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The host cell invasion model suggests that the parasite merozoite goes through three phases to enter the erythrocyte (Figure 8) (reviewed in Topolska et al., 2004).

These phases are: 1) an initial low-affinity interaction between any part of the merozoite and the host cell surface, followed by the reorientation of the merozoite to allow its apical prominence to contact the host cell; 2) irreversible attachment and formation of an electron-dense junction between the apical prominence and the host cell and finally; 3) invasion, in which the parasite propels itself forward into a membrane enclosed compartment called the parasitophorous vacuole, which eventually seals behind the parasite (Dvorak et al., 1975).

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Figure 8: Erythrocyte invasion by P. falciparum.

Diagram showing the three phases of invasion. The merozoite undergoes initial attachment and reorientation; followed by irreversible attachment and junction formation; and parasitophorous vacuole formation and invasion (Kats et al., 2006, originally adapted from Chitnis and Blackman, 2000).

During each phase a specific set of proteins located either on the surface of, or in specific organelles and compartments of the merozoite, are used to recognise and disrupt the erythrocyte (Figure 9). The organelles involved in invasion are the rhoptries, which are made up of two, large, tear-shaped organelles terminating at the apex of the parasite, and the micronemes, which are small tube-shaped structures associated with the rhoptries. Additional proteins are released from the dense granules, which are intermediate in size, spherical, and denser than the micronemes and rhoptries (Langreth et al., 1978).

The first phase of invasion occurs when the merozoite comes into contact with an uninfected erythrocyte. This contact is accidental in nature and results from the recognition of red cell receptors by the coat filaments of the merozoite. These filaments are made up of parasite proteins such as the merozoite surface proteins (MSP-1-10), serine-rich antigen (SERA/SERP) and S antigen. This initial interaction between parasite and host is weak, non-specific and reversible, and merozoites are often seen to dissociate from erythrocytes.

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Figure 9: The P. falciparum merozoite.

Diagram showing the structure and organelles of the P. falciparum merozoite. The merozoite contains secretory organelles involved in invasion, namely the micronemes, rhoptries and dense granules (Bannister et al., 2000).

Invasion continues with the parasite reorientating itself so that its apical end comes into contact with the erythrocyte surface. This reorientation strengthens the interaction between the merozoite surface and the red cell surface and eventually becomes irreversible, and the parasite commits itself to erythrocyte invasion.

After contact, intracellular stores of Ca²⁺ are released within the parasite (Lovett and Sibley, 2003) and this activates the discharge of parasite proteins, such as the erythrocyte binding antigens (EBA-175, EBA-140/BAEBL, EBA-181/JESEBL) and the apical membrane antigen 1 (AMA-1) from the micronemes. The erythrocyte binding antigens form a junction with the erythrocyte membrane receptors, such as Glycophorin A, B, and C/D (Hadley et al., 1987) and these interactions are to some degree redundant and interchangeable (Mayer et al., 2002). The tight junction remains during invasion and is seen as a ring that moves backwards over the parasite surface as the parasite actively invades the erythrocyte (Aikawa et al., 1978). Shortly after junction formation the rhoptries release their parasite proteins, namely the rhoptry associated proteins (RAP-1, RAP-2, RAP-3), high molecular weight rhoptry proteins (RhopH-1, RhopH-2,

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RhopH-3), and proteases (gp76), which aid in the invasion process. The rhoptries also discharge parasite lipids (Etzion et al., 1991). These rhoptry proteins and lipids, together with proteins and lipids from the host cell membrane, are used to form the parasitophorous vacuole membrane (Stewart et al., 1986). This membrane surrounds the parasitophorous vacuole which is a sack-like structure that provides a secure environment for the parasite to reside and grow in. The dense granules are the last organelles to discharge their protein content, such as the ring-infected erythrocyte surface antigen (RESA), ring membrane antigen (RIMA), and the subtilisin-like proteases (PfSUB-1, PfSUB-2). These proteins either integrate themselves into the parasitophorous vacuole or cross the parasitophorous vacuole membrane to interact with the erythrocyte membrane lipid bilayer and erythrocyte membrane skeleton proteins. PfSUB-2 has been implicated in the shedding of MSP-1 and AMA-1 from the parasite surface as it invades the erythrocyte (Howell et al., 2003). Another set of proteases, the rhomboids (PfROM-1 and PfROM-4), cleave AMA-1, thrombospondin-related anonymous protein (TRAP), circumsporozoite protein (CSP) and TRAP-related protein (CTRP), merozoite TRAP homologue (MTRAP), PFF0800c, EBA-175, EBA-140/BAEBL, EBA-181/JESEBL, MAEBL, and the reticulocyte-binding homologues (RH-1, RH-2a, RH-2b, and RH-4), and could therefore be involved in the shedding process (Baker et al., 2006).

Invasion requires the use of an actin/myosin motor complex termed the glideosome (Figure 10) (Keeley and Soldati, 2004, reviewed in Cowman and Crabb, 2006). This complex is located in the parasite’s pellicle, between the parasite plasma membrane and the inner membrane complex (IMC). The IMC is constructed from a single large flattened vesicle that is joined at a single suture line running the length of the long axis of the parasite (Morrissette and Sibley, 2002). The IMC is not found at the apical prominence (Bannister et al., 2000), thereby allowing the micronemes, rhoptries and dense granules to discharge their contents to the external environment. Within the glideosome, Plasmodium myosin (MyoA) (Pinder et al., 1998) attaches to the Myosin Tail Interacting Protein (MTIP) via its light chain and the other end of myosin interacts with actin (Webb

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et al., 1996). MTIP in turn interacts with glideosome-associated protein 45 (GAP45) and glideosome-associated protein 50 (GAP50) to link the glideosome to the IMC (Jones et al., 2006). The glideosome is also present in other invasive stages of Plasmodium, and in sporozoites it has been shown that the glideosome connects to the parasite plasma membrane via aldolase and the adhesin TRAP (Buscaglia et al., 2003). It is therefore thought that the link to the merozoite plasma membrane is formed by the secreted microneme or rhoptry ligands and possibly aldolase. The parasite gains entry into the host cell using the force that is generated by the actin-myosin motor. Because the motor is anchored to the IMC, the generated movement is highly directional and causes the parasite to be propelled forwards into the host cell. During invasion the adhesin-aldolase-actin complex moves towards the posterior end of the parasite and this complex is released from the host cell by proteases such as the subtilisin-like proteases and rhomboids mentioned previously.

Cytoskeleton IMC

EBA-175 ?

Sub-pellicular

Figure 10: The actin-myosin motor of P. falciparum.

Diagram showing the actin-myosin motor found in the P. falciparum glideosome.

The glideosome consists of glideosome-associated protein 50 (Gap50), Myosin Tail Interacting Protein (MTIP), glideosome-associated protein 45 (GAP45) and Plasmodium myosin (MyoA). It attaches to the inner membrane complex (IMC) via GAP50 and interacts with the actin filaments via MyoA. Aldolase and erythrocyte binding antigen-175 (EBA-175) could form the link between actin and the parasite plasma membrane. Myosin moves along the actin filaments and thereby moves the parasite into the host cell (adapted from Jones et al., 2006).

microtubes