Two functional reticulocyte binding-like (RBL) invasion ligands of zoonotic Plasmodium knowlesi exhibit differential adhesion to monkey and human erythrocytes
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Two functional reticulocyte binding-like (RBL) invasion ligands of zoonotic Plasmodium knowlesi exhibit differential adhesion to monkey and human erythrocytes

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Description

Plasmodium knowlesi is a monkey malaria species that is becoming a serious public health concern infecting hundreds and perhaps thousands of humans in Southeast Asia. Invasion of erythrocytes by merozoites entails a cascade of molecular interactions. One step involves the adhesion of Plasmodium reticulocyte binding-like (RBL) proteins. Plasmodium knowlesi merozoites express only two RBL invasion ligands, known as Normocyte Binding Proteins (PkNBPXa and PkNBPXb). Methods Overlapping N-terminal regions of PkNBPXa and PkNBPXb were expressed in COS7 cells and tested for surface expression and adhesion to rhesus monkey erythrocytes. Subsequent tests to study specific receptor ligand interactions included adhesion to a panel of human and non-human primate erythrocytes, enzymatic treatment, and site directed mutagenesis. Results An N-terminal cysteine-rich region of PkNBPXb (PkNBPXb-II) exhibited specific adhesion to rhesus monkey erythrocytes. Mutation of four of five cysteines in PkNBPXb-II interfered with its surface expression on COS7 cells, suggesting disulphide bond conformation is critical for intracellular trafficking. Binding of PkNBPXb-II was abolished when rhesus erythrocytes were pre-treated with chymotrypsin, but not trypsin or neuraminidase. PkNBPXb-II also bound other Old World monkey species and gibbon erythrocytes. However, erythrocytes from other primate species including humans did not bind to PkNBPXb-II or native PkNBPXb. Importantly, unlike PkNBPXb, PkNBPXa bound human erythrocytes, and this binding was independent of the Duffy blood group determinant. Conclusions The data reported here begins to clarify the functional domains of the P. knowlesi RBLs. A binding domain has been identified and characterized in PkNBPXb. Notably, this study demonstrates that unlike PkNBPXb, PkNBPXa can bind to human erythrocytes, suggesting that PkNBPXa may function as a ligand to enable the invasion of P. knowlesi merozoites into human cells.

