Erythrocyte Membrane Protein Research Articles

Analysis of var Gene Transcription Pattern Using DBLα Tags.

The Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) antigens, which are encoded by a multigene family called var genes, are exported and inserted onto the surface of the infected erythrocytes. PfEMP1 plays a key role in the pathogenesis of severe malaria and are major targets of naturally acquired immunity. Studying the expression pattern of var genes in P. falciparum clinical isolates is crucial for understanding disease mechanism and immunity to malaria. However, var genes are highly variable, which makes it difficult to study their expression in clinical isolates obtained directly from malaria patients. In this chapter, we describe an approach for analysis of var gene expression that targets a region referred to as DBLα tag, which is relatively conserved in all var genes.

Assay of Static Adhesion of Plasmodium falciparum-Infected Erythrocytes to Cells, Including Inhibition of the Adhesion.

A feature of the virulent malaria parasite, Plasmodium falciparum, is the sequestration of infected erythrocytes (IEs) to host endothelium. The IEs sequester in the microvasculature by adhesion to host cells resulting in the obstruction of blood flow and often harmful consequences in the host. IEs bind to receptors on host cells with the P. falciparum erythrocyte membrane protein 1 (PfEMP1) that is expressed on the surface of the IEs. The study of parasite cytoadhesion is essential to decipher these ligands, including types of PfEMP1 required for cytoadhesion, the receptors the IEs bind, and how they may be related to the type of malaria disease. An assay for IE adhesion to host cells, including the inhibition of cytoadhesion is described here. The assay involves the purification of IEs with knobs and binding of the IEs to a monolayer of host cells under static conditions. Compounds including proteins, antibodies or drugs can be tested for cytoadhesion inhibitory activity in the assay.

Surface Plasmon Resonance Analysis of PfEMP1 Interaction with Receptors.

A detailed understanding of the interaction between the highly variant Plasmodium falciparum erythrocyte membrane proteins 1 (PfEMP1) and their human binding partners is essential to explain their roles in disease development in malaria, as well as to understand how antibodies can inhibit these interactions and how the parasite manages to evade such an immune response. This chapter focuses on using surface plasmon resonance (SPR) as a reproducible, high-throughput method to quantitatively characterize these interactions. We describe how to utilize protein A or A/G and streptavidin for protein immobilization on SPR sensor chips and provide instructions on how to biotinylate proteins for this purpose and how to use SPR for binding competition assays. Since these experiments rely on recombinant proteins, we also present a method to verify their structural integrity using circular dichroism spectroscopy.

Extraction and Immunoprecipitation of VAR2CSA, the PfEMP1 Associated with Placental Malaria.

The Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) is a key virulence factor for this human malaria parasite. During pregnancy, VAR2CSA is the only PfEMP1 variant expressed on the surface of infected erythrocytes that mediates adhesion to placenta cells and causes severe pregnancy outcomes.In this chapter, we present an optimized protocol to extract and immunoprecipitate endogenous VAR2CSA from the infected erythrocyte membrane phospholipid bilayer environment for subsequent characterization of the central role of VAR2CSA in placental malaria.

Expression of Large Full-Length PfEMP1 Proteins in HEK293 Cells.

Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) is a family of proteins expressed on the surface of red blood cells infected by Plasmodium falciparum. PfEMP1 proteins play a vital role in parasite virulence, and thus are important vaccine candidates to prevent severe disease. VAR2CSA is one specific PfEMP1 essential for pregnancy malaria pathogenesis, and the primary target in pregnancy malaria vaccine development. However, similar to other PfEMP1 proteins, expression of recombinant full-length VAR2CSA is difficult due to its large size, multidomain architecture and high cysteine content. To date, there has been success using higher ordered expression systems (such as mammalian and insect cells) to generate folded and active VAR2CSA. However, recent improvements with mammalian expression systems including cell lines and promoters have pushed the boundaries of yields. Here, we describe a modified protocol beyond current systems that enhances yields of full-length VAR2CSA and can generate higher quantities of material for protein structural and functional characterization.

CRISPR-Cas9 Editing of the Plasmodium falciparum Genome: Special Applications.

