Hypertension Increases Susceptibility to Experimental Malaria in Mice.

Global prevalence of hypertension is on the rise, burdening healthcare, especially in developing countries where infectious diseases, such as malaria, are also rampant. Whether hypertension could predispose or increase susceptibility to malaria, however, has not been extensively explored. Previously, we reported that hypertension is associated with abnormal red blood cell (RBC) physiology and anemia. Since RBC are target host cells for malarial parasite, Plasmodium, we hypothesized that hypertensive patients with abnormal RBC physiology are at greater risk or susceptibility to Plasmodium infection. To test this hypothesis, normotensive (BPN/3J) and hypertensive (BPH/2J) mice were characterized for their RBC physiology and subsequently infected with Plasmodium yoelii (P. yoelii), a murine-specific non-lethal strain. When compared to BPN mice, BPH mice displayed microcytic anemia with RBC highly resistant to osmotic hemolysis. Further, BPH RBC exhibited greater membrane rigidity and an altered lipid composition, as evidenced by higher levels of phospholipids and saturated fatty acid, such as stearate (C18:0), along with lower levels of polyunsaturated fatty acid like arachidonate (C20:4). Moreover, BPH mice had significantly greater circulating Ter119+ CD71+ reticulocytes, or immature RBC, prone to P. yoelii infection. Upon infection with P. yoelii, BPH mice experienced significant body weight loss accompanied by sustained parasitemia, indices of anemia, and substantial increase in systemic pro-inflammatory mediators, compared to BPN mice, indicating that BPH mice were incompetent to clear P. yoelii infection. Collectively, these data demonstrate that aberrant RBC physiology observed in hypertensive BPH mice contributes to an increased susceptibility to P. yoelii infection and malaria-associated pathology.

A single cell atlas of sexual development in Plasmodium falciparum.

The developmental decision made by malaria parasites to become sexual underlies all malaria transmission. Here, we describe a rich atlas of short- and long-read single-cell transcriptomes of over 37,000 Plasmodium falciparum cells across intraerythrocytic asexual and sexual development. We used the atlas to explore transcriptional modules and exon usage along sexual development and expanded it to include malaria parasites collected from four Malian individuals naturally infected with multiple P. falciparum strains. We investigated genotypic and transcriptional heterogeneity within and among these wild strains at the single-cell level, finding differential expression between different strains even within the same host. These data are a key addition to the Malaria Cell Atlas interactive data resource, enabling a deeper understanding of the biology and diversity of transmission stages.

PfCAP-H is essential for assembly of condensin I complex and karyokinesis during asexual proliferation of Plasmodium falciparum.

Condensin I is a pentameric complex that regulates the mitotic chromosome assembly in eukaryotes. The kleisin subunit CAP-H of the condensin I complex acts as a linchpin to maintain the structural integrity and loading of this complex on mitotic chromosomes. This complex is present in all eukaryotes and has recently been identified in Plasmodium spp. However, how this complex is assembled and whether the kleisin subunit is critical for this complex in these parasites are yet to be explored. To examine the role of PfCAP-H during cell division within erythrocytes, we generated an inducible PfCAP-H knockout parasite. We find that PfCAP-H is dynamically expressed during mitosis with the peak expression at the metaphase plate. PfCAP-H interacts with PfCAP-G and is a non-SMC member of the condensin I complex. Notably, the absence of PfCAP-H does not alter the expression of PfCAP-G but affects its localization at the mitotic chromosomes. While mitotic spindle assembly is intact in PfCAP-H-deficient parasites, duplicated centrosomes remain clustered over the mass of unsegmented nuclei with failed karyokinesis. This failure leads to the formation of an abnormal nuclear mass, while cytokinesis occurs normally. Altogether, our data suggest that PfCAP-H plays a crucial role in maintaining the structural integrity of the condensin I complex on the mitotic chromosomes and is essential for the asexual development of malarial parasites. IMPORTANCE: Mitosis is a fundamental process for Plasmodium parasites, which plays a vital role in their survival within two distinct hosts-human and Anopheles mosquitoes. Despite its great significance, our comprehension of mitosis and its regulation remains limited. In eukaryotes, mitosis is regulated by one of the pivotal complexes known as condensin complexes. The condensin complexes are responsible for chromosome condensation, ensuring the faithful distribution of genetic material to daughter cells. While condensin complexes have recently been identified in Plasmodium spp., our understanding of how this complex is assembled and its precise functions during the blood stage development of Plasmodium falciparum remains largely unexplored. In this study, we investigate the role of a central protein, PfCAP-H, during the blood stage development of P. falciparum. Our findings reveal that PfCAP-H is essential and plays a pivotal role in upholding the structure of condensin I and facilitating karyokinesis.

