Reticulocyte-associated protein export machinery and cytoadherence in blood stage malaria parasites

Malaria parasites replicating inside red blood cells export several proteins into the erythrocyte cytosol that modify the host cell to facilitate parasite growth and sequestration. Protein export pathways in Plasmodium falciparum utilize parasite-derived structures in the host cytosol including Maurer's clefts, small vesicles and a tubulovesicular membrane network. Studies on exported proteins, trafficking pathways and cytoadherence in reticulocyte-restricted parasites such as P. vivax are limited. Using the P. yoelii 17X rodent model, we performed a proteomic analysis of infected reticulocyte membrane fraction and identified Plasmodium proteins potentially involved in protein trafficking and cytoadherence including PyExp2 (PY05892), pyst-a (PY04481, PY07631), Py14-3-3 (PY01841), PyEnolase (PY06644), hypothetical protein (PY07648) and a putative adhesin (PY00631). Our initial studies were focused on P. yoelii Exported protein-2 (PyExp2). Immunofluorescence studies showed that PyExp2 was localized to the parasitophorous vacuolar membrane (PVM) but also exported into the host cytoplasm on membrane-bound vesicles budding from the PVM. This is in contrast to P. falciparum where PfExp2 was identified as part of the PTEX translocon exclusively localized to the PVM. Co-localization studies showed that two exported proteins, PY04481 (pyst-a) and PY06203 (PypAg-1), were carried within the PyExp2 positive vesicles indicative of vesicle-mediated transport. PyExp2 positive vesicles were primarily observed in CD71+ reticulocytes but not in mature RBCs infected with P. yoelii, P. berghei or P. falciparum. In transgenic P. yoelii 17X parasites, the association of hemagglutinin-tagged PyExp2 with the PVM and cytosolic vesicles was retained but the pyexp2 gene was refractory to deletion. Our data indicate that in addition to its role as a component of PTEX, Exp2 delineates a distinct vesicular protein trafficking machinery in Plasmodium infected reticulocytes. Preliminary studies showed that the endothelial receptor CD36 bound strongly to 30-32 kDa protein(s) in the P. yoelii 17X infected reticulocyte membrane fraction. Of the Plasmodium proteins identified from the membrane fraction, PY00631, a highly charged conserved protein, bound to CD36. Transgenic P. yoelii 17X parasites expressing a 6xHis-tagged PY00631 was generated to purify the native protein. PY00631 was localized to the host reticulocyte membrane during the schizont stages, strongly supporting a role in the cytoadherence of mature blood stages.