Plasmodium falciparum binding interactions with human brain endothelial cells

Cerebral malaria is the most severe form of malaria and mostly affects children under 5 years causing impaired consciousness, coma and neurological disorders, with life-threatening consequences in affected individuals. A pathological feature of the disease is the sequestration of mature Plasmodium falciparum infected erythrocytes (IEs) in the microvasculature of the brain. P. falciparum Erythrocyte Membrane Protein 1 (PfEMP1) expressed on the surface of IEs is thought to enable the parasites bind to human brain endothelial cells (HBEC) to avoid splenic clearance. An in vitro model of cytoadherence in cerebral malaria has been developed using a human brain endothelial cell line called HBEC-5i, which enables the study of IEs binding to HBEC. Previous work based on three laboratory parasite lines showed that HBEC-binding IEs express a specific subset of the diverse PfEMP1 family which contain sets of cysteine-rich domains called domain cassettes (DC) 8 and 13. Parasites from children diagnosed with cerebral malaria have also been independently shown to express the DC8 and DC13 PfEMP1 types. The adhesion of IEs to HBEC is suggested to occur by binding of a domain of the DC8 and DC13 PfEMP1 variants called CIDRα1, to Endothelial Protein C Receptor (EPCR) on HBEC. However, investigations of the effect of parasite and host environmental factors on adhesion to HBEC are lacking, and further studies are needed to confirm the association of these DC8 and DC13 PfEMP1 to HBEC-binding and the role of EPCR in mediating the cytoadhesion. Therefore, the aim of this thesis was to examine the hypothesis that HBEC-binding would be affected by changes in environmental conditions, and that all IEs that bind to HBEC would express group A-like PfEMP1 containing DC8 and DC13, for binding to EPCR on HBEC. In this study, the effect of pH, parasitaemia, gas, temperature, and serum on cytoadhesion, were investigated using four HBEC-binding parasite lines. Adhesion of IEs to HBEC was found to be pH and parasitaemia -dependent with optimal binding at pH 7.3 and IE adhesion positively correlated with parasitaemia. There was no significant effect of increase in temperature to 39°C and no significant difference between hypoxic and normoxic conditions on adhesion in all parasite lines. Human serum, however, abolished binding of the DC8-expressing parasite line but had minimal effects on adhesion of the DC13-expressing parasite lines. Two Kenyan isolates recently adapted to culture were selected for binding to HBEC and were found to also predominantly express group A-like PfEMP1 including a DC8 PfEMP1 variant and PfEMP1 (s) that contained DBLα1.2 domains. Attempts were made to localise the binding domain within the DC8 and DC13 PfEMP1 variants using recombinant proteins and antibodies. However, the CIDRα1 domain appeared to mediate adhesion of the DC8-expressing parasite line but had no effect on adhesion of the DC13-expressing parasite lines. Only antibodies to the N-terminal domain, known as NTS.DBLα, significantly inhibited binding of all the parasites lines. The role of EPCR and other receptor molecules on endothelial cells including ICAM-1, CD36, CSA, PECAM-1, HABP-1 and heparin, in mediating adhesion to HBEC was also investigated. Using EPCR recombinant protein, monoclonal and polyclonal antibodies, and EPCR-siRNA knockdown in binding assays, EPCR was shown to be involved in adhesion of only the DC8-expressing parasite line and did not affect adhesion of the DC13-expressing parasite lines to HBEC. Binding of DC8-expressing parasite line to HBEC was also inhibited by soluble recombinant PECAM-1. There was no significant adhesion of both types of parasite lines to the other receptor molecules, although minimal binding to HABP-1 was observed. This study expands current knowledge on the parasite binding interactions with HBEC by elucidating some of the environmental factors that affect the binding properties, and gives the optimal conditions for the in vitro model of HBEC-adhesion in cerebral malaria. Findings presented here confirm the association of expression of group A-like PfEMP1 to HBEC-binding and shows that the EPCR-CIDRα1 interaction does not mediate adhesion of all DC8 and DC13- expressing parasite lines to HBEC. Additional receptors, other than EPCR, are therefore required for HBEC-binding in cerebral malaria. The ability of normal human serum to abolish binding of the DC8-expressing parasite line also raises the question of whether IE binding to EPCR is physiologically relevant and suggest that the DC8- expressing parasites associated with cerebral malaria may contribute to the disease in a mechanism other than binding to brain endothelial cell

Deletion of Plasmodium falciparum Protein RON3 Affects the Functional Translocation of Exported Proteins and Glucose Uptake.

The survival of Plasmodium spp. within the host red blood cell (RBC) depends on the function of a membrane protein complex, termed the Plasmodium translocon of exported proteins (PTEX), that exports certain parasite proteins, collectively referred to as the exportome, across the parasitophorous vacuolar membrane (PVM) that encases the parasite in the host RBC cytoplasm. The core of PTEX consists of three proteins: EXP2, PTEX150, and the HSP101 ATPase; of these three proteins, only EXP2 is a membrane protein. Studying the PTEX-dependent transport of members of the exportome, we discovered that exported proteins, such as ring-infected erythrocyte surface antigen (RESA), failed to be transported in parasites in which the parasite rhoptry protein RON3 was conditionally disrupted. RON3-deficient parasites also failed to develop beyond the ring stage, and glucose uptake was significantly decreased. These findings provide evidence that RON3 influences two translocation functions, namely, transport of the parasite exportome through PTEX and the transport of glucose from the RBC cytoplasm to the parasitophorous vacuolar (PV) space where it can enter the parasite via the hexose transporter (HT) in the parasite plasma membrane.IMPORTANCE The malarial parasite within the erythrocyte is surrounded by two membranes. Plasmodium translocon of exported proteins (PTEX) in the parasite vacuolar membrane critically transports proteins from the parasite to the erythrocytic cytosol and membrane to create protein infrastructure important for virulence. The components of PTEX are stored within the dense granule, which is secreted from the parasite during invasion. We now describe a protein, RON3, from another invasion organelle, the rhoptry, that is also secreted during invasion. We find that RON3 is required for the protein transport function of the PTEX and for glucose transport from the RBC cytoplasm to the parasite, a function thought to be mediated by PTEX component EXP2

Complement C1s cleaves PfEMP1 at interdomain conserved sites inhibiting plasmodium falciparum cytoadherence

Cytoadhesion of Plasmodium falciparum-infected erythrocytes (IEs) to the endothelial lining of blood vessels protects parasites from splenic destruction, but also leads to detrimental inflammation and vessel occlusion. Surface display of the P. falciparum erythrocyte membrane protein 1 (PfEMP1) adhesion ligands exposes them to host antibodies and serum proteins. PfEMP1 are important targets of acquired immunity to malaria, and through evolution, the protein family has expanded and diversified to bind a select set of host receptors through antigenically diversified receptor-binding domains. Here, we show that complement component 1s (C1s) in serum cleaves PfEMP1 at semiconserved arginine motifs located at interdomain regions between the receptor-binding domains, rendering the IE incapable of binding the two main PfEMP1 receptors, CD36 and endothelial protein C receptor (EPCR). Bioinformatic analyses of PfEMP1 protein sequences from 15 P. falciparum genomes found the C1s motif was present in most PfEMP1 variants. Prediction of C1s cleavage and loss of binding to endothelial receptors was further corroborated by testing of several different parasite lines. These observations suggest that the parasites have maintained susceptibility for cleavage by the serine protease, C1s, and provides evidence for a complex relationship between the complement system and the P. falciparum cytoadhesion virulence determinant