Browsing by Author "Ecklu-Mensah, G."

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Acquisition of IGG to ICAM-1-Binding DBLΒ Domains in the plasmodium falciparum erythrocyte membrane protein 1 antigen family varies between groups A, B, and C
(Infection and Immunity, 2019-09-19) Ecklu-Mensah, G.; Olsen, R.W.; Bengtsson, A.; Ofori, M.F.; Kusi, K.A.; Koram, K.A.; Hviid, L.; Adams, Y.; Jensen, A.T.R.
Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) is an important malaria virulence factor. The protein family can be divided into clinically relevant subfamilies. ICAM-1-binding group A PfEMP1 proteins also bind endothelial protein C receptor and have been associated with cerebral malaria in children. IgG to these PfEMP1 proteins is acquired later in life than that to group A PfEMP1 not binding ICAM-1. The kinetics of acquisition of IgG to group B and C PfEMP1 proteins binding ICAM-1 is unclear and was studied here. Gene sequences encoding group B and C PfEMP1 with DBL domains known to bind ICAM-1 were used to identify additional binders. Levels of IgG specific for DBL domains from group A, B, and C PfEMP1 binding or not binding ICAM-1 were measured in plasma from Ghanaian children with or without malaria. Seven new ICAM-1-binding DBL domains from group B and C PfEMP1 were identified. Healthy children had higher levels of IgG specific for ICAM-1-binding DBL domains from group A than from groups B and C. However, the opposite pattern was found in children with malaria, particularly among young patients. Acquisition of IgG specific for DBL domains binding ICAM-1 differs between PfEMP1 groups.
Assessment of bacterial diversity in western Accra, Ghana, drinking water samples
(Journal of Water, Sanitation and Hygiene for Development, 2019) Ecklu-Mensah, G.; Sackey, S.T.; Morrison, H.G.; et.al
The design and performance characteristics of municipal drinking water systems can profoundly influence public health. To assess the operational attributes of an Accra, Ghana drinking water distribution system, high-throughput 454 pyrosequencing was employed to characterize its bacterial community composition. Samples from the waterworks and four household sources (one household tap and three polytank storage units) were analyzed within one of Accra’s distribution networks over 4 months. Samples provided between 9,059 and 20,076 reads (average ¼ 13,056) that represented a broad range of bacterial diversity, including rare genera. Minimum Entropy Decomposition (MED) analysis showed that the sequences described four major assemblages. Assemblages 1 and 2 dominated the waterworks and household tap samples while polytank storage unit samples, with one exception, contained assemblages 3 or 4. The considerable bacterial taxonomic difference between different sources suggests that contamination and/or selective growth shapes bacterial community structures after treatment at the waterworks. Of particular interest are the major differences between the poly tank samples following storage and the tap/waterworks samples, suggesting that water storage (stagnation) can select for unique microbial populations
Blood outgrowth endothelial cells (BOECs) as a novel tool for studying adhesion of Plasmodium falciparum-infected erythrocytes
(PLoS ONE, 2018-10) Ecklu-Mensah, G.; Olsen, R.W.; Bengtsson, A.; Ofori, M.F.; Hviid, L.; Jensen, A.T.R.; Adams, Y.
The lack of suitable animal models for the study of cytoadhesion of P. falciparum-infected erythrocytes (IEs) has necessitated in vitro studies employing a range of cell lines of either human tumour origin (e.g., BeWo and C32 cells) or non-human origin (e.g., CHO cells). Of the human cells available, many were isolated from adults, or derived from a pool of donors (e.g., HBEC-5i). Here we demonstrate, for the first time, the successful isolation of blood outgrowth endothelial cells (BOECs) from frozen stabilates of peripheral blood mononuclear cells obtained from small-volume peripheral blood samples from paediatric malaria patients. BOECs are a sub-population of human endothelial cells, found within the peripheral blood. We demonstrate that these cells express receptors such as Intercellular Adhesion Molecule 1 (ICAM-1/CD54), Endothelial Protein C Receptor (EPCR/CD201), platelet/endothelial cell adhesion molecule 1 (PECAM-1/CD31), Thrombomodulin (CD141), and support adhesion of P. falciparum IEs.
