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Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common enzymatic disorder in humans and appears to be protective against falciparum severe malaria. Controversially, it is also thought that Plasmodium vivax has driven the recent selection of G6PD alleles. We use an experimental approach to determine whether G6PD-MahidolG487A variant, a widespread cause of severe G6PD deficiency in Southeast Asia, provides a barrier against vivax malaria. Our results show that the immature reticulocytes (CD71+) targeted by P. vivax invasion are enzymatically normal, even in hemizygous G6PD-Mahidol G487A mutants; thus, allowing the normal growth, development, and high parasite density in severely deficient samples.
Red blood cells (RBCs) begin their circulatory life as reticulocytes (Retics) after their egress from the bone marrow where, as R1 Retics, they undergo significant rearrangements in their membrane and intracellular components, via autophagic, proteolytic, and vesicle-based mechanisms. Circulating, R2 Retics must complete this maturational process, which involves additional loss of significant amounts of membrane and selected membrane proteins. Little is known about the mechanism(s) at the basis of this terminal differentiation in the circulation, which culminates with the production of a stable biconcave discocyte. The membrane of R1 Retics undergoes a selective remodeling through the release of exosomes that are enriched in transferrin receptor and membrane raft proteins and lipids, but are devoid of Band 3, glycophorin A, and membrane skeletal proteins. We wondered whether a similar selective remodeling occurred also in the maturation of R2 Retics. Peripheral blood R2 Retics, isolated by an immunomagnetic method, were compared with mature circulating RBCs from the same donor and their membrane protein and lipid content was analyzed. Results show that both Band 3 and spectrin decrease from R2 Retics to RBCs on a "per cell" basis. Looking at membrane proteins that are considered as markers of membrane rafts, flotillin-2 appears to decrease in a disproportionate manner with respect to Band 3. Stomatin also decreases but in a more proportionate manner with respect to Band 3, hinting at a heterogeneous nature of membrane rafts. High resolution lipidomics analysis, on the contrary, revealed that those lipids that are typically representative of the membrane raft phase, sphingomyelin and cholesterol, are enriched in mature RBCs with respct to Retics, relative to total cell lipids, strongly arguing in favor of the selective retention of at least certain subclasses of membrane rafts in RBCs as they mature from Retics. Our hypothesis that rafts serve as additional anchoring sites for the lipid bilayer to the underlying membrane-skeleton is corroborated by the present results. It is becoming ever more clear that a proper lipid composition of the reticulocyte is necessary for the production of a normal mature RBC.
Plasmodium vivax uses multiple ligand-receptor interactions for preferential invasion of human reticulocytes. Several of these ligands have been identified by in silico approaches based on the role displayed by their orthologs in other Plasmodium species during initial adhesion or invasion. However, the cell adhesion role of proteins that are exclusive to species that specifically invade reticulocytes (as P. vivax and P. cynomolgi) has not been evaluated to date. This study aimed to characterise an antigen shared between Plasmodium species that preferentially infect reticulocytes with a focus on assessing its binding activity to target cells.
Although red blood cell (RBC) transfusions save lives, some patients develop clinically-significant alloantibodies against donor blood group antigens, which then have adverse effects in multiple clinical settings. Few effective measures exist to prevent RBC alloimmunization and/or eliminate alloantibodies in sensitized patients. Donor-related factors may influence alloimmunization; thus, there is an unmet clinical need to identify which RBC units are immunogenic. Repeat volunteer blood donors and donors on iron supplements have elevated reticulocyte counts compared to healthy non-donors. Early reticulocytes retain mitochondria and other components, which may act as danger signals in immune responses. Herein, we tested whether reticulocytes in donor RBC units could enhance RBC alloimmunization. Using a murine model, we demonstrate that transfusing donor RBC units with increased reticulocyte frequencies dose-dependently increased RBC alloimmunization rates and alloantibody levels. Transfusing reticulocyte-rich RBC units was associated with increased RBC clearance from the circulation and a robust proinflammatory cytokine response. As compared to previously reported post-transfusion RBC consumption patterns, erythrophagocytosis from reticulocyte-rich units was increasingly performed by splenic B cells. These data suggest that reticulocytes in a donated RBC unit impact the quality of blood transfused, are targeted to a distinct compartment, and may be an underappreciated risk factor for RBC alloimmunization.
