Plasmodium vivax Tryptophan Rich Antigen PvTRAg36.6 Interacts with PvETRAMP and PvTRAg56.6 Interacts with PvMSP7 during Erythrocytic Stages of the Parasite

Abstract

Plasmodium vivax is most wide spread and a neglected malaria parasite. There is a lack of information on parasite biology of this species. Genome of this parasite encodes for the largest number of tryptophan-rich proteins belonging to 'Pv-fam-a' family and some of them are potential drug/vaccine targets but their functional role(s) largely remains unexplored. Using bacterial and yeast two hybrid systems, we have identified the interacting partners for two of the P. vivax tryptophan-rich antigens called PvTRAg36.6 and PvTRAg56.2. The PvTRAg36.6 interacts with early transcribed membrane protein (ETRAMP) of P.vivax. It is apically localized in merozoites but in early stages it is seen in parasite periphery suggesting its likely involvement in parasitophorous vacuole membrane (PVM) development or maintenance. On the other hand, PvTRAg56.2 interacts with P.vivax merozoite surface protein7 (PvMSP7) and is localized on merozoite surface. Co-localization of PvTRAg56.2 with PvMSP1 and its molecular interaction with PvMSP7 probably suggest that, PvTRAg56.2 is part of MSP-complex, and might assist or stabilize the protein complex at the merozoite surface. In conclusion, the PvTRAg proteins have different sub cellular localizations and specific associated functions during intra-erythrocytic developmental cycle.

Fig 1. Expression of PvTRAg-GFP fusion proteins in transgenic P.falciparum.

Parasite lysates from wild type 3D7, transgenics 3D7_PvTRAg36.6-GFP and 3D7_PvTRAg56.2-GFP were subjected to western blot analyses using monoclonal anti-GFP antibody (upper panels) and anti-Bip antibody as control (lower panels). A: lane 1; wild type 3D7; Lane 2; 3D7_PvTRAg36.6-GFP, B: lane 1; wild type 3D7; lane 2; 3D7_PvTRAg56.2-GFP, Molecular weights of GFP fusion protein and parasite Bip protein are indicated. M shows the protein marker.

Fig 2. Sub cellular localization of PvTRAg36.6 in P.vivax natural infections.

Immunofluorescence images of P.vivax infected red cells. Parasites were labeled with anti- PvTRAg36.6 (green) antibody and DAPI for nuclear staining (blue). Fluorescence pattern observed in ring (R, double infection), trophozoite (T), Schizont (S) stages and in free merozoites (M) are shown. Overlay shows the images merged with bright field.

Fig 3. Co-localization studies of PvTRAg36.6 in P.vivax.

Co-localization images of PvTRAg36.6 with apicoplast, rhoptry and micronemal markers in P.vivax natural infections. (A) Fluorescence pattern observed after co-immuno staining of P.vivax parasite with anti-PvTRAg36.6 (green) and anti-PfClpP (red) recognizing apicoplast in schizont (A, upper panel) as well as in free merozites (A, lower panel). (B) Co-immunostaining of anti-PvTRAg36.6 (green) with anti-PvRII (red) recognizing microneme in a schizont, and (C) Co-immunostaining of anti-PvTRAg36.6 (green) with anti-PvAARP (red) recognizing rhoptry neck in a schizont. The parasite nuclei were stained with DAPI (blue). Overlay shows the images merged with bright field.

Fig 4. Sub cellular localization of PvTRAg56.2 in P.vivax natural infections.

Localization images of PvTRAg56.2 by immuno and co-immuno staining in P.vivax natural infections. (A) Immunofluorescence pattern observed in P.vivax schizont when labeled with anti-PvTRAg56.2 (green). (B) Co-immunostaining of P.vivax schizont with anti-PvTRAg56.2 (green) and PvMSP-1(red). Parasite nuclei were stained with DAPI (blue).

Fig 5. Sub cellular localization of PvTRAg36.6 in P.falciparum transgenic parasites expressing GFP fusion protein.

GFP fluorescence images showing localization of PvTRAg36.6-GFP in trophozoite stages of transgenic parasite line 3D7_ PvTRAg36.6-GFP, B. Images of co-immunostaining between anti-GFP antibody (green) and anti-SBP1 antibody (red). Parasite nuclei were labeled with DAPI (blue). Overlay shows images merged with bright field.

Fig 6. Sub cellular localization of PvTRAg56.2 in P.falciparum transgenic parasites expressing GFP fusion protein.

Images of co-immunostaining between anti-GFP (green) with (A) anti-SERA5 (red) or (B) anti-PfMSP-1 (red) or (C) anti-Spectrin (red) in transgenic parasite line 3D7_ PvTRAg56.2-GFP. Parasite nuclei were labeled with DAPI (blue). Overlay with bright field images are shown.