Ring-infected Erythrocyte Surface Antigen Research Articles

Investigation of liposomal self-adjuvanting peptide epitopes derived from conserved blood-stage Plasmodium antigens.

Malaria is a vector born parasitic disease causing millions of deaths every year. Despite the high mortality rate, an effective vaccine against this mosquito-borne infectious disease is yet to be developed. Up to date, RTS,S/AS01 is the only vaccine available for malaria prevention; however, its efficacy is low. Among a variety of malaria antigens, merozoite surface protein-1(MSP-1) and ring-infected erythrocyte surface antigen (RESA) have been proposed as promising candidates for malaria vaccine development. We developed peptide-based Plasmodium falciparum vaccine candidates that incorporated three previously reported conserved epitopes from MSP-1 and RESA into highly effective liposomal polyleucine delivery system. Indeed, MSP-1 and RESA-derived epitopes conjugated to polyleucine and formulated into liposomes induced higher epitope specific antibody titres. However, immunized mice failed to demonstrate protection in a rodent malaria challenge study with Plasmodium yoelii. In addition, we found that the three reported P. falciparum epitopes did not to share conformational properties and high sequence similarity with P. yoelii MSP-1 and RESA proteins, despite the epitopes were reported to protect mice against P. yoelii challenge.

AGIA Tag System for Ultrastructural Protein Localization Analysis in Blood-Stage Plasmodium falciparum.

Precise subcellular localization of proteins is the key to elucidating the physiological role of these molecules in malaria parasite development, understanding of pathogenesis, and protective immunity. In Plasmodium falciparum, however, detection of proteins in the blood-stage parasites is greatly hampered by the lack of versatile protein tags which can intrinsically label such molecules. Thus, in this study, to develop a novel system that can be used to evaluate subcellular localization of known and novel proteins, we assessed the application of AGIA tag, consisting of 9 amino acids (EEAAGIARP), in P. falciparum blood-stage parasites. Specifically, AGIA-tagged ring-infected erythrocyte surface antigen (RESA-AGIA) was episomally expressed in P. falciparum 3D7 strain. The RESA-AGIA protein was detected by Western blotting and immunofluorescence assay (IFA) using recombinant rabbit anti-AGIA tag monoclonal antibody (mAb) with a high signal/noise ratio. Similarly, AGIA-tagged multidrug resistance protein 1 (MDR1-AGIA), as an example of polyptic transmembrane protein, was endogenously expressed and detected by Western blotting and IFA with anti-AGIA tag mAb. Immunoelectron microscopy of the RESA-AGIA transfected merozoites revealed that mouse anti-RESA and the rabbit anti-AGIA mAb signals could definitively co-localize to the dense granules. Put together, this study demonstrates AGIA tag/anti-AGIA rabbit mAb system as a potentially useful tool for elucidating the subcellular localization of new and understudied proteins in blood-stage malaria parasites at the nanometer-level resolution.

Role of the J Domain Protein Family in the Survival and Pathogenesis of Plasmodium falciparum.

Plasmodium falciparum has dedicated an unusually large proportion of its genome to molecular chaperones (2% of all genes), with the heat shock protein 40 (Hsp40) family (now called J domain proteins, JDPs) exhibiting evolutionary radiation into 49 members. A large number of the P. falciparum JDPs (PfJDPs) are predicted to be exported, with certain members shown experimentally to be present in the erythrocyte cytosol (PFA0660w and PFE0055c) or erythrocyte membrane (ring-infected erythrocyte surface antigen, RESA). PFA0660w and PFE0055c are associated with an exported plasmodial Hsp70 (PfHsp70-x) within novel mobile structures called J-dots, which have been proposed to be dedicated to the trafficking of key membrane proteins such as erythrocyte membrane protein 1 (PfEMP1). Well over half of the PfJDPs appear to be essential, including the J-dot PfJDP, PFE0055c, while others have been found to be required for growth under febrile conditions (e.g. PFA0110w, the ring-infected erythrocyte surface antigen protein [RESA]) or involved in pathogenesis (e.g. PF10_0381 has been shown to be important for protrusions of the infected red blood cell membrane, the so-called knobs). Here we review what is known about those PfJDPs that have been well characterised, and may be directly or indirectly involved in the survival and pathogenesis of the malaria parasite.

Molecular Markers for Sensitive Detection of Plasmodium falciparum Asexual Stage Parasites and their Application in a Malaria Clinical Trial.

