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. 2015 May 26;10(5):e0127500.
doi: 10.1371/journal.pone.0127500. eCollection 2015.

Characterization of Plasmodium vivax Early Transcribed Membrane Protein 11.2 and Exported Protein 1

Affiliations

Characterization of Plasmodium vivax Early Transcribed Membrane Protein 11.2 and Exported Protein 1

Yang Cheng et al. PLoS One. .

Abstract

In Plasmodium, the membrane of intracellular parasites is initially formed during invasion as an invagination of the red blood cell surface, which forms a barrier between the parasite and infected red blood cells in asexual blood stage parasites. The membrane proteins of intracellular parasites of Plasmodium species have been identified such as early-transcribed membrane proteins (ETRAMPs) and exported proteins (EXPs). However, there is little or no information regarding the intracellular parasite membrane in Plasmodium vivax. In the present study, recombinant PvETRAMP11.2 (PVX_003565) and PvEXP1 (PVX_091700) were expressed and evaluated antigenicity tests using sera from P. vivax-infected patients. A large proportion of infected individuals presented with IgG antibody responses against PvETRAMP11.2 (76.8%) and PvEXP1 (69.6%). Both of the recombinant proteins elicited high antibody titers capable of recognizing parasites of vivax malaria patients. PvETRAMP11.2 partially co-localized with PvEXP1 on the intracellular membranes of immature schizont. Moreover, they were also detected at the apical organelles of newly formed merozoites of mature schizont. We first proposed that these proteins might be synthesized in the preceding schizont stage, localized on the parasite membranes and apical organelles of infected erythrocytes, and induced high IgG antibody responses in patients.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Schematic structures, expression and purification of PvETRAMP11.2 and PvEXP1.
A. The PvETRAMP11.2 protein comprises 110 amino acids (AAs), with a calculated molecular mass of 12 kDa. Indicated are the signal peptide (AA positions 1–22) and two transmembrane domains (AAs 7–29 and 52–74, respectively). Truncated PvETRAMP11.2 (AAs 23–110) was constructed for expression. B. The PvEXP1 protein comprises 148 AAs, with a calculated molecular mass of 15 kDa. Indicated are the signal peptide (AAs 1–22) and two transmembrane domains (AAs 4–22 and 77–99, respectively). Truncated PvEXP1 (AAs 23–148) was constructed for expression. Purified recombinant PvETRAMP11.2 (C) and PvEXP1 (D) were resolved by 12% SDS-PAGE. M, protein marker; T, total translation mix; S, supernatant; P, pellet; Ft: flow through; E1, elution fraction 1; E2, elution fraction 2; kDa, kilo Dalton; arrow heads, purified proteins.
Fig 2
Fig 2. Western blot of recombinant PvETRAMP11.2 and PvEXP1 proteins, and P. vivax schizont extracts with specific antibodies.
A. Western blot analysis recombinant proteins using anti-His antibody (His), rabbit immune sera (R), mouse immune sera (M), and vivax-infected human sera (H). a, PvETRAMP11.2; b, PvEXP1. Arrowheads indicate target bands specific to each recombinant protein. B. Anti-PvETRAMP11.2 (lane 2) and anti-PvEXP1 (lane 4), antibodies reacted with P. vivax schizont extracts, however, were not reacted with uninfected erythrocytes (lanes 1 and 3). Arrowheads indicate specific bands to each antibody.
Fig 3
Fig 3. Total IgG against recombinant PvETRAMP11.2 and PvEXP1 proteins using protein microarrays.
Immunoreactivity against each antigen with the sera of malaria patients (Patients) and healthy individual samples (Healthy) from Korea was determined. Each antigen was probed with the sera of 56 malaria patients and 40 healthy individuals. The P values were calculated using Mann-Whitney U-tests. The bar indicates the mean ± standard deviation. There were significant differences in the total prevalence of anti-PvETRAMP11.2, and anti-PvEXP1 IgGs between vivax patients and healthy individuals (p < 0.0001).
Fig 4
Fig 4. IgG responses against recombinant PvETRAMP11.2 and PvEXP1 proteins in immunized mice.
Groups of three PvETRAMP11.2 (Black bar) and PvEXP1 (White bar) mice were immunized with the respective protein constructs, and titers of total IgG were evaluated by ELISA after three immunizations of PvETRAMP11.2 or PvEXP1, respectively. The total IgG titers against PvETRAMP11.2, and PvEXP1 proteins were significantly higher than those of pre-immune mice (p < 0.0001). Results are expressed as means ± SD.
Fig 5
Fig 5. Subcellular localization of PvETRAMP11.2 and PvEXP1 proteins in P. vivax blood-stage parasites.
The parasites were labeled with antisera against immune serum samples: PvETRAMP11.2 and PvMSP1 in ring stage (A), double-labeled with PvETRAMP11.2 and PvEXP1 in ring stage (B) and immature schizont stage (C). Parasites were double-labeled with antisera against PvETRAMP11.2 and Pv12 (D), PvETRAMP11.2 and PvDBP (E) in mature schizont stage, PvETRAMP11.2 and PvEXP1 (F) and PvEXP1 and PvRhopH2 (G), in mature schizont stage. Nuclei were visualized with DAPI in merged images. Scale bar represents 5 μm.
Fig 6
Fig 6. Protein-protein interactions between PvETRAMP11.2 and PvEXP1 were analyzed by in situ proximity ligation assay (PLA).
PvETRAMP11.2 and PvEXP1 interactions were visualized by probing mouse and rabbit immune sera, and staining parasites with probes termed anti-mouse MINUS and anti-rabbit PLUS (A). Hybridization probes were labeled with Texas Red (Red), and nuclei were stained with DAPI (blue). Analysis of different localized proteins, PvETRAMP11.2 and PvMSP1 (B), and the rhoptry organelle localized proteins, PvRALP1 and PvRON2 (C) by PLA assay. Scale bar represents 5 μm.

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