Three Is a Crowd – New Insights into Rosetting in Plasmodium falciparum

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Review

Three [403_TD$IF]Rosetting in Plasmodium falciparum Xue Yan Yam,1,3 Makhtar Niang,2,3 Kripa Gopal Madnani,1,3 and Peter R. Preiser1,* The intracellular malaria parasites extensively modify host erythrocytes to allow nutrient uptake, ensure homeostasis, and evade the host’s immune response. To achieve this, the parasite exports several proteins to the erythrocyte surface. In Plasmodium falciparum, the parasite responsible for the most severe form of human malaria, three major variant surface antigen families – PfEMP1, STEVOR, and RIFIN – have been implicated in contributing to immune evasion, parasite sequestration, and parasite-mediated rosetting of uninfected erythrocytes. Sequestration and rosetting have been linked to parasite-mediated pathology, making the variant surface antigens of P. falciparum major virulence factors. Here we review our current understanding of rosetting mechanism, recent findings of STEVOR, RIFIN-mediated rosetting, and their implication on the severity and pathology of the disease.

Trends Rosetting, the binding of uninfected RBCs to a parasite-infected RBC (iRBC), has been directly linked to the severity of clinical disease. Three parasite protein families, PfEMP1, STEVOR, and RIFIN, mediate rosetting in Plasmodium falciparum. Sequential timing of surface expression of PfEMP1, RIFIN, and STEVOR on iRBC suggests that the parasite has developed three different rosette formation mechanisms, implicating a critical function for parasite survival.

Variant Surface Antigens of Plasmodium Parasites: A Role in Rosetting Plasmodium parasites have a complex life cycle involving a mosquito vector and a mammalian host. The parasite transitions through various stages of growth within the host body, infecting the liver as a sporozoite and undergoing schizogony and gametocytogenesis within the erythrocyte [1]. Disease symptoms in the host occur during the asexual replication of the parasite in the erythrocyte due to the release of inflammatory cytokines, anemia caused by the destruction of erythrocytes, and obstruction of blood flow caused by parasite sequestration [2,3]. During its complex developmental cycle the parasite extensively modifies the surface of the infected red blood cell (iRBC). These surface modifications are directly linked to the ability of the trophozoite and schizont stages to sequester within the deep tissues of the host by binding to endothelial cell receptors and are a significant contributor to the morbidity caused by P[406_TD$IF]. falciparum. As the parasite matures intracellularly, it exports several proteins that are important for its survival and sequestration. Three major variant surface antigen (VSA) families have been identified in P. falciparum: P. falciparum erythrocyte membrane protein 1 (PfEMP1) encoded by the var genes [4], subtelomeric variant open reading frame (STEVOR) encoded by the stevor genes, and repetitive interspersed repeats (RIFIN) encoded by the rif genes [5]. Of these three families, PfEMP1 has been most extensively studied and has been shown to mediate both antigenic variation as well as adhesion of the iRBC [6]. Another striking feature of iRBCs is their ability to bind to uninfected red blood cells (RBCs) forming rosettes. The biological role of rosetting is not clear though some studies have linked it to parasite virulence [7–9]. PfEMP1 has been shown to mediate the binding to a variety of host cell receptors, including CD36, intercellular adhesion molecule 1 (ICAM1) and chrondroitin [407_TD$IF]sulfate A (CSA) (see Glossary). The binding of PfEMP1 to complement receptor 1 (CR1) on the RBC has been shown to lead to rosetting [10]. Recent studies have shown that, in addition to PfEMP1, both STEVOR and RIFIN are able to mediate rosetting by binding to glycophorin C and blood group A antigen, respectively [11,12] (Figure 1). [408_TD$IF]In addition, cumulative evidence from different studies [409_TD$IF]show that the transcription and timing of surface expression of PfEMP1, RIFIN, and STEVOR from

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PfEMP1 mediates rosetting through CR1, heparan sulfate, and blood group A antigen on RBCs. STEVOR mediates rosetting through glycophorin C and, RIFIN mediates rosetting predominantly to blood group A antigen as well as glycophorin A on the surface of the RBC.