.Plasmodium falciparum parasite life stages respond differently to antimalarial drugs. Sensitive stage-specific molecular assays may help to examine parasite dynamics at microscopically detectable and submicroscopic parasite densities in epidemiological and clinical studies. In this study, we compared the performance of skeleton-binding protein 1 (SBP1), ring-infected erythrocyte surface antigen, Hyp8, ring-exported protein 1 (REX1), and PHISTb mRNA for detecting ring-stage trophozoite-specific transcripts using quantitative reverse transcriptase polymerase chain reaction. Markers were tested on tightly synchronized in vitro parasites and clinical trial samples alongside established markers of parasite density (18S DNA and rRNA) and gametocyte density (Pfs25 mRNA). SBP1 was the most sensitive marker but showed low-level expression in mature gametocytes. Novel markers REX1 and PHISTb showed lower sensitivity but higher specificity for ring-stage trophozoites. Using in vivo clinical trial samples from gametocyte-negative patients, we observed evidence of persisting trophozoite transcripts for at least 14 days postinitiation of treatment. It is currently not clear if these transcripts represent viable parasites that may have implications for clinical treatment outcome or transmission potential.

Immunoglobulin response to Plasmodium falciparum RESA proteins in uncomplicated and severe malaria.

BackgroundThe three members of the ring-infected erythrocyte surface antigen (RESA) proteins family share high sequence homologies, which impair the detection and assignment to one or another protein of some pathogenic processes inherent to Plasmodium falciparum malaria. The present study was intended to determine if the antibody and inflammatory responses of children living in a malaria-endemic area varied depending on the RESA-1, RESA-2 or RESA-3 proteins and the severity of the disease, two groups of severe and uncomplicated malaria cases being considered.MethodsTwo synthetic peptides representing predicted B cell epitopes were designed per RESA protein, all located outside of the 3′ and 5′ repetition blocks, in order to allow an antibody detection specific of each member of the family. Recombinant rRESA-1B and rRESA-3B proteins were also engineered. Two groups of Beninese children admitted to hospital in 2009 for either uncomplicated or severe malaria were compared for their plasma levels of IgG specifically recognizing each recombinant RESA protein or synthetic peptide, and for their plasma inflammatory cytokine levels (IFN-γ, TNF-α and IL-10), taking into account host and parasite genetic factors.ResultsThe absence of IgG cross-reactivity between rRESA proteins and their protein carrier as well as between each RESA peptide and a non-epitopic RESA control peptide validated the use of the engineered recombinant proteins and peptides for the measurement of plasma IgG. Taking into account age, fever duration and parasitaemia, a multiple logistic regression performed on children clustered according to their antibody responses’ profiles concluded to an increased risk of severe malaria for P2 (representative of RESA-1) responders (P = 0.007). Increased IL-10 plasma levels were found in children harbouring multiclonal P. falciparum infections on the basis of the T1526G resa2 gene polymorphism (P = 0.004).ConclusionsThis study provided novel tools to dissect the seroreactivity against the three members of the RESA protein family and to describe its relation to protection against malaria. It suggested the measurement of plasma antibodies raised against specific peptides to serve as predictive immunologic markers for disease severity. Lastly, it reinforced previous observations linking the T1526G resa2 gene mutation to severe malaria.Electronic supplementary materialThe online version of this article (doi:10.1186/s12936-015-0799-8) contains supplementary material, which is available to authorized users.

Targeted Disruption of a Ring-infected Erythrocyte Surface Antigen (RESA)-like Export Protein Gene in Plasmodium falciparum Confers Stable Chondroitin 4-Sulfate Cytoadherence Capacity

The Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family proteins mediate the adherence of infected erythrocytes to microvascular endothelia of various organs, including the placenta, thereby contributing to cerebral, placental, and other severe malaria pathogenesis. Several parasite proteins, including KAHRP and PfEMP3, play important roles in the cytoadherence by mediating the clustering of PfEMP1 in rigid knoblike structures on the infected erythrocyte surface. The lack of a subtelomeric region of chromosome 2 that contains kahrp and pfemp3 causes reduced cytoadherence. In this study, microarray transcriptome analysis showed that the absence of a gene cluster, comprising kahrp, pfemp3, and four other genes, results in the loss of parasitized erythrocytes adhering to chondroitin 4-sulfate (C4S). The role of one of these genes, PF3D7_0201600/PFB0080c, which encodes PHISTb (Plasmodium helical interspersed subtelomeric b) domain-containing RESA-like protein 1 expressed on the infected erythrocyte surface, was investigated. Disruption of PFB0080c resulted in increased var2csa transcription and VAR2CSA surface expression, leading to higher C4S-binding capacity of infected erythrocytes. Further, PFB0080c-knock-out parasites stably maintained the C4S adherence through many generations of growth. Although the majority of PFB0080c-knock-out parasites bound to C4S even after culturing for 6 months, a minor population bound to both C4S and CD36. These results strongly suggest that the loss of PFB0080c markedly compromises the var gene switching process, leading to a marked reduction in the switching rate and additional PfEMP1 expression by a minor population of parasites. PFB0080c interacts with VAR2CSA and modulates knob-associated Hsp40 expression. Thus, PFB0080c may regulate VAR2CSA expression through these processes. Overall, we conclude that PFB0080c regulates PfEMP1 expression and the parasite's cytoadherence.

Possible relationship between Plasmodium falciparum ring-infected erythrocyte surface antigen (RESA) and host cell resistance to destruction by chemicals

Repeated incubation of Plasmodium falciparum culture in 0.015% saponin solution for a total of 35 min destroys most of the uninfected cells, leaving only the ring-infected erythrocytes (RIEs). Parasites concentrated by this method can subsequently complete the asexual cycle and infect other erythrocytes. It is possible that resistance to saponin is mediated by one or more of the numerous parasite proteins present in the host erythrocyte membrane. We have found that schizonts are as susceptible as uninfected erythrocytes to saponin, indicating that the protective protein is parasite stage specific. Studies with cultured parasites have shown that ring-infected erythrocyte surface antigen (RESA) strengthens host erythrocyte membrane and protects against destruction. Therefore, we hypothesize that RESA could be involved in resistance to saponin. Here, we have carried out PCR test on RESA gene, using three different primers. One of them showed that P. falciparum isolates collected directly from infected humans and cultured only for a few days, or not at all, have amplicon sizes ranging from 372 to 510 bp. However, the amplicon size changed to 873 bp when in vitro growth was continued for one or more weeks. This genetic transformation precedes acquisition of the ability to confer saponin resistance to RIEs.

Detection of copy number variation and single nucleotide polymorphisms in genes involved in drug resistance and other phenotypic traits in P. falciparum clinical isolates collected from Uganda

There is an increasing interest in mapping the genes of pathogens which underlie important phenotypic traits such as virulence and drug resistance. The Plasmodium falciparum genome exhibits sequence variation that contributes to the pathogenic mechanisms of the parasite. Determining the prevalence of resistance markers could provide a prediction about drug efficacy. Copy number polymorphism (CNP) of genes has been shown to influence important parasite phenotypes. In this work, CNPs within genes involved in drug resistance and other phenotypic traits namely P. falciparum multidrug resistance 1 (pfmdr-1), GTP cyclo hydrolase (gch1), Ring infected erythrocyte surface antigen precursor (resa) and a hypothetical protein coding gene were analyzed by quantitative real time-polymerase reaction (qRT-PCR) among clinical isolates collected from Uganda. The pfmdr-1 codons 86 and 1246 and P. falciparum chloroquine resistance (pfcrt) codon 76 were genotyped for single nucleotide polymorphisms (SNPs) by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), and the proportion of resistance associated mutations were determined among mild and severe malaria cases using the chi-square test. Forty and 42 P. falciparum isolates collected from children with mild and severe malaria respectively were analyzed for CNPs. Seventy five and 81 P. falciparum isolates from children with mild or severe malaria were analyzed for SNPs. No pfmdr-1, gch1 or novel gene amplifications were identified among the P. falciparum clinical isolates. Although chloroquine was officially withdrawn from policy use since 7 years, all P. falciparum isolates presented the associated pfcrt K76T mutation, whatever the clinical status and no specific mutation in the pfmdr-1 gene was associated with disease type. In conclusion, this study provides baseline measures for continued surveillance for changes in copy number and SNP types among genes implicated in drug resistance and other important phenotypes that may have a potential role in parasite virulence mechanisms or drug treatment outcomes.

