Phylum of Protozoa.
Members of this phylum are characterized by the occurrence of the name-giving sporocysts (and/or oocysts) which produce the infectious sporozoites. The life cycles comprise a regular alternation of different sexual and/or asexual generations (Coccidia). Although several multinucleate stages occur during the reproduction of individuals of the different species (Plasmodium/Fig. 2), the typical sporozoan cell is uninucleate. With respect to fine structure the Sporozoa are a relatively uniform group, being provided with a typical apical complex (Pellicle/Fig. 3, Pellicle/Fig. 4, Pellicle/Fig. 5).
Due to these fine-structural features, several groups of parasites have been added to or excluded from the Sporozoa. This led to the systematic concept of Levine et al., which is, however, not generally accepted. Thus, the authors of this chapter do not support the addition of the class Perkinsea, since the fine structure is not identical with that of other sporozoans. Furthermore we find it necessary to include the Piroplasmea (Babesia/Fig. 1, Theileria/Fig. 1) and all blood-parasitizing members of the Adeleidea (genera Karyolysus, Hepatozoon, Haemogregarina, Leucocytozoon simondi/Fig. 1, Karyolysus lacertae/Fig. 1, Hepatozoon/Fig. 1) in the Haemosporida. Thus, in our opinion the following system better reflects the biological and morphological relations among the members of the Sporozoa (Fig. 1).
The major surface proteins of Plasmodium merozoites or Toxoplasma gondii tachyzoites have been shown to be GPI-anchored (glycosylphosphatidylinositols). The Circumsporozoite protein (CSP) of sporozoites and the ookinete surface proteins have the primary sequence requirements for GPI anchoring, although the latter has not been formally demonstrated due to the low amount of material available for study. Although the trimannosyl-glucosaminyl core is common with kinetoplastids, the structure of the GPI anchors differs from the trypanosomes, and among apicomplexans, by the sugar ornamentation of the glycannic core, and it does not undergo remodeling after addition to the protein.
The GPI anchor of Plasmodium spp. has been shown to be a strong immunomodulator responsible for signal transduction in cells of the vertebrate host;
The MSP1 of Plasmodium sp. merozoites undergoes proteolytic processing before reinvasion, and shedding of the cell coat described during invasion coincides with the removal of most of the protein except the C-terminal 19 kDa that is internalized with the parasite inside the red blood cell. In sporozoites, the CSP is deposited on the substrate during motility on serum albumin-coated surfaces, or is capped and shedded when bound to specific antibodies. The mechanism of shedding (release of proteins, cleavage of the anchor, or removal of lipids asscociated with the coat) is not elucidated. The contribution of GPI-anchored proteins to Apicomplexan motility is not known; in addition, exocytosed microneme protein may be transiently inserted into the plasmalemma of the parasites at the invasion stage and therefore be part of the glycocalix during a short period of the parasite cycle.
As described by many investigators, motility of Apicomplexan zoites can be subdivided into three types which are, respectively, gliding, twisting and bending. Only the first type leads to active displacement of the zoite, the two others being involved in changing the direction of motion. Gliding motility is very uncommon among eukaryotic cells; it is probably different from amoeboid locomotion although not sufficiently characterized to identify the possible relation between both processes.
Detailed observations on the gliding motility have been reported in several cases, one of them a gregarine, which is not an intracellular parasite, but has the advantage of being larger and thus more amenable to experimental study; the other models used were Eimeria sp. and Toxoplasma gondii.
In all cases, gliding motility has been described as the backward translocation of a “junction” between a substrate and the zoite surface along the latter's longitudinal axis. The substrate could be detected by small latex beads which, in the case of the gregarine, were translocated and accumulated at the posterior extremity of the organism. The experiments clearly demonstrated that the gliding motility of Apicomplexan zoites was a relative phenomenon in which the moving partner could be the zoite or the substrate depending on their respective size. Physiological studies of these models and others have shown that gliding was temperature- and cytochalasin B-sensitive, this last property being interpreted as a clue to the participation of actin in zoite motility. Indeed, in Toxoplasma gondii, the direct involvement of actin in motility has been demonstrated and myosin is probably also implicated, both molecules being found between the plasmalemma and the inner complex of zoites.