The virulence of Plasmodium falciparum has been attributed in large part to the expression on the surface of infected red blood cells of the variant surface antigen Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1). Different forms of this protein are encoded by individual members of the multicopy gene family called var. Two attributes of the var gene family are key to the pathogenesis of malaria caused by P. falciparum; the hyperrecombinogenic nature of the var gene family that continuously generates antigenic diversity within parasite populations, and the ability of parasites to express only a single var gene at a time and to switch which gene is expressed over the course of an infection. The unique attributes of CRISPR-Cas9 have been applied to help decipher the molecular mechanisms underlying these unusual properties of the var gene family, both as a source of the DNA double strand breaks that initiate var gene recombination and as a way to recruit molecular probes to specific regions of the genome. In this chapter, we describe these somewhat unusual applications of the CRISPR-Cas9 system.

Immunomagnetic Selection of Plasmodium falciparum-Infected Erythrocytes Expressing Particular PfEMP1 Variants.

Plasmodium falciparum expresses a broad range of proteins on the surface of infected erythrocytes (IEs), including members of the P. falciparum erythrocyte membrane protein 1 (PfEMP1) family. This protocol describes an immunomagnetic selection method using PfEMP1-specific antibodies to obtain a parasite clone homogenously expressing a particular PfEMP1 protein. The expression of the corresponding PfEMP1 is later tested by flow cytometry, and the selected parasites can be used for further analysis.

Selecting Plasmodium falciparum-Infected Erythrocytes for Adhesion to Recombinant Receptors Under Flow Conditions.

The var genes encoding the Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) mediating adhesion of infected erythrocytes (IEs) are clonally variant and confer the parasite the ability to evade the host immune response by enabling switching among expression of different PfEMP1 variants. This method of antigenic variation allows the parasite to adhere to a variety of proteins to escape splenic clearance. Enriching IE populations for expression of a specific PfEMP1 variant is crucial for the study of specific var genes PfEMP1 proteins and their role in pathogenicity. Selection of parasites using cell monolayers requires large-volume cultures (>5mL). This chapter describes a method, which rapidly selects for adhesive phenotypes of interest using small-volume cultures (<1mL) under physiological flow conditions.

Analysis by Flow Cytometry of α2-Macroglobulin and Nonimmune IgM-Binding to Plasmodium falciparum-Infected Erythrocytes.

Several members of the Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family can bind human serum proteins such as IgM and α2-macroglobulin (α2M). This binding seems to play a role in pathogenesis and immune evasion by improving the avidity of PfEMP1-mediated binding to erythrocyte receptors and/or by masking antibody epitopes in PfEMP1. In this protocol, we describe a flow cytometry-based protocol to evaluate IgM- and α2M-binding to intact and unfixed mature-stage IEs. The method can be used for laboratory clones and field isolates.

Production of anti-PfEMP1 Polyclonal Antisera in Rats and Mice.

Plasmodium falciparum-infected erythrocytes (IEs) bind various host receptors via members of the P. falciparum erythrocyte membrane protein 1 (PfEMP1) family expressed on the surface of the IEs. Antibody reagents are needed to investigate interactions between specific PfEMP1 proteins and receptors expressed by human endothelial cells. This protocol describes the production of rat and mouse polyclonal anti-PfEMP1 antibodies. Polyclonal antibodies are relatively easy to produce and have advantages compared to monoclonal antibodies (see Chapters 28 - 30 ) for some applications. An ELISA-based method to test the polyclonal antibodies before their use in more advanced procedures is also presented.

Conformational changes in plasma proteins and erythrocytes in puerperal women and strategies of managing the perioperative period

BACKGROUND: Cesarean delivery is associated with prolonged hospitalization compared to spontaneous delivery and the risk of intra- and postoperative complications. The introduction of an accelerated recovery program after a planned cesarean section contributes to the rapid recovery of the patient by optimizing various elements of care.&#x0D; AIM: To study the effect of the components of the accelerated recovery program on the severity of oxidative stress during abdominal delivery at different stages of the perioperative period.&#x0D; MATERIALS AND METHODS: This was a comparative study assessing conformational changes in plasma proteins and erythrocytes in the blood of puerperal women using fluorescence spectroscopy. The study included 81 patients from the perinatal center of Makhachkala who underwent a planned cesarean section under spinal anesthesia. The enrolled women were grouped into the following: control group (n=38), in which perioperative period was traditionally managed, i.e. fasting for 8 h before the operation and introducing an antibiotic after clamping the umbilical cord. Within this group, blood sampling was conducted in all 38 patients at different intervals. In total, 152 samples of the material under study were obtained from control mothers. The 2nd group was the main (n=43) and included women in labor; in this group, perioperative period was managed using accelerated recovery program, by introducing the antibiotic cefazolin and with the intake of a glucose-containing drink 2 h before the operation. In total, 172 samples of the material under study were obtained from the mothers of the 2nd group. Methods for the pre-preparation of biological material and spectral methods of analysis were used in this study.&#x0D; RESULTS: At all stages of preparation of delivery by cesarean section (CS) after spinal anesthesia, minor conformational changes occur in the blood plasma proteins, including umbilical cord blood. In the main group, antibiotic use an hour before delivery increased the oxidative degradation of blood plasma proteins. In the control group, a change in the structural-dynamic parameters of erythrocyte membrane proteins was observed, as indicated by the blue shift in the maximum fluorescence spectrum. This was not observed in the erythrocytes of the main group of puerperal women who received a glucose-containing drink.&#x0D; CONCLUSION: According to total fluorescence data of plasma proteins and umbilical cord blood, it can be assumed that using a glucose-containing drink 2 hours before CS along with an antibiotic helps restore some parameters of the fluorescence of erythrocyte membrane proteins. The data obtained do not indicate any persistent pathological phenomena in mothers body at all stages of preparation for CS delivery using spinal anesthesia against the background of antibiotic use.

