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Vaccine development for Plasmodium vivax, an important human relapsing malaria, is lagging behind. In the case of the most deadly human malaria P. falciparum, unprecedented high levels of protection have been obtained by immunization with live sporozoites under accompanying chemoprophylaxis, which prevents the onset of blood-stage malaria. Such an approach has not been fully evaluated for relapsing malaria. Here, in the P. cynomolgi-rhesus macaque model for relapsing malaria, we employ the parasites' natural relapsing phenotype to self-boost the immune response against liver-stage parasites, following a single-shot high-dose live sporozoite vaccination. This approach resulted in sterile protection against homologous sporozoite challenge in three out of four animals in the group that was also exposed for several days to blood stages during primary infection and relapses. One out of four animals in the group that received continuous chemoprophylaxis to abort blood-stage exposure was also protected from sporozoite challenge. Although obtained in a small number of animals as part of a Proof-of-Concept study, these results suggest that limited blood-stage parasite exposure may augment protection in this model. We anticipate our data are a starting point for further research into correlates of protection and extrapolation of the single-shot approach to develop efficacious malaria vaccines against relapsing human malaria., (© 2022. The Author(s).)

Interleukin (IL)-4 is a cytokine that affects both adaptive and innate immune responses. In the central nervous system, microglia express IL-4 receptors and it has been described that IL-4-exposed microglia acquire anti-inflammatory properties. We here demonstrate that IL-4 exposure induces changes in the cell surface protein expression profile of primary rhesus macaque microglia and enhances their potential to induce proliferation of T cells with a regulatory signature. Moreover, we show that Toll like receptor (TLR)-induced cytokine production is broadly impaired in IL-4-exposed microglia at the transcriptional level. IL-4 type 2 receptor-mediated signaling is shown to be crucial for the inhibition of microglial innate immune responses. TLR-induced nuclear translocalization of NF-κB appeared intact, and we found no evidence for epigenetic modulation of target genes. By contrast, nuclear extracts from IL-4-exposed microglia contained significantly less NF-κB capable of binding to its DNA consensus site. Further identification of the molecular mechanisms that underlie the inhibition of TLR-induced responses in IL-4-exposed microglia may aid the design of strategies that aim to modulate innate immune responses in the brain, for example in gliomas., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Zuiderwijk-Sick, van der Putten, Timmerman, Veth, Pasini, van Straalen, van der Valk, Amor and Bajramovic.)

Malaria remains a serious health concern across the globe. Historically neglected, non- Falciparum human malarias were put back on the agenda by a paradigm shift in the fight against malaria from malaria control to malaria eradication. Here, we review the modeling of the relapsing parasites Plasmodium vivax ( P. vivax ) and Plasmodium ovale ( P. ovale ) in non-human primates with a specific focus on the contribution of these models to our current understanding of the factors that govern parasite-host interactions in P. vivax and P. ovale parasite biology and pathophysiology., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Pasini and Kocken.)

