Characterization of two "Plasmodium falciparum" proteins, MAHRP1 and MAHRP2, involved in host cell refurbishment

Malaria is one of the leading causes of morbidity and death in the world. Responsible for the most virulent form of the disease is the Apicomplexan parasite Plasmodium falciparum transmitted by the female Anopheline mosquito. Today, no vaccine is commercially available. Eradication of malaria failed due to the evolution of drug resistance in the parasite and insecticide resistance in its mosquito vector but is since 2007 again on the agenda of health officials. The understanding of the biology of P. falciparum is limited impeding the identification of new intervention targets.
Disease and death is triggered by blood stages where the parasite undergoes multiple rounds of replication. During this part of the life cycle P. falciparum lives surrounded by a parasitophorous vacuole in the terminally differentiated red blood cell which is metabolically highly reduced lacking compartments, a nucleus, and a protein trafficking machinery. Nutrient supply is also limited. To survive in such an environment the parasite needs to refurbish its host cell inducing remarkable modifications such as the formation of membranous structures termed Maurer’s clefts in the cytosol of the erythrocyte, and knobs on the surface of the red blood cell. The refurbishment processes are initiated by the export of parasite derived proteins beyond the confines of its own plasma membrane, across the parasitophorous vacuolar membrane into the cytosol of the erythrocyte or to the erythrocyte membrane. The major virulence factor PfEMP1 is exported to knobs where it binds as surface exposed molecule to endothelial receptors thereby mediating cytoadherence and sequestration of mature-stage infected erythrocytes in blood capillaries evading clearance by the spleen. This is the key process accounting for clinical symptoms of malaria such as organ failure or cerebral malaria.
Not much is known on how parasite proteins are secreted along such a complex route crossing several membranes. Proteins are thought to be classically secreted into the parasitophorous vacuole. A Plasmodium export element (PEXEL) has been identified in most exported proteins which is recognized by a translocon in
the parasitophorous vacuolar membrane enabling secretion into the erythrocyte cytosol. As an exception to the rule, there are a number of proteins described being exported but lacking such a motif.
In this thesis, we focus on the processes involved in host cell refurbishment as well as on the export mechanism of two of those PEXEL-negative proteins. The aim was to characterize two proteins termed Membrane Associated Histidine-Rich Proteins 1 and 2 (MAHRP1 and MAHRP2) which are exclusively transcribed early during blood stage development when such refurbishment occurs. Both proteins are similar in structure carrying centrally a predicted transmembrane domain. The C-terminal domain of MAHRP1 comprises histidine-rich DHGH repeats while the N-terminal domain of MAHRP2 is histidine-rich. MAHRP1 has previously been shown to localize to Maurer’s clefts whereas nothing was known about MAHRP2.
We generated parasite lines in which the mahrp1 gene was disrupted to investigate possible functions of MAHRP1 in these knock out parasites. In erythrocytes infected with MAHRP1-deficient parasites the major virulence factor PfEMP1 was not exported anymore to the surface of the erythrocyte. It was still produced but was trapped within the confines of the parasite. This resulted in a reduced ability of the infected erythrocytes to bind to the endothelial receptor CD36. The phenotype could be restored by the complementation of the MAHRP1-deficient parasites with episomal expression of the gene. These findings indicate an essential function for MAHRP1 and Maurer’s clefts in the export of the major virulence factor to the surface of the red blood cell.
By immunofluorescence assays and electron microscopy as well as through transfection technology we could show that MAHRP2 is exported to recently described new structures in the infected erythrocyte, called tethers. MAHRP2 is the first and only protein so far specifically localizing to these tubular structures thought to attach Maurer’s clefts to the erythrocyte membrane. Life cell imaging of infected erythrocytes expressing MAHRP2-GFP revealed both mobile and fixed populations of these structures which allowed enrichment by differential centrifugation. Solubilization studies showed that MAHRP2, although having a predicted transmembrane domain, only peripherally associates with membranes
whereas MAHRP1 represents an integral membrane protein. We failed to delete the mahrp2 gene in several attempts indicating an essential function for MAHRP2 in parasite survival. Tagging the mahrp2 gene with the FKBP destabilizing domain, however, resulted in a nearly complete loss of MAHRP2 protein, although with no obvious altered phenotype.
Through pull down experiments and mass spectrometric analyses of the enriched tether fraction obtained by differential centrifugation, we found several potential protein interaction partners of MAHRP2, which subsequently were GFP- or HA-tagged and transfected into parasites for further analyses.
Both MAHRP1 and MAHRP2 are exported despite lacking a classical signal sequence or a PEXEL motif. Trafficking of MAHRP1 and MAHRP2 was ER dependent. Sequences required for export of MAHRP2 were determined using transfectants expressing truncated MAHRP2 fragments. Interestingly, sequence requirements were different from MAHRP1 suggesting alternative export mechanisms. The first 15 amino acids of MAHRP2, the histidine-rich N-terminal region, and the predicted central hydrophobic region were necessary for correct trafficking. Although MAHRP2 is not an integral membrane protein, membrane association seemed to be absolutely essential for the export of MAHRP2.
A better understanding of the function of tethers and Maurer’s clefts which are organelles unique to the most virulent malaria parasite P. falciparum as well as delineation of the export mechanisms of proteins destined for these structures should lead to the development of novel intervention strategies.