Comprehensive analysis of Fc-mediated IgM binding to the Plasmodium falciparum erythrocyte membrane protein 1 family in three parasite clones
(Scientific Reports, 2019-04) Quintana, P.; Ecklu-Mensah, G.; Tcherniuk, S.; Ditlev, S.B.; Oleinikov, A.V.; Hviid, L.; Lopez-Perez, M.
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.
Natural and vaccine-induced acquisition of cross-reactive IgG inhibiting ICAM-1-specific binding of a PfEMP1 subtype associated specifically with cerebral malaria
(Infection and Immunity, 2018-02) Olsen, R.W.; Ecklu-Mensah, G.; Bengtsson, A.; Ofori, M.F.; Lusingu, J.P.A.; Castberg, F.C.; Hviid, L.; Adams, Y.; Jensen, A.T.R.
Cerebral malaria (CM) is a potentially deadly outcome of Plasmodium falciparum malaria that is precipitated by sequestration of infected erythrocytes (IEs) in the brain. The adhesion of IEs to brain endothelial cells is mediated by a subtype of parasite-encoded PfEMP1 that facilitate dual binding to host ICAM-1 and EPCR. The PfEMP1 subtype is characterized by the presence of a particular motif (DBLβ_motif) in the constituent ICAM-1-binding DBLβ domain. The rate of natural acquisition of DBLβ_motif-specific IgG and the ability to induce such antibodies by vaccination are unknown, and the aim of this study was to provide such data. We used ELISA to measure DBLβ-specific IgG in plasma from Ghanaian children with malaria. The ability of human immune plasma and DBLβ-specific rat anti-sera to inhibit the interaction between ICAM-1 and DBLβ was assessed using ELISA and in vitro assays of IE adhesion under flow. Acquisition of DBLβ_motif-specific IgG coincided with age-specific susceptibility to CM. Broadly cross-reactive antibodies inhibiting the interaction between ICAM-1 and DBLβ_motif domains were detectable in immune plasma and in sera of rats immunized with specific DBLβ_motif antigens. Importantly, antibodies against the DBLβ_motif inhibited ICAM-1-specific in vitro adhesion of erythrocytes infected by four of five P. falciparum isolates from cerebral malaria patients. We conclude that natural exposure to P. falciparum as well as immunization with specific DBLβ_motif antigens can induce cross-reactive antibodies that inhibit the interaction between ICAM-1 and a broad range of DBLβ_motif domains. These findings raise hope that a vaccine designed specifically to prevent CM is feasible.
The Role of an Intercellular Adhesion Molecule 1 Binding Motif in Plasmodium Falciparum Erythrocyte Membrane Protein 1 and Other Determinants Involved in Severe Malaria Pathogenesis
(University Of Ghana, 2017-07) Ecklu-Mensah, G.