The essential malaria protein PfCyRPA targets glycans to invade erythrocytes.

Plasmodium falciparum is a human-adapted apicomplexan parasite that causes the most dangerous form of malaria. P. falciparum cysteine-rich protective antigen (PfCyRPA) is an invasion complex protein essential for erythrocyte invasion. The precise role of PfCyRPA in this process has not been resolved. Here, we show that PfCyRPA is a lectin targeting glycans terminating with α2-6-linked N-acetylneuraminic acid (Neu5Ac). PfCyRPA has a >50-fold binding preference for human, α2-6-linked Neu5Ac over non-human, α2-6-linked N-glycolylneuraminic acid. PfCyRPA lectin sites were predicted by molecular modeling and validated by mutagenesis studies. Transgenic parasite lines expressing endogenous PfCyRPA with single amino acid exchange mutants indicated that the lectin activity of PfCyRPA has an important role in parasite invasion. Blocking PfCyRPA lectin activity with small molecules or with lectin-site-specific monoclonal antibodies can inhibit blood-stage parasite multiplication. Therefore, targeting PfCyRPA lectin activity with drugs, immunotherapy, or a vaccine-primed immune response is a promising strategy to prevent and treat malaria.

Inhibition of malaria and babesiosis parasites by putative red blood cell targeting small molecules.

Background: Chemotherapies for malaria and babesiosis frequently succumb to the emergence of pathogen-related drug-resistance. Host-targeted therapies are thought to be less susceptible to resistance but are seldom considered for treatment of these diseases. Methods: Our overall objective was to systematically assess small molecules for host cell-targeting activity to restrict proliferation of intracellular parasites. We carried out a literature survey to identify small molecules annotated for host factors implicated in Plasmodium falciparum infection. Alongside P. falciparum, we implemented in vitro parasite susceptibility assays also in the zoonotic parasite Plasmodium knowlesi and the veterinary parasite Babesia divergens. We additionally carried out assays to test directly for action on RBCs apart from the parasites. To distinguish specific host-targeting antiparasitic activity from erythrotoxicity, we measured phosphatidylserine exposure and hemolysis stimulated by small molecules in uninfected RBCs. Results: We identified diverse RBC target-annotated inhibitors with Plasmodium-specific, Babesia-specific, and broad-spectrum antiparasitic activity. The anticancer MEK-targeting drug trametinib is shown here to act with submicromolar activity to block proliferation of Plasmodium spp. in RBCs. Some inhibitors exhibit antimalarial activity with transient exposure to RBCs prior to infection with parasites, providing evidence for host-targeting activity distinct from direct inhibition of the parasite. Conclusions: We report here characterization of small molecules for antiproliferative and host cell-targeting activity for malaria and babesiosis parasites. This resource is relevant for assessment of physiological RBC-parasite interactions and may inform drug development and repurposing efforts.

The influence of neonatal BCG vaccination on in vitro cytokine responses to Plasmodium falciparum.

BACKGROUND: Bacillus Calmette-Guérin (BCG) vaccination has off-target protective effects against infections unrelated to tuberculosis. Among these, murine and human studies suggest that BCG vaccination may protect against malaria. We investigated whether BCG vaccination influences neonatal in vitro cytokine responses to Plasmodium falciparum. Blood samples were collected from 108 participants in the Melbourne Infant Study BCG for Allergy and Infection Reduction (MIS BAIR) randomised controlled trial (Clinical trials registration NCT01906853, registered July 2013), seven days after randomisation to neonatal BCG (n = 66) or no BCG vaccination (BCG-naïve, n = 42). In vitro cytokine responses were measured following stimulation with P. falciparum-infected erythrocytes (PfIE) or E. coli. RESULTS: No difference in the measured cytokines were observed between BCG-vaccinated and BCG-naïve neonates following stimulation with PfIE or E. coli. However, age at which blood was sampled was independently associated with altered cytokine responses to PfIE. Being male was also independently associated with increased TNF-a responses to both PfIE and E. coli. CONCLUSION: These findings do not support a role for BCG vaccination in influencing in vitro neonatal cytokine responses to P. falciparum. Older neonates are more likely to develop P. falciparum-induced IFN-γ and IFN-γ-inducible chemokine responses implicated in early protection against malaria and malaria pathogenesis.