The parasite surface molecules involved in motility are unknown. Because all surface proteins of Apicomplexa characterized so far are GPI-anchored, they cannot directly transduce a mechanical force originating from the putative actomyosin motor located below the plasma membrane. Instead, transmembrane protein should exist that could operate this transduction. Recent findings concerning Plasmodium sp. have opened a new range of hypotheses: using gene knockout technology, it was shown that the microneme protein TRAP was a major actor of gliding motility of sporozoites. The prevailing hypothesis is now that microneme proteins that have a transmembrane sequence would be exocytosed and inserted into the plasma membrane and their cytoplasmic C terminus sequence would interact with the actomyosin motor whereas their external N terminus would transduce the motility force on the substrate (Fig. 2).
The nature of the interaction between the zoite and the substrate is entirely unknown. A classical receptor-ligand interaction is unlikely since gliding motility has also been observed on glass or on liquid-liquid interfaces and it thus appears that zoites may use physical properties of interfaces and surface tension to interact with substrates or that molecules with broad binding properties on the zoite surface exist.
The orientation of zoite motility, along the axis of the zoite, and its exclusive forward directionality can be related to morphological characteristics of zoites, although no experimental demonstration of a relation between structures and motility has yet been obtained. Indeed, freeze fracture has demonstrated highly organized structures within the pellicle of Apicomplexan zoites: although the outer layer (cytoplasmic membrane) shows little differentiation, the inner membrane complex, which is made of two closely apposed membranes underlying the plasmalemma, is highly organized. This inner complex is in fact a continuous layer of rectangular flattened vesicles (plates) arranged in longitudinal rows except at the anterior extremity where a truncated conical plate (apical cap) is found (Fig. 3). The intra-membranous particles of both membranes of the inner complex are arranged in a dense array of parallel longitudinal lines. Lines of higher particle density are found over the subpellicular microtubules which extend longitudinally under the inner complex (Fig. 4). Similar structures have been observed in all Apicomplexan zoites described so far. This highly organized longitudinal pattern could provide a template for the translocation of the interaction with a substrate along the zoite surface. Moreover, the polarity of these structures (apical cap followed by longitudinal plates) could be correlated with the polarity of gliding. A correlation between these morphological features and the putative actomyosin motor of motility remains to be established.
An interesting phenomenon has been described on apicomplexan zoites which also concerns the surface interactions between these organisms and their surroundings. When zoites are presented with a variety of ligands interacting with their surface (cationized ferritin, surface antigen-specific antibodies), they are at first entirely covered with the ligand but they are able to cap it on their posterior extremity and shed it into the medium. This capping phenomenom shares many common features with gliding motility (polarity, temperature and cytochalasin B sensitivity) and, moreover, seems to be dependent on motility: capping of a ligand has been described as simultaneous to gliding. A peculiar case of capping by Apicomplexan zoites has become famous in being the first explored target of vaccination against malaria: the circumsporozoite precipitation reaction, which is the progressive neutralisation of the capping ability of Plasmodium sporozoites by overwhelming with surface antibodies, subsequently led to the characterization of the circumsporozoite protein, which is a major target of candidate antimalarial vaccines.
If one considers motility of parasites as the way of homing to their host (host-cell in the present case), one would then wonder whether any signal or tropism guides the zoite to its definitive location, which is usually extremely specific. No such phenomenom has been described so far concerning Apicomplexans and in most cases, homing to the vicinity of the target cell seems to be passive, the zoites being driven either by the digestive or the circulatory route depending on the inoculation site. Gliding motility seems to be operational within a short range and to allow accidental contact between zoite and host cell when the organism has been passively carried close to the site of interaction. In the case of malaria sporozoites, the high specificity of recognition between hepatocyte surface proteoglycans and parasite surface proteins seems to be a major actor of organ targeting, probably by inducing binding of sporozoites to the hepatocytes when they are carried in the vicinity of these cells by the blood stream.