Plasma biomarkers of hemoglobin loss in Plasmodium falciparum–infected children identified by quantitative proteomics

AbstractAnemia is common among young children infected with Plasmodium falciparum and severe malarial anemia (SMA) is a major cause of their mortality. Two major mechanisms cause malarial anemia: hemolysis of uninfected as well as infected erythrocytes and insufficient erythropoiesis. In a longitudinal birth cohort in Mali, we commonly observed marked hemoglobin reductions during P falciparum infections with a small proportion that progressed to SMA. We sought biomarkers of these processes using quantitative proteomic analysis on plasma samples from 9 P falciparum-infected children, comparing those with reduced hemoglobin (with or without SMA) vs those with stable hemoglobin. We identified higher plasma levels of circulating 20S proteasome and lower insulin-like growth factor-1 (IGF-1) levels in children with reduced hemoglobin. We confirmed these findings in independent enzyme-linked immunosorbent assay-based validation studies of subsets of children from the same cohort (20S proteasome, N = 71; IGF-1, N = 78). We speculate that circulating 20S proteasome plays a role in digesting erythrocyte membrane proteins modified by oxidative stress, resulting in hemolysis, whereas decreased IGF-1, a critical factor for erythroid maturation, might contribute to insufficient erythropoiesis. Quantitative plasma proteomics identified soluble mediators that may contribute to the major mechanisms underlying malarial anemia. This study was registered at www.clinicaltrials.gov as #NCT01168271.

Identifying Targets of Protective Antibodies against Severe Malaria in Papua, Indonesia, Using Locally Expressed Domains of Plasmodium falciparum Erythrocyte Membrane Protein 1.

Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1), a diverse family of multidomain proteins expressed on the surface of malaria-infected erythrocytes, is an important target of protective immunity against malaria. Our group recently studied transcription of the var genes encoding PfEMP1 in individuals from Papua, Indonesia, with severe or uncomplicated malaria. We cloned and expressed domains from 32 PfEMP1s, including 22 that were upregulated in severe malaria and 10 that were upregulated in uncomplicated malaria, using a wheat germ cell-free expression system. We used Luminex technology to measure IgG antibodies to these 32 domains and control proteins in 63 individuals (11 children). At presentation to hospital, levels of antibodies to PfEMP1 domains were either higher in uncomplicated malaria or were not significantly different between groups. Using principal component analysis, antibodies to 3 of 32 domains were highly discriminatory between groups. These included two domains upregulated in severe malaria, a DBLβ13 domain and a CIDRα1.6 domain (which has been previously implicated in severe malaria pathogenesis), and a DBLδ domain that was upregulated in uncomplicated malaria. Antibody to control non-PfEMP1 antigens did not differ with disease severity. Antibodies to PfEMP1 domains differ with malaria severity. Lack of antibodies to locally expressed PfEMP1 types, including both domains previously associated with severe malaria and newly identified targets, may in part explain malaria severity in Papuan adults.