Extracellular vesicles derived from plasmodium-infected red blood cells alleviate cerebral malaria in plasmodium berghei ANKA-infected C57BL/6J mice.

RTS,S is the first malaria vaccine recommended for implementation among young children at risk. However, vaccine efficacy is modest and short-lived. To mitigate the risk of cerebral malaria (CM) among children under the age of 5, it is imperative to develop new vaccines. EVs are potential vaccine candidates as they obtain the ability of brain-targeted delivery and transfer plasmodium antigens and immunomodulators during infections. This study extracted EVs from BALB/c mice infected with Plasmodium yoelii 17XNL (P.y17XNL). C57BL/6J mice were intravenously immunized with EVs (EV-I.V. + CM group) or subcutaneously vaccinated with the combination of EVs and CpG ODN-1826 (EV + CPG ODN-S.C. + CM group) on days 0 and 20, followed by infection with Plasmodium berghei ANKA (P.bANKA) on day 20 post-second immunization. We monitored Parasitemia and survival rate. The integrity of the Blood-brain barrier (BBB) was examined using Evans blue staining.The levels of cytokines and adhesion molecules were evaluated using Luminex, RT-qPCR, and WB. Brain pathology was evaluated by hematoxylin and eosin and immunohistochemical staining. The serum levels of IgG, IgG1, and IgG2a were analyzed by enzyme-linked immunosorbent assay. Compared with those in the P.bANKA-infected group, parasitemia increased slowly, death was delayed (day 10 post-infection), and the survival rate reached 75 %-83.3 % in the EV-I.V. + ECM and EV + CPG ODN-S.C. + ECM groups. Meanwhile, compared with the EV + CPG ODN-S.C. + ECM group, although parasitemia was almost the same, the survival rate increased in the EV-I.V. + ECM group.Additionally, EVs immunization markedly downregulated inflammatory responses in the spleen and brain and ameliorated brain pathological changes, including BBB disruption and infected red blood cell (iRBC) sequestration. Furthermore, the EVs immunization group exhibited enhanced antibody responses (upregulation of IgG1 and IgG2a production) compared to the normal control group. EV immunization exerted protective effects, improving the integrity of the BBB, downregulating inflammation response of brain tissue, result in reduces the incidence of CM. The protective effects were determined by immunological pathways and brain targets elicited by EVs. Intravenous immunization exhibited better performance than subcutaneous immunization, which perhaps correlated with EVs, which can naturally cross BBB to play a better role in brain protection.

Plasmodium falciparum selectively degrades α-spectrin of infected erythrocytes after invasion.

Remodeling the erythrocyte membrane and skeleton by the malarial parasite Plasmodium falciparum is closely associated with intraerythrocytic development. However, the mechanisms underlying this association remain unclear. In this study, we present evidence that erythrocytic α-spectrin, but not ß-spectrin, was dynamically ubiquitinated and progressively degraded during the intraerythrocytic development of P. falciparum, from the ring to the schizont stage. We further observed an upregulated expression of P. falciparum phosphatidylinositol 3-kinase (PfPI3K) in the infected red blood cells during the intraerythrocytic development of the parasite. The data indicated that PfPI3K phosphorylated and activated erythrocytic ubiquitin-protein ligase, leading to increased α-spectrin ubiquitination and degradation during P. falciparum development. We further revealed that inhibition of the activity of PfPI3K impaired P. falciparum development in vitro and Plasmodium berghei infectivity in mice. These findings collectively unveil an important mechanism of PfPI3K-ubiquitin-mediated degradation of α-spectrin during the intraerythrocytic development of Plasmodium species. Proteins in the PfPI3K regulatory pathway are novel targets for effective treatment of severe malaria. IMPORTANCE: Plasmodium falciparum is the causative agent of severe malaria that causes millions of deaths globally. The parasite invades human red blood cells and induces a cascade of alterations in erythrocytes for development and proliferation. Remodeling the host erythrocytic cytoskeleton is a necessary process during parasitization, but its regulatory mechanisms remain to be elucidated. In this study, we observed that erythrocytic α-spectrin is selectively degraded after P. falciparum invasion, while ß-spectrin remained intact. We found that the α-spectrin chain was profoundly ubiquitinated by E3 ubiquitin ligase and degraded by the 26S proteasome. E3 ubiquitin ligase activity was regulated by P. falciparum phosphatidylinositol 3-kinase (PfPI3K) signaling. Additionally, blocking the PfPI3K-ubiquitin-proteasome pathway in P. falciparum-infected red blood cells reduced parasite proliferation and infectivity. This study deepens our understanding of the regulatory mechanisms of host and malarial parasite interactions and paves the way for the exploration of novel antimalarial drugs.

