Understanding the mechanisms of protein export in Plasmodium berghei liver infection

© 2018 Dr. Pravin RajasekaranThe malaria parasite exports a large repertoire of proteins into the host erythrocyte during blood stage infection. This essential process of host cell remodelling is largely dependent upon the cleavage of a conserved amino acid motif, termed the Plasmodium Export Element (PEXEL). The aspartyl protease plasmepsin V is the enzyme responsible for this cleavage event and previous attempts at genetic deletion have been unsuccessful. Although years of research have revealed the mechanisms behind protein export in erythrocytic infection, less is known about how the parasite exports proteins during the liver stage of malaria infection. This dissertation identifies the molecular mechanisms the parasite requires to facilitate export into the host hepatocyte. Using the Flp-FRT system to conditionally control the expression of plasmepsin V across the life cycle of P. berghei (rodent malaria), we achieved a complete deletion of the 3’UTR of the plasmepsin V gene, specifically in sporozoites. Analysis of this parasite line revealed a defect in liver stage development as the parasites arrested from 6 h following sporozoite infection in mice. Infected mice did not develop patent blood stage infections. Together this suggests an essential role for plasmepsin V during liver stage malaria infection. In order to understand how the parasite exports proteins during liver stage infection, the importance of the PEXEL motif needed to be addressed. We identified several PEXEL-containing protein candidates and examined their export during liver stages to show that their export is dependent upon the presence of a functional PEXEL motif or plasmepsin V. These important findings may help the further identification of exported proteins. It is ultimately important to also contextualise how parasitic infection may change the dynamics of hepatocyte homeostasis, possibly by the result of exported proteins. Recent studies have shown the parasite’s ability to inhibit host hepatocyte apoptotic mechanisms during infection. We examined this using a knockout of key apoptotic inhibitory regulators (cIAP1/2) and found that their absence in mice caused an increase in resistance to liver stage infection. Chemical anatagonism of these apoptotic regulators replicated this effect, highlighting the potential for new drug targets for liver stage malaria. This thesis explores the underlying mechanisms behind protein export in liver stage Plasmodium berghei infection. The results shed light on what is required for the parasite to export proteins to the hepatocyte, for the first time, confirming the importance of plasmepsin V and the PEXEL motif. These discoveries will lead to potential new drug targets and vaccines candidates, aiding the world’s fight to eradicate the debilitating disease burden of malaria