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Expression and co-localization of proteins of Pfs38 complex on Plasmodium merozoites. Co-localization studies were performed by immunofluorescence assays. Merozoites were labelled with (a) mouse anti-Pfs38 and rabbit anti-GLURP or with (b) mouse anti-Pfs38 and rabbit anti-Pfs41 antibodies or with (c) mouse anti-Pfs38 and rabbit anti-Pfs12 antibodies or with (d) mouse anti-Pfs38 and rabbit anti-PfsMSP165 antibodies. Partial co-localization was observed between Pfs38 and other proteins of 6-Cys complex with co-localization coefficient of 0.75, 0.61, 0.82, and 0.83 for a–d, respectively. Td represent bright field images
Source publication
Background
The Plasmodium genome encodes for a number of 6-Cys proteins that contain a module of six cysteine residues forming three intramolecular disulphide bonds. These proteins have been well characterized at transmission as well as hepatic stages of the parasite life cycle. In the present study, a large complex of 6-Cys proteins: Pfs41, Pfs38...
Similar publications
The blood-stage malaria vaccine candidate Plasmodium falciparum apical membrane antigen 1 (PfAMA1) can induce strong parasite growth-inhibitory antibody responses in animals but has not achieved the anticipated efficacy in clinical trials. Possible explanations in humans are the insufficient potency of the elicited antibody responses, as well as th...
Citations
... ELISA-based protein-protein interaction analysis was performed as described previously. 21 Briefly, a 96-well microtiter plate was coated overnight at COC with 50 ng recombinant PfHDP protein. Another unrelated Plasmodium falciparum recombinant protein, Ddi was coated as a negative control. ...
Haemoglobin degradation is crucial for the growth and survival of Plasmodium falciparum in human erythrocytes. Although the process of Hb degradation has been studied in detail, the mechanisms of Hb uptake remain ambiguous to date. Here, we characterized Heme Detoxification Protein (PfHDP); a protein localized in the parasitophorus vacuole, parasite food vacuole and infected erythrocyte cytosol for its role in Hb uptake. Immunoprecipitation of PfHDP‐GFP fusion protein from a transgenic line using GFP trap beads showed the association of PfHDP with Hb as well as with the members of PTEX translocon complex. Association of PfHDP with Hb or Pfexp‐2, a component of translocon complex was confirmed by protein‐protein interaction and immunolocalization tools. Based on these associations, we studied the role of PfHDP in Hb uptake using the PfHDP‐HA‐GlmS transgenic parasites line. PfHDP knock‐down significantly reduced the Hb uptake in these transgenic parasites in comparison to the wild type parasites. Morphological analysis of PfHDP‐HA‐GlmS transgenic parasites in the presence of GlcN showed food vacuole abnormalities and parasite stress, thereby causing a growth defect in the development of these parasites. Transient knockdown of a member of translocon complex, PfHSP101 in HSP101‐DDDHA parasites also showed a decreased uptake of Hb inside the parasite. Together, these results advocate an interaction between PfHDP and the translocon complex at the parasitophorus vacuole membrane and also suggest a role for PfHDP in the uptake of Hb and parasite development. The study thus reveals new insights into the function of PfHDP, making it an extremely important target for developing new antimalarials.
... Individuals with blood group O may be selectively advantaged from the co-existence of anti-A and anti-B antibodies shared by microorganisms [31]. Diversity of surface glycan molecules in red blood cells and ligands such as merozoite surface protein 1, apical membrane antigen 1 and erythrocyte-binding antigen in Plasmodium may also lead to different host susceptibility [11,32,33]. Reduced resetting is potentially another mechanism involved in it [11,34,35]. ...
The association between the ABO blood group and the risk of malaria during pregnancy has not been clearly established. The present study summarised relevant knowledge and reassessed the association through meta-analysis. Articles in MEDICINE and PubMed published before 30 November 2021 were searched. Five studies satisfied the inclusion criteria and were enrolled in the meta-analysis. It was shown that primiparae with different ABO blood group, multiparae with blood group A and non-A, AB and non-AB had a comparable risk of malaria. However, multiparae with blood group B had a significantly higher risk than non-B group [odds ratio (OR) = 1.23, 95% confidence interval (CI) was 1.01 to 1.50, P = 0.04], while multiparae with blood group O had a significantly lower risk than non-O group (OR = 0.78, 95% CI was 0.63 to 0.97, P = 0.03). Therefore, the ABO blood group may not result in a different risk of malaria in primiparae. Blood group B is potentially a risk factor while blood group O is a protective factor for multiparae.
