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A system for functional studies of the major virulence factor of malaria parasites
Pathogen section, Bernhard Nocht Institute for Tropical Medicine, Germany
Interface section, Bernhard Nocht Institute for Tropical Medicine, Germany
Biophysics, Research Center Borstel, Leibniz Lung Center, Germany
Centre for translational Medicine & Parasitology, Department of Immunology and Microbiology, University of Copenhagen and Department of Infectious Diseases, Denmark
Department of Molecular Biology, Faculty of Science, Radboud University, Netherlands
Department of Molecular Biology, Radboud Institute for Molecular Life Sciences, Oncode Institute, Radboud University, Netherlands
Department of Biology, University of Hamburg, Germany
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An unusual trafficking domain in MSRP6 defines a complex needed for Maurer’s clefts anchoring and maintenance in
Alexandra Blancke Soares, Jan Stäcker ... Tobias Spielmann
A system for functional studies of the major virulence factor of malaria parasites
approach using selection linked integration (SLI) to generate
lines expressing single, specific surface adhesins PfEMP1 variants, enabling precise study of PfEMP1 trafficking, receptor binding, and cytoadhesion. By moving the system to different parasite strains and introducing an advanced SLI2 system for additional genomic edits, this work provides
evidence for an innovative and rigorous platform to explore PfEMP1 biology and identify novel proteins essential for malaria pathogenesis including immune evasion.
https://doi.org/10.7554/eLife.103542.3.sa0
: Findings that have theoretical or practical implications beyond a single subfield
: Evidence that features methods, data and analyses more rigorous than the current state-of-the-art
During the peer-review process the editor and reviewers write an eLife Assessment that summarises the significance of the findings reported in the article (on a scale ranging from landmark to useful) and the strength of the evidence (on a scale ranging from exceptional to inadequate).
PfEMP1 is a variable antigen displayed on erythrocytes infected with the malaria parasite
. PfEMP1 mediates binding of the infected cell to the endothelium of blood vessels, a cause of severe malaria. Each parasite encodes ~60 different PfEMP1 variants but only one is expressed at a time. Switching between variants underlies immune evasion in the host and variant-specific severity of disease. PfEMP1 is difficult to study due to expression heterogeneity between parasites which also renders genetic modification approaches ineffective. Here, we used selection-linked integration (SLI) to generate parasites all expressing the same PfEMP1 variant and genome edit the expressed locus. Moving this system from the reference strain 3D7 to IT4 resulted in PfEMP1 expressor parasites with effective receptor binding capacities. We also introduce a second version of SLI (SLI2) to introduce additional genome edits. Using these systems, we study PfEMP1 trafficking, generate cell lines binding to the most common endothelial receptors, survey the protein environment from functional PfEMP1 in the host cell, and identify new proteins needed for PfEMP1-mediated sequestration. These findings show the usefulness of the system to study the key virulence factor of malaria parasites.
A key factor for the pathology of the human malaria parasite
is its capacity to render the infected red blood cells (RBCs) adherent to the endothelium of blood vessels (
). This cytoadhesion allows the parasite to escape spleen-mediated clearance of infected RBCs (
) but causes sequestration of infected RBCs in major organs, which can lead to severe, life-threatening complications including cerebral malaria (
Cytoadhesion is mediated by members of the
erythrocyte membrane protein 1 (PfEMP1) family. PfEMP1s are 150–450 kDa single-pass transmembrane proteins inserted into the membrane of the infected RBC (
genes, with exon 1 encoding the variable extracellular part of PfEMP1 which has diversified to bind different host receptors such as CD36, ICAM-1, EPCR, and CSA through its DBL and CIDR domains (
exon 2 encodes a conserved intracellular C-terminal part, the acidic terminal segment (ATS), which anchors the PfEMP1 underneath the RBC membrane in so-called knobs, parasite-induced elevations of the RBC membrane which contribute to efficient cytoadhesion of the infected RBC (
genes that differ in sequence within and between parasites, but confers each parasite a similar repertoire of human receptor-binding phenotypes (
gene at a given time but can switch to a different
genes between isolates is high, the unique VAR2CSA PfEMP1 binding placental CSA - the cause of the detrimental sequestration leading to pregnancy malaria - is much more conserved between different isolates (
PfEMP1 is the major target for the protective acquired immune response (
gene switching is important to escape immune recognition and a mechanism to establish long-term infection in the host (
). Specific PfEMP1 variants are associated with pathology in the human host and with its immune status (
). Understanding the binding properties of individual PfEMP1 variants, antibody recognition, and switching is therefore critical to understand the pathology of malaria.
How PfEMP1 reaches its final destination at the host cell membrane is only partially understood. Exported parasite proteins are translocated by the PTEX complex into the host cell, but it is not fully clear if this is also true for PfEMP1 (
). Once in the host cell, PfEMP1 is most abundantly found at parasite-induced vesicular cisternae termed Maurer’s clefts, and only a small fraction of all PfEMP1 molecules reach the host cell surface (
). How PfEMP1 is transported within the host cell to reach the surface is unclear, but a number of other exported proteins, for example SBP1 and PTP1-7, are needed for that process (
A key problem in studying PfEMP1 lies in the heterogeneous
gene expression of the parasites in cell culture. This results in a mixed population of cells that have different antigenic and binding properties. Selective enrichment of binding phenotypes through elaborate panning of parasites against receptors or antibodies (
) or the utilization of parasite strains with more stable PfEMP1 expression, such as CS2 (
), has previously been used to circumvent this issue. A further problem is that specific PfEMP1s can be difficult to detect at the protein level. Antibodies against the conserved ATS do not distinguish between PfEMP1 variants and often cross-react with RBC spectrin (
). Extracellular domain-specific antibodies need to be generated for each newly studied PfEMP1 (
). Furthermore, the large size hampers episomal expression, and in some cases, episomally expressed mini-PfEMP1s were used as a surrogate, for example to study PfEMP1 trafficking (
). Finally, research questions needing genetic modification of PfEMP1s pose the problem that the modified locus is only expressed in some of the parasites.
Here, we use selection-linked integration (SLI;
) to generate parasite lines that each predominantly express one specific PfEMP1 (
). This permitted us to generate different parasite lines with binding specificities against all major binding receptors and parasites with modified PfEMP1s. We also introduce SLI version 2 (SLI2) to obtain a second genomic integration in parasites that already have a SLI-based alteration to express a specific tagged PfEMP1. We show that our approach can be used to study mutually exclusive expression of
genes, track the activated PfEMP1 via a small tag, study its trafficking, endothelial receptor binding, its proxiome in living parasites, and identify novel proteins needed for PfEMP1-mediated cytoadhesion.
Activation of specific PfEMP1 in the total cell population in