Members of the genus Plasmodium are eukaryotic microbes. Therefore, the cell and molecular biology of Plasmodium will be similar to other eukaryotes. A unique feature of the malarial parasite is its intracellular lifestyle. Because of its intracellular location the parasite has an intimate relationship with its host cell which can be described at the cellular and molecular levels. In particular, the parasite must enter the host cell, and once inside, it modifies the host cell. The molecular and cellular biology of host-parasite interactions involved in these two processes will be discussed.
Malaria parasites are members of the Apicomplexa. Apicomplexa are characterized by a set of organelles found in some stages of the parasite's life cycle. These organelles, collectively known as apical organelles because of their localization at one end of the parasite, are involved in interactions between the parasite and host. In particular, the apical organelles have been implicated in the process of host cell invasion. In the case of Plasmodium, three distinct invasive forms have been identified: sporozoite, merozoite, and ookinete (see Plasmodium Life Cycle). The following discussion focuses on the cellular biology of merozoites and erythrocyte invasion. References to other Apicomplexa and Plasmodium sporozoites will be made to illustrate common features.
Merozoites rapidly (approximately 20 seconds) and specifically enter erythrocytes. This specificity is manifested both for erythrocytes as the preferred host cell type and for a particular host species, thus implying receptor-ligand interactions. Erythrocyte invasion is a complicated process which is only partially understood at the molecular and cellular levels. However, substantial progress has been made in identifying many of the parasite and host proteins that are important for the invasion process.
Four distinct steps (Gratzer and Dluzewski 1993) in the invasion process can be recognized (Figure):
The initial interaction between the merozoite and the erythrocyte is probably a random collision and presumably involves reversible interactions between proteins on the merozoite surface and the host erythrocyte. Several merozoite surface proteins have been described. The best characterized is merozoite surface protein-1 (MSP-1). Circumstantial evidence implicating MSP-1 in erythrocyte invasion includes its uniform distribution over the merozoite surface and the observation that antibodies against MSP-1 inhibit invasion (Holder 1994). In addition, MSP-1does bind to band 3 (Goel 2003). However, a role for MSP-1 in invasion has not been definitively demonstrated. Similarly, the circumsporozoite protein (CSP) probably plays a role in targeting sporozoites to hepatocytes by interacting with heparin sulfate proteoglycans (Sinnis and Sim 1997).
Another interesting aspect of MSP-1 is the proteolytic processing that is coincident with merozoite maturation and invasion (Cooper 1993). A primary processing occurs at the time of merozite maturation and results in the formation of several polypeptides held together in a non-covalent complex. A secondary processing occurs coincident with merozoite invasion at a site near the C-terminus. The non-covalent complex of MSP-1 polypeptide fragments is shed from the merozoite surface following proteolysis and only a small C-terminal fragment is carried into the erythrocyte. This loss of the MSP-1 complex may correlate with the loss of the 'fuzzy' coat during merozoite invasion. The C-terminal fragment is attached to the merozoite surface by a GPI anchor and consists of two EGF-like modules. EGF-like modules are found in a variety of proteins and are usually implicated in protein-protein interactions. One possibility is that the secondary proteolytic processing functions to expose the EGF-like modules which strengthen the interactions between merozoite and erythrocyte. The importance of MSP-1 and its processing are implied from the following observations:
The exact role(s) which MSP-1 and its processing play in the merozoite invasion process are not known. Other merozoite surface proteins are also involved in the interation of the merozoite with the erythrocyte (reviewed Cowman 2012).
Apical Organelles of
After binding to the erythrocyte, the parasite reorients itself so that the 'apical end' of the parasite is juxtaposed to the erythrocyte membrane. This merozoite reorientation also coincides with a transient erythrocyte deformation. Apical membrane antigen-1 (AMA-1) has been implicated in this reorientation (Mitchell 2004). AMA-1 is a transmembrane protein localized at the apical end of the merozoite and binds erythrocytes. Antibodies against AMA-1 do not interfer with the initial contact between merozoite and erythrocyte thus suggesting that AMA-1 is not involved in merozoite attachement. But antibodies against AMA-1 prevent the reorientation of the merozoite and thereby block merozoite invasion.