1

School of Biological Sciences, Nanyang Technological University, 637551, Singapore 2 Immunology Unit-Pasteur Institute of Dakar, 220 Dakar, Senegal [405_TD$IF]3 These authors contributed equally. *Correspondence: [email protected] (P.R. Preiser).

http://dx.doi.org/10.1016/j.pt.2016.12.012 © 2017 Elsevier Ltd. All rights reserved.

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laboratory-cultured parasite is sequential though RIFIN and PfEMP1 appear on the erythrocyte surface at the same time [13–17] and reviewed in [18,19] (Figure 1A[410_TD$IF]). This suggests that the parasite has developed three different mechanisms that mediate rosette formation suggesting a critical function for parasite survival.

(A) Ring Hours post-invasion Transcripon

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Figure 1. The Three Major Multigene Families in Plasmodium falciparum. (A) Schematic expression profile of var, rif, and stevor multigene families during asexual developmental stage of the laboratory-adapted cultured parasite. Transcription of the var gene starts early in the ring stage parasite followed by the rif and stevor in the late ring/early trophozoite stage. Despite differences in the timing of transcription, both PfEMP1 and RIFIN proteins appear on the infected RBC (iRBC) surface during late ring/early trophozoite parasite stages, while STEVOR appears on the surface later during the late trophozoite/schizont stage. At the late schizont parasite developmental stage, all three of these variant antigens are present on the parasite surface. (B) Rosetting phenotype of PfEMP1, RIFIN, and STEVOR. These variant antigens expressed on the surface of the iRBC mediate rosetting to uninfected RBC using different receptors expressed on the host cell surface. PfEMP1, located at the knobs, mediates rosetting via binding to CR1, heparan sulfate, and blood A antigen on the RBC surface. Variations in the DBL1a domain of PfEMP1 involve interaction with different receptors, resulting in different degrees of rosetting and disease severity. STEVOR appears to cluster at the base of the knobs and mediates rosetting via binding to glycophorin C (Gly C) receptor. RIFIN-mediated rosetting forms giant rosettes via binding to the blood group A antigen on RBC, and forms smaller rosettes via binding to glycophorin A (Gly A) receptor. RIFINmediated rosettes also appear to protect PfEMP1 from antibody recognition [83]. In all, the parasite has developed three different mechanisms that mediate rosette formations, suggesting a critical function for parasite survival. [402_TD$IF]Abbreviations: PfEMP1, Plasmodium falciparum erythrocyte membrane protein 1; RIFIN, repetitive interspersed repeats; STEVOR, subtelomeric variable open reading frame; iRBC, infected red blood cell; DBL, Duffy binding ligand; CR1, Complement receptor 1.

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Glossary CD36: cluster of differentiation 36 is an integral membrane protein found on the surface of many cell types such as blood cells, platelets, spleen cells,and some skin endothelial cells. CD36 binds to a broad range of ligands which can be of proteinaceous and lipidic nature, such as collagen, thrombospondin, lipoprotein, and phospholipids, as well as red blood cells parasitized with Plasmodium falciparum. CR1: complement receptor type 1 (CR1) encoded by the CR1 gene belongs to the regulators of complement activation family. It is a monomeric single-pass type 1 transmembrane glycoprotein expressed on the blood cells, leukocytes, and mircoglia. CR1 is a receptor for complement components C3b/C4b which helps in the regulation and activation of the complement immune system. CR1 is also responsible for Plasmodium falciparum rosetting. CSA: chondroitin sulfate A (CSA) is a sulfated glycosaminoglycan (GAG) composed of a chain of alternating N-acetylgalactosamine and Dglucuronic acid and sulfate residues in equimolar quantities where carbon 4 of the N-acetylgalactosamines is sulfated. It is usually attached to proteins as part of a proteoglycan and found in skin, cornea, cartilage, and umbilical cord. CSA is found to be the receptor for sequestration of Plasmodium falciparum in the placenta. Heparan sulfate (HS): a linear polysaccharide that occurs as a proteoglycan to which two or three HS chains are attached closely. This glycoprotein is found at the cell surface and in the extracellular matrix where HS binds a large number of protein ligands to regulate a range of biological activities. HS also serves as cellular receptor for various viruses and pathogens. ICAM-1: intercellular adhesion molecule-1 (ICAM-1), also known as CD54 (Cluster of Differentiation 54), is a glycoprotein typically expressed on the surface of endothelial cells and leukocytes. It has a single transmembrane domain with an Nterminal extracellular domain and a C-terminal cytoplasmic tail. Thrombospondin: multimeric multidomain glycoprotein found at cell surfaces and in the extracellular

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This review summarizes our current understanding of rosette formation and function with a particular emphasis on the new findings about STEVOR- and RIFIN-mediated rosetting.

