A Novel Erythrocyte Binding Protein of Plasmodium vivax Suggests an Alternate Invasion Pathway into Duffy-Positive Reticulocytes

doi:10.1128/mBio.01261-16

. 2016 Aug 23;7(4):e01261-16.

doi: 10.1128/mBio.01261-16.

A Novel Erythrocyte Binding Protein of Plasmodium vivax Suggests an Alternate Invasion Pathway into Duffy-Positive Reticulocytes

Francis B Ntumngia ¹, Richard Thomson-Luque ², Letícia de Menezes Torres ³, Karthigayan Gunalan ⁴, Luzia H Carvalho ⁵, John H Adams ¹

Affiliations

¹ Center for Global Health and Infectious Diseases Research, College of Public Health, University of South Florida, Tampa, Florida, USA fntumngi@health.usf.edu usfmalaria@gmail.com.
² Center for Global Health and Infectious Diseases Research, College of Public Health, University of South Florida, Tampa, Florida, USA.
³ Center for Global Health and Infectious Diseases Research, College of Public Health, University of South Florida, Tampa, Florida, USA Centro de Pesquisas René Rachou/FIOCRUZ, Belo Horizonte, Minas Gerais, Brazil.
⁴ Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, Maryland, USA.
⁵ Centro de Pesquisas René Rachou/FIOCRUZ, Belo Horizonte, Minas Gerais, Brazil.

PMID: 27555313
PMCID: PMC4999553
DOI: 10.1128/mBio.01261-16

A Novel Erythrocyte Binding Protein of Plasmodium vivax Suggests an Alternate Invasion Pathway into Duffy-Positive Reticulocytes

Francis B Ntumngia et al. mBio. 2016.

. 2016 Aug 23;7(4):e01261-16.

doi: 10.1128/mBio.01261-16.

Authors

Francis B Ntumngia ¹, Richard Thomson-Luque ², Letícia de Menezes Torres ³, Karthigayan Gunalan ⁴, Luzia H Carvalho ⁵, John H Adams ¹

Affiliations

¹ Center for Global Health and Infectious Diseases Research, College of Public Health, University of South Florida, Tampa, Florida, USA fntumngi@health.usf.edu usfmalaria@gmail.com.
² Center for Global Health and Infectious Diseases Research, College of Public Health, University of South Florida, Tampa, Florida, USA.
³ Center for Global Health and Infectious Diseases Research, College of Public Health, University of South Florida, Tampa, Florida, USA Centro de Pesquisas René Rachou/FIOCRUZ, Belo Horizonte, Minas Gerais, Brazil.
⁴ Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, Maryland, USA.
⁵ Centro de Pesquisas René Rachou/FIOCRUZ, Belo Horizonte, Minas Gerais, Brazil.

PMID: 27555313
PMCID: PMC4999553
DOI: 10.1128/mBio.01261-16

Abstract

Erythrocyte invasion by malaria parasites is essential for blood-stage development and an important determinant of host range. In Plasmodium vivax, the interaction between the Duffy binding protein (DBP) and its cognate receptor, the Duffy antigen receptor for chemokines (DARC), on human erythrocytes is central to blood-stage infection. Contrary to this established pathway of invasion, there is growing evidence of P. vivax infections occurring in Duffy blood group-negative individuals, suggesting that the parasite might have gained an alternative pathway to infect this group of individuals. Supporting this concept, a second distinct erythrocyte binding protein (EBP2), representing a new member of the DBP family, was discovered in P. vivax and may be the ligand in an alternate invasion pathway. Our study characterizes this novel ligand and determines its potential role in reticulocyte invasion by P. vivax merozoites. EBP2 binds preferentially to young (CD71(high)) Duffy-positive (Fy(+)) reticulocytes and has minimal binding capacity for Duffy-negative reticulocytes. Importantly, EBP2 is antigenically distinct from DBP and cannot be functionally inhibited by anti-DBP antibodies. Consequently, our results do not support EBP2 as a ligand for invasion of Duffy-negative blood cells, but instead, EBP2 may represent a novel ligand for an alternate invasion pathway of Duffy-positive reticulocytes.

Importance: For decades, P. vivax infections in humans have been defined by a unique requirement for the interaction between the Duffy binding protein ligand of the parasite and the Duffy blood group antigen receptor (DARC). Recent reports of P. vivax infections in Duffy-negative individuals challenge this paradigm and suggest an alternate pathway of infection, potentially using the recently discovered EBP2. However, we demonstrate that EBP2 host cell specificity is more restricted than DBP binding and that EBP2 binds preferentially to Duffy-positive, young reticulocytes. This finding indicates that this DBP paralog does mediate a Duffy-independent pathway of infection.

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Figures

FIG 1

FIG 1

Production and erythrocyte binding properties of recombinant EBP2. (a) Coomassie blue-stained SDS-PAGE gel showing differential mobility of reduced (dithiothreitol-positive [DTT⁺]) and refolded (DTT⁻) recombinant EBP2. Mobility shift is a simple indicator of native conformation of the refolded antigen. (b to d) Recombinant EBP2 (b and c) and recombinant DBPII (d) binding to different red blood cell types and reticulocyte subpopulations, respectively, by flow cytometry. (e) COS7 cell surface-expressed EBP2 and DBPII binding to different Duffy-positive (Fy⁺) and Duffy-negative (Fy⁻) erythrocyte types. (f) COS7 cell surface-expressed EBP2 binding to different reticulocyte subpopulations. Bars show mean percentages of red blood cells with bound antigen and mean numbers of rosettes in 30 microscope fields at a magnification of ×ばつ20 in the flow cytometry and COS7 assays, respectively. Error bars represent ±standard deviations from two independent experiments. All experiments were performed with blood from at least two different donors. DBPII was used as a control antigen.