Turnover of mRNA is a critical step that allows cells to control gene expression. Endoribonucleases, enzymes cleaving RNA molecules internally, are some of the key components of the degradation process. Here we provide a detailed characterization of novel endoribonuclease SLFN14 purified from rabbit reticulocyte lysate. Schlafen genes encode a family of proteins limited to mammals. Their cellular function is unknown or incompletely understood. In reticulocytes, SLFN14 is strongly overexpressed, represented exclusively by the short form, all tethered to ribosomes, and appears to be one of the major ribosome-associated proteins. SLFN14 binds to ribosomes and ribosomal subunits in the low part of the body and cleaves RNA but preferentially rRNA and ribosome-associated mRNA. This results in the degradation of ribosomal subunits. This process is strictly Mg(2+)- and Mn(2+)-dependent, NTP-independent, and sequence nonspecific. However, in other cell types, SLFN14 is a full-length solely nuclear protein, which lacks ribosomal binding and nuclease activities. Mutational analysis revealed the ribosomal binding site and the aspartate essential for the endonucleolytic activity of protein. Only few endoribonucleases participating in ribosome-mediated processes have been characterized to date. Moreover, none of them are shown to be directly associated with the ribosome. Therefore, our findings expand the general knowledge of endoribonucleases involved in mammalian translation control.
Exosomes are 30-100-nm membrane vesicles of endocytic origin that are released after the fusion of multivesicular bodies (MVBs) with the plasma membrane. While initial studies suggested that the role of exosomes was limited to the removal of proteins during the maturation of reticulocytes to erythrocytes, recent studies indicate that they are produced by different types of cells and are involved in promoting inter-cellular communication and antigen presentation. Here, we describe the isolation and characterization of exosomes from peripheral blood of BALB/c mice infected with the reticulocyte-prone non-lethal Plasmodium yoelii 17X strain. Importantly, proteomic analysis revealed the presence of parasite proteins in these vesicles. Moreover, immunization of mice with purified exosomes elicited IgG antibodies capable of recognizing P. yoelii-infected red blood cells. Furthermore, lethal challenge of immunized mice with the normocyte-prone lethal P. yoelii 17XL strain caused a significant attenuation in the course of parasitaemia, increased survival time, and altered the cell tropism to reticulocytes. These results were obtained also when the exosomes were isolated from a P. yoelii-infected reticulocyte culture indicating that reticulocyte-derived exosomes carry antigens and are involved in immune modulation. Moreover, inclusion of CpG ODN 1826 in exosome immunizations elicited IgG2a and IgG2b antibodies and promoted survival, clearance of parasites and subsequent sterile protection of 83% of the animals challenged with P. yoelli 17XL. To our knowledge, this is the first report of immune responses elicited by exosomes derived from reticulocytes opening new avenues for the modulation of anti-malaria responses.
Plasmodium vivax causes approximately 100 million clinical malaria cases yearly1,2. The basis of protective immunity is poorly understood and thought to be mediated by antibodies3,4. Cytotoxic CD8+ T cells protect against other intracellular parasites by detecting parasite peptides presented by human leukocyte antigen class I on host cells. Cytotoxic CD8+ T cells kill parasite-infected mammalian cells and intracellular parasites by releasing their cytotoxic granules5,6. Perforin delivers the antimicrobial peptide granulysin and death-inducing granzymes into the host cell, and granulysin then delivers granzymes into the parasite. Cytotoxic CD8+ T cells were thought to have no role against Plasmodium spp. blood stages because red blood cells generally do not express human leukocyte antigen class I7. However, P. vivax infects reticulocytes that retain the protein translation machinery. Here we show that P. vivax-infected reticulocytes express human leukocyte antigen class I. Infected patient circulating CD8+ T cells highly express cytotoxic proteins and recognize and form immunological synapses with P. vivax-infected reticulocytes in a human leukocyte antigen-dependent manner, releasing their cytotoxic granules to kill both host cell and intracellular parasite, preventing reinvasion. P. vivax-infected reticulocytes and parasite killing is perforin independent, but depends on granulysin, which generally efficiently forms pores only in microbial membranes8. We find that P. vivax depletes cholesterol from the P. vivax-infected reticulocyte cell membrane, rendering it granulysin-susceptible. This unexpected T cell defense might be mobilized to improve P. vivax vaccine efficacy.
