Survival of P. falciparum infected red blood cell aggregates in elongational shear flow.

Rosetting, the formation of red blood cell aggregates, is a life-threatening condition in malaria tropica and not yet fully understood. We study rosette stability using a set of microfluidic stenotic channels, with varied narrowing angle and erythrocytes of blood groups O and A. We find reduced ability of a rosette to pass a stenosis without disruption, the longer the tapered part of the constriction and the narrower the stenosis is. In general, this ability increases with rosette size and is 5-15% higher in blood group A. The experimental results are substantiated by equivalent experiments using lectin-induced red blood cell aggregates and a simulation of the underlying protein binding kinetics.

Red blood cell blood group A antigen level affects the ability of heparin and PfEMP1 antibodies to disrupt Plasmodium falciparum rosettes.

BACKGROUND: The histo-blood group ABO system has been associated with adverse outcomes in COVID-19, thromboembolic diseases and Plasmodium falciparum malaria. An integral part of the severe malaria pathogenesis is rosetting, the adherence of parasite infected red blood cells (RBCs) to uninfected RBCs. Rosetting is influenced by the host's ABO blood group (Bg) and rosettes formed in BgA have previously been shown to be more resilient to disruption by heparin and shield the parasite derived surface antigens from antibodies. However, data on rosetting in weak BgA subgroups is scarce and based on investigations of relatively few donors. METHODS: An improved high-throughput flow cytometric assay was employed to investigate rosetting characteristics in an extensive panel of RBC donor samples of all four major ABO Bgs, as well as low BgA expressing samples. RESULTS: All non-O Bgs shield the parasite surface antigens from strain-specific antibodies towards P. falciparum erythrocyte membrane protein 1 (PfEMP1). A positive correlation between A-antigen levels on RBCs and rosette tightness was observed, protecting the rosettes from heparin- and antibody-mediated disruption. CONCLUSIONS: These results provide new insights into how the ABO Bg system affects the disease outcome and cautions against interpreting the results from the heterogeneous BgA phenotype as a single group in epidemiological and experimental studies.

Direct contact between Plasmodium falciparum and human B-cells in a novel co-culture increases parasite growth and affects B-cell growth.

BACKGROUND: Plasmodium falciparum parasites cause malaria and co-exist in humans together with B-cells for long periods of time. Immunity is only achieved after repeated exposure. There has been a lack of methods to mimic the in vivo co-occurrence, where cells and parasites can be grown together for many days, and it has been difficult with long time in vitro studies. METHODS AND RESULTS: A new method for growing P. falciparum in 5% CO2 with a specially formulated culture medium is described. This knowledge was used to establish the co-culture of live P. falciparum together with human B-cells in vitro for 10 days. The presence of B-cells clearly enhanced parasite growth, but less so when Transwell inserts were used (not allowing passage of cells or merozoites), showing that direct contact is advantageous. B-cells also proliferated more in presence of parasites. Symbiotic parasitic growth was verified using CESS cell-line and it showed similar results, indicating that B-cells are indeed the cells responsible for the effect. In malaria endemic areas, people often have increased levels of atypical memory B-cells in the blood, and in this assay it was demonstrated that when parasites were present there was an increase in the proportion of CD19 + CD20 + CD27 - FCRL4 + B-cells, and a contraction of classical memory B-cells. This effect was most clearly seen when direct contact between B-cells and parasites was allowed. CONCLUSIONS: These results demonstrate that P. falciparum and B-cells undoubtedly can affect each other when allowed to multiply together, which is valuable information for future vaccine studies.

Enhanced virulence of Plasmodium falciparum in blood of diabetic patients.

Rising prevalence of diabetes in sub-Saharan Africa, coupled with continued malaria transmission, has resulted more patients dealing with both communicable and non-communicable diseases. We previously reported that travelers with type 2 diabetes mellitus (T2DM) infected with Plasmodium falciparum were three times more likely to develop severe malaria than non-diabetics. Here we explore the biological basis for this by testing blood from uninfected subjects with type 1 and type 2 diabetes, ex vivo, for their effects on parasite growth and rosetting (binding of infected erythrocytes to uninfected erythrocytes). Rosetting was associated with type 2 diabetes, blood glucose and erythrocyte sedimentation rate (ESR), while parasite growth was positively associated with blood glucose, glycated hemoglobin (HbA1c), body mass index (BMI), fibrinogen and triglycerides. This study establishes a link between diabetes and malaria virulence assays, potentially explaining the protective effect of good glycemic control against severe malaria in subjects with diabetes.