Rosette Formation and Function in P. falciparum Rosetting is defined as the phenomenon during which the iRBC binds to uninfected RBCs, engaging parasite ligands and RBC surface receptors (Figure 2). It is thought that, in this way, the parasite manages to achieve two goals. The first among these is to evade the host immune system by shielding the iRBC and the newly released merozoites from host invasion-inhibitory antibodies [20]. In addition, PfEMP1, RIFIN, and STEVOR are antigenically variant and are encoded by multigene families, further supporting the notion that these proteins are important immune evasion molecules. The second role is to ensure parasite survival and replication by providing a favorable environment for newly released merozoites that can rapidly invade the uninfected RBCs that are part of the rosette [21]. However, these suggested goals are not supported by clear experimental evidence, as there are contradicting reports on whether rosette formation leads to an increase in parasite numbers. Although a single study has shown a correlation between rosetting and increased parasitaemia in African children [22], other studies do not support this conclusion [23]. In addition, one study also showed that the capacity of a parasite to form rosettes in vitro does not positively correlate with the ability to evade invasion-inhibitory antibodies [20]. The fact that field isolates show differences in their capacity to rosette further complicates the issue. However, rosetting has been directly linked to the

matrix milieu. It interacts with a variety of molecules such as collagen, fibronectin, heparin, and blood coagulation and anticoagulant factors. It is also involved in antiangiogenic functions and cell adhesion of Plasmodium falciparuminfected red cells.

Host endothelium

Roseng Blood flow

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Invasion RIFIN/STEVOR interacon

(ii)

(i)

(iv) (iii) Vascular

occulsion

PfEMP1 interacon

Figure 2. Postulated Roles of Variant Surface Antigens of Plasmodium Parasites. The sequential expressions of PfEMP1, RIFIN, and STEVOR on the surface of the infected RBC (iRBC) implicate a critical role for parasite survival. The rosetting ligands of PfEMP1 (CR1 and herapan sulfate) found on the RBC surface are also present on endothelial cells, indicating that endothelial adhesion/sequestration, to avoid splenic clearance of the iRBC, is likely the primary function of PfEMP1, and that PfEMP1-mediated rosettes may be of secondary importance. Hence, this suggests that (i) iRBCs cytoadhere by binding to endothelial receptors using PfEMP1, which is expressed early on the surface of iRBCs, before (ii) RBCs binds to cytoadhered iRBC using either RIFIN or STEVOR, which are expressed on the surface later in the parasite developmental stage. (iii) It is possible that rosetting may then contribute to sequestration in vivo by binding to cytoadhered nonrosetting parasites using either PfEMP1, RIFIN, and/or STEVOR, leading to vascular occlusion and increased disease severity and pathology. In all cases, the rosette would be expected to shield the parasite from the host immune response and (iv) would provide a favorable environment for the newly released merozoites after schizont rupture to ensure efficient invasion. [402_TD$IF]Abbreviations: PfEMP1, Plasmodium falciparum erythrocyte membrane protein 1; RIFIN, repetitive interspersed repeats; STEVOR, subtelomeric variable open reading frame; iRBC, infected red blood cell; CR1, Complement receptor 1.

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ability of P. falciparum to cause complicated disease and severe infection in several studies [7– 9,24]. Moreover, there is evidence that isolates from patients who suffered from complicated malaria have a higher rate of rosetting than those who suffered from mild malaria [9,25]. A model that could explain the fact that rosette formation leads to complicated and severe disease without causing a direct increase in parasitaemia was proposed by Kaul et al. [8]. This model suggests that rosette formation occurs on top of iRBCs that are already bound to the endothelium (sequestered), thereby obstructing blood flow in the thin capillaries of vital organs (Figure 2). This could then lead to the symptoms of severe disease such as coma, respiratory distress, and renal and hepatic failure. In addition, the study also showed that rosetting occurs mainly in the venules of isolated mesocecum vasculature and not in the arterioles, suggesting that the lower venous blood pressure enables the formation of these rosettes. Additional support for a critical role of rosetting in parasite biology comes from the observations that most if not all Plasmodium species are able to rosette [26–29].