Specialized secretory organelles are located at the apical end of the invasive stages of apicomplexan parasites. Three morphologically distinct apical organelles are detected by electron microscopy: micronemes, rhoptries, and dense granules (Table). Dense granules are not always included with the apical organelles and probably represent a heterogeneous population of secretory vesicles.
The contents of the apical organelles are expelled as the parasite invades, thus suggesting that these organelles play some role in invasion. Experiments in Toxoplasma gondii indicate that the micronemes are expelled first and occur with initial contact between the parasite and host (Carruthers and Sibley 1997). An increase in the cytoplasmic concentration of calcium is associated with microneme discharge and likely involves a signal pathway involving phospholipase C, inositoltriphosphate and calcium dependent protein kinases (Sharma and Chitnis 2013).
The rhoptries are discharged immediately after the micronemes and the release of their contents correlate with the formation of the parasitophorous vacuole.
Dense granule contents are released after the parasite has completed its entry, and therefore, are usually implicated in the modification of the host cell. However, subtilisin-like proteases, which are implicated in the secondary proteolytic processing of MSP-1 (discussed above), have also been localized to Plasmodium dense granules (Blackman 1998, Barale 1999). If MSP-1 processing is catalyzed by these proteases, then at least some dense granules must be discharged at the time of invasion.
Following merozoite reorientation and microneme discharge a junction forms between the parasite and host cell. Presumably, microneme proteins are important for junction formation. Proteins localized to the micromenes include:
Of particular note are EBA-175 and DBP which recognize sialic acid residues of the glycophorins and the Duffy antigen, respectively (Table). In other words, these parasite proteins are probably involved in receptor-ligand interactions with proteins exposed on the erythrocyte surface. Disruption of the EBA-175 gene results in the parasite switching from a sialic acid-dependent pathway to a sialic acid-independent pathway (Reed 2000), indicating that there is some redundancy in regards to the receptor-ligand interactions.
Comparison of sequences of EBA-175 and DBP reveal conserved structural features. These include transmembrane domains and receptor-binding domains (Figure, modified from Adams 1992). The receptor-binding activity has been mapped to a domain in which the cysteine and aromatic amino acid residues are conserved between species (blue area in Figure). This putative binding domain is duplicated in EBA-175. The topography of the transmembrane domain is consistent with the parasite ligands being integral membrane proteins with the receptor-binding domain exposed on the merozoite surface following microneme discharge.
|Arrow denotes electron dense junction between merozoite and erythrocyte. Micrograph from Aikawa et al (1978) J. Cell Biol. 77:72.|
EBA-175 and DBP are part of a family of adhesins found in malaria parasites generally called EBL for eyrthrocyte-binding like. Another family of adhesins involved in the binding of merozoites to erthrocytes are the reticulocyte-binding like homologues (Rh). These various adhesins bind to distinct receptors on the erythrocyte and provide redundancy in the ability of the merozoite to form a junction with the erythrocyte (Tham 2012). The basigin receptor may be essential for the invasion of P. falciparum (Crosnier 2011).
Other microneme proteins in the 'TRAP' family have also been implicated in locomotion and/or cell invasion of the sporozoite stage and other apicomplexa (Tomley and Soldati 2001). All of these proteins (EBL, Rh, TRAP families) have domains that are presumably involved in cell-cell adhesion, as well as trans-membrane domains at their C-termini. Microneme release would expose the adhesive domains, which would then bind to receptors on the host cell and thereby form a junction between the invasive form (eg, merozoite) and the host cell (Figure).
Another element of this junction involves a proteins found in the neck of the rhopthry, in particular RON2. RON2 is release from the rhoptry and inserted into the membrane of the erythrocyte. RON2 then binds to AMA-1 which is localized to the surface of the merozoite (Tonkin 2011). The RON2/AMA-1 complex then also contributes to this tight junction formed between the merozoite and erythrocyte. Phosphorylation of AMA-1 may also be involved in parasite entry (Leykauf 2010).
