Monocytes, particularly nonclassical ones, lose their opsonic and nonopsonic phagocytosis capacity during pediatric cerebral malaria.

Introduction: Innate immunity is crucial to reducing parasite burden and contributing to survival in severe malaria. Monocytes are key actors in the innate response and, like macrophages, are plastic cells whose function and phenotype are regulated by the signals from the microenvironment. In the context of cerebral malaria (CM), monocyte response constitutes an important issue to understand. We previously demonstrated that decreased percentages of nonclassical monocytes were associated with death outcomes in CM children. In the current study, we postulated that monocyte phagocytosis function is impacted by the severity of malaria infection. Methods: To study this hypothesis, we compared the opsonic and nonopsonic phagocytosis capacity of circulant monocytes from Beninese children with uncomplicated malaria (UM) and CM. For the CM group, samples were obtained at inclusion (D0) and 3 and 30 days after treatment (D3, D30). The phagocytosis capacity of monocytes and their subsets was characterized by flow cytometry and transcriptional profiling by studying genes known for their functional implication in infected-red blood cell (iRBC) elimination or immune escape. Results: Our results confirm our hypothesis and highlight the higher capacity of nonclassical monocytes to phagocyte iRBC. We also confirm that a low number of nonclassical monocytes is associated with CM outcome when compared to UM, suggesting a mobilization of this subpopulation to the cerebral inflammatory site. Finally, our results suggest the implication of the inhibitory receptors LILRB1, LILRB2, and Tim3 in phagocytosis control. Discussion: Taken together, these data provide a better understanding of the interplay between monocytes and malaria infection in the pathogenicity of CM.

Nilgai antelope display no signs of infection upon experimental challenge with a virulent Babesia bovis strain.

BACKGROUND: Bovine babesiosis is caused by infection with the protozoal parasite Babesia bovis, which is transmitted by Rhipicephalus (Boophilus) spp. It can cause mortality rates up to 90% in immunologically naive Bos taurus cattle. In south Texas, R. (B.) microplus is known to infest nilgai antelope (Boselaphus tragocamelus); however, their susceptibility to infection with B. bovis and their role in the transmission of the parasite remain unknown. In this study, we challenged nilgai antelope with B. bovis and evaluated their susceptibility to infection. METHODS: Nilgai were needle inoculated with ≈108 B. bovis-parasitized erythrocytes (merozoites) or a homogenate of B. bovis-infected larval ticks (sporozoite) delivered intravenously. Bos taurus beef calves were inoculated in parallel, as this strain of B. bovis is lethal to cattle. Temperature and hematocrit were monitored daily over the course of each study, and whole blood was collected for molecular [polymerase chain reaction (PCR)] and serological [indirect enzyme-linked immunosorbent assay (ELISA)] diagnostic evaluation. Histological sections of nilgai cerebral tissue were examined for evidence of infection. Recipient bovine calves were sub-inoculated with blood from nilgai challenged with either stage of the parasite, and they were monitored for clinical signs of infection and evaluated by a PCR diagnostic assay. Red blood cells (RBCs) from prechallenged nilgai and B. taurus beef cattle were cultured with an in vitro B. bovis merozoite culture to examine colonization of the RBCs by the parasite. RESULTS: Nilgai did not display clinical signs of infection upon inoculation with either the merozoite or sporozoite stage of B. bovis. All nilgai were PCR-negative for the parasite, and they did not develop antibodies to B. bovis. No evidence of infection was detected in histological sections of nilgai tissues, and in vitro culture analysis indicated that the nilgai RBCs were not colonized by B. bovis merozoites. Cattle subinoculated with blood from challenged nilgai did not display clinical signs of infection, and they were PCR-negative up to 45 days after transfer. CONCLUSIONS: Nilgai do not appear to be susceptible to infection with a strain of B. bovis that is lethal to cattle. Tick control on these alternative hosts remains a critical priority, especially given their potential to disseminate ticks over long distances.

Plasmodium vivax spleen-dependent protein 1 and its role in extracellular vesicles-mediated intrasplenic infections.