Structure-guided design of VAR2CSA-based immunogens and a cocktail strategy for a placental malaria vaccine.

Placental accumulation of Plasmodium falciparum infected erythrocytes results in maternal anemia, low birth weight, and pregnancy loss. The parasite protein VAR2CSA facilitates the accumulation of infected erythrocytes in the placenta through interaction with the host receptor chondroitin sulfate A (CSA). Antibodies that prevent the VAR2CSA-CSA interaction correlate with protection from placental malaria, and VAR2CSA is a high-priority placental malaria vaccine antigen. Here, structure-guided design leveraging the full-length structures of VAR2CSA produced a stable immunogen that retains the critical conserved functional elements of VAR2CSA. The design expressed with a six-fold greater yield than the full-length protein and elicited antibodies that prevent adhesion of infected erythrocytes to CSA. The reduced size and adaptability of the designed immunogen enable efficient production of multiple variants of VAR2CSA for use in a cocktail vaccination strategy to increase the breadth of protection. These designs form strong foundations for the development of potent broadly protective placental malaria vaccines.

Hemoparasite Analysis and Hematological Parameters of Four Freshwater Turtle Species (Testudines: Podocnemididae) from the Brazilian Amazon.

METHODS: Sampling was performed at three sites in the Tapajós River mouth, Pará State, Brazil, and an aliquot of blood was collected from each turtle by post-occipital dorsal vein puncture. The aliquots were used for the determination of hematological parameters and for the study of hemoparasites in the blood samples. RESULTS: A total of 37 turtles were sampled: 21 Podocnemis expansa, three P. sextuberculata, nine P. unifilis, and four P. erythrocephala. Parasitological analyses showed inclusions in erythrocytes compatible with Haemogregarina sp., representing an overall prevalence of parasitized animals of 64.8% with an average of 94,789 parasites/ml of blood. In this study, the evolutionary forms of Haemogregarina spp. from trophozoites, pre-meronts, meronts, and mature and immature gamonts were observed in three species of freshwater turtle species, and one morphotype of the parasite with high similarity to Haemogregarina podocnemis has been observed. CONCLUSION: Despite recent research on freshwater turtles, this study provides new information on the intensity of blood parasite infection and hematological parameters of free-living animals, highlighting the need for studies on infected and uninfected animals from the same population.

Expression of the MSPDBL2 antigen in a discrete subset of Plasmodium falciparum schizonts is regulated by GDV1 but may not be linked to sexual commitment.

The Plasmodium falciparum merozoite surface protein MSPDBL2 is a polymorphic antigen targeted by acquired immune responses, and normally expressed in only a minority of mature schizonts. The potential relationship of MSPDBL2 to sexual commitment is examined, as variable mspdbl2 transcript levels and proportions of MSPDBL2-positive mature schizonts in clinical isolates have previously correlated with levels of many sexual stage parasite gene transcripts, although not with the master regulator ap2-g. It is demonstrated that conditional overexpression of the gametocyte development protein GDV1, which promotes sexual commitment, also substantially increases the proportion of MSPDBL2-positive schizonts in culture. Conversely, truncation of the gdv1 gene is shown to prevent any expression of MSPDBL2. However, across diverse P. falciparum cultured lines, the variable proportions of MSPDBL2 positivity in schizonts do not correlate significantly with variable gametocyte conversion rates, indicating it is not involved in sexual commitment. Confirming this, examining a line with endogenous hemagglutinin-tagged AP2-G showed that the individual schizonts expressing MSPDBL2 are mostly different from those expressing AP2-G. Using a selection-linked integration system, modified P. falciparum lines were engineered to express an intact or disrupted version of MSPDBL2, showing the protein is not required for sexual commitment or early gametocyte development. Asexual parasite multiplication rates were also not affected by expression of either intact or disrupted MSPDBL2 in a majority of schizonts. Occurring alongside sexual commitment, the role of the discrete MSPDBL2-positive schizont subpopulation requires further investigation in natural infections where it is under immune selection. IMPORTANCE: Malaria parasites in the blood are remarkably variable, able to switch antigenic targets so they may survive within humans who have already developed specific immune responses. This is one of the challenges in developing vaccines against malaria. MSPDBL2 is a target of naturally acquired immunity expressed in minority proportions of schizonts, the end stages of each 2-day replication cycle in red blood cells which contain merozoites prepared to invade new red blood cells. Results show that the proportion of schizonts expressing MSPDBL2 is positively controlled by the expression of the regulatory gametocyte development protein GDV1. It was previously known that expression of GDV1 leads to increased expression of AP2-G which causes parasites to switch to sexual development, so a surprising finding here is that MSPDBL2-positive parasites are mostly distinct from those that express AP2-G. This discrete antigenic subpopulation of mostly asexual parasites is regulated alongside sexually committed parasites, potentially enabling survival under stress conditions.