PfEMP1 and PfEMP1 Mediated Rosetting The var genes constitute a multigene family of approximately 60 members in the reference clone 3D7 that encode the PfEMP1 proteins [30]. They were first identified as high-molecularweight proteins of the iRBC that could be surface-labelled using radioactive iodine [4]. Each protein is of the same basic two-exon structure: exon 1 is composed of an N-terminal segment (NTS), varying combinations of Duffy binding ligand (DBL), cysteine-rich inter-domain regions (CIDRs) domains, and a single transmembrane domain; exon 2 is highly conserved among members of the var gene family and codes for the cytoplasmic acid terminal segment (ATS) domain [31,32]. The domains in exon 1 display a high degree of variability, a feature that contributes to the ability of PfEMP1 to confer different cytoadhesive properties to the iRBCs [33,34] and also to its antigenic variability [35]. Most studies have shown that the var genes are expressed in a mutually exclusive manner at the mRNA as well as protein level [14,36–38], although some recent reports contradicted this widely accepted dogma, showing that multiple vars may be transcribed at a single parasite level or in the early ring stage parasite [14,36,39]. However, through a tightly regulated parasite developmental process, only a single full-length var transcript in a single parasite is maintained and actively transcribed with the silencing of other previously active vars, leading to the mutually exclusive expression of only one type of PfEMP1 protein on the surface of the iRBC [39–41]. Nonetheless, a recent study has also challenged the above accepted paradigm of single expression of PfEMP1 protein by showing two different PfEMP1 proteins on the surface of the same iRBC binding to different host cell endothelial receptors [42]. It is thought that one of the ways in which the parasite evades the host immune response is its capability to alter the antigenicity as well as cytoadhesive properties of the iRBC by switching the expression of the var genes [35,38,43] – which is reviewed in detail in [6,44]. The ATS domain is known to be important for the anchoring of PfEMP1 proteins to membrane protrusions known as knobs [45]. PfEMP1 is extremely prominent on the iRBC surface due to its presentation in the context of the knobs. However, not all PfEMP1 found on the membrane are anchored within the knobs; some reports have shown that the expression of this protein on the iRBC surface is possible in knobless strains. However, in this case, the amount of PfEMP1 on the surface is reduced [46–48]. Different PfEMP1 proteins have also been found to interact with a variety of cell-surface receptors on the iRBC as well as on the endothelial cells – such as heparan sulfate, CD36, CR1, ICAM-1, CSA, thrombospondin, endothelial protein C receptor (EPCR), IgM and a-2 macroglobulin – with different binding affinity [10,49–57] for its efficient cytoadhesion properties. In addition to mediating cytoadhesion of iRBC by binding to specific endothelial receptors, a subset of PfEMP1 is also able to mediate rosetting of the iRBC. Different PfEMP1 variants which

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mediate rosetting have been described (IT4/R29 [10], Palo Alto 89F5 VarO [58], 3D7/ PF13_0003 [59], and IT4/var60 [60]) and belong to a specific subgroup called groupA/UpsA var genes [61]. The N-terminal domain NTS-DBL1a1 of PfEMP1 has been shown to be responsible for mediating rosetting to the RBC in several studies by binding to CR1 [10] as well as heparan sulfate [62–65] on the RBC surface (Figure 1B). Through structural and functional studies, Juillerat et al. demonstrated that the NTS-DBL1a1 of PfEMP1 is directly implicated in rosetting and this interaction is inhibited by heparin in a dose-dependent manner, mimicking heparin-mediated rosette disruption [63]. More precisely, a limited number of basic amino acid residues localized on the surface of subdomains 1 (SD1) and 2 (SD2) of DBL1a of PfEMP1 are shown to be implicated in the heparin-mediated rosetting of the parasite [64]. Rosetting has also been proposed to rely on the carbohydrate moieties on the RBC surface [66–68], and PfEMP1 has been shown to bind to the blood group A antigen [61,69]. Variations in the DBL1a domain of PfEMP1 have also been linked to different degrees of rosetting and disease severity [9,70]. While cytoadhesion and rosetting have often been treated as separate properties of PfEMP1, a recent study has indicated that a single PfEMP1 molecule from P. falciparum rosetting strain can potentially mediate both cytoadhesion and rosetting at the same time [54]. This also suggests that different domains – NTS-DBL1a and DBL2g within a single PfEMP1 variant – can interact with different receptors via specific ligand–receptor interactions to bring about the two phenomena together [54]. More details on PfEMP1and –mediated rosetting can be found in [41_TD$IF]other reviews [6,44].

