Summary

Introduction

Clonal antigenic variation is a strategy employed by many microbial pathogens to vary surface proteins to evade immunity. The human malaria parasite, Plasmodium falciparum, varies a family of adhesion proteins at the infected erythrocyte (IE) surface that it uses to bind to the endothelial lining of blood microvessels (Miller et al., 2002). During blood stage infection, P. falciparum merozoites sequentially invade and egress from red blood cells every 48 hours. As the parasite matures, IEs exhibit reduced deformability and sequester from blood circulation. This allows the parasite to avoid splenic clearance mechanisms, but comes at a cost to the host. Sequestered IEs disrupt microvascular blood flow (Dondorp et al., 2008) and cause localized endothelial dysfunction by damaging endothelial barrier integrity and inducing pro-inflammatory, pro-adhesive, and coagulation pathways (Francischetti et al., 2008; Miller et al., 2013; Moxon et al., 2009). This adhesive phenotype is also associated with organ-specific disease complications from IE adherence in brain (cerebral malaria) and placenta (placental malaria) microvasculature (Miller et al., 2002).

Cytoadhesion is mediated by specific interactions between members of the P. falciparum erythrocyte membrane protein 1 (PfEMP1) family encoded by var genes and receptors on the surfaces of endothelial cells (Rowe et al., 2010). Each parasite genotype encodes ~60 different var genes, which are expressed in a mutually exclusive fashion (Guizetti and Scherf, 2013). Switching between var genes facilitates parasite immune evasion and modifies IE binding specificity (Roberts et al., 1993). A specific PfEMP1 variant, VAR2CSA, interacts with chondroitin sulfate A, which is abundant within the placental intervillous space (Fried and Duffy, 1996; Salanti et al., 2004). In contrast, cerebral binding and severe childhood malaria is associated with specific PfEMP1 variants containing a combination of adhesion domains, termed domain cassettes (DC) 8 and 13 (Avril et al., 2012; Claessens et al., 2012; Lavstsen et al., 2012). The vast majority of P. falciparum infections do not lead to severe malaria, suggesting that IE sequestration is relatively well-adapted to limit host death and favor parasite transmission to mosquitoes. It is therefore interesting that potentially lethal adhesion traits persist in the parasite population. This review covers recent advances in the molecular mechanisms of PfEMP1 binding, integrating findings on protein structure-function, var gene evolution, and adhesion phenotypes associated with severe malaria.

Major adhesion traits of P. falciparum infected erythrocytes

P. falciparum IEs have evolved to bind a diverse array of receptors on different human cell types. Two major adhesion traits that may contribute to severe childhood malaria are endothelial binding and rosetting with uninfected erythrocytes. Adhesion also occurs to platelets, dendritic cells, B cells, monocytes and macrophages to modulate host immune functions, a topic reviewed elsewhere (Chua et al., 2013).

Molecular mechanisms of IE binding

PfEMP1 proteins contain a polymorphic ectodomain, linked through a single transmembrane helix to a relatively conserved cytoplasmic region (Baruch et al., 1995; Smith et al., 1995; Su et al., 1995). The cytoplasmic tail anchors PfEMP1 proteins at parasite-induced knob-like protrusions at the erythrocyte surface (Maier et al., 2009). The PfEMP1 ectodomain is built from a combination of Duffy binding-like (DBL) and cysteine-rich interdomain region (CIDR) adhesion domains (Smith et al., 2000). These binding modules are only found in Plasmodium. The DBL superfamily bridges two important adhesive properties of P. falciparum, having a key role in erythrocyte invasion (DBL-containing erythrocyte binding proteins, DBL-EBPs) and IE cytoadhesion interactions (DBL-PfEMP1) (Miller et al., 2002).

Genomic organization of var genes

Members of the var gene family are concentrated in sub-telomeric regions of all chromosomes and internal regions on chromosomes 4, 7, 8, and 12 (Gardner et al., 2002). Comparison of the 3D7 genome reference isolate and six partial P. falciparum genomes highlights many commonalities between var repertoires (Rask et al., 2010). In particular, the majority of var genes can be classified into three main groups (A, B, and C) on the basis of upstream sequence (Ups), chromosome location, and direction of transcription (Fig. 1). Surprisingly, the var gene repertoire also contains three strain-transcendent variants (var1, var2csa, and type3 var) found in most or all parasite genotypes. It is thought that this genome organization contributes to gene recombination hierarchies and functional specialization of PfEMP1 proteins (Kraemer and Smith, 2006). Intriguingly, UpsA, UpsB, and UpsC var genes and partial var1 and var2csa homologs are present in the highly fragmentary P. reichenowi genome sequence (Rask et al., 2010). This reinforces the ancient origins of the var genome organization and suggests the possibility that functional specialization of proteins may have occurred early.