Identifying immune correlates of protection and mechanisms of immunity accelerates and streamlines the development of vaccines. RTS,S/AS01E, the most clinically advanced malaria vaccine, has moderate efficacy in African children. In contrast, immunization with sporozoites under antimalarial chemoprophylaxis (CPS immunization) can provide 100% sterile protection in naïve adults. We used systems biology approaches to identifying correlates of vaccine-induced immunity based on transcriptomes of peripheral blood mononuclear cells from individuals immunized with RTS,S/AS01E or chemoattenuated sporozoites stimulated with parasite antigens in vitro. Specifically, we used samples of individuals from two age cohorts and three African countries participating in an RTS,S/AS01E pediatric phase 3 trial and malaria-naïve individuals participating in a CPS trial. We identified both preimmunization and postimmunization transcriptomic signatures correlating with protection. Signatures were validated in independent children and infants from the RTS,S/AS01E phase 3 trial and individuals from an independent CPS trial with high accuracies (>70%). Transcription modules revealed interferon, NF-κB, Toll-like receptor (TLR), and monocyte-related signatures associated with protection. Preimmunization signatures suggest that priming the immune system before vaccination could potentially improve vaccine immunogenicity and efficacy. Last, signatures of protection could be useful to determine efficacy in clinical trials, accelerating vaccine candidate testing. Nevertheless, signatures should be tested more extensively across multiple cohorts and trials to demonstrate their universal predictive capacity., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Establishing an in vitro "red blood cell matrix" that would allow uninterrupted access to a stable, homogeneous reticulocyte population would facilitate the establishment of continuous, long-term in vitro Plasmodium vivax blood stage cultures. In this study, we have explored the suitability of the erythroleukemia K562 cell line as a continuous source of such reticulocytes and have investigated regulatory factors behind the terminal differentiation (and enucleation, in particular) of this cell line that can be used to drive the reticulocyte production process. The Duffy blood group antigen receptor (Fy), essential for P. vivax invasion, was stably introduced into K562 cells by lentiviral gene transfer. miRNA-26a-5p and miRNA-30a-5p were downregulated to promote erythroid differentiation and enucleation, resulting in a tenfold increase in the production of reticulocytes after stimulation with an induction cocktail compared with controls. Our results suggest an interplay in the mechanisms of action of miRNA-26a-5p and miRNA-30a-5p, which makes it necessary to downregulate both miRNAs to achieve a stable enucleation rate and Fy receptor expression. In the context of establishing P. vivax-permissive, stable, and reproducible reticulocytes, a higher enucleation rate may be desirable, which may be achieved by the targeting of further regulatory mechanisms in Fy-K562 cells; promoting the shift in hemoglobin production from fetal to adult may also be necessary. Despite the fact that K562 erythroleukemia cell lines are of neoplastic origin, this cell line offers a versatile model system to research the regulatory mechanisms underlying erythropoiesis., (Copyright © 2020 ISEH -- Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.)

Historically neglected, due to its biological peculiarities, the absence of a continuous long-term in vitro blood stage culture system and a propensity towards high morbidity rather than mortality, Plasmodium vivax was put back on the agenda during the last decade by the paradigm shift in the fight against malaria from malaria control to malaria eradication. While the incidence of the deadliest form of malaria, Plasmodium falciparum malaria, has declined since this paradigm shift took hold, the prospects of eradication are now threatened by the increase in the incidence of other human malaria parasite species. Plasmodium vivax is geographically the most widely distributed human malaria parasite, characterized by millions of clinical cases every year and responsible for a massive economic burden. The urgent need to tackle the unique biological challenges posed by this parasite led to renewed efforts aimed at establishing a continuous, long-term in vitro P. vivax blood stage culture. Based on recent discoveries on the role of nutrient sensing in Plasmodium's pathophysiology, this review article critically assesses the extensive body of literature concerning Plasmodium culture conditions with a specific focus on culture media used in attempts to culture different Plasmodium spp. Hereby, the effect of specific media components on the parasite's in vitro fitness and the maturation of the parasite's host cell, the reticulocyte, is analysed. Challenging the wide-held belief that it is sufficient to find the right parasite isolate and give it the right type of cells to invade for P. vivax to grow in vitro, this review contends that a healthy side-by-side maturation of both the parasite and its host cell, the reticulocyte, is necessary in the adaptation of P. vivax to in vitro growth and argues that culture conditions and the media in particular play an essential role in this maturation process.

The primate malaria Plasmodium knowlesi has a long-standing history as an experimental malaria model. Studies using this model parasite in combination with its various natural and experimental non-human primate hosts have led to important advances in vaccine development and in our understanding of malaria invasion, immunology and parasite-host interactions. The adaptation to long-term in vitro continuous blood stage culture in rhesus monkey, Macaca fascicularis and human red blood cells, as well as the development of various transfection methodologies has resulted in a highly versatile experimental malaria model, further increasing the potential of what was already a very powerful model. The growing evidence that P. knowlesi is an important human zoonosis in South-East Asia has added relevance to former and future studies of this parasite species.