... Cependant chez P. falciparum, différentes études ont montré que des anticorps générés contre des protéines recombinantes inhibent en partie l'invasion des érythrocytes par les mérozoïtes et la transmission au moustique (Feller et al., 2013;Paul et al., 2017). Une première expérience utilisant des peptides synthétiques décrit une activité de liaisons de peptides de P38 aux érythrocytes (García et al., 2009 (Rener et al., 1983;van Dijk et al., 2010). ...
... L'interaction de Rh5 avec son récepteur érythrocytaire, la basigine, permet la formation d'un pore déclenchant un signal calcique requis pour les étapes ultérieures de l'invasion (Chen et al., 2011;Volz et al., 2016). Des complexes de protéines parasitaires impliquant des protéines à domaine 6-cystéines ont déjà été décrits chez Plasmodium, notamment Pf41 / Pf12 chez les mérozoïtes (Parker et al., 2015;Paul et al., 2017;Taechalertpaisarn et al., 2012;Tonkin et al., 2013), Pfs230 / Pfs48/45 / PfCCPs chez les gamétocytes (N. Kumar, 1987;Simon et al., 2009) ou encore P36 / P52 chez les sporozoïtes (Arredondo et al., 2018). ...
... ). Plus récemment, une étude par immunoprécipitation a montré que P38 fait partie d'un complexe en surface des mérozoïtes comprenant notamment Pf41 et Pf12(Paul et al., 2017). L'étude a également montré que P38 interagit avec la glycophorine A présente en surface des érythrocytes. ...
L’infection par Plasmodium, parasite responsable du paludisme, débute par l’inoculation de sporozoïtes invasifs par un moustique Anopheles femelle. Les sporozoïtes infectent des cellules du foie pour s’y développer en mérozoïtes qui une fois libérés dans la circulation sanguine vont se développer dans les érythrocytes. L’invasion des cellules implique la sécrétion d’organites apicaux (micronèmes et rhoptries) et la formation d’une structure particulière (jonction mobile) conduisant à l’internalisation du parasite au sein d’une vacuole parasitophore. Les mécanismes moléculaires impliqués dans l’invasion des hépatocytes par les sporozoïtes restent méconnus. Les organites sécrétoires apicaux contiennent des protéines spécifiquement exprimées chez le sporozoïte (dont des protéines à domaine 6-cystéines), et des protéines également exprimées chez les mérozoïtes (comme AMA1, les RONs ou CLAMP) impliquées dans la formation de la jonction mobile. Nos travaux ont révélé le rôle essentiel d’une protéine à domaine 6-cystéines, B9, dans l’invasion des hépatocytes par les sporozoïtes. L’utilisation d’une stratégie de mutagénèse conditionnelle a permis de démontrer le rôle d’AMA1, RON2, RON4 et CLAMP chez les sporozoïtes, pour l’invasion des hépatocytes chez l’hôte mammifère mais également pour la colonisation des glandes salivaires du moustique. Nos résultats confortent l’hypothèse d’une compartimentation fonctionnelle des protéines micronémales chez les sporozoïtes. Ce travail contribue à une meilleure compréhension des mécanismes moléculaires mis en jeu lors de l’invasion des hépatocytes et identifie de nouvelles cibles potentielles pour bloquer l’invasion du foie par Plasmodium.
... The fractionated proteins were transferred from the gel onto a PVDF membrane (Amersham) and the membrane was blocked in blocking buffer (1 × PBS, 0.1% Tween-20, 5% milk powder) for 2 h. The blot was washed and incubated for 1 h with primary antibody [mice anti-GFP (1:1000, Roche-Sigma Aldrich); rabbit anti-Bip (1:2000) [64], rabbit anti-SERA (1:2000) [65]] diluted in dilution buffer (1× PBS, 0.1% Tween-20 and 1% milk powder). Later, the blot was washed and incubated for 1 h with appropriate secondary antibody (anti-rabbit or anti-mouse, 1: 2000, Sigma Aldrich) conjugated to HRP, diluted in dilution buffer. ...
