Abstract:
Plasmodium falciparum is a unicellular protozoan parasite that causes malaria in humans. The parasite is passed to humans through mosquito bites and migrates to the liver before it infects host erythrocytes. It is at the erythrocytic stage of development that the parasite causes malaria pathology. Malaria is characterized by the modification of host erythrocytes making them cytoadherent. This is as a result of formation of protein complexes (knobs) on the surface of the erythrocyte. The knobs that develop on the surface of the erythrocyte are constituted by proteins of host origin as well as some proteins that the parasite ‘exports’ to the host cell surface. Nearly 550 parasite proteins are thought to be exported to the infected erythrocyte. Amongst the exported proteins is P. falciparum heat shock protein 70-x (PfHsp70-x). Hsp70 proteins are known to maintain protein homeostasis. Thus, the export of PfHsp70-x may be important for maintaining protein homeostasis in the host cell. PfHsp70-x is not essential for parasite survival although is implicated in the development of parasite virulence. This is possibly through its role in facilitating the trafficking of parasite proteins to the erythrocyte as well as supporting the formation of protein complexes that constitute the knobs that develop on the surface of the infected erythrocyte. The main objective of the current study was to investigate protein interaction partners of PfHsp70-x. It is generally believed that PfHsp70-x interacts with various proteins of human and parasite origin. Potential candidate interactors include its protein substrates, Hsp70 co-chaperones such as Hsp40 members, and human Hsp70-Hsp90 organizing protein (hHop). The establishment of the PfHsp70-x interactome would highlight the possible role of PfHsp70-x in the development of malaria pathogenicity. Based on bioinformatics analysis, PfHsp70-x was predicted to interact with some exported P. falciparum Hsp40s, hHop and human Hsp90 (hHsp90). Recombinant forms of PfHsp70-x (full length and a truncated form that lacks the C-terminal EEVN motif implicated in co-chaperone binding) were expressed in E. coli BL21 Star (DE3) cells. Recombinant hHop and hHsp70 were expressed in E. coli JM109 (DE3) cells. The proteins were successfully purified using nickel affinity chromatography. Co-affinity chromatography using recombinant PfHsp70-x and immuno-affinity chromatography using PfHsp70-x specific antibody did not confirm the direct interaction of PfHsp70-x with human Hop. However, the direct interaction of hHop and PfHsp70-x has previously been validated in vitro and the current bioinformatics data support
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the existence of such a complex. PfHsp70-x was not stable in the cell lysate that was prepared and this could explain why its interaction with hHop could not be ascertained. However, taken together the evidence from a previous independent study, and the predicted interaction of PfHsp70-x with human chaperones suggests cooperation of chaperone systems which possibly facilitates the folding and function of parasite proteins that are exported to the infected erythrocyte.