These observations suggest that the junction represents a strong connection between the erythrocyte and the merozoite which is mediated by receptor-ligand interactions. Junction formation may be initiated by microneme discharge followed by release of rhoptry neck proteins which exposes the receptor-binding domains of parasite ligands. This mechanism for initiating a tight host-parasite interaction is similar in other invasive stages of apicomplexan parasites.
Apicomplexan parasites actively invade host cells and entry is not due to uptake or phagocytosis by the host cell. This is particularly evident in the case of the erythrocyte which lacks phagocytic capability. Furthermore, the erythrocyte membrane has a 2-dimensional submembrane cytoskeleton which precludes endocytosis. Therefore, the impetus for the formation of the parasitophorous vacuole must come from the parasite. Several events happen during parasite entry including: 1) the disruption of the the submembrane cytoskeleton of the erythrocyte, 2) the formation of the parasitophorous vacuole, and 3) and the shedding of merozoite surface proteins. Parasite entry is driven by an acto-myosin motor complex called the glidosome.
Erythrocyte membrane proteins are redistributed at the time of junction formation so that the contact area is free of erythrocyte membrane proteins. A merozoite serine protease which cleaves erythrocyte band 3 has been described (Braun-Breton 1993). Because of the pivotal role band 3 plays in the homeostatis of the submembrane skeleton, its degradation could result in a localized disruption of the cytoskeleton. Reorganization of the submembrane cytoskeleton and lipid architecture probably accompanies merozoite invasion (Zuccala 2011).
|Micrograph from Aikawa et al (1978) J. Cell Biol. 77:72|
An incipient parasitophorous vacuolar membrane (PVM) forms in the junction area. This membrane invagination is likely derived from both the host membrane and parasite components and expands as the parasite enters the erythrocyte. Connections between the rhoptries and nascent PVM are sometimes observed (Figure, arrow). In addition, the contents of the rhoptries are often lamellar (i.e., multi-layered) membranes and some rhoptry proteins are localized to the PVM following invasion, suggesting that the rhoptries also function in PVM formation (Sam-Yellowe 1996).
Ookinetes lack rhoptries and do not form a parasitophorous vacuole within the mosquito midgut epithelial cells. The ookinetes rapidly pass through the epithelial cells and cause extensive damage as they head toward the basal lamina (Han 2000, Ziegler 2000). Similarly, sporozoites can enter and exit hepatocytes without undergoing exoerythrocytic schizogony. Those parasites which do not undergo schizogony are free in the host cytoplasm, whereas those undergoing schizogony are enclosed within a PVM (Mota 2001). These observations suggest that the PVM is needed for intracellular development and is not necessary for the process of host cell invasion. As the incipient parasitophorous vacuole is being formed, the junction (denoted with C's in figure) between the parasite and host becomes ring-like and the parasite appears to move through this annulus as it enters the expanding parasitophorous vacuole. Instead this moving junction is being pulled from the front of the parasite to the rear resulting in a forward movement of the parasite into the host cell.
As the parasite enters, MSP-1 many of the merozoite surface proteins are shed. This shedding process is mediated by specific proteases and is an ordered process (Boyle 2014).
The invasive forms of apicomplexan parasites are often motile forms that that crawl along the substratum by a type of motility referred to as ‘gliding motility’. Gliding motility, like invasion, also involves the release of micronemal adhesins, attachment to the substratum, and a capping of the adhesins at the posterior end of the zoite. One difference between gliding motility and invasion is that the micronemes must be continuously released as the organism is moving. Thus, gliding motility does not involve this relatively small moving junction, but a continuous formation of new junctions between the zoite and the substratum. In addition, the adhesins are cleaved from the surface of the zoite as the adhesions reach the posterior of the zoite and a trail of the adhesive molecules are left behind the moving zoite on the substratum. The mechanism of motility and invasion are quite similar and thus, during invasion the parasite literally crawls into the host cell through the moving junction. In addition, some apicomplexans use this type of motility to escape from cells and can traverse biological barriers by entering and exiting cells.
