Infect. Dis. 2011; 203: 700-706Crossref PubMed Scopus (119) Google Scholar). MV numbers were increased in patients suffering from severe disease and correlated with peripheral blood parasitemia. After antimalarial treatment, the level of MVs decreased rapidly and continued to decrease further between days 3 and 14 (Nantakomol et al., 2011Nantakomol D. Dondorp A.M. Krudsood S. Udomsangpetch R. Pattanapanyasat K. Combes V. Grau G.E. White N.J. Viriyavejakul P. Day N.P. Chotivanich K. Circulating red cell-derived microparticles in human malaria.J. Infect. Dis. 2011; 203: 700-706Crossref PubMed Scopus (119) Google Scholar). Flow assays using antibodies against the parasite antigen RESA, which is localized underneath the infected RBC (iRBC) membrane, have suggested that this protein is present in MVs from malaria patients (Nantakomol et al., 2011Nantakomol D. Dondorp A.M. Krudsood S. Udomsangpetch R. Pattanapanyasat K. Combes V. Grau G.E. White N.J. Viriyavejakul P. Day N.P. Chotivanich K. Circulating red cell-derived microparticles in human malaria.J. Infect. Dis. 2011; 203: 700-706Crossref PubMed Scopus (119) Google Scholar). Studies in the rodent malaria model (P. berghei) have provided evidence that MVs derived from RBCs (RMVs) induce host inflammatory responses and contribute to pathology during malaria infection. These studies have shown that RMVs are elevated during infection and that they have a potent, Toll-like receptor-mediated proinflammatory effect on macrophages (Couper et al., 2010Couper K.N. Barnes T. Hafalla J.C. Combes V. Ryffel B. Secher T. Grau G.E. Riley E.M. de Souza J.B. Parasite-derived plasma microparticles contribute significantly to malaria infection-induced inflammation through potent macrophage stimulation.PLoS Pathog. 2010; 6: e1000744Crossref PubMed Scopus (172) Google Scholar). Here we demonstrate that RMVs are quantitatively released from iRBCs during development of the human malaria parasite P. falciparum. The majority of RMVs are released very late in the asexual cycle, and they contain both human- and P. falciparum-derived proteins and other cargo. We provide evidence that RMVs are both immunostimulatory and act as messengers between iRBCs. RMVs are transferred among iRBCs and alter the production of transmission stages within a population. Our studies provide a rationale for systematic investigation of the role of RMVs in malaria pathogenesis and as a mediator of cell-cell communication during the parasite life cycle. To characterize the biogenesis, composition, and cellular targets of RMVs in the human malaria parasite P. falciparum, we used an in vitro model in human RBCs. Imaging flow cytometry analysis revealed that a large number of particles present in cell suspension are smaller than RBCs. Microscopic inspection of individual objects clearly supported cytometric classification into three distinct populations based on size differences: clusters of RBCs (“rosettes,” gate M in Figure 1A), single red blood cells and ghosts (S, probably also containing debris), and small particles that looked like vesicles in the corresponding bright field images (RMV, shown in yellow in Figure 1A). We developed a protocol for the purification of RMVs from culture supernatant (i.e., what is also referred to as parasite conditioned medium) based on differential centrifugation, filtration, and a 60% sucrose cushion (Figure S1A available online). The protocol was optimized by analysis of samples from individual purification steps with imaging flow cytometry analysis, microscopy, and western blot. Digestive vacuoles and merozoites were collected in the 3,600 g pellet as demonstrated by Giemsa staining and western blot. The 10,000 g pellet mostly contains membrane debris as suggested by the presence of spectrin and the absence of hemoglobin. The final RMV pellet is enriched in stomatin and hemoglobin (Figure S1B), and imaging flow cytometry analysis of this fraction demonstrated that the purification procedure resulted in enrichment of vesicles to >95% of all detected events (Figure 1A). To further confirm the vesicular nature of these objects, we stained them with calcein-AM and annexin V. (Figure 1B). Calcein-AM is a membrane-permeable MV marker that