Transcriptional changes in compensatory erythropoiesis in sickle cell anemia (SCA) and their disease modulation are unclear. We detected 1226 differentially expressed genes in hemoglobin SS reticulocytes compared to non-anemic hemoglobin AA controls. Assessing developmental expression changes in hemoglobin AA erythroblasts for these genes suggests heightened terminal differentiation in early erythroblasts in SCA that diminishes toward the polychromatic to orthochromatic stage transition. Comparison of reticulocyte gene expression changes in SCA with that in Chuvash erythrocytosis, a non-anemic disorder of increased erythropoiesis due to constitutive activation of hypoxia inducible factors, identified 453 SCA-specific changes attributable to compensatory erythropoiesis. Peripheral blood mononuclear cells (PBMCs) in SCA contain elevated proportions of erythroid progenitors due to heightened erythropoiesis. Deconvolution analysis in PBMCs from 131 SCA patients detected 54 genes whose erythroid expression correlated with erythropoiesis efficiency, which were enriched with SCA-specific changes (OR = 2.9, P = 0.00063) and annotation keyword "ubiquitin-dependent protein catabolic process", "protein ubiquitination", and "protein polyubiquitination" (OR = 4.2, P = 7.5 × 10-5). An erythroid expression quantitative trait locus of one of these genes, LNX2 encoding an E3 ubiquitin ligase, associated with severe pain episodes in 774 SCA patients (OR = 1.7, P = 3.9 × 10-5). Thus, erythroid gene transcription responds to unique conditions within SCA erythroblasts and these changes potentially correspond to vaso-occlusive manifestations.
Human malaria parasites proliferate in different erythroid cell types during infection. Whilst Plasmodium vivax exhibits a strong preference for immature reticulocytes, the more pathogenic P. falciparum primarily infects mature erythrocytes. In order to assess if these two cell types offer different growth conditions and relate them to parasite preference, we compared the metabolomes of human and rodent reticulocytes with those of their mature erythrocyte counterparts. Reticulocytes were found to have a more complex, enriched metabolic profile than mature erythrocytes and a higher level of metabolic overlap between reticulocyte resident parasite stages and their host cell. This redundancy was assessed by generating a panel of mutants of the rodent malaria parasite P. berghei with defects in intermediary carbon metabolism (ICM) and pyrimidine biosynthesis known to be important for P. falciparum growth and survival in vitro in mature erythrocytes. P. berghei ICM mutants (pbpepc-, phosphoenolpyruvate carboxylase and pbmdh-, malate dehydrogenase) multiplied in reticulocytes and committed to sexual development like wild type parasites. However, P. berghei pyrimidine biosynthesis mutants (pboprt-, orotate phosphoribosyltransferase and pbompdc-, orotidine 5'-monophosphate decarboxylase) were restricted to growth in the youngest forms of reticulocytes and had a severe slow growth phenotype in part resulting from reduced merozoite production. The pbpepc-, pboprt- and pbompdc- mutants retained virulence in mice implying that malaria parasites can partially salvage pyrimidines but failed to complete differentiation to various stages in mosquitoes. These findings suggest that species-specific differences in Plasmodium host cell tropism result in marked differences in the necessity for parasite intrinsic metabolism. These data have implications for drug design when targeting mature erythrocyte or reticulocyte resident parasites.
Investigating the role that host erythrocyte proteins play in malaria infection is hampered by the genetic intractability of this anucleate cell. Here we report that reticulocytes derived through in vitro differentiation of an enucleation-competent immortalized erythroblast cell line (BEL-A) support both successful invasion and intracellular development of the malaria parasite Plasmodium falciparum. Using CRISPR-mediated gene knockout and subsequent complementation, we validate an essential role for the erythrocyte receptor basigin in P. falciparum invasion and demonstrate rescue of invasive susceptibility by receptor re-expression. Successful invasion of reticulocytes complemented with a truncated mutant excludes a functional role for the basigin cytoplasmic domain during invasion. Contrastingly, knockout of cyclophilin B, reported to participate in invasion and interact with basigin, did not impact invasive susceptibility of reticulocytes. These data establish the use of reticulocytes derived from immortalized erythroblasts as a powerful model system to explore hypotheses regarding host receptor requirements for P. falciparum invasion.