|Model of the moving junction complex and glidesome driving gliding motility from Besteiro (2011).|
Cytochalasins inhibit merozoite entry, but not attachment, thus suggesting that the force required for parasite invasion and gliding motility is based upon actin-myosin cytoskeletal elements. The ability of myosin, a motor protein, to generate force is well known (eg., muscle contraction). A myosin unique to the Apicomplexa has been identified and is anchored into the inner membrane complex (IMC). The IMC refers to the double membrane lying under the plasma membrane on invasive stages of Apicomplexan parasites. This IMC is further supported by sub-pellicular microtubules that run the length of the parasite. The IMC associated myosin interacts with actin as part of the glidesome. The various adhesins (ie, EBL, Rh, TRAP and AMA-1) making up the moving junction (MJ) complex are then linked to the glidesome (Figure).
Members of the TRAP family and other adhesins have a conserved cytoplasmic domain. This cytoplasmic domain is linked to short actin filaments via aldolase. The actin filaments and myosin are oriented in the space between the inner membrane complex and plasma membrane so that the myosin propels the actin filaments toward the posterior of the zoite. The myosin is anchored into the IMC and does not move. Therefore, the transmembrane adhesins are pulled through the fluid lipid bilayer of the plasma membrane due to their association with the actin filaments. Thus the complex of adhesins and actin filaments is transported towards the posterior of the cell. Since the adhesins are either complexed with receptors on the host cell and anchored to the host cell cytoskeleton, or bound to the substratum, the net result is a forward motion of the parasite (Figure). When the adhesins reach the posterior end of the parasite they are proteolyitcally cleaved and shed from the zoite surface.
In the case of cell invasion the PVM and host cell membrane will need to sealed so that the PVM is intact and surrounding the parasite and the host plasma membrane is also intact. The mechanisms involved in this last step of invasion are not known.
Merozoite invasion is a complex and ordered process. A tentative model of merozoite invasion includes:
Many proteins that are involved in the invasion process have been identified. This will also include signalling events between the various steps of invasion (Santos and Soldati-Favre 2011). However, much still remains to be learned about the cellular and molecular biology of merozoite invasion. A better understanding of the complex process of parasite invasion could lead to the development of novel therapeutic approaches to malaria and other diseases caused by Apicomplexans.
Once inside of the erythrocyte, the parasite undergoes a trophic phase followed by replicative phase. During this intraerythrocytic period, the parasite modifies the host to make it a more suitable habitat. For example, the erythrocyte membrane becomes more permeable to small molecular weight metabolites, presumably reflecting the needs of an actively growing parasite (see Uptake and Permeability).
Another modification of the host cell concerns the cytoadherence of P. falciparum-infected erythrocytes to endothelial cells and the resulting sequestration of the mature parasites in capillaries and post-capillary venules. This sequestration likely leads to microcirculatory alterations and metabolic dysfunctions which could be responsible for many of the manifestations of severe falciparum malaria (see pathogenesis). The cytoadherence to endothelial cells confers at least two advantages for the parasite: 1) a microaerophilic environment which is better suited for parasite metabolism, and 2) avoidance of the spleen and subsequent destruction.
A major structural alteration of the host erythrocyte are electron-dense protrusions, or 'knobs', on the erythrocyte membrane of P. falciparum-infected cells. The knobs are induced by the parasite and several parasite proteins are associated with the knobs (Deitsch and Wellems 1996). Two proteins which might participate in knob formation or affect the host erythrocyte submembrane cytoskeleton and indirectly induce knob formation are the knob-associated histidine rich protein (KAHRP) and erythrocyte membrane protein-2 (PfEMP2), also called MESA. Neither KAHRP nor PfEMP2 are exposed on the outer surface of the erythrocyte, but are localized to the cytoplasmic face of the host membrane (Figure). Their exact roles in knob formation are not known, but may involve reorganizing the submembrane cytoskeleton.