RIFINs and RIFIN-Mediated Rosetting RIFINs constitute the largest known family of variant antigens in P. falciparum, being present in about 150 copies in the 3D7 genome [30]. They are present at the subtelomeric ends of all 14 chromosomes. The encoded proteins are of low molecular weight, about 27–45 kD in size [16]. They may be separated into a cysteine-rich N-terminal segment and a C-terminal region that contains a hypervariable loop region that was initially predicted to be flanked by two transmembrane (TM) regions. Overall, rifins can be categorized into two broad subfamilies, [412_TD$IF]rifA and rif B. The presence of a 25-amino acid insert downstream of the Plasmodium export element (PEXEL) motif, and within the semiconserved region in the A-type RIFINs, distinguishes them from B-type RIFINs [71]. However, recent evidence supports the presence of a single TM domain, with the semiconserved and hypervariable regions of the protein exposed to the extracellular milieu [12,72] for A-type RIFINs, while B-type RIFINs are assumed to possess two TM domains [73]. The A-type RIFINs are trafficked to the iRBC membrane [16,74] via the parasite Maurer’s clefts (MCs), while the B-type RIFINs have been shown to be more internally localized inside the parasite [74]. More recently it was shown that RIFINs are expressed in the merozoite, with A-type RIFINs localized at the apical tip and B-type RIFINs diffused in the cytoplasm of merozoites [74,75]. However, a single unusually transcribed B-type RIFIN is shown to locate at the surface of merozoite and on the surface of the gametes [76]. RIFINs are potentially targets for protective immunity. It was shown that a substantial humoral immune response to RIFINs can be detected through the course of a P. falciparum infection and that this response correlates to milder, asymptomatic infections [77–79]. There is also evidence to show that the rifins are transcribed [413_TD$IF]during gametocyte stages II–V however, whether they are exposed on the iRBC surface at these stages is as yet unclear [80] though some evidence shows that A-type RIFINs are localized as discrete [41_TD$IF]punctate structures at the gametocyte-infected erythrocyte membrane, with B-type RIFINs in the cytoplasm of the parasite [76,81]. The earliest report of rosetting at the asexual stages implicated low-molecular-weight proteins on the surface of the iRBC termed 'Rosettins' [82]. The discovery of RIFINS made them attractive candidates for being these ‘Rosettins’, though this was never formally proven. It was not until very recently that the role of RIFIN in rosetting was confirmed in a study by Goel et al. [12] which showed that one member of the A-type RIFIN family was able to form

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giant rosettes via the binding of the blood group A antigen on RBC. RIFIN is also able to form smaller rosettes with O blood group RBCs by binding to the glycophorin A receptor [12]. Blood group A antigen seems to be the major receptor for RIFIN-mediated rosetting, as A blood group RBCs lacking glycophorin A still bind avidly to iRBCs [12] (Figure 1B). The formation of RIFINmediated rosettes appears to protect PfEMP1 from antibody recognition, further supporting their role as protective antigens [83]. Despite the confirmation study by Goel et al[415_TD$IF]. [12] on the role of one member of A-type RIFIN in rosetting, there are very limited data on the functions of other RIFIN members. It is highly speculative that RIFINs could play multiple different roles in different development stages of the parasite due to its differential localization in the parasite. The B-type RIFINs [416_TD$IF]that remain internally within the parasite could be [417_TD$IF]hypothesized to maintain the parasite integrity and metabolism while the A-type RIFINs are exported to [418_TD$IF]the iRBC cytosol and MCs and translocated to the red cell membrane at the right time and place for its desirable functions. Another plausible role of RIFIN is involved in invasion since RIFINs are detected in merozoites, similar to STEVOR [11]; however, whether A-type or B-type RIFINs play a function remains to be elucidated. Additionally, evidence shows that there is an increase in the expression of rif (A-type) in clinical isolates [75], suggesting that RIFINs might also be involved in some immune evasion mechanism as genes that are over-expressed in vivo may be required for parasite survival in a harsh host immunological environment [75].

