Abstract
More than one-third of the world’s population is exposed to Plasmodium vivax malaria, mainly in Asia1. P. vivax preferentially invades reticulocytes (immature red blood cells)2,3,4. Previous work has identified 11 parasite proteins involved in reticulocyte invasion, including erythrocyte binding protein 2 (ref. 5) and the reticulocyte-binding proteins (PvRBPs)6,7,8,9,10. PvRBP2b binds to the transferrin receptor CD71 (ref. 11), which is selectively expressed on immature reticulocytes12. Here, we identified CD98 heavy chain (CD98), a heteromeric amino acid transporter from the SLC3 family (also known as SLCA2), as a reticulocyte-specific receptor for the PvRBP2a parasite ligand using mass spectrometry, flow cytometry, biochemical and parasite invasion assays. We characterized the expression level of CD98 at the surface of immature reticulocytes (CD71+) and identified an interaction between CD98 and PvRBP2a expressed at the merozoite surface. Our results identify CD98 as an additional host membrane protein, besides CD71, that is directly associated with P. vivax reticulocyte tropism. These findings highlight the potential of using PvRBP2a as a vaccine target against P. vivax malaria.
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Source data are provided with this paper. All other data are available from the corresponding authors upon reasonable request.
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Acknowledgements
We are indebted to C. Chu, R. McGready and the staff of the Mae Sot Malaria Clinic and the clinics associated with SMRU (Tak Province, Thailand) and the patients attending these clinics. We thank M. Mauduit for help at the beginning of the project. We thank the SIgN flow cytometry platform (supported by a grant from the National Research Foundation, Immunomonitoring Service Platform ISP) (NRF2017_SISFP09)). B.R. and B.M. were funded by the Singapore National Medical Research Council (NMRC/CBRG/0047/2013). B.M. was also funded by the Agency for Science, Technology and Research (A*STAR, Singapore) Young Investigator Grant (BMRC YIG grant no: 13/1/16/YA/009), core funds to SIgN from A*STAR, NUHS Start-up grant (NUHSRO/2018/006/SU/01) and MOE Tier 1 (NUHSRO/2018/094/T1/SEED-NOV/04). L.R. was supported by a Singapore National Medical Research Council IRG grant (NMRC/OFIRG/0065/2018), by a Singapore Immunology Network core research grant and by the Horizontal Programme on Infectious Diseases under A*STAR. SMRU is supported by The Wellcome Trust of Great Britain as part of the Oxford Tropical Medicine Research Programme of Wellcome Trust–Mahidol University. R.C. acknowledges funding support through the following grants: T1MOE1702 (MOE Tier 1 Grant through the Singapore University of Technology & Design) and RGUOO180301 (Marsden Grant Sub-award through the University of Otago). W.-H.T. was funded by the Australian Research Council Future Fellowship. Duke-NUS Medical School efforts were supported by Singapore’s Health and Biomedical Sciences (HBMS) Industry Alignment Fund Pre-Positioning (IAF-PP) grant H18/01/a0/018, administered by A*STAR.
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Contributions
B.M. and B.R. carried out the phenotyping characterization of the erythrocytes and the antibody validations for the P. vivax invasion assays. G.C., S.W.H., R.S., V.K. and A.S.M.O. developed the P. vivax library and performed the erythrocyte binding assays. T.T.T.C., A.S. and R.C. performed and analysed the MS data. J.G. and W.-H.T. carried out construct design and protein purification for the PvRBP2a recombinant proteins and the anti-PvRBP2a monoclonal antibodies. Y.C. developed the anti-Duffy antibodies. J.K.Y.C., Y.F. and F.N. were in charge of the management of clinical data. A.E.S., J. Lin, J. Lescar, G.C. and L.F.P.N. managed the biochemistry aspects of the project. S.M.-S. managed the protein modelling of the interaction between CD98 and PvRPB2a. W.N., M.Z.T. and A.-M.C. managed the Octet experiments for CD98 and PvRPB2a interaction measurements. Overall project management was carried out by B.M., B.R. and L.R. The manuscript was prepared by B.M., G.S., B.R. and L.R.
