Members of the genus Plasmodium are eukaryotic microbes. Therefore, the cell and molecular biology of Plasmodium will be similar to other eukaryotes (see powerpoint presentations on basic molecular and cellular biology). 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 (Gratzer and Dluzewski
1993). Four distinct steps 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.
|
|
|
|
|
|
Apical Organelles of Plasmodium Merozoites |
||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
|
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 (Carruthers and Sibley
1999), as is typical of regulated secretion in other eukaryotes.
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. For example, RESA is localized to dense granules in merozoites and is transported to the host erythrocyte membrane shortly after merozoite invasion (Culvenor 1991). 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:
| Receptor/Ligand Interactions | |||||||||
|---|---|---|---|---|---|---|---|---|---|
|
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 domains is consistent with the parasite ligands being integral membrane proteins with the receptor-binding domain exposed on the merozoite surface following microneme discharge.
The other microneme proteins in the 'TRAP' family have also been implicated in locomotion and/or cell invasion (Tomley and Soldati 2001). All of these proteins have domains that are presumably involved in cell-cell adhesion, as well as N-terminal signal sequences and trans-membrane domains at their C-termini.
 |
 |
| Micrograph from Aikawa et al (1978) J. Cell Biol. 77:72 | |
In summary:
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 which exposes the receptor-binding domains of parasite ligands. This mechanism for initiating a tight host-parasite interaction is probably 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.
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.
 |
| 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 (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 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. Cytochalasins inhibit merozoite entry, but not attachment. This inhibition suggests that the force required for parasite invasion is based upon actin-myosin cytoskeletal elements. The ability of myosin to generate force is well characterized (eg., muscle contraction). A myosin unique to the Apicomplexa has been identified and localized to the plasma membrane in Plasmodium and Toxoplasma (Hettmann 2000; Pinder 2000).
Myosin motors, presumably associated with the cytoplasmic portion of the parasite ligands, could move along actin filaments within the parasite and drag the transmembrane ligand/receptor complexes through the fluid lipid bilayer toward the parasite posterior. The movement of the ring-like junction between the parasite and host towards the posterior of the merozoite results in a forward movement of the parasite into the host cell. Once the parasite has completed its entry, the tight junction will disappear and the respective PVM and the host erythrocyte membrane will fuse and separate, thus completing the entry process.
Plasmodium sporozoites and ookinetes, as well as the invasive stages of many other Apicomplexa, are motile organisms that crawl along a substratum. This movement along a substratum, or 'gliding motility', is accompanied by the deposition of a trail of TRAP (Sibley 1998). Gliding motility presumably involves attachment of the parasite ligands (i.e., TRAP) to the substratum. By virtue of their transmembrane domain, the TRAP proteins provide a connection from outside the cell to the intracellular parasite cytoskeleton. Presumably the C-terminal domain interacts with the myosin motor, which then associates with the actin filaments. The C-terminal regions of Plasmodium TRAP and Toxoplasma MIC2 are functionally homologous (Kappe 1999).
The force generated by myosin motors will pull TRAP towards the posterior of the parasite and cause a forward movement of the parasite. TRAP is then deposited on the substratum as it reaches the posterior end of the parasite and forms a trail. Gene disruption studies confirm that TRAP (Sultan 1997; Wengelnik 1999) and CTRP (Yuda 1999; Dessens 1999) play essential roles in both motility and cell invasion. In other words, during invasion the parasite 'crawls' into the host cell. The major difference between cell invasion and gliding motility will be the need for a continuous supply of TRAP and/or other surface proteins during motility.
|
|
|
|
|
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. However, much still remains to be learned about the cellular and molecular biology of merozoite invasion.
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 (Smith 2001).
The 40-50 var genes 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 (Smith 2001). 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.
|
|
|
|
|
|
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.)
| Binding Phenotypes | ||||||||||
|---|---|---|---|---|---|---|---|---|---|---|
|
The different types of DBL domains and CIDR (discussed above) bind to different endothelial cell receptors (Smith 2001; 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). 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.
 |
 |
| 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. |
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).
|
|
|
|
|