becomes fluorescent and trapped in the cytosol upon cleavage by esterases. RMV labeling with calcein-AM dye demonstrated that approximately 80% of events in the final fraction are positive, suggesting presence of esterase activity within RMVs. Colabeling with annexin V confirmed the vesicular nature by binding to phosphatidyl serine on the RMV surface. The final fraction of purified RMVs was analyzed by transmission electron microscopy, demonstrating vesicular shape and size in the range between 100 and 400 nm (Figure 1C). We directly observed the release of RMVs from infected red blood cells during time-lapse imaging experiments in which live parasite cultures were imaged every 2 min over 2 hr (Figure 1D and Movie S1). Imaging revealed that multiple vesicles at different stages of formation exist simultaneously in single iRBCs, suggesting significant RMV production during at least some parts of the parasite cycle. To characterize the properties and function of RMVs, we first purified vesicles from four P. falciparum strains and investigated their protein content by separation of samples on an SDS-PAGE gel followed by Coomassie staining (Figure 2A). We observed a similar protein pattern across RMV fractions derived from all parasite strains, which differed from those of uninfected control samples and from isolated parasite schizont stages (Figure 2A). To detect potential parasite proteins on RMVs, we tested pools of immune sera from malaria patients for reactivity with the same set of samples (Figure 2A). The sera were previously collected from adults in two highly endemic areas in Uganda and Tanzania, as part of the Millennium Village project. Both serum pools strongly reacted with multiple proteins in the infected RMV samples from all parasite strains analyzed, but not with any preparation from uninfected RBCs (uRBCs; Figure 2B). The pattern of reactive bands in RMVs was also different from those present in the schizont preparation. Together, these data suggest that RMVs have a distinct composition and that those derived from iRBCs additionally contain a specific set of parasite antigens. To identify the parasite and host proteins present in RMVs, we characterized purified RMV samples using mass-spectrometry-based proteomic profiling. We analyzed RMVs derived from two culture-adapted parasite strains (3D7 and CS2) and from uRBCs as a control. In all the three preparations, we found that the most abundant RBC proteins were components of RBC lipid rafts such as stomatin and band 3, as well as several carbonic anhydrases (Figure 3A and Table S1), which are known to be enriched in MVs derived from RBCs (Rubin et al., 2008Rubin O. Crettaz D. Canellini G. Tissot J.D. Lion N. Microparticles in stored red blood cells: an approach using flow cytometry and proteomic tools.Vox Sang. 2008; 95: 288-297Crossref PubMed Scopus (137) Google Scholar). To determine whether RMVs are enriched in particular classes of proteins, we stratified the hits from the proteomic analysis by Gene Ontology (GO) localization term enrichment analysis. This analysis revealed that extracellular- and vesicle-associated moieties are the most enriched in RMVs. By both total absolute peptide counts and GO localization, there was no apparent difference in RBC protein content between infected and uninfected RMVs (Figures 3A and S2). We identified more than 30 parasite proteins in the RMV preparations from 3D7 and CS2 parasite strains (Figure 3B and Table S1). These proteins mainly belong to two classes: proteins associated with RBC membranes and proteins involved in parasite invasion into RBCs (Figure 3C). The first class is represented by components of the Maurer’s clefts (SBP1, Rex1/2, MAHRP1/2, and PfMC-2TM), proteins linked to the RBC surface membrane (Clag3.1/2, RESA, and MESA), and proteins associated with the parasitophorous vacuole membrane (PVM; Exp-2 and Etramp2). The second class is represented by erythrocyte binding antigens (EBA-175 and EBA-181, which bind to glycophorins during merozoite invasion before being shed) and rhoptry proteins (RhopH2/H3 and Rap2). To further corroborate the enrichment of some of the proteins found in RMVs, we performed immunoblots of RMV samples using specific antibodies (Figure 3D). These experiments confirmed enrichment of stomatin and partial depletion of spectrin, as well as presence of the secreted parasite proteins RESA, SBP1, and, at relatively lower abundance, the PVM marker Exp-1. Importantly, we did not identify any markers for components of the parasite-induced knob complex on the iRBC surface, including KAHRP and PfEMP1, or resident parasite proteins such as the ER marker BIP. Together with the presence of RBC lipid raft proteins on RMVs, this finding implies that RMVs arise by blebbing from specific subdomains within the RBC membrane. We also consistently detected the same parasite markers across the two genetically diverse reference parasite strains, 3D7 and CS2, suggesting that the composition of RMVs is conserved in P. falciparum. To determine the distribution and orientation of host and parasite proteins within RMVs, we performed enzyme protection assays with purified RMVs. RMVs were treated with the detergent Triton X-100 (TX-100) and with either trypsin or proteinase K. These experiments demonstrated that glycophorin C is present on the RMV surface, as can be expected if RMVs bleb off the RBC surface. We found that stomatin and two parasite proteins, Exp-1 and SBP1, were protected (Figure 3E). It has been previously shown that the C-terminal tail of the Maurer’s cleft protein SBP1 faces the RBC cytoplasm, whereas the N terminus is located in the lumen (Blisnick et al., 2000Blisnick T. Morales Betoulle M.E. Barale J.C. Uzureau P. Berry L. Desroses S. Fujioka H. Mattei D. Braun Breton C. Pfsbp1, a Maurer’s cleft Plasmodium falciparum protein, is associated with the erythrocyte skeleton.Mol. Biochem. Parasitol. 2000; 111: 107-121Crossref PubMed Scopus (187) Google Scholar). The absence of any processed product in our protection assays therefore suggests that the Maurer’s cleft membrane is present within the RMV, and it independently confirms RMV integrity in the preparation. We also observed that EBA-175 and EBA-181, two of the most abundant parasite proteins identified by RMV proteomics, are efficiently digested by both trypsin and proteinase K even in the absence of TX-100. This demonstrates that the proteins are only peripherally associated with RMVs upon shedding. The large immunogenic EBA ectodomains face the inside of the microneme and would therefore be protected from enzyme digestion in the case of microneme contamination in our preparation. In conclusion, proteomic profiling demonstrates that RMVs from iRBCs contain (1) a set of enriched RBC proteins and (2) parasite antigens derived from the RBC surface and internalized membranes, in particular from the Maurer’s clefts. In other systems, microvesicles have been defined as vesicular particles with a specific density in sucrose gradients and a size range from 0.1 to 1 μm (Muralidharan-Chari et al., 2010Muralidharan-Chari V. Clancy J.W. Sedgwick A. D’Souza-Schorey C. Microvesicles: mediators of extracellular communication during cancer progression.J. Cell Sci. 2010; 123: 1603-1611Crossref PubMed Scopus (712) Google Scholar). To determine whether our preparations from iRBCs and RBCs represent homogenous populations of microvesicles, we prepared culture supernatants by ultracentrifugation and layered pellets on a continuous linear sucrose gradient. We obtained ten fractions and analyzed them for protein content by BCA and western blot analysis with the subset of parasite and host markers described above. We demonstrated that both protein content and specific RMV markers peaked in fractions 3 and 4 (Figures 4A and 4B ). These fractions represent a density of 1.221–1.198 g/cm3, which is within the range of densities for MV preparations from other cell types and is higher than exosome density (1.08–1.22 g/cm3) (Raposo et al., 1996Raposo G. Nijman H.W. Stoorvogel W. Liejendekker R. Harding C.V. Melief C.J. Geuze H.J. B lymphocytes secrete antigen-presenting vesicles.J. Exp. Med. 1996; 183: 1161-1172Crossref PubMed Scopus (2444) Google Scholar). Probing with the merozoite marker AMA-1 was negative, again confirming absence of merozoites and homogeneity of the vesicular population in the preparations. Immunoblotting demonstrated the presence of the putative resident RMV