The recent research efforts to establish a Plasmodium vivax continuous, long-term blood-stage culture have focused on the ideal host cell type. However, this is only part of the story, as the P. vivax intraerythrocytic life cycle is complex. A successful, long-term, robust culture system will depend on a multifaceted approach combining the ideal cell type and parasite isolates, and the culture conditions., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Background: Plasmodium cynomolgi, a non-human primate malaria parasite species, has been an important model parasite since its discovery in 1907. Similarities in the biology of P. cynomolgi to the closely related, but less tractable, human malaria parasite P. vivax make it the model parasite of choice for liver biology and vaccine studies pertinent to P. vivax malaria. Molecular and genome-scale studies of P. cynomolgi have relied on the current reference genome sequence, which remains highly fragmented with 1,649 unassigned scaffolds and little representation of the subtelomeres.  Methods: Using long-read sequence data (Pacific Biosciences SMRT technology), we assembled and annotated a new reference genome sequence, PcyM, sourced from an Indian rhesus monkey. We compare the newly assembled genome sequence with those of several other Plasmodium species, including a re-annotated P. coatneyi assembly., Results: The new PcyM genome assembly is of significantly higher quality than the existing reference, comprising only 56 pieces, no gaps and an improved average gene length. Detailed manual curation has ensured a comprehensive annotation of the genome with 6,632 genes, nearly 1,000 more than previously attributed to P. cynomolgi . The new assembly also has an improved representation of the subtelomeric regions, which account for nearly 40% of the sequence. Within the subtelomeres, we identified more than 1300 Plasmodium interspersed repeat ( pir ) genes, as well as a striking expansion of 36 methyltransferase pseudogenes that originated from a single copy on chromosome 9., Conclusions: The manually curated PcyM reference genome sequence is an important new resource for the malaria research community. The high quality and contiguity of the data have enabled the discovery of a novel expansion of methyltransferase in the subtelomeres, and illustrates the new comparative genomics capabilities that are being unlocked by complete reference genomes., Competing Interests: Competing interests: None of the authors declared competing interest.

Many variant proteins encoded by Plasmodium-specific multigene families are exported into red blood cells (RBC). P. falciparum-specific variant proteins encoded by the var, stevor and rifin multigene families are exported onto the surface of infected red blood cells (iRBC) and mediate interactions between iRBC and host cells resulting in tissue sequestration and rosetting. However, the precise function of most other Plasmodium multigene families encoding exported proteins is unknown. To understand the role of RBC-exported proteins of rodent malaria parasites (RMP) we analysed the expression and cellular location by fluorescent-tagging of members of the pir, fam-a and fam-b multigene families. Furthermore, we performed phylogenetic analyses of the fam-a and fam-b multigene families, which indicate that both families have a history of functional differentiation unique to RMP. We demonstrate for all three families that expression of family members in iRBC is not mutually exclusive. Most tagged proteins were transported into the iRBC cytoplasm but not onto the iRBC plasma membrane, indicating that they are unlikely to play a direct role in iRBC-host cell interactions. Unexpectedly, most family members are also expressed during the liver stage, where they are transported into the parasitophorous vacuole. This suggests that these protein families promote parasite development in both the liver and blood, either by supporting parasite development within hepatocytes and erythrocytes and/or by manipulating the host immune response. Indeed, in the case of Fam-A, which have a steroidogenic acute regulatory-related lipid transfer (START) domain, we found that several family members can transfer phosphatidylcholine in vitro. These observations indicate that these proteins may transport (host) phosphatidylcholine for membrane synthesis. This is the first demonstration of a biological function of any exported variant protein family of rodent malaria parasites., Competing Interests: The authors have declared that no competing interests exist.

