Genetic validation of the function of PfEMP1 in Plasmodium falciparum rosette formation

Abstract

Malaria remains a significant global health concern, affecting half of the world’s population. Severe malaria, a life-threatening form of the disease, results from the adhesion of Plasmodium falciparum-infected erythrocytes to host cell receptors. One such adhesion phenotype is rosetting, where infected erythrocytes bind to uninfected ones, forming clusters known as rosettes. This adhesion process is mediated by parasite surface antigens, particularly the P. falciparum erythrocyte membrane protein 1 (PfEMP1), encoded by the var multigene family (~60 distinct copies per parasite genome transcribed in a mutually exclusive manner). Current understanding of PfEMP1 function primarily stems from studies involving recombinant proteins, but rarely by reverse genetics. Studying PfEMP1's role in adhesion within live P. falciparum-infected erythrocytes has been challenging due to difficulties in genetically manipulating P. falciparum and maintaining the expression of a single PfEMP1 variant. My thesis therefore aimed to use CRISPR/Cas9 genome technology for functional studies of PfEMP1 in live parasites.

Firstly, I used CRISPR/Cas9 genome editing to achieve two main objectives: generating a parasite population expressing a single var gene and incorporating epitope tags into PfEMP1. By integrating the drug resistance gene ‘BSD’ into the locus of the rosetting “IT4var60” var gene, I generated homogenous parasite population that exclusively expressed IT4VAR60 PfEMP1, under blasticidin pressure. Profiling var gene transcription in the transgenic parasites revealed IT4var60 as the predominant transcript. Additionally, I successfully introduced epitope tags at the N-terminus of IT4VAR60 PfEMP1, confirmed by recognition by anti-tag antibodies.

Secondly, I used CRISPR/Cas9 to generate a series of IT4VAR60 mutants, aiming to investigate the role of IT4VAR60 and specific amino acid residues in rosette formation in IT4 P. falciparum strain. I generated an IT4var60-knockout mutant, where rosetting was completely abolished, which confirmed the role of IT4VAR60 PfEMP1 in mediating rosette in IT4 strain. Previous studies using recombinant proteins have identified residues within the IT4VAR60 DBL⍺ domain that are essential for erythrocyte binding. My transgenic mutants of these residues revealed their importance in rosetting in IT4 parasites, although my results show that they were not the sole residues involved, suggesting the involvement of additional residues.

Thirdly, I targeted a different rosetting variant in another parasite strain and modified PfEMP1 variants with other adhesion phenotypes. I successfully generated transgenic parasites for rosetting variant HB3VAR06 in the HB3 strain, and for non-rosetting variants HB3VAR03 and IT4VAR19, which adhere to human brain endothelial cells. Additionally, I investigated the role of the DBL⍺ domain in rosetting by creating a chimeric “domain-swap” transgenic IT4var19[IT4var60-DBL⍺]. Genome editing was successful in each case, and immunofluorescent staining and flow cytometry using PfEMP1 domain-specific antibodies, showed surface expression of the respective PfEMP1.

Overall, I have demonstrated the effectiveness and versatility of CRISPR/Cas9 technology in manipulating PfEMP1 variants in live infected cells. This work presents a time-efficient approach for obtaining a homogenous population of parasites expressing a specific PfEMP1 variant. This approach can be potentially to be applied to any variant in any P. falciparum genotype, thereby enabling the study of a wide range of host-parasite adhesion interactions. This is crucial for enhancing the understanding of severe malaria and developing targeted interventions.