Exploration of factors that influence Plasmodium falciparum fitness and virulence

Abstract

Malaria is an ancient disease that still has profound impact on human population. The virulence of the most lethal malaria parasite, Plasmodium falciparum, can be attributed to several features of the parasite. P. falciparum is known for its indiscriminate red blood cell (RBC) invasion and aptitude for cytoadherence. The latter is associated with various disease pathologies. Thisthesis explores factors that influence the virulence and fitness of P. falciparum, both from the host and parasite perspective.

The association between ABO blood groups and protection from severe malaria has sparked many studies, and blood group O has emerged as protective against severe disease. This protection has been attributed to the binding of uninfected RBCs (uRBC) by the parasitized RBC (pRBC), a mechanism known as rosetting. Using a robust high-throughput flow cytometric method, we characterized rosetting for six parasite strains/isolates in all four major ABO blood groups. Rosettes formed in non-O blood shielded the major parasite surface antigen (PfEMP1) from antibody recognition. As blood group A is further subdivided based on qualitative and quantitative properties of the A-antigen, we found that levels of A-antigen on RBCs were positively correlated with rosette sturdiness against disruption by heparin and antibodies.

RIFINs, another large family of surface antigens, has been implicated in blood group A rosetting. Members of this family can be divided into A- and B-RIFINS, depending on cellular localization and parasite stage expression. To set the scene for future studies of RIFINS, we generated and validated antibodies for various antibody-based methods. We identified two nonrosetting RIFIN-expressing parasite lines that had not been characterized before. Their dominant rif transcripts were identified by RNA sequencing.

As PfEMP1s along with RIFINs and other surface adhesins must be trafficked and inserted into the pRBC membrane to fulfil their cytoadhesive function, we hypothesized that his process might be affected by varied conditions in the host. Here, we describe the loss of pRBC’s adhesive capacities in acidified environment for rosetting and placental binding parasite stains. The reduction was associated with the loss of surface exposed PfEMP1 due to disturbances in the last steps of PfEMP1 trafficking and membrane insertion.

Heparin-derivatives, including sevuparin, have sparked interest as possible adjunctive therapeutics in severe malaria treatment. Here, we investigated the mechanisms behind the invasion inhibition by clinically well-tolerated sevuparin and explored the additional antiparasitic properties of this compound. Sevuparin severely affected parasite intracellular development with delayed schizogony and reduced parasitemia after drug removal. The metabolic disturbances manifested in abnormal morphology, abundant extracellular parasites, and reduction of PfEMP1 on the pRBC surface. Inhibition by sevuparin was distinct from classical plasmodial surface anion channel (PSAC) inhibitors, suggesting the involvement of other channels or transporters. Using protein pull-downs from membranes of pRBCs and uRBCs, we identified putative sevuparin interactomes. Due to the identification of multiple human proteins linked to cation homeostasis and haemolysis, we measured cellular sodium levels. Upon treatment with sevuparin, cellular sodium levels were increased in pRBCs, whereas no differences were noted in uRBCs.

In conclusion, we found that A-antigen levels on RBCs affect rosette characteristics, which should be considered in future studies investigating associations between blood group A and risk to develop severe malaria. We have validated tools for the study of RIFIN family of proteins and their possible function in disease pathogenesis. In addition, we demonstrated that PfEMP1 trafficking to the surface is pH sensitive. Finally, we showed that sevuparin has multimodal activity against malaria parasites.