Safety, infectivity and immunogenicity of a genetically attenuated blood-stage malaria vaccine

BackgroundThere is a clear need for novel approaches to malaria vaccine development. We aimed to develop a genetically attenuated blood-stage vaccine and test its safety, infectivity, and immunogenicity in healthy volunteers. Our approach was to target the gene encoding the knob-associated histidine-rich protein (KAHRP), which is responsible for the assembly of knob structures at the infected erythrocyte surface. Knobs are required for correct display of the polymorphic adhesion ligand P. falciparum erythrocyte membrane protein 1 (PfEMP1), a key virulence determinant encoded by a repertoire of var genes.MethodsThe gene encoding KAHRP was deleted from P. falciparum 3D7 and a master cell bank was produced in accordance with Good Manufacturing Practice. Eight malaria naïve males were intravenously inoculated (day 0) with 1800 (2 subjects), 1.8 × 105 (2 subjects), or 3 × 106 viable parasites (4 subjects). Parasitemia was measured using qPCR; immunogenicity was determined using standard assays. Parasites were rescued into culture for in vitro analyses (genome sequencing, cytoadhesion assays, scanning electron microscopy, var gene expression).ResultsNone of the subjects who were administered with 1800 or 1.8 × 105 parasites developed parasitemia; 3/4 subjects administered 3× 106 parasites developed significant parasitemia, first detected on days 13, 18, and 22. One of these three subjects developed symptoms of malaria simultaneously with influenza B (day 17; 14,022 parasites/mL); one subject developed mild symptoms on day 28 (19,956 parasites/mL); and one subject remained asymptomatic up to day 35 (5046 parasites/mL). Parasitemia rapidly cleared with artemether/lumefantrine. Parasitemia induced a parasite-specific antibody and cell-mediated immune response. Parasites cultured ex vivo exhibited genotypic and phenotypic properties similar to inoculated parasites, although the var gene expression profile changed during growth in vivo.ConclusionsThis study represents the first clinical investigation of a genetically attenuated blood-stage human malaria vaccine. A P. falciparum 3D7 kahrp– strain was tested in vivo and found to be immunogenic but can lead to patent parasitemia at high doses.Trial registrationAustralian New Zealand Clinical Trials Registry (number: ACTRN12617000824369; date: 06 June 2017).

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

BackgroundThe 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.MethodsAn 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.ResultsAll 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.ConclusionsThese 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.Graphical

PfEMP1-Specific Immunoglobulin G Reactivity Among Beninese Pregnant Women With Sickle Cell Trait.

BackgroundSickle cell trait (HbAS) protects against severe Plasmodium falciparum malaria but not against placental malaria (PM). In this study, P falciparum erythrocyte membrane protein (PfEMP1)-specific antibodies were measured in HbAA and HbAS Beninese pregnant women as a proxy of exposure to specific PfEMP1 variants.MethodsPlasma samples collected at delivery from 338 HbAA and 63 HbAS women were used to measure immunoglobulin (Ig)G levels to 6 recombinant PfEMP1 proteins and 3 corresponding native proteins expressed on the infected erythrocyte (IE) surface. Immunoglobulin G-mediated inhibition of VAR2CSA+ IEs adhesion to chondroitin sulfate A (CSA) was also tested.ResultsLevels of PfEMP1-specific IgG were similar in the 2 groups, except for native IT4VAR09 on IEs, where IgG levels were significantly higher in HbAS women. Adjusted odds ratios for women with positive IgG to HB3VAR06 and PFD1235w suggest a lower risk of infection with these virulent variants among HbAS individuals. The percentage of IEs binding to CSA did not differ between HbAA and HbAS women, but it correlated positively with levels of anti-VAR2CSA and parity. Women with PM had lower levels of anti-VAR2CSA-specific IgG and lower IgG-mediated inhibition of IE adhesion to CSA.ConclusionsThe findings support similar malaria exposure in HbAA and HbAS women and a lack of HbAS-dependent protection against placental infection among pregnant women.

A systematic review on genetic diversity of var gene DBL1α domain from different geographical regions in Plasmodium falciparum isolates.

Background The major variant surface antigen (VSA) in Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) encoded by var gene family has an important role in cytoadhesion/sequestration and rosetting by adhesion of uninfected erythrocytes to infected erythrocytes leading to disease severity. DBL1α domain in the PfEMP-1, protein is crucial in the cytoadhesion phenomena in P. falciparum infections and this review aims to analyse the genetic diversity of DBL1α domain sequences in PfEMP-1 from different geographical regions globally. Methods All available DBL1α sequence data was reviewed by using the electronic database PubMed, ResearchGate, Google, Google scholar, MEDLINE with the following Keywords-Plasmodium falciparum", "var gene", "DBL1α", "field isolate", "diversity", "polymorphism", "Africa", "America", "Asia" and "Caribbean" from different geographical regions across the world. Results A total of 240 studies were identified initially but only 20 studies qualified for this systematic review. The overall ratio of distinct sequences DBL1α domain was 24.62/1167 the highest in African region (33.59/766 isolates) and lowest in South America (5.6/215 isolates). In the 18 included studies, the presence of distinct DBL1α sequences was the highest in Oceania 55.32% (1186/2144) followed by Africa (38.43%), Asia (22.45%) and South America (16.48%), though the sample size in Oceania was comparatively smaller to that of Africa and South America. Conclusion This review highlights the ratio and percentage of distinct sequences of DBL1α domain of var gene in different geographical regions giving an idea of the existing diversity prevalent in this potential vaccine target gene which may contribute to designing the preventive measures towards disease severity.