Depleted circulatory complement-lysis inhibitor (CLI) in childhood cerebral malaria returns to normal with convalescence.

BACKGROUND: Cerebral malaria (CM), is a life-threatening childhood malaria syndrome with high mortality. CM is associated with impaired consciousness and neurological damage. It is not fully understood, as yet, why some children develop CM. Presented here is an observation from longitudinal studies on CM in a paediatric cohort of children from a large, densely-populated and malaria holoendemic, sub-Saharan, West African metropolis. METHODS: Plasma samples were collected from a cohort of children with CM, severe malarial anaemia (SMA), uncomplicated malaria (UM), non-malaria positive healthy community controls (CC), and coma and anemic patients without malaria, as disease controls (DC). Proteomic two-dimensional difference gel electrophoresis (2D-DIGE) and mass spectrometry were used in a discovery cohort to identify plasma proteins that might be discriminatory among these clinical groups. The circulatory levels of identified proteins of interest were quantified by ELISA in a prospective validation cohort. RESULTS: The proteome analysis revealed differential abundance of circulatory complement-lysis inhibitor (CLI), also known as Clusterin (CLU). CLI circulatory level was low at hospital admission in all children presenting with CM and recovered to normal level during convalescence (p < 0.0001). At acute onset, circulatory level of CLI in the CM group significantly discriminates CM from the UM, SMA, DC and CC groups. CONCLUSIONS: The CLI circulatory level is low in all patients in the CM group at admission, but recovers through convalescence. The level of CLI at acute onset may be a specific discriminatory marker of CM. This work suggests that CLI may play a role in the pathophysiology of CM and may be useful in the diagnosis and follow-up of children presenting with CM.

Blood group and size dependent stability of P. falciparum infected red blood cell aggregates in capillaries.

For Plasmodium falciparum related malaria (B50), one of the outstanding host factors for the development of severe disease is the ABO blood group of malaria patients, where blood group O reduces the probability of severe disease as compared to individuals of groups A, B, or AB. In this report, we investigate the stability of rosette aggregates in malaria caused by Plasmodium falciparum in microflows. These flows are created in microfluidic channels with stenosis-like constrictions of different widths down to ones narrower as the rosette's diameter. High speed videos were recorded and analyzed by a MATLAB© based tracking software (SURF: SUrvival of Rosettes in Flow). We find a correlation of rosette size, channel diameter, and blood group regarding the mobility of the rosettes. Following the concept of a thermodynamic model, we find a critical width of the stenosis for rosette rupture during their passage. Our data reveal that under physiologically relevant conditions, rosettes in blood group A have a higher rosette frequency and stability as compared to blood group O (BG O), which constitutes a crucial factor promoting the observed protection in BG O individuals against severe malaria in non-O individuals.

Membrane integration and topology of RIFIN and STEVOR proteins of the Plasmodium falciparum parasite.

The malarial parasite Plasmodium exports its own proteins to the cell surfaces of red blood cells (RBCs) during infection. Examples of exported proteins include members of the repetitive interspersed family (RIFIN) and subtelomeric variable open reading frame (STEVOR) family of proteins from Plasmodium falciparum. The presence of these parasite-derived proteins on surfaces of infected RBCs triggers the adhesion of infected cells to uninfected cells (rosetting) and to the vascular endothelium potentially obstructing blood flow. While there is a fair amount of information on the localization of these proteins on the cell surfaces of RBCs, less is known about how they can be exported to the membrane and the topologies they can adopt during the process. The first step of export is plausibly the cotranslational insertion of proteins into the endoplasmic reticulum (ER) of the parasite, and here, we investigate the insertion of three RIFIN and two STEVOR proteins into the ER membrane. We employ a well-established experimental system that uses N-linked glycosylation of sites within the protein as a measure to assess the extent of membrane insertion and the topology it assumes when inserted into the ER membrane. Our results indicate that for all the proteins tested, transmembranes (TMs) 1 and 3 integrate into the membrane, so that the protein assumes an overall topology of Ncyt-Ccyt. We also show that the segment predicted to be TM2 for each of the proteins likely does not reside in the membrane, but is translocated to the lumen.