Recent studies indicate that human spleen contains over 95% of the total parasite biomass during chronic asymptomatic infections caused by Plasmodium vivax. Previous studies have demonstrated that extracellular vesicles (EVs) secreted from infected reticulocytes facilitate binding to human spleen fibroblasts (hSFs) and identified parasite genes whose expression was dependent on an intact spleen. Here, we characterize the P. vivax spleen-dependent hypothetical gene (PVX_114580). Using CRISPR/Cas9, PVX_114580 was integrated into P. falciparum 3D7 genome and expressed during asexual stages. Immunofluorescence analysis demonstrated that the protein, which we named P. vivax Spleen-Dependent Protein 1 (PvSDP1), was located at the surface of infected red blood cells in the transgenic line and this localization was later confirmed in natural infections. Plasma-derived EVs from P. vivax-infected individuals (PvEVs) significantly increased cytoadherence of 3D7_PvSDP1 transgenic line to hSFs and this binding was inhibited by anti-PvSDP1 antibodies. Single-cell RNAseq of PvEVs-treated hSFs revealed increased expression of adhesion-related genes. These findings demonstrate the importance of parasite spleen-dependent genes and EVs from natural infections in the formation of intrasplenic niches in P. vivax, a major challenge for malaria elimination.

Evaluating Plasmodium falciparum automatic detection and parasitemia estimation: A comparative study on thin blood smear images.

Malaria is a deadly disease that is transmitted through mosquito bites. Microscopists use a microscope to examine thin blood smears at high magnification (1000x) to identify parasites in red blood cells (RBCs). Estimating parasitemia is essential in determining the severity of the Plasmodium falciparum infection and guiding treatment. However, this process is time-consuming, labor-intensive, and subject to variation, which can directly affect patient outcomes. In this retrospective study, we compared three methods for measuring parasitemia from a collection of anonymized thin blood smears of patients with Plasmodium falciparum obtained from the Clinical Department of Parasitology-Mycology, National Reference Center (NRC) for Malaria in Paris, France. We first analyzed the impact of the number of field images on parasitemia count using our framework, MALARIS, which features a top-classifier convolutional neural network (CNN). Additionally, we studied the variation between different microscopists using two manual techniques to demonstrate the need for a reliable and reproducible automated system. Finally, we included thin blood smear images from an additional 102 patients to compare the performance and correlation of our system with manual microscopy and flow cytometry. Our results showed strong correlations between the three methods, with a coefficient of determination between 0.87 and 0.92.

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.

APEX2-based proximity proteomic analysis identifies candidate interactors for Plasmodium falciparum knob-associated histidine-rich protein in infected erythrocytes.

The interaction of Plasmodium falciparum-infected red blood cells (iRBCs) with the vascular endothelium plays a crucial role in malaria pathology and disease. KAHRP is an exported P. falciparum protein involved in iRBC remodelling, which is essential for the formation of protrusions or "knobs" on the iRBC surface. These knobs and the proteins that are concentrated within them allow the parasites to escape the immune response and host spleen clearance by mediating cytoadherence of the iRBC to the endothelial wall, but this also slows down blood circulation, leading in some cases to severe cerebral and placental complications. In this work, we have applied genetic and biochemical tools to identify proteins that interact with P. falciparum KAHRP using enhanced ascorbate peroxidase 2 (APEX2) proximity-dependent biotinylation and label-free shotgun proteomics. A total of 30 potential KAHRP-interacting candidates were identified, based on the assigned fragmented biotinylated ions. Several identified proteins have been previously reported to be part of the Maurer's clefts and knobs, where KAHRP resides. This study may contribute to a broader understanding of P. falciparum protein trafficking and knob architecture and shows for the first time the feasibility of using APEX2-proximity labelling in iRBCs.

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.

On-Chip Impedance Spectroscopy of Malaria-Infected Red Blood Cells.

Malaria is a disease that affects millions of people worldwide, particularly in developing countries. The development of accurate and efficient methods for the detection of malaria-infected cells is crucial for effective disease management and control. This paper presents the electrical impedance spectroscopy (EIS) of normal and malaria-infected red blood cells. An EIS microfluidic device, comprising a microchannel and a pair of coplanar electrodes, was fabricated for single-cell measurements in a continuous manner. Based on the EIS results, the aim of this work is to discriminate Plasmodium falciparum-infected red blood cells from the normal ones. Different from typical impedance spectroscopy, our measurement was performed for the cells in a low-conductivity medium in a frequency range between 50 kHz and 800 kHz. Numerical simulation was utilized to study the suitability parameters of the microchannel and electrodes for the EIS experiment over the measurement frequencies. The measurement results have shown that by using the low-conductivity medium, we could focus on the change in the conductance caused by the presence of a cell in the sensing electrode gap. The results indicated a distinct frequency spectrum of the conductance between the normal and infected red blood cells, which can be further used for the detection of the disease.