proteins EXP-1, RESA, and SBP-1, as well as stomatin and band 3 in fractions 3 to 5. To independently confirm particle abundance across the sucrose fractions and to determine whether RMVs were also homogenous in size, we analyzed RMVs using a nanoparticle tracking technology (NanoSight), which quantifies particles between 0.1 and 1 μm. This analysis confirmed that RMVs peak in sucrose fractions 3 and 4 in preparations from both RBCs and iRBCs, with the majority of RMVs between 100–150 nm in size. The iRBC preparation showed a longer tail in the size distribution, suggesting existence of an additional subset of RMVs with slightly larger size, between 150 and 250 nm, but with the same density. A similar size distribution was obtained by flow cytometry with size beads and calcein-AM staining (data not shown). To determine whether vesicles are constitutively released or whether their release is linked to a particular phase in the parasite cycle, we performed a series of kinetic experiments. We collected supernatants from highly synchronized parasite cultures every 12 hr starting at the ring stage and isolated vesicles for total particle quantification by size (using NanoSight), protein content (using BCA) and protein composition (using western blot analysis). These analyses suggested that RMV release increases steadily during the parasite cycle and peaks late during schizogony or shortly thereafter. This dynamic of release coincided with the emergence of a prominent vesicular subpopulation of 150–250 nm in the iRBC preparation only. Immunoblotting revealed concomitant peak levels of parasite RMV markers such as RESA and SBP1 and the host markers spectrin and band 3 in the vesicular fraction from late schizogony (Figure 5A). To further distinguish between vesicle release during parasite development or during egress, which can include release of parasite-derived organelles, we used two complementary approaches to either genetically or chemically inhibit parasite egress. It was recently shown that conditional knockdown of the P. falciparum calcium-dependent protein kinase 5 (PfCDPK5) results in a block of parasite egress (Dvorin et al., 2010Dvorin J.D. Martyn D.C. Patel S.D. Grimley J.S. Collins C.R. Hopp C.S. Bright A.T. Westenberger S. Winzeler E. Blackman M.J. et al.A plant-like kinase in Plasmodium falciparum regulates parasite egress from erythrocytes.Science. 2010; 328: 910-912Crossref PubMed Scopus (229) Google Scholar). Using this conditional knockdown line, we collected supernatants and analyzed vesicle production over time in the presence or absence of CDPK5 protein. Our results strongly suggest that RMVs are released before and not during parasite egress (Figure 5B). Importantly, similar experiments with the cysteine protease inhibitor e64, an inhibitor of parasite egress (Millholland et al., 2011Millholland M.G. Chandramohanadas R. Pizzarro A. Wehr A. Shi H. Darling C. Lim C.T. Greenbaum D.C. The malaria parasite progressively dismantles the host erythrocyte cytoskeleton for efficient egress.Mol. Cell. Proteomics. 2011; 10 (M111.010678)Crossref PubMed Scopus (62) Google Scholar), could phenocopy this effect (Figure 5B). Altogether, these data demonstrate that the peak release of RMVs from iRBCs occurs shortly before egress (i.e., within the last 6–8 hr of the parasite asexual cycle). RMVs are therefore distinct from recently described postrupture vesicles that are released upon egress of parasites from the red blood cell (Millholland et al., 2011Millholland M.G. Chandramohanadas R. Pizzarro A. Wehr A. Shi H. Darling C. Lim C.T. Greenbaum D.C. The malaria parasite progressively dismantles the host erythrocyte cytoskeleton for efficient egress.Mol. Cell. Proteomics. 