Background
Plasmodium falciparum is the pathogen responsible for the most devastating form of human malaria. As it replicates asexually in the erythrocytes of its human host, the parasite feeds on haemoglobin uptaken from these cells. Heme, a toxic by-product of haemoglobin utilization by the parasite, is neutralized into inert hemozoin in the food vacuole of the parasite. Lipid homeostasis and phospholipid metabolism are crucial for this process, as well as for the parasite’s survival and propagation within the host. P. falciparum harbours a uniquely large family of phospholipases, which are suggested to play key roles in lipid metabolism and utilization.
Results
Here, we show that one of the parasite phospholipase ( P. falciparum lysophospholipase, Pf LPL1) plays an essential role in lipid homeostasis linked with the haemoglobin degradation and heme conversion pathway. Fluorescence tagging showed that the Pf LPL1 in infected blood cells localizes to dynamic vesicular structures that traffic from the host-parasite interface at the parasite periphery, through the cytosol, to get incorporated into a large vesicular lipid rich body next to the food-vacuole. Pf LPL1 is shown to harbour enzymatic activity to catabolize phospholipids, and its transient downregulation in the parasite caused a significant reduction of neutral lipids in the food vacuole-associated lipid bodies. This hindered the conversion of heme, originating from host haemoglobin, into the hemozoin, and disrupted the parasite development cycle and parasite growth. Detailed lipidomic analyses of inducible knock-down parasites deciphered the functional role of Pf LPL1 in generation of neutral lipid through recycling of phospholipids. Further, exogenous fatty-acids were able to complement downregulation of Pf LPL1 to rescue the parasite growth as well as restore hemozoin levels.
Conclusions
We found that the transient downregulation of Pf LPL1 in the parasite disrupted lipid homeostasis and caused a reduction in neutral lipids essentially required for heme to hemozoin conversion. Our study suggests a crucial link between phospholipid catabolism and generation of neutral lipids (TAGs) with the host haemoglobin degradation pathway.
... Several lines of evidence have suggested that MSP3 is a strong vaccine candidate antigen. First, seroepidemiological studies have demonstrated that levels of MSP3specific antibodies are associated with protection against clinical malaria (9,18,(24)(25)(26)(27)(28)(29)(30)(31). ...
... For the interaction analysis, the C-terminal region of MSP6 consisting of C-terminal residues 161 to 373 (MSP6C 161-372 ), MSP7C 195-352 , and residues 22 to 298 of RAP2 (RAP2 ) were produced in Escherichia coli (see Fig. S2 in the supplemental material) and subsequently used to generate specific antisera in mice and rabbits. Fragments of MSP1 protein consisting of aa 1052 to 1664, designated MSP1 65 , and SERA5 (serine repeat antigen 5) were generated as described previously (28,34). The five antisera were used to study the colocalization of these proteins on merozoites using immunofluorescence assay (IFA). ...
... Although PfMSP3 is abundantly expressed on the merozoite surface, it is still uncertain how this protein is associated with the merozoite surface. To establish the PfMSP3-associated network on the Plasmodium merozoite surface, we carried out a detailed protein-protein interaction analysis by performing immuno-pulldown of parasite lysate proteins using antibodies against an rMSP3 N-terminal fragment, a technique previously used to identify proteinprotein interactions (28,34,39,40). Five parasite proteins, MSP1, MSP6, MSP7, RAP2, and SERA5, which may be directly or indirectly involved in the invasion process were identified together with MSP3 in four independent LC-MS/MS experiments. ...
Plasmodium falciparum merozoite surface protein 3 (MSP3) is an abundantly expressed secreted merozoite surface protein and a leading malaria vaccine candidate antigen. However, it is unclear how MSP3 is retained on the surface of merozoites without a GPI anchor or a transmembrane domain. In the present study, we identified an MSP3 associated network on the Plasmodium merozoite surface by Immunoprecipitation of Plasmodium merozoite lysate using anti-MSP3N antibodies followed by mass spectrometry analysis. The results suggested the association of MSP3 with other merozoite surface proteins; MSP1, MSP6, MSP7, RAP2 and SERA5. Protein-protein interaction studies by ELISA binding and SPR analysis showed that MSP3 complex consists of MSP1, MSP6 and MSP7 proteins, Immunological characterization of MSP3 revealed that MSP3N is strongly recognized by hyper immune sera from African and Asian populations. Furthermore, we demonstrate that human antibodies affinity-purified against rMSP3N promote opsonic phagocytosis of merozoites in cooperation with monocytes. At non-physiological concentrations, anti-MSP3N antibodies inhibited the growth of P. falciparum in-vitro . Together the data suggests that MSP3 and especially its N-terminal region containing known B/T-cell epitopes is a target of naturally acquired immunity against malaria and is also an important candidate for a multi-subunit malaria vaccine.