Malaria is one of the most significant tropical diseases, and of the Plasmodium species that cause human malaria, P. vivax is the most geographically widespread. However, P. vivax remains a relatively neglected human parasite since research is typically limited to laboratories with direct access to parasite isolates from endemic field settings or from non-human primate models. This restricted research capacity is in large part due to the lack of a continuous P. vivax in vitro culture system, which has hampered the ability for experimental research needed to gain biological knowledge and develop new therapies. Consequently, efforts to establish a long-term P. vivax culture system are confounded by our poor knowledge of the preferred host cell and essential nutrients needed for in vitro propagation. Reliance on very heterogeneous P. vivax field isolates makes it difficult to benchmark parasite characteristics and further complicates development of a robust and reliable culture method. In an effort to eliminate parasite variability as a complication, we used a well-defined Aotus-adapted P. vivax Sal-1 strain to empirically evaluate different short-term in vitro culture conditions and compare them with previous reported attempts at P. vivax in vitro culture Most importantly, we suggest that reticulocyte enrichment methods affect invasion efficiency and we identify stabilized forms of nutrients that appear beneficial for parasite growth, indicating that P. vivax may be extremely sensitive to waste products. Leuko-depletion methods did not significantly affect parasite development. Formatting changes such as shaking and static cultures did not seem to have a major impact while; in contrast, the starting haematocrit affected both parasite invasion and growth. These results support the continued use of Aotus-adapted Sal-1 for development of P. vivax laboratory methods; however, further experiments are needed to optimize culture conditions to support long-term parasite development.

Malaria is estimated to kill 438 000 people annually, mostly due to severe malaria, which is closely associated with microcirculatory vasculopathy, although its pathogenesis remains incompletely understood. Here, we propose that the largely ignored glycocalyx of the vascular endothelium plays an important role in facilitating the pathogenesis of severe malaria., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Background: Evidence suggests that Plasmodium knowlesi malaria in Sarawak, Malaysian Borneo remains zoonotic, meaning anti-malarial drug resistance is unlikely to have developed in the absence of drug selection pressure. Therefore, adequate response to available anti-malarial treatments is assumed., Methods: Here the ex vivo sensitivity of human P. knowlesi isolates in Malaysian Borneo were studied, using a WHO schizont maturation assay modified to accommodate the quotidian life cycle of this parasite. The in vitro sensitivities of P. knowlesi H strain adapted from a primate infection to in vitro culture (by measuring the production of Plasmodium lactate dehydrogenase) were also examined together with some assays using Plasmodium falciparum and Plasmodium vivax., Results: Plasmodium knowlesi is uniformly highly sensitive to artemisinins, variably and moderately sensitive to chloroquine, and less sensitive to mefloquine., Conclusions: Taken together with reports of clinical failures when P. knowlesi is treated with mefloquine, the data suggest that caution is required if using mefloquine in prevention or treatment of P. knowlesi infections, until further studies are undertaken.

Background: Malaria is a major health and socio-economical problem in tropical and sub-tropical areas of the world. Several methodologies have been used to assess parasite viability during the adaption of field strains to culture or the assessment of drug potential, but these are in general not able to provide an accurate real-time assessment of whether parasites are alive or dead., Methods: Different commercial dyes and kits were assessed for their potential to allow for the real-time detection of whether a blood stage malaria parasite is dead or alive., Results: Here, a methodology is presented based on the potential-sensitive mitochondrial probe JC-1, which allows for the real-time visualization of live (red staining) and/or dead (absence of red staining) blood stage parasites in vitro and ex vivo. This method is applicable across malaria parasite species and strains and allows to visualize all parasite blood stages including gametocytes. Further, this methodology has been assessed also for use in drug sensitivity testing., Conclusions: The JC-1 staining approach is a versatile methodology that can be used to assess parasite viability during the adaptation of field samples to culture and during drug treatment. It was found to hold promise in the assessment of drugs expected to lead to delayed death phenotypes and it currently being evaluated as a method for the assessment of parasite viability during the adaptation of patient-derived Plasmodium vivax to long-term in vitro culture.

