Novel components used for protein export and functionality in Plasmodium falciparum
Creators: name: Charnaud, Sarah Caroline
Publisher: name: Monash University
Year: 2017
Subjects: Uncategorized

description: BACKGROUND: Plasmodium parasites, the causative agents of malaria, amplify in the erythrocytes of their host animals. To thrive the parasites remodel their host cells, to import plasma nutrients and evade the immune system. The most pathogenic human parasite is Plasmodium falciparum (Pf), which causes severe morbidity and mortality due to a surface exposed exported protein, PfEMP1, which allows the infected erythrocytes (IE) to bind endothelial cells and avoid splenic clearance. This can cause severe pathogenesis as IE occlude microvasculature, and when this occurs in the brain can result in coma and death. In order to remodel their host cells the intra-erythrocytic parasites export effector proteins into their host cells. These proteins must first traverse the parasite’s plasma membrane and then the parasitophorous vacuole (PV) space and PV membrane (PVM) which envelop the parasite. Proteins cross the PVM in an unfolded state via a proteinaceous pore termed PTEX. There are five currently known components of PTEX including a Hsp100 chaperone. Hsp100s typically interact with other chaperone components, including Hsp70/Hsp40 pairs that can unfold and refold complex proteins. The aim of my thesis was to identify the co-chaperones and accessory proteins that co-operate with PTEX to facilitate protein export. RESULTS: Interrogation of the genome identified a Pf specific Hsp70 chaperone termed Hsp70-x. Antibodies made to Hsp70-x indicated it was first secreted into the PV and then exported into the IE. Although Hsp70-x lacks a typical export motif, genetic knockdown of PTEX expression greatly reduced Hsp70-x export demonstrating PTEX is its exporter. Microscopy of IE indicated that Hsp70-x co-localised with Hsp40 rich J-dots, and also with PfEMP1 implying that the chaperones aid PfEMP1 to reach the host surface. To further resolve Hsp70-x’s function, its gene was deleted creating a ∆hsp70-x mutant. The mutant, although viable, had a slightly longer cell cycle and proliferated more slowly than the parental line under limiting nutrient conditions. ∆hsp70-x also overexpressed a number of exported and chaperone proteins, perhaps as compensation for the loss of the Hsp70-x chaperone. Some of these included structural proteins and resulted in the IE becoming more rigid. PfEMP1 puncta were reduced during export although surface expression of PfEMP1 ultimately reached normal levels. Despite normal PfEMP1 surface display, ∆hsp70-x parasites were deficient at binding to endothelial cell receptors under flow conditions. To identify other proteins associated with PTEX, radiolabelled pulse chase and immunoprecipitation of PTEX identified PV1, which is a 50 kDa essential protein with no homology suggestive of function. A number of additional binding experiments indicated PV1 associated with PTEX and also with certain exported cargoes suggesting that it may aid the export of cargo proteins through PTEX. CONCLUSIONS: Hsp70-x is present in the PV where it could interact with PTEX to aid protein export, and in the host where it could help refold proteins that have been exported via PTEX in an unfolded state. Parasites lacking Hsp70-x grow more slowly, and the IE are more rigid and poorer at binding endothelial cell receptors. Some of these changes could be in response to altered transcription and translation of various exported proteins. The function of Hsp70-x appears to be diverse probably because it aids in the transport and efficient folding of many exported proteins required for nutrient acquisition and cytoadherance. Inhibition of Hsp70-x could therefore result in parasites with greatly reduced in vivo fitness and hence it is a potential drug target that would reduce parasite virulence. Identification of PV1, a novel member of PTEX, has increased our understanding of the mechanism of protein export, and this could also ultimately open new avenues for therapeutics.