Host Erythrocyte Environment Influences the Localization of Exported Protein 2, an Essential Component of the Plasmodium Translocon

Abstract

Malaria parasites replicating inside red blood cells (RBCs) export a large subset of proteins into the erythrocyte cytoplasm to facilitate parasite growth and survival. PTEX, the parasite-encoded translocon, mediates protein transport across the parasitophorous vacuolar membrane (PVM) in Plasmodium falciparum-infected erythrocytes. Proteins exported into the erythrocyte cytoplasm have been localized to membranous structures, such as Maurer's clefts, small vesicles, and a tubovesicular network. Comparable studies of protein trafficking in Plasmodium vivax-infected reticulocytes are limited. With Plasmodium yoelii-infected reticulocytes, we identified exported protein 2 (Exp2) in a proteomic screen of proteins putatively transported across the PVM. Immunofluorescence studies showed that P. yoelii Exp2 (PyExp2) was primarily localized to the PVM. Unexpectedly, PyExp2 was also associated with distinct, membrane-bound vesicles in the reticulocyte cytoplasm. This is in contrast to P. falciparum in mature RBCs, where P. falciparum Exp2 (PfExp2) is exclusively localized to the PVM. Two P. yoelii-exported proteins, PY04481 (encoded by a pyst-a gene) and PY06203 (PypAg-1), partially colocalized with these PyExp2-positive vesicles. Further analysis revealed that with P. yoelii, Plasmodium berghei, and P. falciparum, cytoplasmic Exp2-positive vesicles were primarily observed in CD71؉ reticulocytes versus mature RBCs. In transgenic P. yoelii 17X parasites, the association of hemagglutinintagged PyExp2 with the PVM and cytoplasmic vesicles was retained, but the pyexp2 gene was refractory to deletion. These data suggest that the localization of Exp2 in mouse and human RBCs can be influenced by the host cell environment. Exp2 may function at multiple points in the pathway by which parasites traffic proteins into and through the reticulocyte cytoplasm.

Plasmodium species: master renovators of their host cells

“…Proteins that are resident in the parasitophorous vacuole and exported proteins seem to be excluded from these structures in P. falciparum 15,56,61 , but evidence suggests that related structures in P. berghei may contain components of the PTEX 60 . EXP2 has also been observed in membranous vesicles in reticulocytes that are infected with P. falciparum and the rodent parasites, Plasmodium yoelii and P. berghei 62 . These vesicles are usually absent from infected mature erythrocytes, which indicates that the localization of EXP2 may be determined by the environment of the host cell 62 .…”

“…EXP2 has also been observed in membranous vesicles in reticulocytes that are infected with P. falciparum and the rodent parasites, Plasmodium yoelii and P. berghei 62 . These vesicles are usually absent from infected mature erythrocytes, which indicates that the localization of EXP2 may be determined by the environment of the host cell 62 . Whether these vesicles represent membranous structures that have budded from the PVM and have a role in protein export remains to be determined.…”

In Silico Characterization of Plasmodium falciparum and P. yoelii Translocon and Exoribonuclease II (RNase II) Identified in the Merozoite Rhoptry Proteome

“…However, there is no evidence to suggest that P. yoelii contains Maurer's clefts. Intracellular membrane networks similar to the Maurer's cleft as well as intracytoplasmic vesicles have been described in the cytoplasm of rodent Plasmodium infected cells [25]. Experiments to isolate intracytoplasmic membranes and vesicles for investigating protein distribution will identify differences, if they exist, in the distribution of proteins involved in intracellular trafficking across the PVM.…”

“…Experiments to isolate intracytoplasmic membranes and vesicles for investigating protein distribution will identify differences, if they exist, in the distribution of proteins involved in intracellular trafficking across the PVM. The differences in the localization of P. yoelii EXP-2 within cytoplasmic vesicles and the PVM, suggests that proteins participating in the export of proteins across the PVM may have different distribution with the host cell cytoplasm of different Plasmodium species [25].…”

“…The RNAse II and translocon genes were amplified by polymerase chain reaction (PCR) from Plasmodium genomic DNA using primers designed from DNA sequences obtained from PlasmoDB [18] for use in recombinant DNA construction. Due to the differences observed in localization of proteins involved in parasitophorous vacuole membrane (PVM) transport and exported proteins [23][24][25], gene mining approaches and bioinformatics were employed to examine data present in PlasmoDB [18], regarding gene and protein annotations for P. falciparum genes PF3D7_0906000 and PF3D7_1436300. Predicted structural characteristics, functional domains and motifs from annotated data within the PlasmoDB [18] database were compared, in an attempt to develop testable experimental approaches that can identify specific conserved features that will expand our understanding of asexual stage host cell invasion and parasitism, identify new vaccine and drug targets and new diagnostic biomarkers.…”