Malaria parasites actively remodel the infected red blood cell (irbc) by exporting proteins into the host cell cytoplasm. The human parasite Plasmodium falciparum exports particularly large numbers of proteins, including proteins that establish a vesicular network allowing the trafficking of proteins onto the surface of irbcs that are responsible for tissue sequestration. Like P. falciparum, the rodent parasite P. berghei ANKA sequesters via irbc interactions with the host receptor CD36. We have applied proteomic, genomic, and reverse-genetic approaches to identify P. berghei proteins potentially involved in the transport of proteins to the irbc surface. A comparative proteomics analysis of P. berghei non-sequestering and sequestering parasites was used to determine changes in the irbc membrane associated with sequestration. Subsequent tagging experiments identified 13 proteins (Plasmodium export element (PEXEL)-positive as well as PEXEL-negative) that are exported into the irbc cytoplasm and have distinct localization patterns: a dispersed and/or patchy distribution, a punctate vesicle-like pattern in the cytoplasm, or a distinct location at the irbc membrane. Members of the PEXEL-negative BIR and PEXEL-positive Pb-fam-3 show a dispersed localization in the irbc cytoplasm, but not at the irbc surface. Two of the identified exported proteins are transported to the irbc membrane and were named erythrocyte membrane associated proteins. EMAP1 is a member of the PEXEL-negative Pb-fam-1 family, and EMAP2 is a PEXEL-positive protein encoded by a single copy gene; neither protein plays a direct role in sequestration. Our observations clearly indicate that P. berghei traffics a diverse range of proteins to different cellular locations via mechanisms that are analogous to those employed by P. falciparum. This information can be exploited to generate transgenic humanized rodent P. berghei parasites expressing chimeric P. berghei/P. falciparum proteins on the surface of rodent irbc, thereby opening new avenues for in vivo screening adjunct therapies that block sequestration.

Adherence of parasite-infected red blood cells (irbc) to the vascular endothelium of organs plays a key role in the pathogenesis of Plasmodium falciparum malaria. The prevailing hypothesis of why irbc adhere and sequester in tissues is that this acts as a mechanism of avoiding spleen-mediated clearance. Irbc of the rodent parasite Plasmodium berghei ANKA sequester in a fashion analogous to P. falciparum by adhering to the host receptor CD36. To experimentally determine the significance of sequestration for parasite growth, we generated a mutant P. berghei ANKA parasite with a reduced CD36-mediated adherence. Although the cognate parasite ligand binding to CD36 is unknown, we show that nonsequestering parasites have reduced growth and we provide evidence that in addition to avoiding spleen removal, other factors related to CD36-mediated sequestration are beneficial for parasite growth. These results reveal for the first time the importance of sequestration to a malaria infection, with implications for the development of strategies aimed at reducing pathology by inhibiting tissue sequestration.

Since its discovery in the 17th century, the red blood cell, recognized in time as the critical cell component for survival, has been the focus of much attention. Its unique role in gas exchange (oxygen/CO(2) transport) and its distinct characteristics (absence of nucleus; biconcave cell shape) together with an - in essence - unlimited supply lead to extensive targeted biochemical, molecular and structural studies. A quick PubMed query with the word "erythrocyte" results in 198 013 scientific articles of which 162 are red blood cell proteomics studies, indicating that this new technique has been only recently applied to the red blood cell and related fields. Standard and comparative proteomics have been widely used to study different blood components. A growing body of proteomics literature has since developed, which deals with the characterization of red blood cells in health and disease. The possibility offered by proteomics to obtain a global snapshot of the whole red blood cell protein make-up, has provided unique insights to many fields including transfusion medicine, anaemia studies, intra-red blood cell parasite biology and translational research. While the contribution of proteomics is beyond doubt, a full red blood cell understanding will ultimately require, in addition to proteomics, lipidomics, glycomics, interactomics and study of post-translational modifications. In this review we will briefly discuss the methodology and limitations of proteomics, the contribution it made to the understanding of the erythrocyte and the advances in red blood cell-related fields brought about by comparative proteomics., (Copyright 2010 Elsevier Masson SAS. All rights reserved.)