FIG 2

FIG 2

Immunogenicity and functional analysis of anti-EBP2 antibodies. (a and b) Antisera raised in mice (a) and rabbits (b) against recombinant EBP2 were tested by endpoint dilution for reactivity with the homologous antigen (Ag). Antigen preparations were allowed to adsorb onto wells of a microtiter plate and allowed to react with different dilutions of the antisera. Mouse anti-DBPII-Sal1 and mAb-3D10 were used as negative-control antibodies in the ELISA. Each point on the curves represents the mean OD from duplicate wells, while error bars represent ±standard deviations. (c and d) Mouse anti-EBP2 IgG (c) or anti-DBPII IgG (d) was tested against homologous and heterologous antigens in either the COS7 or flow cytometry binding assay for inhibition of EBP2 and DBPII reticulocyte binding. Each bar represents percent binding to reticulocytes in the presence of immune IgG relative to preimmune IgG. Error bars represent ±standard deviations.

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References

1. Mendis K, Sina BJ, Marchesini P, Carter R. 2001. The neglected burden of Plasmodium vivax malaria. Am J Trop Med Hyg 64:97–106. - PubMed
1. Baird JK, Snow RW. 2007. Acquired immunity in a holoendemic setting of Plasmodium falciparum and P. vivax malaria. Am J Trop Med Hyg 76:995–996. - PMC - PubMed
1. Miller LH, Mason SJ, Clyde DF, McGinniss MH. 1976. The resistance factor to Plasmodium vivax in blacks. The Duffy-blood-group genotype, FyFy. N Engl J Med 295:302–304. doi:10.1056/NEJM197608052950602. - DOI - PubMed
1. Welch SG, McGregor IA, Williams K. 1977. The Duffy blood group and malaria prevalence in Gambian West Africans. Trans R Soc Trop Med Hyg 71:295–296. - PubMed
1. Adams JH, Sim BK, Dolan SA, Fang X, Kaslow DC, Miller LH. 1992. A family of erythrocyte binding proteins of malaria parasites. Proc Natl Acad Sci U S A 89:7085–7089. doi:10.1073/pnas.89.15.7085. - DOI - PMC - PubMed

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[1] Mendis K, Sina BJ, Marchesini P, Carter R. 2001. The neglected burden of Plasmodium vivax malaria. Am J Trop Med Hyg 64:97–106. - PubMed

[2] Mendis K, Sina BJ, Marchesini P, Carter R. 2001. The neglected burden of Plasmodium vivax malaria. Am J Trop Med Hyg 64:97–106. - PubMed

[3] Baird JK, Snow RW. 2007. Acquired immunity in a holoendemic setting of Plasmodium falciparum and P. vivax malaria. Am J Trop Med Hyg 76:995–996. - PMC - PubMed

[4] Baird JK, Snow RW. 2007. Acquired immunity in a holoendemic setting of Plasmodium falciparum and P. vivax malaria. Am J Trop Med Hyg 76:995–996. - PMC - PubMed

[5] Miller LH, Mason SJ, Clyde DF, McGinniss MH. 1976. The resistance factor to Plasmodium vivax in blacks. The Duffy-blood-group genotype, FyFy. N Engl J Med 295:302–304. doi:10.1056/NEJM197608052950602. - DOI - PubMed

[6] Miller LH, Mason SJ, Clyde DF, McGinniss MH. 1976. The resistance factor to Plasmodium vivax in blacks. The Duffy-blood-group genotype, FyFy. N Engl J Med 295:302–304. doi:10.1056/NEJM197608052950602. - DOI - PubMed

[7] Welch SG, McGregor IA, Williams K. 1977. The Duffy blood group and malaria prevalence in Gambian West Africans. Trans R Soc Trop Med Hyg 71:295–296. - PubMed

[8] Welch SG, McGregor IA, Williams K. 1977. The Duffy blood group and malaria prevalence in Gambian West Africans. Trans R Soc Trop Med Hyg 71:295–296. - PubMed

[9] Adams JH, Sim BK, Dolan SA, Fang X, Kaslow DC, Miller LH. 1992. A family of erythrocyte binding proteins of malaria parasites. Proc Natl Acad Sci U S A 89:7085–7089. doi:10.1073/pnas.89.15.7085. - DOI - PMC - PubMed

[10] Adams JH, Sim BK, Dolan SA, Fang X, Kaslow DC, Miller LH. 1992. A family of erythrocyte binding proteins of malaria parasites. Proc Natl Acad Sci U S A 89:7085–7089. doi:10.1073/pnas.89.15.7085. - DOI - PMC - PubMed

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A Novel Erythrocyte Binding Protein of Plasmodium vivax Suggests an Alternate Invasion Pathway into Duffy-Positive Reticulocytes

Affiliations

A Novel Erythrocyte Binding Protein of Plasmodium vivax Suggests an Alternate Invasion Pathway into Duffy-Positive Reticulocytes

Authors

Affiliations

Abstract

Figures

References

MeSH terms

Substances

Grants and funding

LinkOut - more resources

Full Text Sources

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