Most studies on malaria-parasite digestion of hemoglobin (Hb) have been performed using P. falciparum maintained in mature erythrocytes, in vitro. In this study, we examine Plasmodium Hb degradation in vivo in mice, using the parasite P. berghei, and show that it is possible to create mutant parasites lacking enzymes involved in the initial steps of Hb proteolysis. These mutants only complete development in reticulocytes and mature into both schizonts and gametocytes. Hb degradation is severely impaired and large amounts of undigested Hb remains in the reticulocyte cytoplasm and in vesicles in the parasite. The mutants produce little or no hemozoin (Hz), the detoxification by-product of Hb degradation. Further, they are resistant to chloroquine, an antimalarial drug that interferes with Hz formation, but their sensitivity to artesunate, also thought to be dependent on Hb degradation, is retained. Survival in reticulocytes with reduced or absent Hb digestion may imply a novel mechanism of drug resistance. These findings have implications for drug development against human-malaria parasites, such as P. vivax and P. ovale, which develop inside reticulocytes.
Control genes, which are often referred to as housekeeping genes, are frequently used to normalise mRNA levels between different samples. However, the expression level of these genes may vary among tissues or cells and may change under certain circumstances. Thus, the selection of housekeeping genes is critical for gene expression studies. To address this issue, 7 candidate housekeeping genes including several commonly used ones were investigated in isolated human reticulocytes. For this, a simple DeltaCt approach was employed by comparing relative expression of 'pairs of genes' within each sample. On this basis, stability of the candidate housekeeping genes was ranked according to repeatability of the gene expression differences among 31 samples.
Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is a rare autosomal metabolic disorder caused by thymidine phosphorylase (TP) deficiency. Successful therapeutic interventions for this disease rely on a means for efficient and long-lasting circulation of the TP enzyme. In this study we exploit lentiviral transduction of hematopoietic stem cells and an erythroid cell line (BEL-A) to generate reticulocytes that contain active TP. Significant loss of overexpressed TP during erythroid differentiation can be reduced by addition of the ubiquitination inhibitor MG132. However, the ubiquitination sites are located in the substrate binding site in human TP, and their removal abolished enzyme activity. Examination of the TP structure and mechanism suggested that these sites are only exposed in the absence of substrate. We show that supplementation of culture media with thymidine during differentiation reduces enzyme degradation, doubling the amount of TP retained in reticulocytes. This study provides proof of principle that therapeutic reticulocytes expressing TP can be generated in vitro and that ubiquitin-mediated degradation can be subverted through masking ubiquitination sites to ensure retention of human TP in reticulocytes following erythroid differentiation.
Despite significant advances, many changes occurring in the tumor microenvironment during acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) remain unclear. The surface of immature reticulocytes was examined by atomic force microscopy (AFM) to determine specific changes during the development of ALL and AML. In patients with ALL the surface area of reticulocytes increased by 18.5 %, volume by 8.7 %, the width of invaginations by 18 %, and cell height decreased by 7.8 %. In patients with AML, the volume increased by 12.6 %, roughness by 35.5 %, the height of protrusions by 36.2 %, the depth of invaginations by 24.8 %, their width by 18.2 %, and the maximum height difference of the surface by 31.9 %. The obtained data1 has important prognostic value in studying the bone marrow activity during acute leukemia.
A very large biomass of intact asexual-stage malaria parasites accumulates in the spleen of asymptomatic human individuals infected with Plasmodium vivax. The mechanisms underlying this intense tropism are not clear. We hypothesised that immature reticulocytes, in which P. vivax develops, may display high densities in the spleen, thereby providing a niche for parasite survival.
Plasmodium vivax parasites preferentially invade reticulocyte cells in a multistep process that is still poorly understood. In this study, we used ex vivo invasion assays and population genetic analyses to investigate the involvement of complement receptor 1 (CR1) in P. vivax invasion. First, we observed that P. vivax invasion of reticulocytes was consistently reduced when CR1 surface expression was reduced through enzymatic cleavage, in the presence of naturally low-CR1-expressing cells compared with high-CR1-expressing cells, and with the addition of soluble CR1, a known inhibitor of P. falciparum invasion. Immuno-precipitation experiments with P. vivax Reticulocyte Binding Proteins showed no evidence of complex formation. In addition, analysis of CR1 genetic data for worldwide human populations with different exposure to malaria parasites show significantly higher frequency of CR1 alleles associated with low receptor expression on the surface of RBCs and higher linkage disequilibrium in human populations exposed to P. vivax malaria compared with unexposed populations. These results are consistent with a positive selection of low-CR1-expressing alleles in vivax-endemic areas. Collectively, our findings demonstrate that CR1 availability on the surface of RBCs modulates P. vivax invasion. The identification of new molecular interactions is crucial to guiding the rational development of new therapeutic interventions against vivax malaria.