The knobs are believed to play a role in the sequestration of infected erythrocytes since they are points of contact between the infected erythrocyte and vascular endothelial cells and parasite species which express knobs exhibit the highest levels of sequestration. In addition, disruption of the KAHRP results in loss of knobs and the ability to cytoadhere under flow conditions (Crabb 1997). A polymorphic protein, called PfEMP1, has also been localized to the knobs and is exposed on the host erythrocyte surface. The translocation of PfEMP1 to the erythrocyte surface depends in part on another erythrocyte membrane associated protein called PfEMP3 (Waterkeyn 2000). PfEMP1 probably functions as a ligand which binds to receptors on host endothelial cells. Other proposed cytoadherence ligands include a modified band-3, called pfalhesin (Sherman 1995), sequestrin, rifins and clag9 (Craig and Scherf 2001).
PfEMP1 is a member of the var gene family (Hviid 2015). Each parasite has an estimated 40-60 var genes which exhibit a high degree of variability, but have a similar overall structure (Figure). PfEMP1 has a large extracellular N-terminal domain, a transmembrane region and a C-terminal intracellular domain. The C-terminal region is conserved between members of the var family and is believed to anchor PfEMP1 to the erythrocyte submembrane cytoskeleton. In particular, this acidic C-terminal domain may interact with the basic KAHRP of the knob (Waller 1999) as well as spectrin and actin (Oh 2000).
The extracellular domain is characterized by 1-5 copies of Duffy-binding like (DBL) domains. These DBL domains are similar to the receptor-binding region of the ligands involved in merozoite invasion (discussed above). The DBL domains exhibit a conserved spacing of cysteine and hydrophobic residues, but otherwise show little homology. Phylogenetic analysis indicates that there are five distinct classes (designated as a, b, g, d, and e) of DBL domains (Hviid 2015). The first DBL is always the same type (designated a) and this is followed by a cysteine-rich interdomain region (CIDR). A variable number of DBL in various orders make up the rest of the extracellular domain of PfEMP-1.
During each mitotic cycle the var genes undergo recombination leading to the continuous generation of additional variants (Claessens 2014). Interestingly, the structure of the var gene family has been present since before the P. falciparum and P. reichenowi divergence which occurred more than two million years ago (Zilversmit 2013).
Possible Receptors Identified
Several possible endothelial receptors (Box) have been identified by testing the ability of infected erythrocytes to bind in static adherence assays (Beeson and Brown 2002). One of the best characterized among these is CD36, an 88 kDa integral membrane protein found on monocytes, platelets and endothelial cells. Infected erythrocytes from most parasite isolates bind to CD36 and the binding domain has been mapped to the CIDR of PfEMP1 (see Figure). However, CD36 has not been detected on endothelial cells of the cerebral blood vessels and parasites from clinical isolates tend to adhere to both CD36 and intracellular adhesion molecule-1 (ICAM1). ICAM1 is a member of the immunoglobulin superfamily and functions in cell-cell adhesion. In addition, sequestration of infected erythrocytes and ICAM1 expression has been co-localized in the brain (Turner 1994).
Chondroitin sulfate A (CSA) has been implicated in the cytoadherence within the placenta and may contribute to the adverse affects of P. falciparum during pregnancy. The role of some of the other potential receptors is not clear. For example, adherence to thrombospondin exhibits a low affinity and cannot support binding under flow conditions. Binding to VCAM1, PECAM1 and E-selectin appears to be rare and questions about their constitutive expression on endothelial cells have been raised. However, cytoadherence could involve multiple receptor/ligand interactions.
Rosetting is another adhesive phenomenon exhibited by P. falciparum-infected erythrocytes. Infected erythrocytes from some parasite isolates will bind mutiple uninfected erythrocytes and PfEMP1 appears to have a role in at least some rosetting. Possible receptors include complement receptor-1 (CR1), blood group A antigen, or glycosaminoglycan moieties on an unidentified proteoglycan. (See figure depicting possible receptor-ligand interactions involved in rosetting on another webpage.)
The different types of DBL domains and CIDR (discussed above) bind to different endothelial cell receptors (Craig and Scherf 2001). For example, DBLa, which comprises the first domain, binds to many of the receptors associated with rosetting. The binding of the CIDR to CD36 may account for the abundance of this particular binding phenotype among parasite isolates.