Heparinoid sevuparin inhibits Streptococcus-induced vascular leak through neutralizing neutrophil-derived proteins.

Acute lung injury (ALI) and respiratory distress can develop as a consequence of sepsis with pathogens such as group A Streptococcus (GAS). In the pathogenesis of sepsis-associated ALI, endothelial barrier disruption brought on by phagocyte activation is considered a causative factor. Here, we find that sevuparin, a heparinoid with low anticoagulant activity, prevents neutrophil-induced lung plasma leakage in a murine model of systemic inflammation evoked by heat-killed GAS (hkGAS). Furthermore, using human neutrophils and endothelial cell monolayers, we demonstrate that sevuparin inhibits hkGAS-induced endothelial barrier disruption by neutralizing the activity of neutrophil-derived proteins. By mass spectrometry of neutrophil secretion, we identify proteins, including serprocidins, S100 proteins, and histone H4, that interact with sevuparin and that are responsible for the disruptive effect on endothelial integrity. Collectively, our results demonstrate the critical role of neutrophil-derived proteins in vascular hyperpermeability caused by GAS and suggest sevuparin as a potential therapeutic in acute neutrophilic inflammation.-Rasmuson, J., Kenne, E., Wahlgren, M., Soehnlein, O., Lindbom, L. Heparinoid sevuparin inhibits Streptococcus-induced vascular leak through neutralizing neutrophil-derived proteins.

Effect of ABO blood group on asymptomatic, uncomplicated and placental Plasmodium falciparum infection: systematic review and meta-analysis.

BACKGROUND: Malaria clinical outcomes vary by erythrocyte characteristics, including ABO blood group, but the effect of ABO blood group on asymptomatic, uncomplicated and placental Plasmodium falciparum (P. falciparum) infection remains unclear. We explored effects of ABO blood group on asymptomatic, uncomplicated and placental falciparum infection in the published literature. METHODS: A systematic review and meta-analysis was performed using the preferred reporting items for systematic reviews and meta-analyses guidelines. Articles in Pubmed, Embase, Web of Science, CINAHL and Cochrane Library published before February 04, 2017 were searched without restriction. Studies were included if they reported P. falciparum infection incidence or prevalence, stratified by ABO blood group. RESULTS: Of 1923 articles obtained from the five databases (Embase = 728, PubMed = 620, Web of Science = 549, CINAHL = 14, Cochrane Library = 12), 42 met criteria for systematic review and 37 for meta-analysis. Most studies (n = 30) were cross-sectional, seven were prospective cohort, and five were case-control studies. Meta-analysis showed similar odds of uncomplicated P. falciparum infection among individuals with blood group A (summary odds ratio [OR] 0.96, 15 studies), B (OR 0.89, 15 studies), AB (OR 0.85, 10 studies) and non-O (OR 0.95, 17 studies) as compared to those with blood group O. Meta-analysis of four cohort studies also showed similar risk of uncomplicated P. falciparum infection among individuals with blood group non-O and those with blood group O (summary relative risk [RR] 1.03). Meta-analysis of six studies showed similar odds of asymptomatic P. falciparum infection among individuals with blood group A (OR 1.05), B (OR 1.03), AB (OR 1.23), and non-O (OR 1.07) when compared to those with blood group O. However, odds of active placental P. falciparum infection was significantly lower in primiparous women with non-O blood groups (OR 0.46, 95% confidence interval [CI] 0.23 - 0.69, I2 0.0%, three studies), particularly in those with blood group A (OR 0.41, 95% CI 0.003 - 0.82, I2 1.4%, four studies) than those with blood group O. CONCLUSIONS: This study suggests that ABO blood group may not affect susceptibility to asymptomatic and/or uncomplicated P. falciparum infection. However, blood group O primiparous women appear to be more susceptible to active placental P. falciparum infection.

Effect of the ABO blood group on susceptibility to severe malaria: A systematic review and meta-analysis.

