Dataset Information

Comprehensive analysis of Fc-mediated IgM binding to the Plasmodium falciparum erythrocyte membrane protein 1 family in three parasite clones.

ABSTRACT: PfEMP1 is a family of adhesive proteins expressed on the surface of Plasmodium falciparum-infected erythrocytes (IEs), where they mediate adhesion of IEs to a range of host receptors. Efficient PfEMP1-dependent IE sequestration often depends on soluble serum proteins, including IgM. Here, we report a comprehensive investigation of which of the about 60 var gene-encoded PfEMP1 variants per parasite genome can bind IgM via the Fc part of the antibody molecule, and which of the constituent domains of those PfEMP1 are involved. We erased the epigenetic memory of var gene expression in three distinct P. falciparum clones, 3D7, HB3, and IT4/FCR3 by promoter titration, and then isolated individual IEs binding IgM from malaria-unexposed individuals by fluorescence-activated single-cell sorting. The var gene transcription profiles of sub-clones measured by real-time qPCR were used to identify potential IgM-binding PfEMP1 variants. Recombinant DBL and CIDR domains corresponding to those variants were tested by ELISA and protein arrays to confirm their IgM-binding capacity. Selected DBL domains were used to raise specific rat anti-sera to select IEs with uniform expression of candidate PfEMP1 proteins. Our data document that IgM-binding PfEMP1 proteins are common in each of the three clones studied, and that the binding epitopes are mainly found in DBLε and DBLζ domains near the C-terminus.

SUBMITTER: Quintana MDP

PROVIDER: S-EPMC6465264 | biostudies-literature | 2019 Apr

REPOSITORIES: biostudies-literature

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Comprehensive analysis of Fc-mediated IgM binding to the Plasmodium falciparum erythrocyte membrane protein 1 family in three parasite clones.

Quintana Maria Del Pilar MDP Ecklu-Mensah Gertrude G Tcherniuk Sergey O SO Ditlev Sisse Bolm SB Ditlev Sisse Bolm SB Oleinikov Andrew V AV Hviid Lars L Lopez-Perez Mary M

Scientific reports 20190415 1

PfEMP1 is a family of adhesive proteins expressed on the surface of Plasmodium falciparum-infected erythrocytes (IEs), where they mediate adhesion of IEs to a range of host receptors. Efficient PfEMP1-dependent IE sequestration often depends on soluble serum proteins, including IgM. Here, we report a comprehensive investigation of which of the about 60 var gene-encoded PfEMP1 variants per parasite genome can bind IgM via the Fc part of the antibody molecule, and which of the constituent domains ...[more]

PMID: 30988351

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Project description:Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) is a variant surface antigen family expressed on infected red blood cells that plays a role in immune evasion and mediates adhesion to vascular endothelium. PfEMP1s are potential targets of protective antibodies as suggested by previous seroepidemiology studies. Here, we used previously reported proteomic analyses of PfEMP1s of clinical parasite isolates collected from Malian children to identify targets of immunity. We designed a peptide library representing 11 PfEMP1 domains commonly identified on clinical isolates by membrane proteomics and then examined peptide-specific antibody responses in Malian children. The number of previous malaria infections was associated with development of PfEMP1 antibodies to peptides from domains CIDRα1.4, DBLγ11, DBLβ3, and DBLδ1. A zero-inflated negative binomial model with random effects (ZINBRE) was used to identify peptide reactivities that were associated with malaria risk. This peptide selection and serosurvey strategy revealed that high antibody levels to peptides from DBLγ11 and DBLδ1 domains correlated with decreased parasite burden in future infections, supporting the notion that specific PfEMP1 domains play a role in protective immunity. IMPORTANCE Plasmodium infection causes devastating disease and high mortality in young children. Immunity develops progressively as children acquire protection against severe disease, although reinfections and recrudescences still occur throughout life in areas of endemicity, partly due to parasite immunoevasion via switching of variant proteins such as Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) expressed on the infected erythrocyte surface. Understanding the mechanisms behind antibody protection can advance development of new therapeutic interventions that address this challenge. PfEMP1 domain-specific antibodies have been linked to reduction in severe malaria; however, the large diversity of PfEMP1 domains in circulating parasites has not been fully investigated. We designed representative peptides based on B cell epitopes of PfEMP1 domains identified in membranes of clinical parasite isolates and surveyed peptide-specific antibody responses among young Malian children in a longitudinal birth cohort. We examined previous infections and age as factors contributing to antibody acquisition and identified antibody specificities that predict malaria risk.