Skeleton binding protein 1 localizes to the Maurer's cleft and interacts with PfHSP70-1 and PfHSP70-x in Plasmodium falciparum gametocyte-infected erythrocytes.

Plasmodium falciparum accounts for the majority of malaria deaths, due to pathology provoked by the ability of infected erythrocytes to adhere to vascular endothelium within deep tissues. The parasite recognizes endothelium by trafficking and displaying protein ligands on the surface of asexual stage infected erythrocytes, such as members of the large family of pathogenic proteins, P. falciparum erythrocyte membrane protein 1 (PfEMP1). Parasite-encoded skeleton binding protein 1 (SBP1) plays an important role in the transport of these binding-related surface proteins, via cleft-like membranous structures termed Maurer's clefts, which are present within the cytoplasm of infected erythrocytes. Erythrocytes infected with gametocyte stages accumulate in the extravascular compartment of bone marrow; and it was suggested that their surface-expressed adhesion molecule profile and protein trafficking mechanisms might differ from those in asexual stage parasites. Protein trafficking mechanisms via Maurer's clefts have been well investigated in asexual stage parasite-infected erythrocytes; but little is known regarding the gametocyte stages. In this study, we characterized SBP1 during gametocyte maturation and demonstrated that SBP1 is expressed and localizes to dot-like Maurer's cleft structures in the cytoplasm of gametocyte-infected erythrocytes. Co-immunoprecipitation and mass spectrometry assays indicated that SBP1 interacts with the molecular chaperones PfHSP70-1 and PfHSP70-x. Localization analysis suggested that some PfHSP70-1 and/or PfHSP70-x localize in a dot-like pattern within the cytoplasm of immature gametocyte-infected erythrocytes. These findings suggest that SBP1 may interact with HSP70 chaperones in the infected erythrocyte cytoplasm during the immature gametocyte stages.

P. falciparum Invasion and Erythrocyte Aging.

Plasmodium parasites need to find red blood cells (RBCs) that, on the one hand, expose receptors for the pathogen ligands and, on the other hand, maintain the right geometry to facilitate merozoite attachment and entry into the red blood cell. Both characteristics change with the maturation of erythrocytes. Some Plasmodia prefer younger vs. older erythrocytes. How does the life evolution of the RBC affect the invasion of the parasite? What happens when the RBC ages? In this review, we present what is known up until now.

Prodomain-driven enzyme dimerization: a pH-dependent autoinhibition mechanism that controls Plasmodium Sub1 activity before merozoite egress.