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 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.

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.

Correlations between the degree of infection by wild strain of Toxoplasma gondii in vitro and porcine hematological parameters.

The apicomplexa Toxoplasma gondii is capable of actively proliferating in numerous types of nucleated cells, and therefore has a high potential for dissemination and resistance. Thus, the present work aimed to correlate the inoculum concentrations and amount of post-infection parasites with porcine hematological parameters (including biochemistry) through in vitro culture. Porcine blood was incubated with different concentrations of parasites (1.2 × 107, 6/3/1.5 × 106 cells/mL), then the concentrations of red blood cells (RBC) and their morphology, total and differential leukocytes, and free peptides were evaluated. In addition, eight different blood samples analyzed before inoculation, where subsequent multivariate analysis was applied to correlate different variables with trophozoite concentration. The results showed no significant variation (p < 0.05) in the relative levels of free peptides, or the relative percentage of RBC at all the parasite concentrations tested. However, the normalized percentages of leukocytes and neutrophils showed a significant reduction, while those of lymphocytes, eosinophils and monocytes showed the opposite behavior. Semi-automatic processing of images exhibited significant microcytosis and hypochromia. The multivariate analysis revealed a positive correlation between the amount number of protozoa (AP) and the variables: "Red cells" and "Neutrophils", an indifference between the AP and the content of free peptides, and the concentration of monocytes in the samples; and a negative correlation for AP and the percentages of lymphocytes and eosinophils. Our results suggest that specific changes in hematological parameters may be associated with different degrees of parasitemia, demanding a thorough diagnostic process and adequate treatment.

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.

Description of an intramonocytic haemoparasite, Hepatozoon lainsoni sp. nov. (Apicomplexa: Adeleorina: Hepatozoidae), infecting Ameiva ameiva lizard (Reptilia: Squamata: Teiidae) in northern Brazil.

Haemogregarine (Apicomplexa: Adeleorina) parasites are considered to be the most common and widespread haemoparasites in reptiles. The genus Hepatozoon (Apicomplexa: Adeleorina: Hepatozoidae) can be found parasitizing a broad range of species and, in reptiles, they infect mainly peripheral blood erythrocytes. The present study detected and characterized a haemogregarine isolated from the lizard species, Ameiva ameiva, collected from the municipality of Capanema, Pará state, north Brazil. Blood smears and imprints from lungs, brain, heart, kidney, liver, bone marrow and spleen were observed using light microscopy and the parasite was genetically identified by molecular analysis. Morphological, morphometric and molecular data were obtained. Parasite gamonts were found in 49.5% (55/111) of the blood smears from A. ameiva, and were characterized as oval, averaging 12.0 ± 0.8 × 5.9 ± 0.6 µm2 in size, which displaced the nuclei of parasitized monocytes laterally. Parasite forms resembling immature gamonts were observed in the spleen and bone marrow of the lizards. Furthermore, phylogenetic analyses of 18S rRNA sequences did not reveal gene similarity with other Hepatozoon spp. sequences from reptiles. Thus, morphological and molecular analyses have identified a new species of Hepatozoon parasite, Hepatozoon lainsoni sp. nov., which infects monocytes of the A. ameiva lizard.

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.

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.

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.

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.

Amino acid supplementation confers protection to red blood cells before Plasmodium falciparum bystander stress.

ABSTRACT: Malaria is a highly oxidative parasitic disease in which anemia is the most common clinical symptom. A major contributor to the malarial anemia pathogenesis is the destruction of bystander, uninfected red blood cells (RBCs). Metabolic fluctuations are known to occur in the plasma of individuals with acute malaria, emphasizing the role of metabolic changes in disease progression and severity. Here, we report conditioned medium from Plasmodium falciparum culture induces oxidative stress in uninfected, catalase-depleted RBCs. As cell-permeable precursors to glutathione, we demonstrate the benefit of pre-exposure to exogenous glutamine, cysteine, and glycine amino acids for RBCs. Importantly, this pretreatment intrinsically prepares RBCs to mitigate oxidative stress.