During invasion of erythrocytes, malaria parasites produce and encase themselves within a parasitophorous vacuole and export proteins into the host cell to remodel it. Protein export allows parasites to develop and evade immune responses and is directly linked to virulence that causes death of the host. Export of most proteins requires proteolytic cleavage of a conserved motif (RxLxE/Q/D) in the N-terminus of exported proteins called the Plasmodium export element (PEXEL). An aspartic protease, Plasmepsin V, is responsible for cleavage of the PEXEL motif, which enables translocation of proteins across the vacuole membrane. The PEXEL motif and Plasmepsin V are conserved in all Plasmodium species and attempts to genetically disrupt the plasmespsin V gene in P. falciparum and P. berghei have been unsuccessful, suggesting it is essential for parasite survival in the host. P. berghei is known to export proteins to the infected erythrocyte and possibly the hepatocyte; however, the role of Plasmepsin V in this species is unknown.
We have generated transgenic P. berghei expressing Plasmepsin V fused to GFP and show it is expressed during erythrocytic infection. Affinity purification of P. berghei Plasmepsin V and assessment of its activity revealed it cleaves the PEXEL motif with the same specificity as the P. falciparum enzyme and could be inhibited with a small molecule Plasmepsin V inhibitor we developed.
We aim to investigate whether Plasmepsin V also plays a role in the liver-stage of infection using the small molecule inhibitor as well as the flp-FRT recombinase system to conditionally delete the gene in mosquito-stage parasites. Finally, we have generated parasites expressing various PEXEL reporter proteins to assess export during hepatocyte infection. Results towards these aims will also be presented.