Project description:Plasmodium falciparum malaria remains one of the most deadly infections worldwide. The pathogenesis of the infection results from the sequestration of infected erythrocytes (IRBC) in vital organs, including the brain, with resulting impairment of blood flow, hypoxia, and lactic acidosis. Sequestration occurs through the adhesion of IRBC to host receptors on microvascular endothelium by Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1), a large family of variant surface antigens, each with up to seven extracellular domains that can bind to multiple host receptors. Consequently, antiadhesive therapies directed at single endothelial adhesion molecules may not be effective. In this study, we demonstrated that the serine protease thrombin, which is pivotal in the activation of the coagulation cascade, cleaved the major parasite adhesin on the surface of IRBC. As a result, adhesion under flow was dramatically reduced, and already adherent IRBC were detached. Thrombin cleavage sites were mapped to the Duffy binding-like δ1 (DBLδ1) domain and interdomains 1 and 2 in the PfEMP1 of the parasite line IT4var19. Furthermore, we observed an inverse correlation between the presence of thrombin and IRBC in cerebral malaria autopsies of children. We investigated a modified (R67A) thrombin and thrombin inhibitor, hirugen, both of which inhibit the binding of substrates to exosite I, thereby reducing its proinflammatory properties. Both approaches reduced the barrier dysfunction induced by thrombin without affecting its proteolytic activity on PfEMP1, raising the possibility that thrombin cleavage of variant PfEMP1 may be exploited as a broadly inhibitory antiadhesive therapy. Plasmodium falciparum malaria is the third leading cause of mortality due to a pathogen, with 214 million people infected and 438,000 deaths annually. The adhesion of Plasmodium falciparum-infected erythrocytes (IRBC) to microvascular endothelium is a major pathological process in severe malaria. While the recent implementation of artemisinin-based antimalarial therapy for severe malaria improves patient survival by targeting all parasite stages, antiparasite drugs alone may not immediately reverse pathophysiological processes in occluded vessels. Here we show that thrombin, an enzyme intimately involved in the clotting process, cleaves the main parasite adhesin expressed on the surface of IRBC, thereby preventing and reversing the binding of IRBC to endothelial cells. This beneficial effect of thrombin can be achieved by modified thrombins that cause significantly less clotting and vessel leakage while preserving the ability to cleave the parasite protein. Our results provide the basis for using modified thrombins as adjunctive therapy in severe malaria.

Project description:BackgroundIn areas of high transmission people often harbour multiple clones of Plasmodium falciparum, but even PCR-based diagnostic methods can only detect a fraction (the detectability, q) of all clones present in a host. Accurate measurements of detectability are desirable since it affects estimates of multiplicity of infection, prevalence, and frequency of breakthrough infections in clinical drug trials. Detectability can be estimated by typing repeated samples from the same host but it has been unclear what should be the time interval between the samples and how the data should be analysed.MethodsA longitudinal molecular study was conducted in the Kassena-Nankana district in northern Ghana. From each of the 80 participants, four finger prick samples were collected over a period of 8 days, and tested for presence of different Merozoite Surface Protein (msp) 2 genotypes. Implications for estimating q were derived from these data by comparing the fit of statistical models of serial dependence and over-dispersion.ResultsThe distribution of the frequencies of detection for msp2 genotypes was close to binomial if the time span between consecutive blood samples was at least 7 days. For shorter intervals the probabilities of detection were positively correlated, i.e. the shorter the interval between two blood collections, the more likely the diagnostic results matched for a particular genotype. Estimates of q were rather insensitive to the statistical model fitted.ConclusionsA simple algorithm based on analysing blood samples collected 7 days apart is justified for generating robust estimates of detectability. The finding of positive correlation of detection probabilities for short time intervals argues against imperfect detection being directly linked to the 48-hour periodicity of P. falciparum. The results suggest that the detectability of a given parasite clone changes over time, at an unknown rate, but fast enough to regard blood samples taken one week apart as statistically independent.

Dataset Information

Comprehensive analysis of Fc-mediated IgM binding to the Plasmodium falciparum erythrocyte membrane protein 1 family in three parasite clones.

Publications

Comprehensive analysis of Fc-mediated IgM binding to the Plasmodium falciparum erythrocyte membrane protein 1 family in three parasite clones.

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