Malaria symptoms are associated with the asexual multiplication of Plasmodium falciparum within human red blood cells (RBCs) and fever peaks coincide with the egress of daughter merozoites following the rupture of the parasitophorous vacuole (PV) and the RBC membranes. Over the last two decades, it has emerged that the release of competent merozoites is tightly regulated by a complex cascade of events, including the unusual multi-step activation mechanism of the pivotal subtilisin-like protease 1 (Sub1) that takes place in three different cellular compartments and remains poorly understood. Following an initial auto-maturation in the endoplasmic reticulum (ER) between its pro- and catalytic domains, the Sub1 prodomain (PD) undergoes further cleavages by the parasite aspartic protease plasmepsin X (PmX) within acidic secretory organelles that ultimately lead to full Sub1 activation upon discharge into the PV. Here, we report the crystal structure of full-length P. falciparum Sub1 (PfS1FL) and demonstrate, through structural, biochemical, and biophysical studies, that the atypical Plasmodium-specific Sub1 PD directly promotes the assembly of inactive enzyme homodimers at acidic pH, whereas Sub1 is primarily monomeric at neutral pH. Our results shed new light into the finely tuned Sub1 spatiotemporal activation during secretion, explaining how PmX processing and full activation of Sub1 can occur in different cellular compartments, and uncover a robust mechanism of pH-dependent subtilisin autoinhibition that plays a key role in P. falciparum merozoites egress from infected host cells.IMPORTANCEMalaria fever spikes are due to the rupture of infected erythrocytes, allowing the egress of Plasmodium sp. merozoites and further parasite propagation. This fleeting tightly regulated event involves a cascade of enzymes, culminating with the complex activation of the subtilisin-like protease 1, Sub1. Differently than other subtilisins, Sub1 activation strictly depends upon the processing by a parasite aspartic protease within acidic merozoite secretory organelles. However, Sub1 biological activity is required in the pH neutral parasitophorous vacuole, to prime effectors involved in the rupture of the vacuole and erythrocytic membranes. Here, we show that the unusual, parasite-specific Sub1 prodomain is directly responsible for its acidic-dependent dimerization and autoinhibition, required for protein secretion, before its full activation at neutral pH in a monomeric form. pH-dependent Sub1 dimerization defines a novel, essential regulatory element involved in the finely tuned spatiotemporal activation of the egress of competent Plasmodium merozoites.

Plasmodium falciparum Glutamic Acid-Rich Protein-Independent Polyclonal Antibodies Inhibit Malaria Parasite Growth in Human Erythrocytes.

Plasmodium falciparum glutamic acid-rich protein (PfGARP) is a recently characterized cell surface antigen encoded by Plasmodium falciparum, the causative agent of severe human malaria pathophysiology. Previously, we reported that the human erythrocyte band 3 (SLC4A1) serves as a host receptor for PfGARP. Antibodies against PfGARP did not affect parasite invasion and growth. We surmised that PfGARP may play a role in the rosetting and adhesion of malaria. Another study reported that antibodies targeting PfGARP exhibit potent inhibition of parasite growth. This inhibition occurred without the presence of any immune or complement components, suggesting the activation of an inherent density-dependent regulatory system. Here, we used polyclonal antibodies against PfGARP and a monoclonal antibody mAb7899 to demonstrate that anti-PfGARP polyclonal antibodies, but not mAb7899, exerted potent inhibition of parasite growth in infected erythrocytes independent of PfGARP. These findings suggest that an unknown malaria protein(s) is the target of growth arrest by polyclonal antibodies raised against PfGARP.

Infected erythrocytes and plasma proteomics reveal a specific protein signature of severe malaria.

Cerebral malaria (CM), the most lethal complication of Plasmodium falciparum severe malaria (SM), remains fatal for 15-25% of affected children despite the availability of treatment. P. falciparum infects and multiplies in erythrocytes, contributing to anemia, parasite sequestration, and inflammation. An unbiased proteomic assessment of infected erythrocytes and plasma samples from 24 Beninese children was performed to study the complex mechanisms underlying CM. A significant down-regulation of proteins from the ubiquitin-proteasome pathway and an up-regulation of the erythroid precursor marker transferrin receptor protein 1 (TFRC) were associated with infected erythrocytes from CM patients. At the plasma level, the samples clustered according to clinical presentation. Significantly, increased levels of the 20S proteasome components were associated with SM. Targeted quantification assays confirmed these findings on a larger cohort (n = 340). These findings suggest that parasites causing CM preferentially infect reticulocytes or erythroblasts and alter their maturation. Importantly, the host plasma proteome serves as a specific signature of SM and presents a remarkable opportunity for developing innovative diagnostic and prognostic biomarkers.

Dantu Blood Group Erythrocytes Form Large Plasmodium falciparum Rosettes Less Commonly.