Understanding how ABO blood group interacts with Plasmodium falciparum (P. falciparum) infection may facilitate development of antimalarial treatments and vaccines. This study systematically summarizes information on the relationship of ABO blood group with severe P. falciparum infection, level of parasitemia and haemoglobin. A total of 1923 articles were retrieved from five databases. After removal of duplicates, and two levels of screening, 21 articles were selected for inclusion in the meta-analysis. A meta-analysis of the studies showed an increased odds of severe P. falciparum infection among individuals with blood group A, B, AB or non-O compared with blood group O. However, the difference in the level of P. falciparum parasitemia was not significant among individuals with blood group A or non-O compared with blood group O. The difference in haemoglobin level among P. falciparum infected individuals was also not significant between those with blood group A, B or AB versus those with blood group O.

Low plasma haptoglobin is a risk factor for life-threatening childhood severe malarial anemia and not an exclusive consequence of hemolysis.

Severe Malarial Anemia (SMA), a life-threatening childhood Plasmodium falciparum malaria syndrome requiring urgent blood transfusion, exhibits inflammatory and hemolytic pathology. Differentiating between hypo-haptoglobinemia due to hemolysis or that of genetic origin is key to understand SMA pathogenesis. We hypothesized that while malaria-induced hypo-haptoglobinemia should reverse at recovery, that of genetic etiology should not. We carried-out a case-control study of children living under hyper-endemic holoendemic malaria burden in the sub-Saharan metropolis of Ibadan, Nigeria. We show that hypo-haptoglobinemia is a risk factor for childhood SMA and not solely due to intravascular hemolysis from underlying schizogony. In children presenting with SMA, hypo-haptoglobinemia remains through convalescence to recovery suggesting a genetic cause. We identified a haptoglobin gene variant, rs12162087 (g.-1203G > A, frequency = 0.67), to be associated with plasma haptoglobin levels (p = 8.5 × 10-6). The Homo-Var:(AA) is associated with high plasma haptoglobin while the reference Homo-Ref:(GG) is associated with hypo-haptoglobinemia (p = 2.3 × 10-6). The variant is associated with SMA, with the most support for a risk effect for Homo-Ref genotype. Our insights on regulatory haptoglobin genotypes and hypo-haptoglobinemia suggest that haptoglobin screening could be part of risk-assessment algorithms to prevent rapid disease progression towards SMA in regions with no-access to urgent blood transfusion where SMA accounts for high childhood mortality rates.

Levels of human proteins in plasma associated with acute paediatric malaria.

BACKGROUND: The intimate interaction between the pathophysiology of the human host and the biology of the Plasmodium falciparum parasite results in a wide spectrum of disease outcomes in malaria. Development of severe disease is associated with a progressively augmented imbalance in pro- and anti-inflammatory responses to high parasite loads and sequestration of parasitized erythrocytes. Although these phenomena collectively constitute common denominators for the wide variety of discrete severe malaria manifestations, the mechanistic rationales behind discrepancies in outcome are poorly understood. Exploration of the human pathophysiological response by variations in protein profiles in plasma presents an excellent opportunity to increase the understanding. This is ultimately required for better prediction, prevention and treatment of malaria, which is essential for ongoing elimination and eradication efforts. RESULTS: An affinity proteomics approach was used to analyse 541 paediatric plasma samples collected from community controls and patients with mild or severe malaria in Rwanda. Protein profiles were generated with an antibody-based suspension bead array containing 255 antibodies targetting 115 human proteins. Here, 57 proteins were identified with significantly altered levels (adjusted p-values < 0.001) in patients with malaria compared to controls. From these, the 27 most significant proteins (adjusted p-values < 10-14) were selected for a stringent analysis approach. Here, 24 proteins showed elevated levels in malaria patients and included proteins involved in acute inflammatory response as well as cell adhesion. The remaining three proteins, also implicated in immune regulation and cellular adhesivity, displayed lower abundance in malaria patients. In addition, 37 proteins (adjusted p-values < 0.05) were identified with increased levels in patients with severe compared to mild malaria. This set includes, proteins involved in tissue remodelling and erythrocyte membrane proteins. Collectively, this approach has been successfully used to identify proteins both with known and unknown association with different stages of malaria. CONCLUSION: In this study, a high-throughput affinity proteomics approach was used to find protein profiles in plasma linked to P. falciparum infection and malaria disease progression. The proteins presented herein are mainly involved in inflammatory response, cellular adhesion and as constituents of erythrocyte membrane. These findings have a great potential to provide increased conceptual understanding of host-parasite interaction and malaria pathogenesis.