Regarding the host cell recognition process, two groups seem to exist among Sporozoa: one in which a highly specific recognition (and adhesion) step occurs which concerns the blood parasites (Hemosporidia, Piroplasms), and another in which little if any specific recognition of the host cell concerning the Coccidia can be demonstrated. This difference is better understood when one realizes that a Plasmodium merozoite is only able to invade the red blood cells of an extremely narrow range of hosts whereas a Toxoplasma tachyzoite readily invades almost any type of cells it gets in contact with. The difference between the two groups could also be interpreted as the need for receptor ligand mediated adhesion as a prerequisite to invasion by blood parasites whereas this step would be unnecessary to Coccidia whose host cell and tissue specificity in situ could be due to other mechanisms. However, another possibility is that binding always occurs before invasion but its specificity varies depending on the parasite and allows a variable range of invasion that may or may not need further specific interaction to determine development or escape and search for a more suitable host cell. Although the surface proteins of the invasive stages seem to be involved in the recognition binding to the host cell, another set of proteins stored in micronemes and translocated on the surface of the parasite upon interaction with host cells appear to be essential in parasite-host cell interaction in all Apicomplexa. The present status of knowledge does not clearly differentiate between recognition binding and motility of Apicomplexan zoites.
In the case of blood parasites (Plasmodium, Babesia), receptor-ligand types of interaction have been shown to occur between zoites and erythrocytes. These events seem to be involved in the adhesion of the parasite to its target cell which holds the partners together after accidental contact and allows invasion to proceed. Phase contrast microscopic observations of red blood cell invasion by merozoites of Plasmodium knowlesi or Plasmodium falciparum have shown that initial contact could occur between any two points of the surfaces of the two cells. Then the merozoite reorients itself in such a way that its apical end faces the erythrocyte membrane. Moving junction formation and invasion then occur. Reorientation has been suggested to be effected by a gradient of receptors directed towards the apical end of the zoite. The respective part of receptor-mediated interaction in initial contact, reorientation and moving junction formation is still unclear because of the difficulty of manipulating these different events in vitro. Thus the following applies to these interactions as a whole and we will only describe the Plasmodium falciparum model.
Recognition events concern interactions between merozoite receptors and erythrocytic ligands. Soluble molecules acting as bridges between both surfaces may also be involved. Most of the data obtained on these interactions come from in vitro experiments in which a Plasmodium falciparum culture was subjected to various treatments (use of variant red cell phenotypes, enzymatic alteration of erythrocyte surface, competition with soluble molecules) or from affinity of parasite molecules for erythrocytes (erythrocyte-binding antigens) or erythrocyte fractions (Glycophorin).
Concerning the erythrocyte, Glycophorin A (major sialoglycoprotein) is the major ligand in interaction with merozoites. More precisely, O-linked sialylated polysaccharides of the molecule seem to be directely involved in recognition. However, in addition, sialic acid-independent recognition and invasion can occur in certain strains, which suggests the existence of alternate pathways allowing the parasite to survive if one way fails to operate.
The main merozoite receptors described so far are molecules having affinity for the erythrocyte surface that were initially described as erythrocyte-binding antigens (EBA). These molecules are located in micronemes and are believed to be translocated in a transmembrane orientation on the merozoite surface where they play a major role in host cell recognition-invasion. A highly conserved family of EBA proteins expressed in the merozoite stages of various Plasmodium sp has been characterized; they all contain a conserved N-terminal cystein-rich domain that is involved in recognition. The way these microneme proteins that are stored in the lumen of an organelle become transmembrane in the parasite surface is unknown.
Although Plasmodium knowlesi shares a part of its erythrocyte host range with Plasmodium falciparum, the recognition events seem to be distinct from the ones involved for the latter species. The Duffy blood group antigen has been described to play a key role in P. knowlesi invasion, in association with the DBL, which is a member of the Erythrocyte-Binding ligands. The Duffy antigen is also involved in P. vivax invasion.
The role of merozoite surface proteins in erythrocyte invasion is unclear; the major merozoite surface protein (MSP1) may participate in initial binding, and antibodies that inhibit its proteolytic cleavage occurring during invasion block cell entry, but the receptor for this interaction is unknown. This surface protein may be acting as an early ligand of low affinity that allows close contact between both partners and is then strenghthened by the EBL-glycophorin interaction.
Whether the receptor-ligand recognition that precedes invasion is maintained in the moving junction that powers invasion is unknown; however, observations on the interaction between P. knowlesi and Duffy negative red cells where the junction does not form suggest that the DBL protein may be involved in the junction.