... The amplified PCR fragments were cloned in pGEMT vector (Promega) and later subcloned in pET28a expression vector between BamHI and XhoI restriction sites. PfMSP-1 19 , PfMSP-1 65 , and PfMSP-3 (amino acids 21-238) genes were cloned as described earlier [24][25][26]. ...
... The expression and purification of PfMSP-1 65 and PfMSP-1 19 was performed as described earlier [24,25]. ...
... Antibody titres in serum samples were quantified by enzyme-linked immunosorbent assay (ELISA). Antibodies against PfMSP-1 19 and PfMSP-1 65 were raised according to the protocol as described earlier [24,25]. IgG (immunoglobulin) purification was performed with Protein-A sepharose beads according to the manufacturer's protocol. ...
Plasmodium falciparum
Merozoite surface protein (MSP) 1 has been studied extensively as a vaccine candidate antigen. PfMSP-1 undergoes proteolytic processing into four major products; p83, p30, p38 and p42 that are associated in the form of non-covalent complex(s) with other merozoite surface proteins. To delineate MSP1 regions involved in the interaction with other MSPs, here we expressed recombinant proteins (PfMSP-165) encompassing part of p38 and p42 regions and PfMSP-119PfMSP-165 interacted strongly with PfMSP-3, PfMSP-6, PfMSP-7, PfMSP-9, whereas PfMSP-119did not interact with any of these proteins. Since MSP-1 complex binds human erythrocytes, we examined the ability of these proteins to bind human erythrocyte. Among the proteins of MSP-1 complex, PfMSP-6 and PfMSP-9 bound to human erythrocytes. Serological studies showed that PfMSP-165was frequently recognized by sera from malaria endemic regions whereas this was not the case for PfMSP-119In contrast, antibodies against PfMSP-119 showed much higher inhibition of merozoite invasion compared to antibodies against the larger PfMSP-165fragment. Importantly, anti-PfMSP-119antibodies recognized both recombinant proteins; PfMSP-119 as well as PfMSP-165, however anti-PfMSP-165antibody failed to recognize the PfMSP-119 protein. Together these results demonstrate that PfMSP-1 sequences upstream of the 19kDa C-terminal region is involved in molecular interactions with other merozoite surface proteins and these sequences may probably serve as a smoke screen to evade antibody response to the membrane bound C-terminal 19 kDa region.
... The glutamate-rich protein (GLURP), a 220 kDa antigen expressed throughout the life-cycle of the malaria parasites in a mammalian host [12,13], is localized to the Plasmodium falciparum Pfs38 protein complex on the merozoite surface, and the protein contains a module of six cysteine residues forming three intramolecular disulphide bonds. The Pfs38 complex binds to host erythrocytes directly via glycophorin A as a receptor [14]. The GLURP protein consists of the three defined regions of an N-terminal non-repetitive region (R0), a central repetitive sequence (R1) and a C-terminal repetitive region (R2). ...
... The limited amino acid substitutions in the AAU likely reflects the functional constraints of the GLURP protein. A recent study reported that GLURP formed the Pfs38 protein complex on the merozoite surface that binds to erythrocytes via glycophorin A and participates in the parasite invasion of erythrocytes [14]. This is consistent with the three neutrality tests (Tajima's D, Fu and Li's D* and Fu and Li's F* tests) that all gave non-significant values, suggesting that the conserved AAU sequences were maintained under negative purifying selection. ...
... Frequency, subtypes and size of the P. falciparum GLURP polypeptide in global database. Size indicates the number of amino acid residues detected between blocks[4][5][6][7][8][9][10][11][12][13][14][15] No. of repeat units n GLURP subtype Subtype name Size ...
Background:
The glutamate-rich protein (GLURP) of the malaria parasite Plasmodium falciparum is a key surface antigen that serves as a component of a clinical vaccine. Moreover, the GLURP gene is also employed routinely as a genetic marker for malarial genotyping in epidemiological studies. While extensive size polymorphisms in GLURP are well recorded, the extent of the sequence diversity of this gene is rarely investigated. The present study aimed to explore the genetic diversity of GLURP in natural populations of P. falciparum.