ABO Blood Group Antigen Decorated Giant Unilamellar Vesicles Exhibit Distinct Interactions with Plasmodium falciparum Infected Red Blood Cells.

Severe malaria is considered to be the deadliest disease of this century, primarily among children in sub-Saharan Africa. It stems from infection by the virulent parasite Plasmodium falciparum. The pathogenesis of the disease is based on the rosetting phenomenon, which occurs during the life cycle of the parasite in red blood cells (RBCs) and promotes the binding of parasitized RBCs to healthy ones. The role of the ABO blood group antigens in relation to the phenomenon has previously only been investigated in clinical isolates obtained from malaria patients. Here, we aim to clarify their role using synthetic ABO-decorated giant unilamellar vesicles (GUVs), which serve as simple biomimetic models of RBC-size cell membranes. Our results suggest clearly and for the first time that the blood group A and O antigens have a direct impact on receptor-specific rosetting phenomena when compared to the B antigen, which only participates in rosetting to an insignificant degree. Thus, glycodecorated GUVs represent a practical tool for studying cell-surface interactions.

SURGE complex of Plasmodium falciparum in the rhoptry-neck (SURFIN4.2-RON4-GLURP) contributes to merozoite invasion.

Plasmodium falciparum invasion into red blood cells (RBCs) is a complex process engaging proteins on the merozoite surface and those contained and sequentially released from the apical organelles (micronemes and rhoptries). Fundamental to invasion is the formation of a moving junction (MJ), a region of close apposition of the merozoite and the RBC plasma membranes, through which the merozoite draws itself before settling into a newly formed parasitophorous vacuole (PV). SURFIN4.2 was identified at the surface of the parasitized RBCs (pRBCs) but was also found apically associated with the merozoite. Using antibodies against the N-terminus of the protein we show the presence of SURFIN4.2 in the neck of the rhoptries, its secretion into the PV and shedding into the culture supernatant upon schizont rupture. Using immunoprecipitation followed by mass spectrometry we describe here a novel protein complex we have named SURGE where SURFIN4.2 forms interacts with the rhoptry neck protein 4 (RON4) and the Glutamate Rich Protein (GLURP). The N-terminal cysteine-rich-domain (CRD) of SURFIN4.2 mediates binding to the RBC membrane and its interaction with RON4 suggests its involvement in the contact between the merozoite apex and the RBC at the MJ. Supporting this suggestion, we also found that polyclonal antibodies to the extracellular domain (including the CRD) of SURFIN4.2 partially inhibit merozoite invasion. We propose that the formation of the SURGE complex participates in the establishment of parasite infection within the PV and the RBCs.

Exploring parasite heterogeneity using single-cell RNA-seq reveals a gene signature among sexual stage Plasmodium falciparum parasites.

The malaria parasite has a complex lifecycle, including several events of differentiation and stage progression, while actively evading immunity in both its mosquito and human hosts. Important parasite gene expression and regulation during these events remain hidden in rare populations of cells. Here, we combine a capillary-based platform for cell isolation with single-cell RNA-sequencing to transcriptionally profile 165 single infected red blood cells (iRBCs) during the intra-erythrocytic developmental cycle (IDC). Unbiased analyses of single-cell data grouped the cells into eight transcriptional states during IDC. Interestingly, we uncovered a gene signature from the single iRBC analyses that can successfully discriminate between developing asexual and sexual stage parasites at cellular resolution, and we verify five, previously undefined, gametocyte stage specific genes. Moreover, we show the capacity of detecting expressed genes from the variable gene families in single parasites, despite the sparse nature of data. In total, the single parasite transcriptomics holds promise for molecular dissection of rare parasite phenotypes throughout the malaria lifecycle.

Factors influencing the induction of high affinity antibodies to Plasmodium falciparum merozoite antigens and how affinity changes over time.