Background: Macaques are the closest evolutionary relatives of humans routinely used in basic and applied biomedical research. Their genetic, physiological, immunological and metabolic similarity to humans, second only to that of the great apes, makes them invaluable models of human disease. These similarities also mean that macaques are often the only experimental models available for evaluating increasingly specific drugs in development, and as a proof-of-concept bridge can help reduce the numbers of compounds that fail in clinical pharmaceutical research. In vertebrates, red blood cells (RBCs) diseases are frequently severe as their role as sole gas transporter makes them indispensable to survival; much research has therefore focused on an in-depth understanding of the functioning of the RBC. RBCs also host malaria, babesia and other parasites. Recently, we presented an in-depth proteome for the human RBC and a comparative human/mouse RBC proteome., Material and Methods: Here, we present directly comparable data for the human, mouse and rhesus RBC proteomes. All proteins were identified, validated and categorized in terms of sub-cellular localization, protein family and function and, in comparison with the human and mouse RBC, were classified as orthologues, family-related or unique. Splice isoforms were identified and polypeptides migrating with anomalous apparent molecular weights were grouped into putatively ubiquitinylated or partially degraded complexes., Results and Discussion: Overall there was close concordance between mouse, human and rhesus proteomes, confirming the unexpected RBC complexity. Several novel findings in the human and mouse proteomes have been confirmed here. This comparison sheds light on several open issues in RBC biology and provides a departure point for more comprehensive understanding of RBC function.

Membrane proteomics offers unprecedented possibilities to compare protein expression in health and disease leading potentially to the identification of markers, of targets for therapeutics and to a better understanding of disease mechanisms. From transfusion medicine to infectious diseases, from cardiovascular affections to diabetes, comparative proteomics has made a contribution to the identification of proteins unique to RBCs of patients with specific illnesses shedding light on possible RBC markers for systemic diseases. In this review we will provide a short overview of some of the main achievements obtained by comparative proteomics in the field of RBC-related local and systemic diseases and suggest some additional areas of RBCs research to which comparative proteomics approaches could be fruitfully applied or extended in combination with biochemical techniques., ((c) 2009 Elsevier B.V. All rights reserved.)

Membrane proteomics is concerned with accurately and sensitively identifying molecules involved in cell compartmentalisation, including those controlling the interface between the cell and the outside world. The high lipid content of the environment in which these proteins are found often causes a particular set of problems that must be overcome when isolating the required material before effective HPLC-MS approaches can be performed. The membrane is an unusually dynamic cellular structure since it interacts with an ever changing environment. A full understanding of this critical cell component will ultimately require, in addition to proteomics, lipidomics, glycomics, interactomics and study of post-translational modifications. Devoid of nucleus and organelles in mammalian species other than camelids, and constantly in motion in the blood stream, red blood cells (RBCs) are the sole mammalian oxygen transporter. The fact that mature mammalian RBCs have no internal membrane-bound organelles, somewhat simplifies proteomics analysis of the plasma membrane and the fact that it has no nucleus disqualifies microarray based methods. Proteomics has the potential to provide a better understanding of this critical interface, and thereby assist in identifying new approaches to diseases., ((c) 2009 Elsevier B.V. All rights reserved.)

Plasmodium knowlesi is an intracellular malaria parasite whose natural vertebrate host is Macaca fascicularis (the 'kra' monkey); however, it is now increasingly recognized as a significant cause of human malaria, particularly in southeast Asia. Plasmodium knowlesi was the first malaria parasite species in which antigenic variation was demonstrated, and it has a close phylogenetic relationship to Plasmodium vivax, the second most important species of human malaria parasite (reviewed in ref. 4). Despite their relatedness, there are important phenotypic differences between them, such as host blood cell preference, absence of a dormant liver stage or 'hypnozoite' in P. knowlesi, and length of the asexual cycle (reviewed in ref. 4). Here we present an analysis of the P. knowlesi (H strain, Pk1(A+) clone) nuclear genome sequence. This is the first monkey malaria parasite genome to be described, and it provides an opportunity for comparison with the recently completed P. vivax genome and other sequenced Plasmodium genomes. In contrast to other Plasmodium genomes, putative variant antigen families are dispersed throughout the genome and are associated with intrachromosomal telomere repeats. One of these families, the KIRs, contains sequences that collectively match over one-half of the host CD99 extracellular domain, which may represent an unusual form of molecular mimicry.

