**26. Specific interactions and junction formation**

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 glycohorins and the Duffy antigen, respectively. In other words, these parasite protein are probably involved in receptor-ligand interaction with proteins exposed on the erythrocyte surface. Disruption of the EBA-175 gene results in the parasite switchingfrom a sialic aciddependentpathway to a sialic acid-independent pathway [64], indicating that there is some redundancy with regards to the receptor – ligand interactions.

Comparison of sequences of EBA-175 and DBP reveal conserved structural features. These include trans-membrane domains and receptor-binding domains.[65] The receptor-binding activity has been mapped to a domain in which the cysteine and aromatic amino acid residues are conserved between species. This putative binding domain is duplicated in EBA-175. The topography of the trans-membrane domain 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 protein in the 'TRAP' family have also been implicated in locomotion and / or cell invasion.[66] All of these proteins have domains that are presumably involved in cell-cell adhesion, as N-terminal single sequences and trans-membrane domains at their C- terminal.

Biology of Malaria Parasites 27

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 adhesion reach the posterior of the zotie and a trail of the adhesive molecules are left behind the moving zoite on the substratum. However, 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. 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 inner membrane complex.[72] This myosin is part of a motor complex which is linked to the adhesins. Members of the TRAP family and other adhesins have a conserved cytoplasmic domain. This cytoplamic 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 filament toward the posterior of the zoite. The myosin is anchored into the inner membrane complex 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 filament. Thus the complex of adhesins and actin filaments is transported towards the posterior of the cell. Since the adhesins are either complexes with receptor on the host cell or bound to the substratum, the net result is a forward motion of the zoite. When the adhesins reach the posterior end of the parasite

they are proteolyitcally cleaved and shed from the zoite surface.

being juxtaposed to the erythrocyte membrane.

proteins exposed by microneme discharge.

6. Closure of the PVM and erythrocyte membrane

Merozoite invasion is a complex and ordered process. A tentative model of merozoite

1. Initial merozotie binding involves reversible interactions between merozotie surface proteins and the host erythrocyte. The exact roles of MSPI and other merozoite surface

2. Reorientation by an unknown mechanism result in the apical end of the merozotie

3. Discharge of the micronemes is coincident with the formation of a tight junction between the host and parasite. The tight junction is mediated by receptor-ligand interactions between erythrocyte surface proteins and integral parasite membrane

4. localized clearing of the erythrocyte submembrane cytoskeleton and formation of the incipient parasitophorous vacuole. PVM formation is correlated with the discharge of

5. movement of the merozoite through the ring-shaped tight junction formed by the receptor/ligand complex. The force is generated by myosin motor associated with the trans-membrane parasite ligands moving along actin filaments within the parasite.

**29. Summary** 

invasion includes:

the rhoptries.

proteins are not known.

### **27. In summary**


These observations suggest that the junction represents a strong connection between the erythrocyte and the merozoite which is mediated by receptor-ligand interaction. 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.

## **28. Parasite entry**

Apicomplexan parasite 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.[67]. Because of the pivotal role band 3 plays in the homeostasis of the submembrane skeleton, its degradation could result in a localized disruption of the cytoskeleton. An incipient parasitophorous vacular membrane (PVM) forms in the junction area. This membrane invagination is likely derived from both the host membrane and parasite component and expands as the parasite enters the erythrocyte. Connection between the rhoptries and nascent PVM are sometimes observed. In addition, the contents of the rhoptries are often lamellar (i.e. multilayered) membrane and some rhoptry proteins are localized to the PVM following invasion, suggesting that the rhoptries function in PVM formation.[68]

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.[69,70] Similarly, sporozoites can enter and exit hepatocytes without undergoing exoerythrocytic schizogony. Those parasites which do not undergo schzogony are free in the host cytoplasm, whereas those undergoing schizogony are enclosed within a PVM.[71] 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 between the parasite and host becomes ring-like and the parasite appear to move through this annulus as it enters the expanding parasitophotous vacuole.

Apicomplexan parasite activity invade host cell and entry is not due to uptake or phagocytosis by the host cell. In addition, the zoites are often motile forms 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 adhesions at the posterior end of the zoite. One difference between gliding motility and invasion is that the

• An electron-dense junction forms between the apical end of the merozoite and host

These observations suggest that the junction represents a strong connection between the erythrocyte and the merozoite which is mediated by receptor-ligand interaction. 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

Apicomplexan parasite 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.[67]. Because of the pivotal role band 3 plays in the homeostasis of the submembrane skeleton, its degradation could result in a localized disruption of the cytoskeleton. An incipient parasitophorous vacular membrane (PVM) forms in the junction area. This membrane invagination is likely derived from both the host membrane and parasite component and expands as the parasite enters the erythrocyte. Connection between the rhoptries and nascent PVM are sometimes observed. In addition, the contents of the rhoptries are often lamellar (i.e. multilayered) membrane and some rhoptry proteins are localized to the PVM following invasion, suggesting that the

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.[69,70] Similarly, sporozoites can enter and exit hepatocytes without undergoing exoerythrocytic schizogony. Those parasites which do not undergo schzogony are free in the host cytoplasm, whereas those undergoing schizogony are enclosed within a PVM.[71] 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 between the parasite and host becomes ring-like and the parasite appear to move through this

Apicomplexan parasite activity invade host cell and entry is not due to uptake or phagocytosis by the host cell. In addition, the zoites are often motile forms 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 adhesions at the posterior end of the zoite. One difference between gliding motility and invasion is that the

• Tight junction formation and microneme release occur at about the same time • Proteins localized to the micronemes bind to receptors on the erythrocyte surface

erythrocyte membrane immediately after reorientation.

probably similar in other invasive stages of apicomplexan parasites.

**27. In summary** 

**28. Parasite entry** 

rhoptries function in PVM formation.[68]

annulus as it enters the expanding parasitophotous vacuole.

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 adhesion reach the posterior of the zotie and a trail of the adhesive molecules are left behind the moving zoite on the substratum. However, 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. 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 inner membrane complex.[72] This myosin is part of a motor complex which is linked to the adhesins. Members of the TRAP family and other adhesins have a conserved cytoplasmic domain. This cytoplamic 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 filament toward the posterior of the zoite. The myosin is anchored into the inner membrane complex 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 filament. Thus the complex of adhesins and actin filaments is transported towards the posterior of the cell. Since the adhesins are either complexes with receptor on the host cell or bound to the substratum, the net result is a forward motion of the zoite. When the adhesins reach the posterior end of the parasite they are proteolyitcally cleaved and shed from the zoite surface.