Systematic Analysis of FKBP Inducible Degradation Domain Tagging Strategies for the Human Malaria Parasite Plasmodium falciparum

Targeted regulation of protein levels is an important tool to gain insights into the role of proteins essential to cell function and development. In recent years, a method based on mutated forms of the human FKBP12 has been established and used to great effect in various cell types to explore protein function. The mutated FKBP protein, referred to as destabilization domain (DD) tag when fused with a native protein at the N- or C-terminus targets the protein for proteosomal degradation. Regulated expression is achieved via addition of a compound, Shld-1, that stabilizes the protein and prevents degradation. A limited number of studies have used this system to provide powerful insight into protein function in the human malaria parasite Plasmodium falciparum. In order to better understand the DD inducible system in P. falciparum, we studied the effect of Shld-1 on parasite growth, demonstrating that although development is not impaired, it is delayed, requiring the appropriate controls for phenotype interpretation. We explored the quantified regulation of reporter Green Fluorescent Protein (GFP) and luciferase constructs fused to three DD variants in parasite cells either via transient or stable transfection. The regulation obtained with the original FKBP derived DD domain was compared to two triple mutants DD24 and DD29, which had been described to provide better regulation for C-terminal tagging in other cell types. When cloned to the C-terminal of reporter proteins, DD24 provided the strongest regulation allowing reporter activity to be reduced to lower levels than DD and to restore the activity of stabilised proteins to higher levels than DD29. Importantly, DD24 has not previously been applied to regulate proteins in P. falciparum. The possibility of regulating an exported protein was addressed by targeting the Ring-Infected Erythrocyte Surface Antigen (RESA) at its C-terminus. The tagged protein demonstrated an important modulation of its expression.

Modulating effects of plasma containing anti-malarial antibodies on in vitro anti-malarial drug susceptibility in Plasmodium falciparum

BackgroundThe efficacy of anti-malarial drugs is determined by the level of parasite susceptibility, anti-malarial drug bioavailability and pharmacokinetics, and host factors including immunity. Host immunity improves the in vivo therapeutic efficacy of anti-malarial drugs, but the mechanism and magnitude of this effect has not been characterized. This study characterized the effects of 'immune' plasma to Plasmodium falciparumon the in vitro susceptibility of P. falciparum to anti-malarial drugs.MethodsTitres of antibodies against blood stage antigens (mainly the ring-infected erythrocyte surface antigen [RESA]) were measured in plasma samples obtained from Thai patients with acute falciparum malaria. 'Immune' plasma was selected and its effects on in vitro parasite growth and multiplication of the Thai P. falciparum laboratory strain TM267 were assessed by light microscopy. The in vitro susceptibility to quinine and artesunate was then determined in the presence and absence of 'immune' plasma using the 3H-hypoxanthine uptake inhibition method. Drug susceptibility was expressed as the concentrations causing 50% and 90% inhibition (IC50 and IC90), of 3H-hypoxanthine uptake.ResultsIncubation with 'immune' plasma reduced parasite maturation and decreased parasite multiplication in a dose dependent manner. 3H-hypoxanthine incorporation after incubation with 'immune' plasma was decreased significantly compared to controls (median [range]; 181.5 [0 to 3,269] cpm versus 1,222.5 [388 to 5,932] cpm) (p= 0.001). As a result 'immune' plasma reduced apparent susceptibility to quinine substantially; median (range) IC50 6.4 (0.5 to 23.8) ng/ml versus 221.5 (174.4 to 250.4) ng/ml (p = 0.02), and also had a borderline effect on artesunate susceptibility; IC50 0.2 (0.02 to 0.3) ng/ml versus 0.8 (0.2 to 2.3) ng/ml (p = 0.08). Effects were greatest at low concentrations, changing the shape of the concentration-effect relationship. IC90 values were not significantly affected; median (range) IC90 448.0 (65 to > 500) ng/ml versus 368.8 (261 to 501) ng/ml for quinine (p > 0.05) and 17.0 (0.1 to 29.5) ng/ml versus 7.6 (2.3 to 19.5) ng/ml for artesunate (p = 0.4).Conclusions'Immune' plasma containing anti-malarial antibodies inhibits parasite development and multiplication and increases apparent in vitro anti-malarial drug susceptibility of P. falciparum. The IC90 was much less affected than the IC50 measurement.