Mice have close genetic/physiological relationships to humans, breed rapidly, and can be genetically modified, making them the most used mammal in biomedical research. Because the red blood cell (RBC) is the sole gas transporter in vertebrates, diseases of the RBC are frequently severe; much research has therefore focused on RBC and cardiovascular disorders of mouse and humans. RBCs also host malaria parasites. Recently we presented an in-depth proteome for the human RBC. Here we present directly comparable data for the mouse RBC as membrane-only, soluble-only, and combined membrane-bound/soluble proteomes (comprising, respectively, 247, 232, and 165 proteins). All proteins were identified, validated, and categorized in terms of subcellular localization, protein family, and function, and in comparison with the human RBC, were classified as orthologs, family-related, or unique. Splice isoforms were identified, and polypeptides migrating with anomalous apparent molecular weights were grouped into putatively ubiquitinated or partially degraded complexes. Overall there was close concordance between mouse and human proteomes, confirming the unexpected RBC complexity. Several novel findings in the human proteome have been confirmed here. This comparison sheds light on several open issues in RBC biology and provides a departure point for more comprehensive understanding of RBC function.

In addition to transporting oxygen and carbon dioxide to and from the tissues, a range of other functions are attributed to red blood cells (RBCs) of vertebrates. Diseases compromising RBC performance in any of these functions warrant in-depth study. Furthermore, the human RBC is a vital host cell for the malaria parasite. Much has been learned from classical biochemical approaches about RBC composition and membrane organization. Here, we use mass spectrometry (MS)-based proteomics to characterize the normal RBC protein profile. The aim of this study was to obtain the most complete and informative human RBC proteome possible by combining high-accuracy, high-sensitivity protein identification technology (quadrupole time of flight and Fourier transform MS) with selected biochemical procedures for sample preparation. A total of 340 membrane proteins and 252 soluble proteins were identified, validated, and categorized in terms of subcellular localization, protein family, and function. Splice isoforms of proteins were identified, and polypeptides that migrated with anomalously high or low apparent molecular weights could be grouped into either ubiquitinylated, partially degraded, or ester-linked complexes. Our data reveal unexpected complexity of the RBC proteome, provide a wealth of data on its composition, shed light on several open issues in RBC biology, and form a departure point for comprehensive understanding of RBC functions.

New drugs are urgently needed to combat the growing problem of drug resistance in Plasmodium falciparum malaria. The infected erythrocyte is a multicompartmental system, and its transporters are of interest as drug targets in their own right and as potential routes for the delivery of antimalarial drugs. Choline is an important nutrient that penetrates infected erythrocyte membranes through the endogenous carrier and through parasite-induced permeability pathways, but nothing is known about its transport into the intracellular parasite. Here we present the first characterization of choline transport across the parasite membrane. Transport exhibits Michaelis-Menten kinetics with an apparent K(m) of 25.0 +/- 3.5 muM for choline. The carrier is inhibitor-sensitive, temperature-dependent, and Na(+)-independent, and it is driven by the proton-motive force. Highly active bis-amidine and bis-quaternary ammonium compounds are also known to penetrate the host erythrocyte membrane through parasite-induced permeability pathways. Here, we demonstrate that the parasite choline transporter mediates the delivery of these compounds to the intracellular parasite. Thus, the induced permeability pathways in the host erythrocyte membrane and the parasite choline transporter described here form a cooperative transport system that shows great promise for the selective targeting of new agents for the chemotherapy of malaria.