Results:
The polymorphic C-terminal repetitive R2 region of GLURP sequences from 65 P. falciparum isolates in Thailand were generated and combined with the data from 103 worldwide isolates to generate a GLURP database. The collection was comprised of 168 alleles, encoding 105 unique GLURP subtypes, characterized by 18 types of amino acid repeat units (AAU). Of these, 28 GLURP subtypes, formed by 10 AAU types, were detected in P. falciparum in Thailand. Among them, 19 GLURP subtypes and 2 AAU types are described for the first time in the Thai parasite population. The AAU sequences were highly conserved, which is likely due to negative selection. Standard Fst analysis revealed the shared distributions of GLURP types among the P. falciparum populations, providing evidence of gene flow among the different demographic populations.
Conclusions:
Sequence diversity causing size variations in GLURP in Thai P. falciparum populations were detected, and caused by non-synonymous substitutions in repeat units and some insertion/deletion of aspartic acid or glutamic acid codons between repeat units. The P. falciparum population structure based on GLURP showed promising implications for the development of GLURP-based vaccines and for monitoring vaccine efficacy.
... The merozoite surface consists of membrane proteins such as the AMA1 or GPI anchored proteins such as the MSP1, which collectively constitute the integral merozoite surface proteins. Additionally, peripheral surface proteins such as the MSP3 and GLURP are attached through protein-protein interactions with integral merozoite surface proteins [18,19]. ...
... Complete sequencing of the P. falciparum genome revealed that both GLURP and MSP3 (along with other members of MSP3like gene family) are located on the same locus of chromosome 10 adjacent to each other [72]. Both MSP3 and GLURP are secreted upon schizogony and have been found to associate with the merozoite surface through protein-protein interactions [18,19]. While, MSP3 has been found to associate into dimmers and tetramers through the leucine zipper motif toward its C-terminal end [73], forming amyloid-like fibrilar structure [74] which may bind to the erythrocyte surface, the exact function of GLURP remains unknown. ...
Introduction: GMZ2 is a recombinant protein consisting of conserved domains of GLURP and MSP3, two asexual blood-stage antigens of Plasmodium falciparum, and is designed with the aim of mimicking naturally acquired anti-malarial immunity. The rationale for combining these two antigens is based on a series of immune epidemiological studies from geographically diverse malaria endemic regions; functional in vitro studies; and pre-clinical studies in rodents and New World monkeys. GMZ2 adjuvanted with alhydrogel® (alum) was well tolerated and immunogenic in three phase 1 studies. The recently concluded phase 2 trial of GMZ2/alum, involving 1849 participants 12 to 60 month of age in four countries in West, Central and Eastern Africa, showed that GMZ2 is well tolerated and has some, albeit modest, efficacy in the target population.
Areas covered: PubMed (www.ncbi.nlm.nih.gov/pubmed) was searched to review the progress and future prospects for clinical development of GMZ2 sub-unit vaccine. We will focus on discovery, naturally acquired immunity, functional activity of specific antibodies, sequence diversity, production, pre-clinical and clinical studies.
Expert commentary: GMZ2 is well tolerated and has some, albeit modest, efficacy in the target population. More immunogenic formulations should be developed.
Background
Malaria remains a major global health priority, and monoclonal antibodies (mAbs) are emerging as potential new tools to support efforts to control the disease. Recent data suggest that Fc-dependent mechanisms of immunity are important mediators of protection against the blood stages of the infection, but few studies have investigated this in the context of mAbs. We aimed to isolate mAbs agnostic to cognate antigens that target whole merozoites and simultaneously induce potent neutrophil activity measured by the level of reactive oxygen species (ROS) production using an antibody-dependent respiratory burst (ADRB) assay.
Methods
We used samples from semi-immune adults living in coastal Kenya to isolate mAbs that induce merozoite-specific ADRB activity. We then tested whether modifying the expressed IgG1 isotype to an IgG–IgA Fc region chimera would enhance the level of ADRB activity.
Results
We isolated a panel of nine mAbs with specificity to whole merozoites. mAb J31 induced ADRB activity in a dose-dependent fashion. Compared to IgG1, our modified antibody IgG–IgA bi-isotype induced higher ADRB activity across all concentrations tested. Further, we observed a negative hook effect at high IgG1 mAb concentrations (i.e., >200 µg/mL), but this was reversed by Fc modification. We identified MSP3.5 as the potential cognate target of mAb J31.
Conclusions
We demonstrate an approach to engineer mAbs with enhanced ADRB potency against blood-stage parasites.