Dantu erythrocytes, which express a hybrid glycophorin B/A protein, are protective against severe malaria. Recent studies have shown that Dantu impairs Plasmodium falciparum invasion by increasing erythrocyte membrane tension, but its effects on pathological host-parasite adhesion interactions such as rosetting, the binding of uninfected erythrocytes to P. falciparum-infected erythrocytes, have not been investigated previously. The expression of several putative host rosetting receptors-including glycophorin A (GYPA), glycophorin C (GYPC), complement receptor 1 (CR1), and band 3, which complexes with GYPA to form the Wrightb blood group antigen-are altered on Dantu erythrocytes. Here, we compare receptor expression, and rosetting at both 1 hour and 48 hours after mixing with mature trophozoite-stage Kenyan laboratory-adapted P. falciparum strain 11019 parasites in Dantu and non-Dantu erythrocytes. Dantu erythrocytes showed lower staining for GYPA and CR1, and greater staining for band 3, as observed previously, whereas Wrightb and GYPC staining did not vary significantly. No significant between-genotype differences in rosetting were seen after 1 hour, but the percentage of large rosettes was significantly less in both Dantu heterozygous (mean, 16.4%; standard error of the mean [SEM], 3.2) and homozygous donors (mean, 15.4%; SEM, 1.4) compared with non-Dantu erythrocytes (mean, 32.9%; SEM, 7.1; one-way analysis of variance, P = 0.025) after 48 hours. We also found positive correlations between erythrocyte mean corpuscular volume (MCV), the percentage of large rosettes (Spearman's rs = 0.5970, P = 0.0043), and mean rosette size (rs = 0.5206, P = 0.0155). Impaired rosetting resulting from altered erythrocyte membrane receptor expression and reduced MCV might add to the protective effect of Dantu against severe malaria.

Trio fluorophore-based phenotypic assay for the detection of artemisinin-induced growth-arrested Plasmodium falciparum in human erythrocytes.

Artemisinin combination therapy remains effective for the treatment of falciparum malaria. However, Plasmodium falciparum can escape the effects of artemisinin by arresting their growth. The growth-arrested parasites cannot be distinguished from nonviable parasites with standard microscopy techniques due to their morphological similarities. Here, we demonstrated the efficacy of a new laboratory assay that is compatible with the artemisinin susceptibility test. As a result of the differential cell permeabilities of two DNA-binding fluorophores, growth-arrested P. falciparum can be distinguished from parasites killed by artemisinin, since the latter lose cell membrane permeability. This fluorescence-based assay increased the sensitivity and specificity of the ring survival assay in the assessment of artemisinin susceptibility. When combined with a third fluorophore-conjugated anti-human leukocyte antibody, this trio fluorophore assay became more useful in identifying growth-arrested parasites in mock human blood samples. This novel assay is a simple and rapid technique for monitoring artemisinin resistance with greater sensitivity and accuracy compared with morphology-based observations under a light microscope.

Plasmodium falciparum ZIP1 Is a Zinc-Selective Transporter with Stage-Dependent Targeting to the Apicoplast and Plasma Membrane in Erythrocytic Parasites.

Replication of the malarial parasite in human erythrocytes requires massive zinc fluxes, necessitating the action of zinc transporters across the parasite plasma and organellar membranes. Although genetic knockout studies have been conducted on a few "orphan" zinc transporters in Plasmodium spp., none of them have been functionally characterized. We used the recombinant Plasmodium falciparum Zrt-/Irt-like protein (PfZIP1) and specific antibodies generated against it to explore the subcellular localization, function, metal-ion selectivity, and response to cellular zinc levels. PfZIP1 expression was enhanced upon the depletion of cytosolic Zn2+. The protein transitioned from the processed to unprocessed form through blood stages, localizing to the apicoplast in trophozoites and to the parasite plasma membrane in schizonts and gametocytes, indicating stage-specific functional role. The PfZIP1 dimer mediated Zn2+ influx in proteoliposomes. It exhibited preferential binding to Zn2+ compared to Fe2+, with the selectivity for zinc being driven by a C-terminal histidine-rich region conserved only in primate-infecting Plasmodium species.

Identification of a non-exported Plasmepsin V substrate that functions in the parasitophorous vacuole of malaria parasites.

IMPORTANCE: In the manuscript, the authors investigate the role of the protease Plasmepsin V in the parasite-host interaction. Whereas processing by Plasmepsin V was previously thought to target a protein for export into the host cell, the authors now show that there are proteins cleaved by this protease that are not exported but instead function at the host-parasite interface. This changes the view of this protease, which turns out to have a much broader role than anticipated. The result shows that the protease may have a function much more similar to that of related organisms. The authors also investigate the requirements for protein export by analyzing exported and non-exported proteins and find commonalities between the proteins of each set that further our understanding of the requirements for protein export.