Background
Malaria is an infectious disease caused by several species of protozoan parasites belonging to the
Plasmodium genus, although just one species,
Plasmodium falciparum, is responsible for the vast majority of human mortality and morbidity. About half of the world’s population live in regions that are described as being at risk of malaria, with approximately one million deaths attributed to this disease annually [
1].
Plasmodium parasites are transmitted to the human host through the bite of an infected mosquito and, after a largely asymptomatic liver stage, are released as merozoites, initiating the blood stage of infection. It is during this stage that the symptoms of malaria are exhibited. Once within the blood,
P. falciparum merozoites recognize and invade host erythrocytes where they multiply and, after rupturing the host cell, are released to begin another cycle of invasion. Since erythrocyte invasion by the
P. falciparum merozoite is an essential step in the parasite lifecycle, considerable research has been aimed at understanding the molecular mechanisms for the invasion process [
2] with the ultimate aim of developing anti-malarial drugs and vaccines [
2-
4].
It is known that the invasion of erythrocytes by the merozoite involves a series of extracellular molecular recognition events between host receptors and parasite ligands. For
P. falciparum, several host-parasite receptor-ligand interactions have been described which include the interactions between glycophorin A (GYPA) and erythrocyte binding antigen (EBA)175 [
5-
8] and those between basigin (BSG) and reticulocyte binding-like homologue (RH)5 [
9]. Despite this progress, the molecular mechanisms of invasion are not completely understood and further progress will be facilitated by the application of new methods, especially since the strategies used to date have largely relied on erythrocytes from patients with rare blood groups [
6,
10-
12]. Working with erythrocyte cell surface proteins, however, is technically challenging due to the genetic intractability of the anucleate erythrocyte and difficulties in solubilizing amphipathic membrane proteins in their native conformation. Furthermore, extracellular protein interactions can be highly transient – often having half-lives of just fractions of a second [
13]. To address these challenges, a method called AVEXIS (for AVidity-based EXtracellular Interaction Screen) was developed which detects even very weak interactions between the entire ectodomains of receptor proteins expressed as soluble recombinant proteins in mammalian cells [
14]. This approach, together with other advances in the expression of full-length, active
P. falciparum recombinant proteins [
15,
16], allowed the successful identification of two new erythrocyte-merozoite interactions [
9,
17]. This progress aside, the majority of the proteins located on the surface of the
P. falciparum merozoite have no known binding partner [
2].
The lack of knowledge about these parasite proteins can be largely attributed to technical limitations of existing approaches that modify the interacting species, or require removing the membrane protein from its native environment [
18]. Surface plasmon resonance (SPR) and biolayer interferometry (BLI) have exhibited only a limited number of successes with membrane proteins due to the need to immobilize one of the binding partners and their inherent mass sensitivity. Micro-scale thermophoresis (MST) [
19] normally requires removing the protein from the native environment, and the AVEXIS technique is not suitable for proteins where the extracellular region cannot be expressed as a soluble recombinant protein - such as those that span the membrane multiple times; for example, transporters or channels. With over 50 different multi-span membrane proteins detectable on human erythrocytes [
20], many potential host receptors are therefore excluded from the AVEXIS approach. Also, AVEXIS, SPR, BLI, and MST are not generally suitable to detect interactions with receptors that are formed by two or more proteins within the membrane. Finally, and as demonstrated here, the requirement to express the proteins recombinantly in a heterologous cell line can result in the absence of cell-specific, post-translational modifications that are essential for ligand binding. With this in mind, a technique that can sensitively and quantitatively measure interactions between
Plasmodium proteins and their receptors on the surface of intact erythrocytes would be particularly useful.
It has been shown recently that BSI is a highly sensitive, label-free and free-solution assay technology that is compatible with a wide array of complex matrices [
21]. This unique interferometer is based on illuminating a 100 × 210-μm dimensioned semicircular channel in a microfluidic chip with coherent light from a laser to create a pattern of light that is interrogated in the backscatter configuration [
22]. This pattern of light - the interference pattern - has periodic dim and bright spots (fringes) whose positions are related to the refractive index (RI) of the fluid in the channel. When molecules interact, the compound formed has a distinct and different RI than either of, or the sum of, the RI for the reactants. These changes in RI are a consequence of the conformation, hydration and electron density changing when the ligand binds to the receptor, and induce measurable changes in the position of the interference fringe pattern. BSI has been used to quantify binding affinities on systems with
K
D values ranging from a few picomolar (pM) to tens of millimolar (mM). BSI is a universal detection method which uses control and reference samples to enable the quantification of specific binding reactions, even within highly complex matrices, such as vesicles prepared from cell membranes [
21].