2011; 10 (M111.010678)Crossref PubMed Scopus (62) Google Scholar). This conclusion is supported by the absence of markers for these postrupture vesicles such as the parasite antigens Sera-5 and Sera-6 in RMVs by proteomics (Table S1). To quantify RMV release from iRBCs and RBCs, we designed an experiment using two lipophilic membrane dyes, which are readily incorporated into RBCs: PKH26, which emits red light, and PKH67, which emits green light. This combination of dyes allows tracking of distinct particle or cell populations. We labeled highly synchronized trophozoite stage iRBCs with PKH67 and mixed them with uRBCs that we labeled with PKH26. Using flow cytometry and fluorescence microscopy, we monitored release of red and green RMVs during the remainder of the parasite cycle and after reinvasion, in the presence or absence of e64, (see Figure 5C, panel I, for the experimental setup). Using different mixtures of labeled iRBCs and RBCs, we determined the relative contribution of iRBC-derived RMVs to total RMV production. This demonstrated that iRBCs release about ten times more RMVs than uRBCs, and e64 experiments independently confirmed that these RMVs are released before egress (Figures 5C and S3). So far we have provided a thorough examination of the composition, biophysical properties, and release kinetics of RMVs. In the next series of experiments, we aimed to examine their potential physiological role(s). Recent studies in the rodent malaria model have demonstrated that RMVs derived from P. berghei iRBCs strongly activate the innate immune response through macrophage stimulation, suggesting a role of iRMVs in malaria pathology (Couper et al., 2010Couper K.N. Barnes T. Hafalla J.C. Combes V. Ryffel B. Secher T. Grau G.E. Riley E.M. de Souza J.B. Parasite-derived plasma microparticles contribute significantly to malaria infection-induced inflammation through potent macrophage stimulation.PLoS Pathog. 2010; 6: e1000744Crossref PubMed Scopus (172) Google Scholar). To investigate the potential of Plasmodium falciparum RMVs to modulate the innate immune response in human malaria, we analyzed their effect on human peripheral blood mononuclear cells (PBMCs), macrophages, and neutrophils derived from healthy, malaria-naive donors. PBMCs were incubated with RMVs from uninfected and infected RBCs to determine which cell type was activated and whether pro- or anti-inflammatory cytokines were induced. We measured the cell markers CD3 (T cells), CD19 (B cells), and CD14 (monocytes) in combination with the activation markers CD54, CD25, CD40 CD163, CD86, and CD36 by flow cytometry; cell viability was assessed by annexin V and propidium iodide staining. These experiments demonstrated that monocytes (CD14+) are the main targets of RMVs (Figures 6A and S4A–S4C). Specifically, they showed upregulation of the activation markers CD40, CD54, and CD86 and downregulation of CD163 upon stimulation with RMVs from iRBCs but not from uRBCs. For assessment of the effect of RMVs on human macrophages, monocytes were isolated from PBMCs, differentiated into macrophages, and activated with RMVs. Quantitative RT-PCR (qRT-PCR) analysis demonstrated that RMVs from iRBCs can activate the proinflammatory cytokines interleukin-6 (IL-6), IL-12, and IL-1β and the anti-inflammatory cytokine IL-10 in a dose-dependent manner (Figure S4D). We confirmed these results on a protein level via ELISA, measuring IL-10 and tumor necrosis factor alpha (TNF-α) in culture supernatants (Figure 6B). Likewise, qRT-PCR and confirmatory ELISA demonstrated that RMVs induce the expression of IL-10 and TNF-α in PBMCs (Figures S4B and S4C). Importantly, macrophage activation depends on the active uptake of RMVs, as demonstrated by microscopy of RMV uptake (Figure 6C), and cytokine induction was inhibited in presence of cytochalasin D, an inhibitor of phagocytosis (Figure 6D). To probe the role of iRBC-derived RMVs on human neutrophils, we incubated freshly isolated human neutrophils with purified RMVs inside a microfluidic device with small migration channels (Butler et al., 2010Butler K.L. Ambravaneswaran V. Agrawal N. Bilodeau M. Toner M. Tompkins R.G. Fagan S. Irimia D. Burn injury reduces neutrophil directional migration speed in microfluidic devices.PLoS ONE. 2010; 5: e11921Crossref PubMed Scopus (98) Google Scholar). Brief exposure (30 min) to RMVs from iRBCs but not from uRBCs activated neutrophils to spontaneously move, even in the absence of any guiding chemotactic gradients (Figure 6E). We also found that neutrophils preincubated with RMVs from uRBCs migrated at a slower rate compared those preincubated with RMVs from iRBCs or untreated controls. Together, these data demonstrate that RMVs from iRBCs but not from