Afucosylated Plasmodium falciparum-specific IgG is induced by infection but not by subunit vaccination

Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family members mediate receptor- and tissue-specific sequestration of infected erythrocytes (IEs) in malaria. Antibody responses are a central component of naturally acquired malaria immunity. PfEMP1-specific IgG likely protects by inhibiting IE sequestration and through IgG-Fc Receptor (FcγR) mediated phagocytosis and killing of antibody-opsonized IEs. The affinity of afucosylated IgG to FcγRIIIa is up to 40-fold higher than fucosylated IgG, resulting in enhanced antibody-dependent cellular cytotoxicity. Most IgG in plasma is fully fucosylated, but afucosylated IgG is elicited in response to enveloped viruses and to paternal alloantigens during pregnancy. Here we show that naturally acquired PfEMP1-specific IgG is strongly afucosylated in a stable and exposure-dependent manner, and efficiently induces FcγRIIIa-dependent natural killer (NK) cell degranulation. In contrast, immunization with a subunit PfEMP1 (VAR2CSA) vaccine results in fully fucosylated specific IgG. These results have implications for understanding protective natural- and vaccine-induced immunity to malaria.

Molecular cloning and characterization of a novel aspartyl aminopeptidase from Trichinella spiralis

Trichinellosis is an important zoonotic parasitic disease worldwide and is principally caused by ingesting animal meat containing Trichinella infective larvae. Aspartyl aminopeptidase is an intracytoplasmic metalloproteinase that specifically hydrolyzes the N-terminus of polypeptides free of acidic amino acids (aspartic acid and glutamate), and plays an important role in the metabolism, growth and development of organisms. In this study, a novel T. spiralis aspartyl aminopeptidase (TsAAP) was cloned and expressed, and its biological properties and roles in worm growth and development were investigated. The results revealed that TsAAP transcription and expression in diverse T. spiralis stages were detected by RT-PCR and Western blotting, and primarily localized at cuticle, stichosome and intrauterine embryos of this nematode by immunofluorescence test. rTsAAP has the enzymatic activity of native AAP to hydrolyze the substrate H-Glu-pNA. There was a specific binding between rTsAAP and murine erythrocyte, and the binding site was localized in erythrocyte membrane proteins. Silencing of TsAAP gene by specific dsRNA significantly reduced the TsAAP expression, enzymatic activity, intestinal worm burdens and female fecundity. The results demonstrated that TsAAP participates in the growth, development and fecundity of T. spiralis and it might be a potential target molecule for anti-Trichinella vaccines.

Computational Insights into the Interaction between Cytoadherence Receptor gC1qR and the DBLβ12 Domain of a Plasmodium falciparum PfEMP1 Ligand.

Human receptor gC1qR is a 32 kD protein that mediates the cytoadherence of Plasmodium falciparum-infected erythrocytes (IEs) to human brain microvascular endothelial cells (HBMEC) and platelets. The cytoadherence of IEs to gC1qR has been associated with severe malaria symptoms. The cytoadherence to gC1qR is mediated by the Duffy binding-like β12 (DBLβ12) domain of Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1), PFD0020c. Here, we report the structural insights into the binding of the DBLβ12 domain of PfEMP1 with the human receptor gC1qR using computational methods. A molecular model of the DBLβ12 domain was generated and used for protein–protein docking with the host receptor gC1qR. The protein–protein docking revealed that the DBLβ12 asymmetrically interacts with two subunits of the gC1qR trimer at the solution face of gC1qR. A total of 21 amino acid residues of DBLβ12 interact with 26 amino acid residues in the gC1qR trimer through 99 nonbonding interactions and 4 hydrogen bonds. Comparative analysis of binding sites on the DBL domain fold for the two receptors gC1qR and ICAM1 showed that the two sites are distinct. This is the first study that provides structural insights into DBLβ12 binding with its receptor gC1qR and may help in designing novel antisevere malaria interventions.