Here, it is shown for the first time that BSI can be used to detect and quantify low-affinity interactions between recombinant Plasmodium proteins and their known receptors natively embedded within the membrane of whole unmodified erythrocytes. These data demonstrate that composition and environment are critical when attempting to define the molecular mechanisms involved in erythrocyte invasion. BSI enables binding studies to be performed on the intact cell, such that the membrane protein receptors are unaltered and can form complexes, as they would in vivo. The results presented here indicate that this BSI-based methodology represents a new approach to identify and quantify interactions involved in erythrocyte invasion by P. falciparum, particularly those that contain erythrocyte-specific post-translational modifications.
Discussion
The data presented here demonstrate that BSI is well suited for the detection and quantification of interactions between
P. falciparum merozoite proteins and host receptors at the surface of intact, unmodified erythrocytes. BSI has numerous advantages over other approaches. First, it is easy to establish whether the interaction is saturable (and therefore specific), and does not require any washing or separation steps that may preclude the detection of transient interactions (
K
D ~ μM range, t½ <1 second), a typical feature of extracellular interactions between membrane-tethered proteins [
13]. Second, BSI can be used to measure interactions with unaltered cells, providing erythrocyte receptors embedded in the native membrane. This advantage is particularly important for investigating erythrocyte receptors that are very difficult to work with biochemically, particularly those that are formed from multi-chain protein complexes and/or contain multiple transmembrane-spanning regions. As demonstrated here, some erythrocyte receptors contain functionally critical post-translational modifications that are difficult to faithfully recapitulate using popular expression systems, necessitating the use of the native protein. Third, BSI is a label-free technique, so query ligands do not need to be derivatized with reporter molecules, such as fluorophores, protein tags or radioactive isotopes. These reporters lengthen assay time, increase cost and may interfere with interactions. Fourth, BSI is typically performed as a free-solution or homogeneous assay, obviating the requirement for any complex immobilization chemistry. Finally, because it is based on a microfluidic channel that has small dimensions (100 × 210μm) protein sample consumption is constrained, with required quantities in the nanomole to picomole range. Taken collectively, BSI could contribute to malaria research by both facilitating the use of recombinant, soluble
P. falciparum merozoite protein resources [
15,
16] to systematically screen panels of
Plasmodium proteins for their ability to bind erythrocytes, and performing further characterization of their receptors so as to define their mechanism of action.
One of the main motivations for exploring the use of BSI to detect interactions with receptors on the surface of erythrocytes was the difficulty in detecting binding between recombinantly expressed GYPA and PfEBA175. This is most likely due to the undersialylation of GYPA produced in HEK293E cells. Even with the additional measures taken to increase sialylation, such as co-expression with sialyltransferases, recombinant GYPA was undersialylated in comparison to the native protein, and was unable to bind to PfEBA175. Others have shown that GYPA expressed in HEK293 cells can bind to PfEBA175 [
29] and so it can be speculated that the high level expression system based on EBNA1/oriP amplification used here [
30] may have saturated the glycosylation pathway leading to undersialylation.
In regard to the PfEBA175-GYPA interaction, a five-fold higher equilibrium binding affinity on the surface of erythrocytes was measured using BSI relative to that measured using GYPA extracted from cells, biotinylated and immobilized on a streptavidin-coated surface for SPR experiments [
8]. This higher binding affinity may be due to the increased mobility of GYPA within the erythrocyte membrane to form a PfEBA175 receptor. This observation is also consistent with both structural [
31] and biochemical studies on PfEBA175 [
8] which show that PfEBA175 is capable of forming dimers in solution, and that GYPA forms reversible, non-covalent dimers within the erythrocyte membrane [
32,
33]. These findings have led to a model whereby a GYPA dimer induces the dimerization of PfEBA175 at the merozoite surface which may be important for invasion [
34]. The data presented here are consistent with this model, and suggest that the higher affinity of the PfEBA175-GYPA interaction, as measured at the erythrocyte surface rather than when tethered to a solid surface, could be due to the increased mobility of GYPA within the erythrocyte membrane, allowing it to form avid dimers.
The affinity determined here by BSI for the PfRH5-BSG interaction, with the receptor natively presented on whole erythrocytes compares very favorably to the values reported using purified recombinant soluble proteins and SPR [
9,
35,
36]. This observation might suggest that the PfRH5 binding site on the erythrocyte surface is entirely contained within the BSG ectodomain and does not depend on other erythrocyte proteins to act as co-receptors, such as the BSG-associated monocarboxylate transporter, MCT1 [
37].
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Competing interests
DJB and AK have a financial interest in a company that is commercializing BSI. The other authors declare that they have no competing interests.
Authors’ contributions
PS performed the BSI assays under the guidance of AK and DJB. AJP, SJB and MW produced and characterized recombinant proteins. AJP, PS, AK, DJB and GJW wrote the manuscript. DJB and GJW conceived and managed the research. All authors read and approved the final manuscript.