Understanding the functional characteristics of naturally acquired antibodies against P. falciparum merozoite antigens is crucial for determining the protective functions of antibodies. Affinity (measured as kd) of naturally acquired antibodies against two key targets of acquired immunity, EBA175 and PfRh2, was determined using Surface Plasmon Resonance (SPR) in a longitudinal survey in Nigeria. A majority of the participants, 79% and 67%, maintained stable antibody affinities to EBA175 and PfRh2, respectively, over time. In about 10% of the individuals, there was a reciprocal interaction with a reduction over time in antibody affinity for PfRh2 and an increase for EBA175. In general, PfRh2 elicited antibodies with higher affinity compared to EBA175. Individuals with higher exposure to malaria produced antibodies with higher affinity to both antigens. Younger individuals (5-15 years) produced comparable or higher affinity antibodies than adults (>15 years) against EBA175, but not for PfRh2. Correlation between total IgG (ELISA) and affinity varied between individuals, but PfRh2 elicited antibodies with a higher correlation in a majority of the participants. There was also a correlation between antibody inhibition of erythrocyte invasion by merozoites and PfRh2 affinity. This work gives new insights into the generation and maintenance of antibody affinity over time.

A Thioredoxin Homologous Protein of Plasmodium falciparum Participates in Erythrocyte Invasion.

Invasion of erythrocytes by merozoites is required in the life cycle of malarial parasites. Proteins derived from the invasive merozoites are essential ligands for erythrocyte recognition and penetration. In this study, we report a novel protein that possesses a Trx domain-like structure of the thioredoxin family and is expressed on the surface of merozoites of the malaria parasite Plasmodium falciparum This protein, namely, PfTrx-mero protein, displayed a mutated sequence character at the Trx domain, but with a specific binding activity to human erythrocytes. Specific antibodies to the protein inhibited merozoite invasion into human erythrocytes. Immunization with a homologous protein of Plasmodium berghei strain ANKA also showed significant protection against lethal infection in mice. These results suggested that the novel PfTrx-like-mero protein expressed on the surface of merozoites is an important ligand participating in erythrocyte invasion and a potential vaccine candidate.

Characterization of the Catalytic Subunits of the RNA Exosome-like Complex in Plasmodium falciparum.

The eukaryotic ribonucleic acid (RNA) exosome is a versatile multiribonuclease complex that mediates the processing, surveillance, and degradation of virtually all classes of RNA in both the nucleus and cytoplasm. The complex, composed of 10 to 11 subunits, has been widely described in many organisms. Bioinformatic analyses revealed that there may be also an exosome-like complex in Plasmodium falciparum, a parasite of great importance in public health, with eight predicted subunits having high sequence similarity to their counterparts in yeast and human. In this work, the putative RNA catalytic components, designated as PfRrp4, PfRrp41, PfDis3, and PfRrp6, were identified and systematically analyzed. Quantitative polymerase chain reaction (QPCR) analyses suggested that all of them were transcribed steadily throughout the asexual stage. The expression of these proteins was determined by Western blot, and their localization narrowed to the cytoplasm of the parasite by indirect immunofluorescence. The recombinant proteins of PfRrp41, PfDis3, and PfRrp6 exhibited catalytic activity for single-stranded RNA (ssRNA), whereas PfRrp4 showed no processing activity of both ssRNA and dsRNA. The identification of these putative components of the RNA exosome complex opens up new perspectives for a deep understanding of RNA metabolism in the malarial parasite P. falciparum.

Antibodies in children with malaria to PfEMP1, RIFIN and SURFIN expressed at the Plasmodium falciparum parasitized red blood cell surface.

Naturally acquired antibodies to proteins expressed on the Plasmodium falciparum parasitized red blood cell (pRBC) surface steer the course of a malaria infection by reducing sequestration and stimulating phagocytosis of pRBC. Here we have studied a selection of proteins representing three different parasite gene families employing a well-characterized parasite with a severe malaria phenotype (FCR3S1.2). The presence of naturally acquired antibodies, impact on rosetting rate, surface reactivity and opsonization for phagocytosis in relation to different blood groups of the ABO system were assessed in a set of sera from children with mild or complicated malaria from an endemic area. We show that the naturally acquired immune responses, developed during malaria natural infection, have limited access to the pRBCs inside a blood group A rosette. The data also indicate that SURFIN4.2 may have a function at the pRBC surface, particularly during rosette formation, this role however needs to be further validated. Our results also indicate epitopes differentially recognized by rosette-disrupting antibodies on a peptide array. Antibodies towards parasite-derived proteins such as PfEMP1, RIFIN and SURFIN in combination with host factors, essentially the ABO blood group of a malaria patient, are suggested to determine the outcome of a malaria infection.