Isolation of Theileria parva ring-infected erythrocyte surface antigen (RESA) homolog

East coast fever (ECF) is a severe lymphoproliferative disease of cattle caused by the intracellular protozoan Theileria parva from the phylum Apicomplexa. Gene homologs encoding antigens from other apicomplexan parasites constitute a source of vaccine candidate antigens. The ring-infected erythrocyte surface antigen (RESA) protein is a vaccine candidate for malaria caused by Plasmodium falciparum and should be evaluated for inclusion in a subunit vaccine against ECF. Here we isolated and sequenced the T. parva RESA2 homolog and compared it to that of the malaria parasite, P. falciparum. The deduced amino acid sequence of the T. parva RESA2 gene is very similar to the P. falciparum RESA protein (38% identity). Key words: Theileria parva, vaccine, ring-infected erythrocyte surface antigen 2 (RESA2), apicomplexan antigens, homolog screen.

Molecular cloning and characterisation of the RESA gene, a marker of genetic diversity of Plasmodium falciparum

To identity immunodiagnostic antigen genes, a Plasmodium falciparum (Dd2 clone) expression library was screened using human immune sera. The ring-infected erythrocyte surface antigen (RESA) was isolated: this antigen of the resistant clone presents repeat tandem sequences like the 3D7 clone, albeit in different numbers. RESA has been studied as a marker of genetic diversity, with different sizes being observed in different isolates and clones of Plasmodium falciparum. The native protein was localised in cultures by western-blot and immuno-transmission electron microscopy. The antigenicity of RESA was evaluated by ELISA, using the carboxy-terminal repeat region as antigen. The assay’s sensitivity and specificity were 78.2 and 94% respectively.

Quantification of antibody-secreting lymphocytes that react with Pf155/RESA from Plasmodium falciparum: an ELISPOT assay for field studies.

We have adapted the enzyme-linked immunospot assay (ELISPOT) to enumerate the cells from Plasmodium falciparum-primed donors that produce IgG in vitro in response to malaria antigens. In vitro activation of cell cultures with two synthetic peptides (EENVEHDA)4, and (LGRSGGDIIKMQTL) corresponding to immunodominant T cell epitopes of the ring-infected erythrocyte surface antigen (Pf155/RESA) gave specific antibody-secreting cells (ASC) in five and six of the 15 P. falciparum-primed donors from Cameroon. Antibodies produced after a stimulation by synthetic peptides reacted also with total parasite proteins. However, crude P. falciparum antigen did not trigger a higher number of cells than did synthetic peptides. The absence of significant relation between the presence of sera antibodies and in vitro ASC against the same peptide suggests that the kinetics of circulating primed lymphocytes and antibodies are different. We evaluated 0.04-0.29% of peripheral blood B cells to be the frequency of memory cells specific to a single Pf155/RESA epitope in these donors. This study suggests that the ELISPOT assay should permit the analysis of B cell responses to malarial antigens at the single-cell level and its applicability to epidemiological field studies. This assay should be well suited to the identification of T helper epitopes capable of inducing the production of antibodies by human B cells, and will constitute an important tool for the selection of immunogens to be included in a subunit vaccine.

The ring-infected erythrocyte surface antigen (RESA) of Plasmodium falciparum stabilizes spectrin tetramers and suppresses further invasion

AbstractThe malaria parasite Plasmodium falciparum releases the ring-infected erythrocyte surface antigen (RESA) inside the red cell on entry. The protein migrates to the host cell membrane, where it binds to spectrin, but neither the nature of the interaction nor its functional consequences have previously been defined. Here, we identify the binding motifs involved in the interaction and describe a possible function. We have found that spectrin binds to a 108–amino acid fragment (residues 663-770) of RESA, and that this RESA fragment binds to repeat 16 of the β-chain, close to the labile dimer-dimer self-association site. We further show that the RESA fragment stabilizes the spectrin tetramer against dissociation into its constituent dimers, both in situ and in solution. This is accompanied by enhanced resistance of the cell to both mechanical and thermal degradation. Resealed erythrocytes containing RESA663-770 display resistance to invasion by merozoites of P falciparum. We infer that the evolutionary advantage of RESA to the parasite lies in its ability to prevent invasion of cells that are already host to a developing parasite, as well as possibly to guard the cell against thermal damage at the elevated body temperatures prevailing in febrile crises.

Pitting of malaria parasites and spherocyte formation.

