Identification and characterization of a novel Plasmodium falciparum merozoite apical protein involved in erythrocyte binding and invasion - PubMed (original) (raw)
Identification and characterization of a novel Plasmodium falciparum merozoite apical protein involved in erythrocyte binding and invasion
Thilan Wickramarachchi et al. PLoS One. 2008.
Abstract
Proteins that coat Plasmodium falciparum merozoite surface and those secreted from its apical secretory organelles are considered promising candidates for the vaccine against malaria. In the present study, we have identified an asparagine rich parasite protein (PfAARP; Gene ID PFD1105w), that harbors a predicted signal sequence, a C-terminal transmembrane region and whose transcription and translation patterns are similar to some well characterized merozoite surface/apical proteins. PfAARP was localized to the apical end of the merozoites by GFP-targeting approach using an inducible, schizont-stage expression system, by immunofluorescence assays using anti-PfAARP antibodies. Immuno-electron microsopic studies showed that PfAARP is localized in the apical ends of the rhoptries in the merozoites. RBC binding assays with PfAARP expressed on COS cells surface showed that it binds to RBCs through its N-terminal region with a receptor on the RBC surface that is sensitive to trypsin and neuraminidase treatments. Sequencing of PfAARP from different P. falciparum strains as well as field isolates showed that the N-terminal region is highly conserved. Recombinant protein corresponding to the N-terminal region of PfAARP (PfAARP-N) was produced in its functional form in E. coli. PfAARP-N showed reactivity with immune sera from individuals residing in P. falciparum endemic area. The anti-PfAARP-N rabbit antibodies significantly inhibited parasite invasion in vitro. Our data on localization, functional assays and invasion inhibition, suggest a role of PfAARP in erythrocyte binding and invasion by the merozoite.
Conflict of interest statement
Competing Interests: The authors have declared that no competing interests exist.
Figures
Figure 1. (A) Schematic representation of structure of PfAARP (Gene ID PFD1105w) gene showing location of signal sequence (SS) and trans-membrane (TM) region.
The locations of asparagine rich region and conserved proline repeat region are also marked, respective amino acid positions are also indicated. (B) Amino acid sequence alignment of PfAARP with that of four homologs from P. berghei (PB402266.00.0) P. chabaudi (PC401501.00.0) P. vivax strain SaI-1 (Pv090210) and P. yoelii yoelii strain 17XNL (PY06454). Amino acids that are identical in at least three of five species (>60%) are shown in dark, amino acids that are similar in at least three of five species (>60%) or to those shown in dark, are shaded light grey. Transmembrane region is indicated by solid bar.
Figure 2. Stage specific expression of PfAARP in asexual blood stage parasites.
(A) Relative transcription of PfAARP assessed by real-time-RT-PCR using total RNA extracted from tightly synchronized parasite cultures at early ring (ER), late ring (LR), trophozoite (T), early schizont (ES) and late schizont (LS) stages (8, 16, 30, 40 and 48 h after invasion). Stage specific expression of EBA-175 and Falcipain 2 was analyzed as controls. (B) Northern blot analysis of total RNAs isolated from synchronized parasite cultures at ring (lane 1), trophozoite (lane 2), and schizont (lane 3) stage hybridized with labeled PfAARP probe. Equal loading of RNA in all the wells was confirmed by ethidium bromide staining of rRNAs in the gels (lower panel). (C) Western blots analyses of equal number of highly synchronized parasites at ring (lane 1), trophozoite (lane 2) and schizont (lane 3) with anti-PfAARP antibodies. Anti-HRPII antibodies were used to probe a blot ran in parallel to show equal loading in each wells (lower panel).
Figure 3. Localization of PfAARP to the apical end of the merozoites.