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Fig. 1

Var gene organization and protein domain architectures. A. Chromosomal organization of var genes. Group B and A var genes are located in the subtelomeric regions and transcribed in opposite orientations. Group C are typically found on central chromosome clusters. B. There is a recombination hot spot between subdomains 2 and 3 in the DBL domain of the PfEMP1 head structure. DC8 is an unusual chimeric gene between a group B and group A var gene. C. Different types of DBL and CIDR domains are located at the N- and C-terminus of proteins. Small PfEMP1 have four extracellular domains, large PfEMP1 have additional DBL domains after the head structure or the second DBL-CIDR structure.

Structure-Function of PfEMP1 proteins

The modular architecture of PfEMP1s and arrangement of ligand binding domains

Although PfEMP1 differ in size and number of extracellular domains, nearly all proteins have a tandem DBL-CIDR domain at the N-terminus, called the semi-conserved head structure. In small proteins, this is followed by a second DBL-CIDR tandem forming a 4 domain extracellular unit, while large proteins have additional types of DBL domains (Fig. 1). By sequence similarity, DBL and CIDR domains are classified into major types (α, β, γ, δ, etc.) and sub-types (e.g. CIDRα1.1) (Rask et al., 2010; Smith et al., 2000). Sequence classification likely reflects structural and functional specialization of domains. For instance, the PfEMP1 head structure is nearly always comprised of a DBLα-CIDRα tandem and other DBL and CIDR sequence types (β, γ, δ, etc.) are located C-terminal to these (Fig. 1) (Rask et al., 2010). Furthermore, group A head structures have diverged in sequence and binding properties from group B and C (Robinson et al., 2003) (Fig. 2).

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Fig. 2

Model of PfEMP1 head structure divergence. The PfEMP1 head structure has diverged into three major binding groups under selection for EPCR-binding, CD36-binding, or to form rosettes with uninfected erythrocytes. The proportion of different head structure types in the 3D7 reference genome isolate and their predicted binding properties is shown. Note there may be some binding exceptions (e.g. only a subset of CIDRα1 domains bind EPCR, see Fig. 1). CD36 binding is the most common PfEMP1 adhesion trait and is associated with mild malaria. EPCR-binding and rosetting is linked to group A head structures. Group A PfEMP1 tend to be expressed in early childhood infections or malaria naïve, and are also associated with severe malaria. Under normal circumstances, protein C (PC) binds to EPCR and is activated by the thrombin (T)/thrombomodulin (TM) complex. Activated protein C (APC) that is released into the plasma has anti-coagulant activity and the APC/EPCR complex activates the protease activated receptor 1 (PAR1) to mediate intracellular signaling. The protein C-EPCR signalling pathway has anti-inflammatory, anti-thrombotic and endothelial cytoprotective activities that help maintain vascular integrity (Mosnier et al., 2007). The loss of EPCR at sites of P. falciparum IE sequestration (Moxon et al., 2013) and EPCR-binding parasites (Turner et al., 2013) may combine to interfere or subvert these pathways and contribute to disease pathogenesis.

The modular nature of PfEMP1s naturally led to the suggestion that they operate as strings of distinct ligand binding modules and that DBL-CIDR domains may form a structure-function unit. This prediction is supported in that many binding properties have been ascribed to single domains, with for example, ICAM1 binding to DBLβ domains (Howell et al., 2008) and CD36 to CIDRα in the PfEMP1 head structure (Baruch et al., 1997). However, specific adhesion traits, such as CSA binding, appear to involve a combination of domains (Clausen et al., 2012; Srivastava et al., 2011). Recent computational analysis identified 21 tandem domain arrangements of two or more domains (e.g. DC8 or DC13) (Fig. 1), which are highly conserved across parasite genotypes (Rask et al., 2010). This conservation suggests that DCs may be maintained as larger structure-function units, encoding one or more adhesion properties.