STEVOR and STEVOR-Mediated Rosetting Stevors constitute the third largest multigene family of P. falciparum [30]. This family is comprised of 33 members in the reference clone 3D7. They are located at the subtelomeric ends of all 14 chromosomes, in proximity to the vars and the rifs, and they encode proteins of 35–40 kD in size [5]. STEVORs are predicted to have two-transmembrane (2TM) topology and an intermediate hypervariable region (HVR) predicted to be surface exposed [84,85]. However, experimental evidence supports a model with a single TM, a short C-terminal cytoplasmic domain and an external N-terminal semiconserved and hypervariable region [72,86]. About two or three members of stevor have been shown to be transcribed at a single parasite level, and multiple variants of the protein are also detectable [86–90]. Studies have shown that certain stevor variants are conserved across strains, indicating that these members may be under specific selective pressure [91]. The presence of the PEXEL motif at the N-terminus of STEVOR was an early indication that it is trafficked beyond the confines of the parasite and into the iRBC cytosol. At the trophozoite stage, the protein is located at the MCs of the parasite [13,88,92] and can subsequently be detected on the iRBC membrane at late schizont stage, with some detected in the developing merozoites [72,75,86,88,89]. Detailed studies using atomic force microscropy (AFM) clearly established that STEVOR export to the iRBC surface continues throughout the development of the parasite, starting at the trophozoite stage and peaking at the schizont stage [93]. STEVOR appears to cluster at the base of the knobs, indicating a potential interplay between PfEMP1 and STEVOR on the iRBC surface [93] (Figure 1B). Different members of STEVOR can be expressed on the surface of the iRBC and this contributes to different antigenic properties of the iRBC [86]. While STEVOR is recognized by the immune sera obtained from malaria-infected adults, there is limited evidence on whether these can be linked to immune protection against the parasite or reduced pathology [94]. The location on the surface of the iRBC, along with its hypervariable region, suggested that STEVOR plays an important role in immune evasion; at the same time, the presence of a semiconserved region indicates some functional constraints. The recent findings that STEVOR binds glycophorin C on the uninfected RBC, resulting in PfEMP1-independent rosetting, was the

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first demonstration that additional parasite-derived proteins on the iRBC surface could mediate host receptor binding activity [11], and this was subsequently demonstrated for RIFIN [12]. Interestingly, STEVOR, similarly to RIFIN, is also present at the apical tip and surface of the merozoite [11,88,90]. The observation of a second peak of stevor transcription in merozoites suggests that a new set of STEVOR protein is expressed in the developing or free merozoites [88], though this still remains to be formally demonstrated. Whether STEVOR expressed on the merozoite has different functional properties than those expressed on the iRBC surface still needs to be established. Recent data also appear to indicate that STEVOR may function both in relation to immune evasion and in facilitating invasion [11,90] where anti-STEVOR antibodies were shown to inhibit merozoite invasion in vitro as well as block STEVOR erythrocyte binding [11]. Additionally, the study by Niang et al. [11] showed that there is a link between stevor transcript levels and invasion efficiency of rosetting as compared to nonrosetting laboratory parasite strains. Importantly, they also show that STEVOR-mediated rosetting provides the parasite with a growth advantage and protects merozoites from invasion-inhibitory antibodies in the rosetting parasite as compared to the nonrosetting parasite. These data also suggest that disruption of STEVOR-mediated rosettes significantly enhances the efficiency of invasion-inhibitory antibodies [11], which is in line with a report demonstrating the association of rosetting and enhanced parasite invasion [95]. In addition to its function of RBC binding, STEVOR has also been shown to contribute to increased iRBC membrane rigidity at the late trophozoite and schizont stages, during which it is expressed on the iRBC membrane [96]. However, the mechanism underlying STEVOR’s effect on membrane rigidity remains unknown. STEVOR expression at the sexual gametocytes stages [419_TD$IF]iRBC membrane has also been shown to have an impact on its membrane rigidity [97]. Recently, Naissant et al. [98] has revealed that the mechanism of switching in the deformability of immature and mature gametocytes is regulated by protein kinase A through the cytoplasmic tail of STEVOR. This change in phosphorylation will affect the interaction of STEVOR with the cytoskeletal ankyrin complex, leading to changes in rigidity and deformability of the infected cells which may facilitate gametocyte sequestration. At this stage, a similar mechanism can be proposed to be exploited by the parasite in the asexual blood stage for STEVOR-mediated membrane rigidity. However, the question of whether or not STEVOR is also involved in host endothelial cell binding and sequestration at these stages remains unanswered.