A central pathogenic feature in Plasmodium falciparum cerebral malaria (CM) is the sequestration of infected erythrocytes (IEs) which results in the obstruction of blood flow, inflammation and damage to the endothelial cell barrier integrity. Adhesion is mediated by a family of approximately 60 highly variant parasite proteins called P. falciparum erythrocyte membrane protein 1 (PfEMP1), which are displayed on the surface of infected cells. All PfEMP1 contain multiple extracellular domains of Duffy binding like (DBL) and cysteine-rich interdomain region (CIDR) proteins that mediate adhesion to various host endothelial receptors such as intercellular adhesion molecule 1 (ICAM-1). An important unanswered question in malaria pathogenesis has always been whether a specific binding phenotype can be linked to any of the different severe malaria phenotypes. The broad objectives of this study were to investigate the involvement of an ICAM-1 binding motif in PfEMP1 in the development of CM, as well as to evaluate the contributions of the immune response and other determinants driving severe malaria complications. A highly conserved motif in DBLβ domain of group A PfEMP1 molecules was identified using sequence information. IEs expressing PfEMP1 containing the motif had a dual binding affinity for endothelial protein C receptor (EPCR) and ICAM-1 when tested under in vitro flow adhesion assays. Quantitative real time PCR (qPCR) showed higher transcription of this motif in parasites isolated from children with CM compared with non-cerebral severe malaria (p=0.020). The sequences defining the motif were exceptionally conserved and thus suggested its ability to induce cross-reactive responses. Broadly reactive cross-inhibitory antibodies against the motif were acquired following P. falciparum natural infections, in addition to being experimentally induced. Such cross-reactive antibodies interfered with ICAM-1 binding of recombinant motif-containing DBLβ domains and IEs from paediatric CM patients. These findings make the motif an attractive candidate to be included in a vaccine cocktail that seeks to prevent CM complications. Markers of endothelial activation (sICAM-1, sEPCR, Ang-2, HMGB1) in Ghanaian children with severe or uncomplicated malaria and their levels following resolution of infection were determined using ELISA. The levels were higher in malaria patients in comparison with healthy controls (p < 0.001). These levels declined significantly in convalescent patients except for high mobility group box 1 protein (HMGB1) which remained high to a similar degree as observed during infection (p > 0.05), an observation reported for the first time in malaria. Blood outgrowth endothelial cells (BOECs) from peripheral blood mononuclear cells of Ghanaian malaria patients were isolated and characterised as physiologically relevant cells to potentially study falciparum-infected erythrocyte adhesive interactions. Flow cytometry analysis showed that BOECs expressed several endothelial surface markers including malaria parasite adhesion proteins ICAM-1 and EPCR. Thus, future use of these cells could advance our understanding of cytoadhesion involving both receptors. Very little is known about the frequency of IgM-binding PfEMP1 in the P. falciparum genome, a phenotype that has been associated with severe disease. Quantitative real time PCR was used to assess the var transcript levels of 3D7/NF54 parasites selected for IgM binding. This study uncovered at least six new var/PfEMP1 candidates that could potentially mediate binding to IgM. Overall, this study expands current knowledge on the contributions made by both host and parasite factors to severe malaria pathogenesis. Detailed knowledge of these factors is crucially important for effective management of malaria.
Structure-Guided Identification of a Family of Dual Receptor-Binding PfEMP1 that Is Associated with Cerebral Malaria
(Cell Host and Microbe, 2017-03) Lennartz, F.; Adams, Y.; Bengtsson, A.; Olsen, R.W.; Turner, L.; Ndam, N.T.; Ecklu-Mensah, G.; Moussiliou, A.; Ofori, M.F.; Gamain, B.; Lusingu, J.P.; Petersen, J.E.V.; Wang, C.W.; Nunes-Silva, S.; Jespersen, J.S.; Lau, C.K.Y.; Theander, T.G.; Lavstsen, T.; Hviid, L.; Higgins, M.K.; Jensen, A.T.R.
Cerebral malaria is a deadly outcome of infection by Plasmodium falciparum, occurring when parasite-infected erythrocytes accumulate in the brain. These erythrocytes display parasite proteins of the PfEMP1 family that bind various endothelial receptors. Despite the importance of cerebral malaria, a binding phenotype linked to its symptoms has not been identified. Here, we used structural biology to determine how a group of PfEMP1 proteins interacts with intercellular adhesion molecule 1 (ICAM-1), allowing us to predict binders from a specific sequence motif alone. Analysis of multiple Plasmodium falciparum genomes showed that ICAM-1-binding PfEMP1s also interact with endothelial protein C receptor (EPCR), allowing infected erythrocytes to synergistically bind both receptors. Expression of these PfEMP1s, predicted to bind both ICAM-1 and EPCR, is associated with increased risk of developing cerebral malaria. This study therefore reveals an important PfEMP1-binding phenotype that could be targeted as part of a strategy to prevent cerebral malaria. © 2017 The Author(s)