Diallo, N. | bioRxiv Science

bioRxiv2026

CRISPR/Cas9 genome editing to generate single variant Plasmodium falciparum lines and enable reverse genetic studies of PfEMP1 function in live parasites

Adhesion interactions between Plasmodium falciparum infected erythrocytes (IEs) and human cells bring about microvascular sequestration and contribute to severe malaria pathology. Parasite adhesion molecules on the IE surface are members of the P. falciparum erythrocyte membrane protein 1 (PfEMP1) family, encoded by var genes, which interact with receptors on human cells. Progress in understanding PfEMP1-host receptor interaction is hindered by the lack of genetic tools for PfEMP1 functional studies in live parasites and the spontaneous switching of var gene transcription in culture leading to change in adhesion phenotype. We developed a CRISPR/Cas9 genome editing strategy that takes advantage of var gene mutually exclusive expression to generate single variant P. falciparum lines and enable reverse genetic studies of PfEMP1 function. A drug resistance gene and 2A peptide enabling bi-cistronic transcription were inserted between the promoter and exon I of the it4var60 gene encoding a PfEMP1 variant that mediates the virulence-associated rosetting phenotype. After genome editing and drug selection, only it4var60-transcribing parasites survived, and >90% of IEs expressed IT4VAR60-PfEMP1 on their surface and formed rosettes. When drug pressure was removed, switching to other variants occurred. The approach was adapted to generate epitope tagged-PfEMP1 allowing immunofluorescent detection with commercial antibodies, and modifications of the homology directed repair template enabled investigation of PfEMP1 function including point mutations and a gene knockout that abolished adhesion. These methods can be applied to any var gene in any P. falciparum genotype and are potentially transformative for functional studies of multi-gene family members in live parasites.

microbiology↗

bioRxiv2024

Identification of novel PfEMP1 variants containing domain cassettes 11, 15 and 8 that mediate the Plasmodium falciparum virulence-associated rosetting phenotype

Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) is a diverse family of variant surface antigens, encoded by var genes, that mediates binding of infected erythrocytes to human cells and plays a key role in parasite immune evasion and malaria pathology. The increased availability of parasite genome sequence data has revolutionised the ability to study PfEMP1 diversity across multiple P. falciparum isolates. However, making functional sense of genomic data relies on the ability to infer binding phenotype from var gene sequence. For P. falciparum rosetting, the binding of infected erythrocytes to uninfected erythrocytes, the analysis of var gene/PfEMP1 sequences encoding the phenotype is limited, with only eight rosette-mediating PfEMP1 variants described to date. These known rosetting PfEMP1 variants fall into two types, characterised by N- terminal domains known as "domain cassette" 11 (DC11) and DC16. Here we test the hypothesis that DC11 and DC16 are the only PfEMP1 types in the P. falciparum genome that mediate rosetting, by examining a set of thirteen recent culture-adapted Kenyan parasite lines. We first analysed the var gene/PfEMP1 repertoires of the Kenyan lines and identified an average of three DC11 or DC16 PfEMP1 variants per genotype. In vitro rosette selection of the parasite lines yielded four with a high rosette frequency, and analyses of their var gene transcription, infected erythrocyte PfEMP1 surface expression, rosette disruption and erythrocyte binding function identified four novel rosette- mediating PfEMP1 variants. Two of these were of the predicted DC11 type (one showing the dual rosetting/IgM-Fc-binding phenotype), whereas two contained DC15 (DBL1.2-CIDR1.5b) a PfEMP1 type not previously associated with rosetting. We also showed that a Thai parasite line expressing a DC8-like PfEMP1 binds to erythrocytes to form rosettes. Hence, these data expand current knowledge of rosetting mechanisms and emphasize that the PfEMP1 types mediating rosetting are more diverse than previously recognised. Author summary Malaria is an infectious tropical disease caused by the parasite Plasmodium falciparum that kills more than half a million people every year, mostly young children in sub-Saharan Africa. Life- threatening episodes of malaria are characterised by huge numbers of parasitised red blood cells in the infected host, many of which bind to blood vessel walls and block blood flow, causing tissue damage and organ failure. Sometimes parasitised red cells also bind to uninfected red cells to form clusters of cells called rosettes, which make the blockage of blood flow in vital organs even worse. Previous research has begun to decipher how parasitised red cells bind to uninfected red cells to form rosettes, but little is yet known about the process. Here we identify some new versions of the "sticky proteins" (adhesion molecules) that are made by malaria parasites and displayed on the surface of infected red cells to bring about rosette formation. The rosette-mediating adhesion molecules are members of a large family, and we identify here a few characteristic types within this family that mediate rosetting. This work is an important step towards the goal of understanding how malaria parasite rosettes form rosettes in order to develop preventions or treatments to reverse rosetting and reduce the number of people dying from severe malaria.

microbiology↗