BackgroundA high prevalence of spherocytes was detected in blood smears of children enrolled in a case control study conducted in the malaria holoendemic Lake Victoria basin. It was speculated that the spherocytes reflect intraerythrocytic removal of malarial parasites with a concurrent removal of RBC membrane through a process analogous to pitting of intraerythrocytic inclusion bodies. Pitting and re-circulation of RBCs devoid of malaria parasites could be a host mechanism for parasite clearance while minimizing the anaemia that would occur were the entire parasitized RBC removed. The prior demonstration of RBCs containing ring-infected erythrocyte surface antigen (pf 155 or RESA) but no intracellular parasites, support the idea of pitting.MethodsAn in vitro model was developed to examine the phenomenon of pitting and spherocyte formation in Plasmodium falciparum infected RBCs (iRBC) co-incubated with human macrophages. In vivo application of this model was evaluated using blood specimens from patients attending Kisumu Ditrict Hospital. RBCs were probed with anti-RESA monoclonal antibody and a DNA stain (propidium iodide). Flow cytometry and fluorescent microscopy was used to compare RBCs containing both the antigen and the parasites to those that were only RESA positive.ResultsCo-incubation of iRBC and tumor necrosis factor-alpha activated macrophages led to pitting (14% ± 1.31% macrophages with engulfed trophozoites) as opposed to erythrophagocytosis (5.33% ± 0.95%) (P < 0.01). Following the interaction, 26.9% ± 8.1% of the RBCs were spherocytes as determined by flow cytometric reduction in eosin-5-maleimide binding which detects RBC membrane band 3. The median of patient RBCs with pitted parasites (RESA+, PI-) was more than 3 times (95,275/μL) that of RESA+, PI+ RBCs (28,365/μL) (P < 0.01). RBCs with pitted parasites showed other morphological abnormalities, including spherocyte formation.ConclusionIt is proposed that in malaria holoendemic areas where prevalence of asexual stage parasites approaches 100% in children, RBCs with pitted parasites are re-circulated and pitting may produce spherocytes.

Construction of a human functional single‐chain variable fragment (scFv) antibody recognizing the malaria parasitePlasmodium falciparum

Falciparum malaria is one of the most deadly and profound human health problems around the tropical world. Antimalarial drugs are now considered to be a powerful treatment; however, there are drugs currently being used that are resistant to Plasmodium falciparum parasites spreading in different parts of the world. Although the protective immune response against intraerythrocytic stages of the falciparum malaria parasite is still not fully understood, immune antibodies have been shown to be associated with reduced parasite prevalence. Therefore antibodies of the right specificity present in adequate concentrations and affinity are reasonably effective in providing protection. In the present study, VH (variable domain of heavy chain) and VL (variable domain of light chain) were isolated from human blood lymphocytes of P. falciparum in one person who had high serum titre to RESA (ring-infected erythrocyte surface antigen). Equal amounts of VH and VL were assembled together with universal linker (G4S)3 to generate scFvs (single-chain variable fragments). The scFv antibodies were expressed with a phage system for the selection process. Exclusively, an expressed scFv against asynchronous culture of P. falciparum-infected erythrocytes was selected and characterized. Sequence analysis of selected scFv revealed that this clone could be classified into a VH family-derived germline gene (VH1) and Vkappa family segment (Vkappa1). Using an indirect immunofluorescence assay, we could show that soluble expressed scFv reacted with falciparum-infected erythrocytes. The results encourage the further study of scFvs for development as a potential immunotherapeutic agent.

Impact of red blood cell polymorphisms on the antibody response to Plasmodium falciparum in Senegal

The evidence of protection afforded by red blood cell polymorphisms against either clinical malaria or Plasmodium falciparum blood levels varies with the study site and the type of malaria transmission. Nevertheless, no clear implication of an antibody-related effect has yet been established in the protection related to red blood cell polymorphisms. We performed a prospective study, where plasma IgG and IgG subclasses directed to recombinant proteins from the merozoite surface protein 2 (MSP2/3D7 and MSP2/FC27) and the ring-infected erythrocyte surface antigen (RESA) were determined in a cohort of 413 Senegalese children before the annual malaria transmission season. The antibody response was dependent on age, and to a lesser extent, on the village of residence. IgG3 responders to all proteins, IgG responders to RESA and MSP2/3D7, as well as IgG2 to RESA and IgG1 responders to MSP2/3D7, presented enhanced mean values of parasite density, as evaluated during an 18-month follow-up. The levels of IgG and IgG3 to MSP2/3D7 were negatively associated with the risk of occurrence of a malaria attack during the following transmission season. Compared to normal children, sickle cell trait carriers presented lower levels of IgG to MSP2/3D7. Similarly, G6PD A− girls had lower levels of IgG and IgG3 to MSP2/FC27 than did G6PD normal girls. The impact of these particular genetic polymorphisms on the modulation of the antibody response is discussed.