In the sporozoite stage of the malarial parasite life cycle, receptor-ligand recognition has been postulated to drive the interaction with the target cell, which is the hepatocyte. Although Plasmodium sporozoites have been shown to invade a rather large range of cells in vitro, their very efficient homing to the hepatocytes in vivo is probably driven by the affinity between the circumsporozoite surface protein CSP and the hepatocyte surface heparan sulfate proteoglycans.
Another system of recognition, which is found in Plasmodium sporozoites and is conserved in many Apicomplexa is the TRAP microneme protein family. In Plasmodium sporozoites, this protein also binds sulfated sugar-containing domains on the hepatocyte surface. Toxoplasma gondii and Eimeria tenella contain TRAP-like proteins in their micronemes, but their putative receptors have not been identified.
Micronemes of T. gondii and Cryptosporidium sp. also contain proteins with cell-binding properties that probably play a role in host cell recognition, although yet unidentified; in Sarcocystis sp. a microneme protein with lectin properties has been described.
Due to their medical significance, host cell invasion by Apicomplexan zoites has mainly been studied with malaria parasites and to a lesser extent with the Coccidia Toxoplasma, Eimeria, Sarcocystis or with piroplasms.
In all cases in which invasion has been described in detail, Apicomplexan zoites enter the host cell apical end first, i.e. the area where the so-called apical complex is located and especially where the rhoptry peduncles reach the zoite membrane. The zoite then gains access into an intracellular compartment surrounded by a membrane which is continuous with the host cell plasmalemma during invasion. The fate of that membrane has long been controversial since some authors have described its disintegration during the invasion, arguing that the parasitophorous vacuole appeared later around the cytoplasmic parasite. These conclusions were based on preparation artifacts due to the peculiar nature of the developing parasitophorous vacuole membrane which is poorly stabilized by the electron microscope preparation.
A very precise description of invasion has been given for Plasmodium knowlesi, this species being more amenable to experimental study of invasion than any other malarial parasite because of the relatively long shelf life of its merozoites. The initial step of internalization is a close contact between the apical end of the merozoite and the red blood cell plasmalemma. This latter becomes thicker on its inner side under this contact. Initiated as a segment of a sphere, this “junction” turns into a ring which moves backward on the zoite surface, whereas the host membrane area previously involved in the junction becomes the developing parasitophorous vacuole. The ring travels along the zoite to its posterior end where the junction ends up as a segment of a sphere, at which level the vacuole closes and separates from the host plasmalemma. The surface coat of the merozoite has been shown to be “shaved off” at the level of the junction as this latter went along the zoite surface. A 60-kDa protein has been described in the Plasmodium falciparum moving junction.
Associated with this process are the formation of vesicles facing the zoite apex on the cytoplasmic side of the developing parasitophorous vacuole, and a clear decrease in the electron density of the rhoptry contents. The rhoptries open at the tip of the zoite and their contents are in contact with the parasitophorous vacuole membrane. Membrane whorls (also called myelinic figures) have also been described in the vacuole as well as in “discharged rhoptries” especially when using an enhancing procedure based on tannic acid fixation. Freeze fracture electron microscopy has provided complementary information on the phenomenon: a major discovery was the total depletion of intramembranous particles of the developing parasitophorous vacuole compared to the erythrocyte plasmalemma, which indicated a very low proteinic content for this membrane. A second observation was that the moving junction had a peculiar freeze fracture structure as a rhomboedric network of “particles” in the erythrocytic side, which indicated that the junction was more than a close apposition of membranes but that the internal structure of the host cell membrane was altered at this level. The possible involvement of micronemes in moving junction formation has to be further evaluated
From a physiological point of view, the major finding was that cytochalasin B inhibited the invasion although the junction was initiated at the apex of the zoite and some vesicles appeared on the cytoplasmic side of the junction. Protease inhibitors were also found to block the phenomenon, which suggests that proteases may be involved in the process.