The malarial parasite's invasion is complex, active and coordinated, involving many low and high affinity interactions with receptors on target cell membrane. Proteomics analysis has described around 40 proteins in P. vivax which could be involved in reticulocyte invasion; few have been studied with the aim of elucidating how many of them establish specific interactions with their respective host cells. Given the importance of knowing which of the parasite's protein regions are functionally important for invasion, minimum regions mediating specific interaction between Plasmodium vivax apical membrane antigen 1 (PvAMA-1) and its host cell were here elucidated. The region covering PvAMA-1 domains I and II (PvAMA-DI-II) specifically bound to the CD71+ red blood cell subpopulation. A 20 residue-long region (81EVENAKYRIPAGRCPVFGKG100) located in domain I was capable of inhibiting PvAMA-DI-II recombinant protein binding to young reticulocytes (CD71+CD45-) and rosette formation. This conserved peptide specifically interacted with high affinity with reticulocytes (CD71+) through a neuraminidase- and chymotrypsin-treatment sensitive receptor. Such results showed that, despite AMA-1 having universal functions during late Plasmodium invasion stages, PvAMA-1 had reticulocyte-preferring binding regions, suggesting that P. vivax target cell selection is not just restricted to initial interactions but maintained throughout the erythrocyte invasion cycle, having important implications for designing a specific anti-P. vivax vaccine.
The interactions between Plasmodium parasites and human erythrocytes are prime targets of blood stage malaria vaccine development. The reticulocyte binding protein 2-P1 (RBP2-P1) of Plasmodium vivax, a member of the reticulocyte binding protein family, has recently been shown to be highly antigenic in several settings endemic for malaria. Yet, its functional characteristics and the relevance of its antibody response in human malaria have not been examined. In this study, the potential function of RBP2-P1 as an invasion ligand of P. vivax was evaluated. The protein was found to be expressed in schizonts, be localized at the apical end of the merozoite, and preferentially bind reticulocytes over normocytes. Human antibodies to this protein also exhibit erythrocyte binding inhibition at physiologically relevant concentrations. Furthermore, RBP2-P1 antibodies are associated with lower parasitemia and tend to be higher in asymptomatic carriers than in patients. This study provides evidence supporting a role of RBP2-P1 as an invasion ligand and its consideration as a vaccine target.
Plasmodium parasites' invasion of their target cells is a complex, multi-step process involving many protein-protein interactions. Little is known about how complex the interaction with target cells is in Plasmodium vivax and few surface molecules related to reticulocytes' adhesion have been described to date. Natural selection, functional and structural analysis were carried out on the previously described vaccine candidate P. vivax merozoite surface protein 10 (PvMSP10) for evaluating its role during initial contact with target cells. It has been shown here that the recombinant carboxyl terminal region (rPvMSP10-C) bound to adult human reticulocytes but not to normocytes, as validated by two different protein-cell interaction assays. Particularly interesting was the fact that two 20-residue-long regions (388DKEECRCRANYMPDDSVDYF407 and 415KDCSKENGNCDVNAECSIDK434) were able to inhibit rPvMSP10-C binding to reticulocytes and rosette formation using enriched target cells. These peptides were derived from PvMSP10 epidermal growth factor (EGF)-like domains (precisely, from a well-defined electrostatic zone) and consisted of regions having the potential of being B- or T-cell epitopes. These findings provide evidence, for the first time, about the fragments governing PvMSP10 binding to its target cells, thus highlighting the importance of studying them for inclusion in a P. vivax antimalarial vaccine.
Different proteins derived from the membrane or the apical organelles become involved in malarial parasite invasion of host cells. Among these, the rhoptry neck proteins (RONs) interact with a protein component of the micronemes to enable the formation of a strong bond which is crucial for the parasite's successful invasion. The present study was aimed at identifying and characterizing the RON5 protein in Plasmodium vivax and evaluating its ability to bind to reticulocytes.
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