Background: Malaria is a devastating parasitic disease that kills 400,000 people per year. Resistance to current antimalarials is rising, and therefore, it is an urgent need to better understand the biological functions of the essential blood-stage genes in Plasmodium falciparum, as these may serve as future drug targets. P. falciparum RhopH complex is essential for parasite survival and contains 3 proteins that localise to the host red blood cell (RBC) membrane that are involved in importing nutrients from the serum to support the rapid growth of the parasite. Currently, the mechanism by which the complex moves from the parasite to the RBC surface is unknown and is the focus of this study.
Methods: To determine the role of PTEX in trafficking RhopH complex, one of the PTEX components was conditionally knocked down using PfHSP101-HAglmS parasite line and the trafficking of the RhopH protein components was determined using immunofluorescence (IFA) techniques. Sequential solubilisation of parasite-infected RBCs was used to determine the localisation of the RhopH complex at different lifecycle stages. In parallel, split GFP technology was used to image RhopH components by fusing one component of GFP to a RhopH component, and the other component to a protein with a known localisation. When both components were in close proximity, an increase in fluorescence was used to infer localisation.
Results: IFA results showed that RhopH proteins reach the RBC surface after the knockdown of an essential PTEX component. Our results suggest that RhopH protein complex traffics to the RBC membrane via a PTEX-independent manner. Preliminary sequential solubilisation studies identify that RhopH components reside on the cytosolic side of the parasite vacuole membrane, and further investigations are being done to confirm this. A stable parasite line containing RhopH components tagged with GFP1-10 has been created and GFP11 has been introduced into proteins known to localise in the parasite vacuole and exported to the RBC surface.
Conclusion: Using molecular and biochemical techniques, these studies will reveal how RhopH components traffic from the parasite to the RBC surface and will be an important tool for discovering how malaria parasites import nutrients into the host RBC.