The data obtained on other species of malarial parasites suggest a very similar internalization process for these organisms (Fig. 5). This is also true for the piroplasm Babesia sp. which enters a red blood cell by moving junction and with surface coat shedding. A unique feature of Babesia is the disintegration of the parasitophorous vacuole membrane soon after invasion, the parasite then lying directly within the erythrocytic stroma, whereas Plasmodium sp. completes its schizogony within a parasitophorous vacuole.
Host cell invasion by Coccidia has been studied both in vitro and in vivo. Because of the larger size of the zoites and the more complex organization of the host cells, the results obtained by electron microscopy were not as clear as for Plasmodium. Moreover, the host cells chosen for experimental studies were in some cases professional phagocytes (monocytes, macrophages), which raised additional questions concerning the active part played by zoites in entry. To summarize the present status of knowledge, one can however assume that host cell invasion by Coccidia proceeds as described for Plasmodium, i.e. by a moving junction gliding backward on the zoite which enters a parasitophorous vacuole, the membrane of which is continuous with the plasmalemma of the host cell but depleted of intramembranous particles (Fig. 4, Fig. 5). The freeze fracture features of the moving junction are less conspicuous but, when described, are identical to the ones found on red blood cell membranes in Plasmodium.
Coccidian zoites do not show a thick surface coat but when covered with monoclonal antibodies (which do not induce capping), shedding of this layer occurs during invasion. Abundant membrane whorls are found in the parasitophorous vacuole or in “empty rhoptries” and vesicles are found on the cytoplasmic side of the parasitophorous vacuole.
Invasion of professional phagocytes by Toxoplasma gondii (Fig. 6, Fig. 7)has long been a matter of controversy between authors who claimed that active invasion occurred and others claiming that phagocytosis occurred, followed by inhibition of fusion of the phagosome with lysosomes. It is now clear that successful invasion by Toxoplasma gondii is always an active process by the parasite, whatever the host cell, but that interaction with phagocytes, especially in the context of the immune response of the host, leads to a competition between active entry and phagocytosis, which in turn leads to either parasite survival or killing.
Thus, even if some uncertainties persist, the invasion process is probably identical for all intracellular Apicomplexa, characterized by a moving junction and the development of the parasitophorous vacuole. Though the moving junction is indeed generated by the zoite, we know nothing of it beyond morphological observations. Its physiological features (antero-posterior polarity, cytochalasin B block, coat shedding) are very similar to those described for motility of zoites and these two processes are probably related but this remains to be investigated. A schematic view of the invasion process is shown in Fig. 8.
The origin of the parasitophorous vacuole membrane is still controversial. The respective contributions (qualitative and quantitative) of the host cell plasma membrane and of rhoptry products are not fully determined, although both are probably involved. Ward et al., using fluorescent lipids or electrophysiological procedures, have shown in both Plasmodium sp. and Toxoplasma gondii that host cell membrane components (essentially lipids) contribute a major part of the early parasitophorous membrane.
The contribution of the apical secretory organelles to invasion is not clearly understood. They undergo sequential exocytosis during the process in Toxoplasma gondii and probably also in others. Micronemes are likely to exocytose molecules involved in recognition-adhesion motility at an early stage of interaction. Rhoptries that contribute proteins which are integrated into the parasitophorous vacuole membrane are also believed to secrete enzymes such as proteases or phospholipases that would modify the host cell plasmalemma and facilitate vacuole formation (for example, the cleavage of the eythrocyte protein band 3 by a serine protease of Plasmodium is believed to lead to disorganisation of the spectrin network of the red blood cell as a prerequisite to red cell invasion). Dense granules are exocytosed into the vacuole after invasion and their role is not well understood, yet they are probably involved in rendering the vacuole contents and membrane efficient to sustain parasite metabolic needs. In P. falciparum, a dense granule protein (RESA) is translocated under the erythrocyte membrane and associates with spectrin.
A peculiar process has been described for the piroplasm Theileria sp.: invasion of mononuclear lymphocytes by sporozoites seems to involve a zipper mechanism in a receptor-mediated endocytosis due to specific receptors on the zoite surface. These findings are very unique among Apicomplexa and may represent an exception to the moving junction process. In addition, in this model, as in the other piroplasm Babesia (which invades red cells in a manner very similar to Plasmodium), the vacuole membrane is disintegrated after invasion and the parasite develops in the cytoplasm of the host cell.