Several lines of evidence suggest PfEMP1 ectodomains have higher order organization. For instance, the structure of a DBL1α-CIDR1γ tandem domain reveals close packing between the DBL and CIDR domains to generate a rigid structural unit (Vigan-Womas et al., 2012). In addition, two small angle x-ray scattering (SAXS) studies show that full-length PfEMP1 ectodomains can adopt different shapes. Whereas full-length VAR2CSA ectodomain folds back on itself into a compact structure (Clausen et al., 2012; Srivastava et al., 2010), a CD36 and ICAM-1 binding PfEMP1 variant has an extended structure (Brown et al., 2013). These different arrangements may facilitate specific PfEMP1-receptor interactions and align pRBCs with cell surface receptors. Despite the availability of isolated DBL and CIDR structures, a major unanswered question is how PfEMP1 proteins recognize different protein or carbohydrate substrates. Additional sequencing and analysis of var genes paired with studies of PfEMP1 domain and ectodomain structures in complex with host receptors will be needed to fully understand which surface determinants are needed for PfEMP1 binding specificity and how PfEMP1 ectodomains are organized to present these binding surfaces to host receptors.

Group A var genes and severe childhood malaria

African children with severe malaria suffer from four main complications — coma (cerebral malaria), respiratory distress, hypoglycaemia and severe anaemia. There is immunoepidemiological data that group A PfEMP1 variants are associated with early childhood infections (Cham et al., 2009; Jensen et al., 2004; Lavstsen et al., 2012) and that severe childhood malaria infections are linked to the expression of a restricted subset of PfEMP1 (Bull et al., 2000; Nielsen et al., 2002). The continuous release of var sequence data has enabled increasingly more sophisticated molecular tools to solve the challenge of detecting and typing transcript expression of this diverse gene family. Although different approaches are used for var profiling making it difficult to compare between studies, a consensus is emerging that group A-like var genes are increased in severe malaria infections. Within the group A, rosetting parasites were more strongly linked to respiratory distress, and non-rosetting, group A-like genes with cerebral malaria in Kenya (Warimwe et al., 2012). Furthermore, using new primers sets that are specific to different PfEMP1 domain types, DC8 and DC13 var genes (Lavstsen et al., 2012) and EPCR binding (Turner et al., 2013) are strongly linked to cerebral malaria and other severe malaria complications. Taken together, these findings suggest that PfEMP1 proteins have diverged into CD36 binding and non-CD36 binding subsets (Fig. 2). The non-CD36 binding group A subset may confer a growth and transmission advantage in malaria naïve hosts, although this is balanced by a greater risk of severe malaria outcomes. An important question is whether all EPCR-binding or rosetting variants carry the same risk of severe outcomes, or if the combination of adhesion traits associated with specific EPCR-binding or rosetting PfEMP1 variants influences the extent of cerebral sequestration or disease severity. Currently, most studies have used broadly cross-reactive DBLα primers for var profiling and the new PfEMP1 domain type specific primers (e.g. DC8) have been only been evaluated on patient isolates from Tanzania (Lavstsen et al., 2012). The two approaches amplify different regions of var genes and are subject to different limitations in detecting different var types (Lavstsen et al., 2012). In the future, further application of these combined molecular tools for profiling var gene expression in combination with EPCR binding and rosetting assays are needed to fully resolve the question of which PfEMP1 subtypes are most likely to cause severe disease in patients from different regions of the world.

Conclusions

Despite the extensive diversification of var genes, recent work suggests that different var gene repertoires encode similar compositions of proteins, which are under selection for different host or microvascular niches. These protein groups can be classified by chromosome location and domain composition and appear to be maintained by ancient gene recombination hierarchies. Analysis of the PfEMP1 head structure indicates it has diverged into CD36-binding, EPCR-binding, and rosetting variants, which have different roles in mild versus severe malaria. Intriguingly, many of the known parasite cytoadhesion receptors are shared between endothelial cells and leukocytes, and thus it will be important to understand if there is selection on PfEMP1s between strong endothelial adhesion and host inflammatory properties. The finding that DC8 and DC13 variants encode EPCR binding activity has important implications for malaria pathogenesis. The ability of DC8 and DC13 variants to bind avidly to diverse endothelial cell types (Avril et al., 2013) may benefit parasites by conferring a growth and transmission advantage in malaria naïve hosts. However, this adhesion trait may also interfere with protein C-EPCR signaling pathways (Moxon et al., 2013; Turner et al., 2013), resulting in deregulated inflammation and coagulation pathways and contribute to severe malaria complications. To design disease interventions, it will be important to better understand how IE-host receptor binding interactions may be manipulating the host environment to the parasite benefit.

Acknowledgements

Footnotes

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