Rosetting in P. falciparum: A Three-Pronged Approach? The P. falciparum parasite radically modifies the RBC within which it matures by establishing various subcellular compartments and protein trafficking organelles [99]. The membrane of the iRBC is also modified by the presence of several parasite proteins, many of which are exposed to the immune system of the host. In P. falciparum the three main variant antigen families are generally transcribed in chronological order during the asexual blood stage cycle, with var being transcribed first, then rif, followed by stevor (Figure 1A) in the laboratory parasite strain with some exceptions. Despite these differences in timing, both PfEMP1 and RIFIN seem to appear on the iRBC surface at the early trophozoite stages of parasite growth [16]. [420_TD$IF]By contrast, STEVOR appears on [421_TD$IF]the iRBC surface during the late trophozoite stage [13,86,88]. At the late schizont stages of development, all three of these variant antigens are therefore present on the parasite surface. The fact that the parasite has developed three different mechanisms, and has dedicated a significant proportion of its genetic capacity to facilitate rosette formation, would indicate that rosetting plays a critical role in parasite survival. This raises the question of whether and how the different gene families interact with each other to mediate rosetting, and how the parasite modulates the expression of variants of all three families based on host receptor availability.

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While STEVOR rosette formation is mediated via binding to glycophorin C [11], RIFIN rosettes are formed by binding predominantly to the blood group A antigen [12]. Work done much earlier has also shown that PfEMP1 is able to mediate rosetting of the iRBC by binding to CR1 on the red cell surface [10]. Thus, all three variant antigens are capable of mediating the same function on the iRBC, while using different receptors on the surface of the host cell (Figure 1B). It is of note that the rosetting ligands on the host cell surface are largely glycosylated [65,100], suggesting that the parasite ligands may contain lectin-like domains. While glycophorin C and blood group A antigen are exclusively found on the RBC, the rosetting ligands recognized by PfEMP1 (heparan sulfate and CR1) are also found on endothelial cells. This could indicate that PfEMP1-mediated rosettes are of secondary importance to the parasite and that endothelial adhesion is the critical function mediated by PfEMP1, which can interact with many different surface receptors on multiple cell types. This may explain why there is no direct evidence to date that PfEMP1-mediated rosettes provide a growth advantage for the parasite. In vivo circulating rosettes would be expected to be trapped in the spleen and lead to parasite clearance. The sequential appearance of PfEMP1, RIFIN, and STEVOR on the surface of the iRBC ensures that sequestration, which is critical for the parasite to avoid splenic clearance in vivo, would happen before rosette formation. The early expression of PfEMP1 would therefore ensure that parasites first adhere to the endothelial receptors before RBC binding occurs via either RIFIN or STEVOR. In a recent study it was shown that STEVOR can mediate rosette formation on bound iRBCs under flow conditions, and that the binding strength of STEVOR to RBCs is stronger than the interaction between PfEMP1 and some of its receptors [93]. Hence this suggests that rosetting may in vivo contribute to sequestration by binding to cytoadhere nonrosetting parasites, leading to augmented vascular occlusion resulting in increased disease severity and pathology (Figure 2). STEVOR-mediated rosettes are capable of conferring a selective advantage on the growth of the parasite by shielding merozoites from invasion-inhibitory antibodies [11]. Similarly, RIFINmediated rosettes appear to protect the iRBC from immune recognition, though whether this also leads to increased parasitaemia is not known [83]. Unlike for PfEMP1, both RIFIN and STEVOR are also expressed on the merozoite surface and apical tip [11,76,88–90], suggesting a potential role in merozoite invasion or immune evasion besides rosetting. It is an attractive hypothesis that RIFIN and STEVOR specifically recruit RBCs for rosette formations that are also suitable for subsequent merozoite invasion. In such an event, the released merozoites would be able to immediately engage with highly suitable RBCs via the apical or surface-expressed RIFIN and STEVOR. This would explain that, while anti-STEVOR antibody can block merozoite invasion by preventing initial interaction with the RBC, it is not essential for the parasite to survive in culture [11]. A striking but so far unexplored observation is that some of the receptors used for rosetting (CR1, glycophorin C, and glycophorin A) are also known invasion ligands that interact with reticulocyte binding protein homolog 4 (RH4) [101], erythrocyte binding antigen (EBA) 140 [102], and EBA 175 [103], respectively. Thus, the same receptor is engaged by parasite proteins during the process of rosetting and invasion. It is important to note that while all three proteins (PfEMP1s, RIFINs, and STEVORs) have been implicated in rosetting [10–12], only STEVORs have been conclusively shown to participate in both rosetting and invasion [11]. Whether this is a matter of evolution where the invasion proteins, such as the RHs family, have expanded and developed into these variant gene families, or whether these three variant proteins are complementary or substitutions to the invasion ligands for parasite invasion require further investigations. It will be of interest to also further study the binding regions and kinetics of these different sets of receptor–ligand interactions involved in rosetting in order to obtain some perspective on why the parasite uses multiple receptors for the same function.