(A webpage assembled by Hagai Ginsburg contains detailed figures depicting many aspects of host-parasite interactions, including: knob composition and receptor-ligand interactions, PfEMP-1 structure and binding specificities, endothelial cell receptors and rosetting. Sherman et al (2003) has reviewed the mechanisms of cytoadherence.)
The encoding of the cytoadherence ligand by a highly polymorphic gene family presents a paradox in that receptor/ligand interactions are generally considered highly specific. Interestingly, selection for different cytoadherent phenotypes result in a concommitant change in the surface antigenic type (Biggs 1992). Similarly, examination of clonal parasite lines revealed that changes in the surface antigenic type correlated with differences in binding to CD36 and ICAM1. For example, the parental line (A4) adhered equally well to CD36 and ICAM1, whereas one of the A4-derived clones (C28) exhibited a marked preference for CD36 (Figure, modified from Roberts 1992). Binding to ICAM1 was then reselected by panning the infected erythrocytes on ICAM1. All three parasite clones (A4, C28, C28-I) exhibited distinct antigenic types as demonstrated by agglutination with hyper-immune sera.
The expression of a particular PfEMP1 will result in a parasite with a distinct cytoadherent phenotype and this may also affect pathogenesis and disease outcome. For example, binding to ICAM-1 is usually implicated in cerebral pathology. Therefore, parasites expressing a PfEMP1 which binds to ICAM1 may be more likely to cause cerebral malaria. In fact, higher levels of transcription of particular var genes are found in cases of severe malaria as compared to uncomplicated malaria (Rottmann 2006). Similarly, a higher proportion of isolates which bind to CSA are obtained from the placenta as compared to the peripheral circulation of either pregnant women or children (Figure, modified from Beeson 1999). Furthermore, placental malaria is frequently associated with higher levels of transcription of a particular var gene which binds CSA (Duffy 2006). This phenomenon is not restricted to the placenta in that there is a dominant expression of particular var genes in the various tissues (Figure, from Montgomery 2007). Subsequent work has confirmed that the various variants of PfEMP1 have different tropisms for different tissues (Smith 2014).
|Figure, modified from Beeson 1999. Shows the proportions of isolates that bind to CSA, CD36, or ICAM-1. Infected erythrocytes were collected from the placenta, peripheral circulation of the mother, or peripheral circulation of the child.||Figure, from Montgomery 2007. Shows the proportion of the various types of PfEMP1 (designated as groups 1-6) expressed in different tissues (brain, lung, heart and spleen) from 3 different patients. PM30 died of severe malaria anemia. PM32 was diagnosed with both cerebral malaria and severe anemia. PM55 was diagnosed with only cerebral malaria.|
More recently it has been demonstrated that a distinct subset of var genes are highly transcribed following selection on human brain endothelial cells and that these same distinct subtypes are associated with cerebral malaria (Aird 2014; Cunnington 2013; Smith 2013). This tissue specific expression of particular var genes implies that different tissues are selecting out different parasite populations based on the particular PfEMP1 being expressed on the surface of the infected erythrocyte.
Although sequestration offers many advantages to the parasite, the expression of antigens on the surface of the infected erythrocyte provides a target for the host immune system. The parasite counters the host immune response by expressing antigenically distinct PfEMP1 molecules on the erythrocyte surface. This allows the parasite to avoid clearance by the host immune system, but yet maintain the cytoadherent phenotype. This antigenic switching may occur as frequently as 2% per generation in the absence of immune pressure (Roberts 1992). The molecular mechanism of antigenic switching is not known. Experimental evidence indicates that the mechanism is not associated with duplicative transposition into specific expression-linked sites as found in African trypanosomes. Only a single var gene is expressed at a time (i.e., allelic exclusion). The non-expressed genes are kept silent by proteins which bind to the promoter region. A gene can become activated by repositioning to a particular location in the nucleus and is associated with chromatin modification. This expression spot can only accommodate a single active gene promoter. Thus the var promoter is sufficient for both the silencing and the mono-allelic transcription of a PfEMP1 allele (Voss 2006; Guizetti 2013).