The role of PHIST proteins in cytoadherence of P.falciparum

Plasmodium falciparum, the causative agent of malaria, is responsible for over half a million deaths each year and approximately 50% of the world population lives in malaria endemic areas. Despite strategies to reduce the burden of infection like transmission control and development of drugs and vaccines, malaria remains a major public health concern.
A characteristic of P. falciparum infected red blood cells (iRBC) is the ability to avoid splenic clearance by adhering to the vascular endothelium. This pathologic feature is a major contributor to the severity of malaria tropica and as a consequence of the cytoadherence of iRBCs, a high number of parasites are sequestered to different tissues leading to vascular occlusion and inflammation. The major ligand for this cytoadhesion is the P. falciparum erythrocyte membrane protein 1 (PfEMP1), anchored in the erythrocyte membrane in knob structures. The semi-conserved intracellular acidic terminal segment (ATS) domain anchors PfEMP1 to the host cell, whereas the highly variable ectodomain is responsible for endothelial receptor binding of iRBCs. Recently, the ATS domain of PfEMP1 was found to be a conserved protein interaction epitope and was shown to interact in vitro with the PHIST domain of PFI1780w, a member of the Plasmodium helical interspersed sub-telomeric (PHIST) protein family. The initial identification of this large gene family counted 72 paralogs in P. falciparum, which are organized into three subgroups (PHISTa, PHISTb, PHISTc). All PHIST proteins contain a conserved domain of approximately 150 amino acids, predicted to consist of four consecutive alpha helices. It is proposed that PHIST domains facilitate protein interactions and that the semi-conserved ATS epitope may be involved in the parasite`s cytoadherence.
To date, little is known about the role of PHIST proteins but recent data indicate that they might be implicated in knob formation, in altered host cell rigidity, in transport of PfEMP1 and in adhesion of iRBCs in the brain microvasculature. Moreover, members of the PHIST family were found to localize to the iRBC periphery, to bind to cytoskeletal components of the host cell, and were found in detergent-resistant membrane fractions indicating an important role of PHIST proteins in host cell refurbishment.
The aim of this thesis was to gain insight into the functional role of a subset of exported PHIST proteins with a focus on PFE1605w, a protein of the PHISTb subclass which showed significant higher binding affinity to PfEMP1 than PFI1780w. By immunofluorescence and immunoelectron microscopy we were able to show that PFE1605w is exported to the RBC membrane, co-migrates with PfEMP1 and localizes to knobs. NMR and fluorescence polarization experiments revealed that its PHIST domain binds directly to the C-terminus of the ATS. Polarization experiments using PFE1605w and a set of ATS domains from different PfEMP1 molecules showed substantial variation in affinity across the different ATS domains, suggesting that different PHIST proteins might have been optimized for interacting with different PfEMP1 variants. Moreover, in collaboration we resolved the first crystallographic structure of a PHIST domain and derived a partial model of the PHIST-PfEMP1 interaction from nuclear magnetic resonance measures.
Inducible down regulation of PFE1605w levels using the FKBP destabilisation domain but also controlled tethering at Maurer’s clefts with the knocksideways technique resulted in absence of PFE1605w in knobs and led to strongly reduced adhesion properties of iRBC to the endothelial receptor CD36. To assess the specific selection of a PHIST protein for a particular PfEMP1 molecule, we selected iRBCs through binding to different host receptors thus selectively switching to different PfEMP1 molecules. Interestingly, adhesion to other endothelial receptors was less affected or even unaltered by PFE1605w depletion, suggesting that PFE1605w is optimized for a particular subset of PfEMP1 molecules. Moreover, absence of PFE1605w in knobs did not ablate PfEMP1 surface exposure, thus suggesting no role of PFE1605w in PfEMP1 transport.
Co-immunoprecipitation (Co-IP) assays with two constructs which covered only the C-terminal ATS fragment of each of the two main subtypes of PfEMP1 molecules but lacked a TM domain allowed the determination of any in vivo interaction of PFE1605w with both ATS-C fragments. In a next step, Co-IP experiments with the full-length PFE1605w-HA fusion protein revealed a small number of host integral membrane proteins and components of the erythrocyte cytoskeleton as putative protein interaction partners of PFE1605w. These findings allowed us to perform reverse Co-IP experiments with specific antibodies against several of the detected host cell proteins. Reverse Co-IP experiments with antibodies against band 4.2 identified other components of the band 3 complex, including band 3, band 4.2 and α- and β-chains of spectrin, and ankyrin but no other P. falciparum protein except PFE1605w, clearly suggesting that PFE1605w interacts with one or several components of the band 3 complex. From this, it would be possible to map the exact interaction epitope where PFE1605w is interacting with the band 3 complex.
On a side-line of this project we investigated the var gene expression and binding phenotypes of 3D7 parasites selected to bind to ICAM-1 and showed that ICAM-1 binding selects for parasites expressing PFL0020w and PF07_0050, both group B PfEMP1 molecules. With a single PfEMP1 expressing parasite population we were able to show that PFL0020w binds recombinant ICAM-1 through the DBLβ domain. Furthermore, a dual binding affinity of PFL0020w to different endothelial receptors was detected.
In summary, in this thesis we show for the first time that a member of the PHIST protein family exercises its functional role in knobs and interacts both with key molecules of the cytoadherence complex and the host cytoskeleton. We therefore propose that the functional role of PFE1605w is to anchor a variety of PfEMP1 molecules to the host cytoskeleton. It remains to be elucidated how other PHIST proteins and other key molecules of the cytoadherence complex further contribute to anchoring of PfEMP1 within the knob structure. These results clearly demonstrate the important role of the expanded PHIST protein family in P. falciparum and suggest avenues for innovative interactions.