(A) Schematic diagram of pTGFP-AARP plasmid construct containing selectable marker (human DHFR) under calmodulin promoter (5′ CAM), transactivator Tati-3 under MSP-2 promoter (5′ MSP-2) and chimeric gene consisting of secreted GFP (with signal sequence) and PfAARP under the control of Tet-responsive promoter. Expression of fusion gene is induced when ahydrotetracycline (ATc) is removed from the cultures. (B) Fluorescent microscopic images of transgenic parasites at schizont stages showing localization of PfAARP fused to a GFP reporter and expressed in an inducible system using schizont stage specific promoter. The parasite nuclei were stained with DAPI (blue). Enlarged images of selected individual free merozoite are shown in the insets. (C) Immuno-fluorescence assay to localize PfAARP in the schizont/merozoite stage parasites using anti-PfAARP (green) antibodies. The parasite nuclei were stained with DAPI (blue) and slides were visualized by fluorescence microscope. S, schizont and M, free merozoites.
Figure 4. Immunofluorescence assay to localize PfAARP by coimmuno-staining of P. falciparum parasites with anti-PfAARP (green) and anti-MSP-1 (red) antibodies.
The parasite nuclei were stained with DAPI (blue) and slides were visualized by fluorescence microscope. The apical ends of the merozoites have dense structure. MSP-1 staining was found around the merozoites and the PfAARP was localized at the apex of the merozoites. MS, mid schizont; LS, late schizont and M, free merozoites.
Figure 5. Spatial localization of PfAARP by co-immunostaining studies with microneme resident proteins AMA-1 (A) and EBA-175 (B).
P. falciparum parasites were co-immunostained with anti-PfAARP (green) and anti-AMA-1 or anti-EBA-175 (red) antibodies. The parasite nuclei were stained with DAPI (blue). Both the microneme markers and PfAARP showed punctate staining in the schizonts. In the late schizonts and merozoites, AMA-1 was present over the entire surface of the merozoites but is most densely distributed at their apical tip, whereas EBA-175 staining was restricted to the apical ends. Enlarged image of selected individual merozoite is shown in the inset. MS, mid schizont; LS, late schizont and M, free merozoites.
Figure 6. Spatial localization of PfAARP by co-immunostaining studies with rhoptry resident protein Clag3.1.
P. falciparum parasites were coimmuno-stained with anti- PfAARP (green) and anti-Clag3.1 (red) antibodies. The parasite nuclei were stained with DAPI (blue). Clag3.1 staining was present in the rhoptry bulb in the free and invading merozoites (lower panel). PfAARP was localized just above these two rhoptry bulbs towards the apex of the merozoites. Enlarged image of merozoite invading in the host erythrocyte is shown in the inset. LS, late schizont and M, free merozoites.
Figure 7. Localization of PfAARP by immuno-electron microscopy.
Ultra thin sections of P. falciparum parasites at schizont/merozoite stages were labeled with anti-PfAARP antibody and gold labeled secondary antibody. Labeling was observed in the apical end of the rhoptries in merozoite. Scale bar = 250 nm.
Figure 8. (A) Anti-PfAARP antibodies are present in human immune sera from P. falciparum endemic area.
Western blot analysis showing reactivity of recombinant PfAARP-N (lane 1) with human immune sera, PfMSP-119 (lane 2) was kept as a positive control. (B) Coomassie blue stain SDS- poly acryl amide gel ran in parallel.
Figure 9. Erythrocyte binding assay using recombinant PfAARP-N and binding inhibition by anti-PfAARP-N antibodies.
(A) Western blot using monoclonal anti penta histidine antibodies showing detection of recombinant proteins in elutes from the RBC binding assays of protein PfDH60 (negative control; lane 1), PvRII (lane 2) and PfAARP-N (lane 3) using untreated human RBC, and in elutes from similar binding assays of PfAARP-N using human RBCs treated with trypsin (lane 4), neurminidase (lane 5) and chymotrypsin (lane 6). (B) Western blot of elutes from the RBC binding assays with the recombinant PfAARP-N protein pre-incubated in RPMI alone (lane 1) or with antibodies purified from pre-immune sera (100 µg, lane 2) and anti- PfAARP antibodies purified from rabbit immune sera (10, 25, 50 and 100 µg, lane 3–6). Equal amount of recombinant protein was used in each assay.
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