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Publié le 01 janvier 2012
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Semenya et al. Malaria Journal 2012, 11:228
http://www.malariajournal.com/content/11/1/228
RESEARCH Open Access
Two functional reticulocyte binding-like (RBL)
invasion ligands of zoonotic Plasmodium knowlesi
exhibit differential adhesion to monkey and
human erythrocytes
1,4 1,5 1 2 1,3*Amma A Semenya , Tuan M Tran , Esmeralda VS Meyer , John W Barnwell and Mary R Galinski
Abstract
Background: Plasmodium knowlesi is a monkey malaria species that is becoming a serious public health concern
infecting hundreds and perhaps thousands of humans in Southeast Asia. Invasion of erythrocytes by merozoites
entails a cascade of molecular interactions. One step involves the adhesion of Plasmodium reticulocyte binding-like
(RBL) proteins. Plasmodium knowlesi merozoites express only two RBL invasion ligands, known as Normocyte
Binding Proteins (PkNBPXa and PkNBPXb).
Methods: Overlapping N-terminal regions of PkNBPXa and PkNBPXb were expressed in COS7 cells and tested for
surface expression and adhesion to rhesus monkey erythrocytes. Subsequent tests to study specific receptor ligand
interactions included adhesion to a panel of human and non-human primate erythrocytes, enzymatic treatment,
and site directed mutagenesis.
Results: An N-terminal cysteine-rich region of PkNBPXb (PkNBPXb-II) exhibited specific adhesion to rhesus monkey
erythrocytes. Mutation of four of five cysteines in PkNBPXb-II interfered with its surface expression on COS7 cells,
suggesting disulphide bond conformation is critical for intracellular trafficking. Binding of PkNBPXb-II was abolished
when rhesus erythrocytes were pre-treated with chymotrypsin, but not trypsin or neuraminidase. PkNBPXb-II also
bound other Old World monkey species and gibbon erythrocytes. However, erythrocytes from other primate
species including humans did not bind to PkNBPXb-II or native PkNBPXb. Importantly, unlike PkNBPXb, PkNBPXa
bound human erythrocytes, and this binding was independent of the Duffy blood group determinant.
Conclusions: The data reported here begins to clarify the functional domains of the P. knowlesi RBLs. A binding
domain has been identified and characterized in PkNBPXb. Notably, this study demonstrates that unlike PkNBPXb,
PkNBPXa can bind to human erythrocytes, suggesting that PkNBPXa may function as a ligand to enable the
invasion of P. knowlesi merozoites into human cells.
Keywords: Plasmodium knowlesi, Malaria, Erythrocyte, Merozoite, Adhesion, Binding protein, Reticulocyte, Invasion
ligand, RBLs
* Correspondence: mary.galinski@emory.edu
1
Emory Vaccine Center and Yerkes National Primate Research Center, Emory
University, Atlanta, GA, USA
3
Department of Medicine, Division of Infectious Diseases, Emory University
School of Medicine, Atlanta, GA, USA
Full list of author information is available at the end of the article
© 2012 Semenya et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited.Semenya et al. Malaria Journal 2012, 11:228 Page 2 of 12
http://www.malariajournal.com/content/11/1/228
Background mutations or are incomplete gene fragments and may be
Malaria is an ancient disease that continues to affect non-functional pseudogenes (Meyer, Galinski, Barnwell,
several hundred million people annually in countries unpublished data). Based on initial RBP binding data
of Africa, Asia, and South and Central America. Four [12] and parasite biology, the RBLs have been predicted
species of protozoan parasites of the genus Plasmo- to be critical in the initial selection and apical
attachdium, Plasmodium falciparum, Plasmodium vivax, ment of a merozoite to a potential host cell leading to a
Plasmodium ovale and Plasmodium malariae, have commitment to invasion by the subsequent release of
long been known to cause the disease with most of the Duffy Binding-Like/Erythrocyte Binding-Like (DBL/EBL)
nearly one million malaria-associated deaths attributable invasion ligand proteins from the microneme organelles
to P. falciparum [1]. In the last decade, a fifth species, and the formation of a tight junction [12,16].
Plasmodium knowlesi, a well-known cause of monkey A family of six paralogous rbl genes are now
recogmalaria, has emerged as a potential cause of severe and nized in the human malaria species P. falciparum [17],
fatal malaria in humans with more than 700 documented six in the chimpanzee parasite, Plasmodium reichenowi
cases [2-5]; unreported cases are likely to be in the thou- [18], two in P. knowlesi [19,20], at least two in
Plasmosands in Southeast Asia. dium cynomolgi (also a simian malaria species) [19,20],
As obligate, intracellular parasites, Plasmodium uti- and 14 in the rodent parasite Plasmodium yoelii [21].
lizes various strategies to efficiently gain entry into host The two rbl genes characterized so far in P. cynomolgi
erythrocytes and evade host immune responses, thereby are orthologous to rbp-1 and rbp-2 of P. vivax [19]. It is
achieving the ability to replicate and survive. The asex- likely there will be other rbl genes identified in P.
cynoual erythrocytic stage of the Plasmodium life cycle is the molgi, as in P. vivax, but this remains to be determined
cause of all clinical symptomology of malaria, and suc- with the completion of genome sequences for this
kincessful merozoite invasion is essential for the mainten- dred species.
ance of a malaria infection and propagation of the Studies on the RBL ligands in P. falciparum, also
parasites. An improved understanding of these processes known as the reticulocyte binding protein homologs or
is important for devising prophylactic and therapeutic Rh proteins, have been extensive in recent years, with
strategies against the various species that cause human these in mind as vaccine candidates [17]. Putative
malaria. erythrocyte binding domains have been reported to date
Plasmodium knowlesi has traditionally been instru- within the five expressed P. falciparum RBL proteins,
mental as a model parasite species in studies of erythro- PfRh1, PfRh2a, PfRh2b, PfRh4 and PfRh5 [22-26].
Gencyte invasion and has served as a stringent model for etic manipulation and invasion assays aiming to study
pre-clinical, proof-of-principle blood-stage malaria vac- the function of the various Rh paralogs have also given
cine testing in rhesus monkeys [5-10]. Like P. falcip- rise to the hypothesis that like the EBL invasion ligands,
arum, and in contrast to P. vivax, P. knowlesi merozoites the RBL family may present alternative invasion
pathare not restricted to invading reticulocytes but also in- ways for P. falciparum parasites [27,28].
vade mature erythrocytes. Merozoite invasion is a com- This study specifically sought to investigate the RBLs
plex, multi-component process, involving a series of using P. knowlesi to better understand their role as
parasite-host molecular interactions that are not com- adhesins in the intricate process of erythrocyte invasion,
pletely defined. In studies using P. knowlesi, after initial and their potential as a target for vaccine development.
steps of surface adhesion, apical reorientation and host This laboratory has previously reported the
identificacell selection, the merozoite forms an irreversible tion of two intact rbl genes in P. knowlesi, and showed
electron-dense tight junction with the membrane of its that they become expressed in the microneme
orgatarget erythrocyte [11]. The Reticulocyte Binding nelles as large proteins of approximately 300 kDa; a
Protein-Like (RBL) superfamily of ligands expressed at small relic pseudogene orthologous to rbp-1 of P. vivax
the apical end of merozoites has been implicated in the was also confirmed in this species [20]. The P. knowlesi
attachment of the apical end of this invasive stage to the RBLs were named as normocyte binding proteins,
surface of erythrocytes and its commitment to entering PkNBPXa and PkNBPXb, and it was confirmed that
the selected host cell [10,12-14]. both these proteins adhere to rhesus RBCs in traditional
Two Reticulocyte Binding Proteins (RBP-1 and RBP-2), erythrocyte binding assays [20]. Immuno-electron
mifrom which the RBL name is derived, were originally croscopy further showed that the P. knowlesi RBLs
apdefined in P. vivax and shown to be expressed late in pear to be released as merozoites attach to and invade
schizogony as large proteins (>300 kDa) that specifically erythrocytes [20]. Given the limited number of only two
bound to reticulocyte host cells [12]. Up to 10 paralo- functioning rbl family members confirmed in the P.
gous rbl genes have since been identified in the P. vivax knowlesi genome, and predictably the restricted
potengenome [15], a number of which have frameshift tial for alternative RBL invasion pathways, it wasSemenya et al. Malaria Journal 2012, 11:228 Page 3 of 12
http://www.malariajournal.com/content/11/1/228
speculated that these proteins, and particularly the Macaca mulatta (Indian and Chinese origin), long-tailed
binding domains, could serve as effective immunogens macaques (Macaca fascicularis), pigtail macaques
for pre-clinical, proof-of-principle efficacy studies in (Macaca nemestrina), sooty mangabeys (Cercocebus
rhesus monkeys. atys) and white mice was collected at the Yerkes
NaIn the current study, the functiona

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