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Peer review information Nature Microbiology thanks Tuan Tran, Sanjeeva Srivastava and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.
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Extended data
Extended Data Fig. 1 Reticulocyte phenotyping.
Trypsin resistance profile of different markers expressed at the surface of cord blood reticulocytes. The black and yellow histograms represent the level of expression before and after trypsin treatment respectively. The trypsin sensitive proteins are annotated in red and the resistant ones in black. The isotype antibody used as control is represented in grey. We used human cord blood samples pre-enriched with reticulocytes and that thus still harbour some normocytes, hence the double peak observed for some proteins such as CD71 and CD98.
Extended Data Fig. 2 Flow cytometry analysis of adult peripheral blood, cord blood and P. vivax isolates.
a, Flow cytometry profile of CD98 and CD71 expression on thiazole orange (TO) negative, low and high erythrocytes for three different human adult peripheral blood samples (forward scatter (FSC) for x-axis and side scatter (SSC) for y-axis). The TO high subset represents the most immature reticulocyte population compared to low subset. b, Flow cytometry histogram of CD98 expression (right) at the surface of P. vivax rings, early infected reticulocyte forms, gated on Hoechst-positive cells (left), side scatter (SSC) for y-axis. The infected cells positive for Hoechst (a DNA stain) are in blue and the uninfected red blood cells (uRBC), which do not contain DNA, are in red. c, Comparison of delta of geometric mean fluorescence intensity (MFI) between immature reticulocytes from three cord blood samples and three vivax infected patients (ring stage), Values are expressed as mean ± SD, unpaired Student t-test.
Extended Data Fig. 3 Binding assay of erythrocytes to HEK cells expressing P. vivax genes.
a, Schematic description of the development of the library and its use in the erythrocyte binding assay: (1) cloning of P. vivax genes in the pDisplay plasmid, (2) transfection of HEK cells, (3) expression of the protein containing MYC and HA tags at the surface of HEK cells and (5) binding assay with erythrocytes. b, Schematic representation of full-length PvRBP2a and of its recombinant protein fragments used in this study (right). Signal peptide (SP), transmembrane helix (TM) and nucleotide-binding domain (green) are indicated. The ticks indicated for nonsynonymous SNPs with alternate allele frequency >20%. c, Representative images from 3 independent experiments of binding of reticulocytes (CD71 + ) or normocytes (CD71-) loaded with the fluorescent dye CFSE to HEK cells expressing the PvRBP2a 1315-1650 (negative control), Duffy binding protein II (DBPII) fragment or PvRBP2a23-767 in the presence or absence of anti-CD98 antibodies. The scale represents 50 μm. d, The binding of reticulocytes to HEK transfected with the PvRBP2a23–767 fragment was done in octuplicate (eight independent experiments) and was shown to follow a normal distribution as determine by the D’Agostino’s K-squared test, and differed significantly from binding to non-transfected HEK cells, Values are expressed as mean ± SD, Student t-test. e, Binding assays of reticulocytes to HEK cells expressing PvRBP2b481-1530 in the presence or absence of anti-CD98 antibodies. (three independent experiments). Values are expressed as mean ± SD, no significant differences were observed. e, Binding assays of normocytes to HEK cells expressing PvRBP2a23-767 in the presence or absence of anti-CD98 antibodies (three independent experiments). Values are expressed as mean ± SD, no significant differences were observed.
Extended Data Fig. 4 Antigen specificity of anti-PvRBP2a antibodies.
Mouse 1C3 and 3A11 mAbs recognize specifically the PvRBP2a fragment, PvRBP2a23–767, expressed by HEK cells when tested by flow cytometry. As positive control, rabbit anti-myc antibodies were used. Secondary anti-mouse IgG antibodies coupled with eFluor 660 or mouse anti-rabbit immunoglobulin coupled with Alexa 674 were used as negative control.
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Malleret, B., El Sahili, A., Tay, M.Z. et al. Plasmodium vivax binds host CD98hc (SLC3A2) to enter immature red blood cells. Nat Microbiol 6, 991–999 (2021). https://doi.org/10.1038/s41564-021-00939-3
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DOI: https://doi.org/10.1038/s41564-021-00939-3
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