uRBCs can strongly stimulate cells of the innate immune system. In the PKH experiments described in Figure 5C, we noted that in some cases fluorescence from PKH67-labeled vesicles could also be found inside iRBCs, suggesting that they had been incorporated into the cell. To further investigate this observation, we performed a series of RMV uptake experiments and phenotypic assays. First we investigated uptake of labeled RMVs by microscopy, immunoelectron microscopy, and flow cytometry. Live fluorescence microscopy revealed that PKH67-labeled RMVs are efficiently incorporated into iRBCs and eventually accumulate in the parasite and at its nuclear periphery (Figure 7A, panel I). Uptake appears to be specific to iRBCs, since RMVs were found mostly bound to the surface of uRBCs (Figure 7A, panel II). Quantification of uptake with PKH67-labeled RMVs also demonstrated that those derived from iRBCs are incorporated at significantly higher rates than those from uRBCs (Figure 7A, panel III). Notably, only a subset of iRBCs internalizes RMVs even at very high concentrations, suggesting that not all iRBCs are equally receptive for uptake. To visualize RMV uptake on an ultrastructural level, we performed immune electron microscopy of RBCs after incubation with biotinylated RMVs from infected RBCs. In concordance with live microscopy, we observed the presence of labeled vesicles in the host cell cytoplasm and in the parasite (Figure 7B). These internalized RMVs are surrounded by additional membranes, suggesting that phagocytosis-like mechanisms are operational in infected RBCs. To determine whether RMV uptake has a phenotypic effect on parasite growth, we treated ring-stage parasites with a serial dilution of purified RMVs and investigated parasitemia after one replication cycle (Figure 7C). While we did not observe a significant alteration in growth rates after 48 hr, we noted the emergence of increased numbers of gametocytes in the parasite culture at later time points. Sexual stages, or gametocytes, are formed from asexual parents at low rates of <0.1%–15% per reinvasion round in vitro. In vivo gametocyte production appears to cover an equally large range (Alano, 2007Alano P. Plasmodium falciparum gametocytes: still many secrets of a hidden life.Mol. Microbiol. 2007; 66: 291-302Crossref PubMed Scopus (92) Google Scholar). Upon maturation, gametocytes are transmitted to a mosquito vector during a blood meal, where they undergo fertilization and further development. It has been reported that parasite-conditioned medium (i.e., the parasite culture supernatants that we use for RMV isolation) can increase the proportion of gametocytes formed (Dyer and Day, 2003Dyer M. Day K.P. Regulation of the rate of asexual growth and commitment to sexual development by diffusible factors from in vitro cultures of Plasmodium falciparum.Am. J. Trop. Med. Hyg. 2003; 68: 403-409PubMed Google Scholar), and such conditioned medium is commonly used as a stimulus to increase gametocyte production under in vitro conditions (Fivelman et al., 2007Fivelman Q.L. McRobert L. Sharp S. Taylor C.J. Saeed M. Swales C.A. Sutherland C.J. Baker D.A. Improved synchronous production of Plasmodium falciparum gametocytes in vitro.Mol. Biochem. Parasitol. 2007; 154: 119-123Crossref PubMed Scopus (157) Google Scholar). The factor(s) responsible for this effect have not yet been identified. To test whether RMVs from conditioned medium have a gametocyte-inducing effect, we quantified gametocyte production in 3D7 parasites upon addition of conditioned medium or purified RMVs derived from the same conditioned medium. These experiments revealed that RMVs derived from iRBCs stimulated increased gametocyte production in a titrable fashion and similar to conditioned medium from late stage parasite cultures, whereas RMVs" @default.
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• W2018122290 title "Malaria-Infected Erythrocyte-Derived Microvesicles Mediate Cellular Communication within the Parasite Population and with the Host Immune System" @default.
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• W2018122290 doi "https://doi.org/10.1016/j.chom.2013.04.009" @default.
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