Distinct Protein Classes Including Novel Merozoite Surface Antigens in Raft-like Membranes of Plasmodium falciparum

Glycosylphosphatidylinositol (GPI)-anchored proteins coat the surface of extracellular Plasmodium falciparum merozoites, of which several are highly validated candidates for inclusion in a blood-stage malaria vaccine. Here we determined the proteome of gradient-purified detergent-resistant membranes of mature blood-stage parasites and found that these membranes are greatly enriched in GPI-anchored proteins and their putative interacting partners. Also prominent in detergent-resistant membranes are apical organelle (rhoptry), multimembrane-spanning, and proteins destined for export into the host erythrocyte cytosol. Four new GPI-anchored proteins were identified, and a number of other novel proteins that are predicted to localize to the merozoite surface and/or apical organelles were detected. Three of the putative surface proteins possessed six-cysteine (Cys6) motifs, a distinct fold found in adhesive surface proteins expressed in other life stages. All three Cys6 proteins, termed Pf12, Pf38, and Pf41, were validated as merozoite surface antigens recognized strongly by antibodies present in naturally infected individuals. In addition to the merozoite surface, Pf38 was particularly prominent in the secretory apical organelles. A different cysteine-rich putative GPI-anchored protein, Pf92, was also localized to the merozoite surface. This insight into merozoite surfaces provides new opportunities for understanding both erythrocyte invasion and anti-parasite immunity.

A role for the Plasmodium falciparum RESA protein in resistance against heat shock demonstrated using gene disruption

During erythrocyte invasion, the Plasmodium falciparum Ring-infected erythrocyte surface antigen (RESA) establishes specific interactions with spectrin. Based on analysis of strains with a large chromosome 1 deletion, RESA has been assigned several functions, none of which is firmly established. Analysis of parasites with a disrupted resa1 gene and isogenic parental or resa3-disrupted controls confirmed the critical role of RESA in the surface reactivity of immune adult sera on glutaraldehyde-fixed ring stages. Absence of RESA did not influence merozoite invasion or erythrocyte membrane rigidity, was associated with a modest increase of cytoadhesion to CD36 under conditions of flow, but resulted in marked susceptibility to heat shock. resa1-KO-infected erythrocytes were prone to heat-induced vesiculation like uninfected erythrocytes, whereas parental or resa3-KO infected erythrocytes remained undamaged. Furthermore, a 6 h exposure of ring stages at 41 degrees C resulted in 33% culture inhibition of resa1-KO parasites while marginally impacting parental and resa3-KO parasite growth. This points to a role for RESA in protecting the infected erythrocyte cytoskeleton during febrile episodes. Infection patterns of resa1-KO and parental parasites in Saimiri sciureus indicated that RESA does not, at least on its own, modulate virulence in the squirrel monkey, as had been previously suggested.

Revisiting Putative Functional Properties of the Plasmodium falciparum Pf155/RESA Protein Using Genetically Engineered Parasites

Pf155/RESA (Ring Infected Erythrocyte Surface Antigen), is a Plasmodium falciparum protein discharged by the dense granules of merozoites during invasion and exported to the inner face of the erythrocyte membrane, where it interacts with spectrin in the young stages. Chromosome 1 subtelomeric deletion, eliminating amongst others the resa gene, may occur during adaptation of parasite isolates to in vitro culture. This is accompanied by erythrocyte membrane modifications of the red blood cell, such as increased adhesion and effect on membrane rigidity. It has been proposed that RESA was likely to contribute to these functional and rheological modifications. To assess this, we have constructed resa knock‐out parasites in the FUP/CB strain. This results in negativating the erythrocyte membrane immunofluorescence of glutaraldehyde‐fixed red blood cells (EMIF), indicating that RESA is critical for EMIF reactivity of hyperimmune human sera. Phenotypic and functional analysis of resa k.o. parasites indicated that loss of RESA expression neither affects membrane rigidity nor CD36 binding under flow conditions. Furthermore, infection of Saimiri sciureus monkeys showed that resa gene deletion does not account on its own for the greater adaptation and parasite virulence in this model.