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Taken together, the currently available data would suggest that iRBCs first cytoadhere by binding to many different endothelial receptors using PfEMP1. Subsequently, the bound iRBCs would bind to RBCs using either PfEMP1, RIFIN, and/or STEVOR, resulting in vascular occlusion and increased pathology (Figure 2). In all cases, the rosette would be expected to shield the parasite from immune attack. It is not yet clear whether RIFIN- and STEVORmediated binding to RBCs is sequential or complementary, where RBCs that are bound by both ligands would provide the greatest benefit for the parasite. RIFIN- and STEVOR-mediated rosettes would provide a favorable environment for the merozoites that are released after schizont rupture, thereby ensuring efficient invasion. At this stage, both RIFIN and STEVOR have been shown to bind to specific RBC receptors, and no evidence exists on whether they also engage with endothelial receptors. It will be interesting to study whether the RIFINs and STEVORs also play a role in cytoadhesion.

Concluding Remarks Rosetting is a conserved phenomenon in most, if not all, primate and rodent Plasmodium species, such as P. ovale [28], P. vivax [104], P. fragile [105], P. coatneyi [106], and P. chabaudi [26], and has been linked to disease pathology. Members of the Plasmodium interspersed repeats (pir) superfamily have been shown to be present in all human and rodent species of Plasmodium [107,108], with stevor and rif thought to be representative members in P. falciparum [19]. Recent evidence shows that P. vivax rosettes via binding to glycophorin C [109][42_TD$IF]. Additionally, vivax interspersed repeats (VIRs) from P. vivax and chabaudi interspersed repeats (CIR) [423_TD$IF]from rodent parasite P. chabaudi have [42_TD$IF]also been suggested as the ligand involved in rosetting in P. vivax and P. chabaudi respectively [110,111]. In P. falciparum three multigene families have now been identified that mediate rosetting. While extensive studies have so far been carried out on the role of PfEMP1 and adhesion, there is an increased understanding of the biological role of STEVOR and RIFIN. The demonstration that both RIFIN and STEVOR are important rosetting ligands and provide immune protection to the parasite now highlights the need to study these families in more detail (see Outstanding Questions). Acknowledgments This work was supported by the Singapore Ministry of Education Academic Research Fund Tier 1 grant (RG 20/12).

Outstanding Questions Is there a coordinated interplay between the variant antigens PfEMP1, STEVOR, and RIFIN on the surface of the infected red cell in mediating rosetting? Does the parasite modulate the expression of variants of all three families based on host receptor availability and host immune pressure? What are the binding regions and kinetics of these different sets of receptor–ligand interactions, and does this provide insights on why the parasite uses multiple receptors for the same function? Do STEVOR and RIFIN play a role in parasite sequestration/cytoadhesion? How important are STEVOR and RIFIN in mediating immune evasion? Do STEVOR and RIFIN mediate rosetting in clinical isolates, and does this contribute to disease severity? Do rosette-disrupting antibodies against variant surface antigens provide any clinical protection against the severe disease? Are STEVOR/RIFIN immune targets, and can they be used in combination as potential malaria vaccine candidates?

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