G 35 51g

Labyrithine

Solid

Solid

Labyrithine

Numerous Numerous Few

Numerous

Numerous

Absent

Numerous each other in the number of apical organelles, the shape and electron-density of the rhoptries, the location of the nucleus, and the presence or absence of polysaccharide granules. The nucleus is more centrally located in the tachyzoite (Figure 2.1 A) and merozoite (Figure 2.11B) and more basally located in the bradyzoite (Figure 2.23B) and sporozoite (Figure 2.21B). An additional cytoplasmic structure not described above is the polysaccharide granule. The polysaccharide granules are ovoid structures (250-180 nm) of variable electron-density located in both the apical and basal cytoplasm. They contain an unusual form of carbohydrate which is biochemically more similar to plant amylopectin than animal glycogen (Coppin et al., 2005). These granules are rarely found in tachyzoites or merozoites, but are present in large numbers in sporozoites and bradyzoites (Figures 2.21B, 2.23B). The granules appear to represent a stored energy source, which would be consistent with a possible requirement for the long-term survival of the bradyzoites and sporozoites or the extra energy needs during transmission between hosts. The most marked variations are in the apical organelles (see review by Dubey et al., 1998). There are relatively few micronemes in the tachyzoite and merozoite, but these are more numerous in the bradyzoite and sporozoite. In the case of the dense granules, these are numerous (5-12) in the tachyzoite and sporozoite, with fewer in the bradyzoite and very few (2-3) in the merozoite. In the case of the rhop-tries, there are differences in the number, shape, and electron-density between stages. The number of rhoptries is relatively similar (5-12) for the tachyzoite, bradyzoite, and sporozoite, with fewer in the merozoite (3-5). The rhoptries in the tachyzoite and sporozoite appear to have an elongated swelling with a labyrithine appearance, in comparison with the more bulbous and electron-dense swelling in the merozoite and bradyzoite. These differences are summarized in Table 2.1.

2.2.3 Host-cell invasion

Observations of T. gondii invasion have been performed in cell cultures and on red blood cells (which appear to be a possible, although unusual, abortive host-cell for this parasite) (Michel et al., 1979). Invasion is operated by a moving junction, which has the same morphological features as the one described for Plasmodium knowlesi (Aikawa et al., 1981), both in thin section and in freeze fracture. Interestingly, T. gondii makes the same junction with nucleated cells and with red blood cells (Porchet-Hennere and Torpier, 1983). It is a very close apposition of the parasite and host plasma membrane (Figures 2.3B, 2.4B), with thickening of the host side, and an accumulation of rhom-boidally organized intramembranous particles on the protoplasmic face of the host plasma membrane (Figure 2.3C). This forms a very tight junction, which excludes small electron-dense tracers such as Ruthenium Red. The molecular organization of the moving junction is still unclear, but recent data have shown that it involves proteins derived from the rhoptry neck in association with

FIGURE 2.3 Host-cell invasion in vitro.

(A) Serial section though the apical area of a tachyzoite at an early stage of Hela cell invasion. The moving junction is covering the apex of the tachyzoite (arrow); an empty rhoptry (eR) has exocytosed its contents in the neighboring host-cell cytoplasm as small vesicles (v). Bar = 0.1 pm.

(B) Freeze-fracture image of the apical area of an invading tachyzoite at a stage corresponding to Figure 2.3A. The typical structure of the moving junction in the protoplasmic face (Pv) of the host-cell plasmalemma (which will turn into the parasitophorous vacuole membrane) is visible (arrows) below the parasite apical exoplasmic plasmalemmal face (Ee). HC, host cell. Arrow lower right represents angle of shadowing. Bar = 0.1 pm.

(C) Freeze-fracture image of the apex of an invading tachyzoite at a similar stage of invasion as Figure 2.3B, but corresponding to the complementary fracture faces, showing the pit (arrow) in the parasitophorous vacuole membrane (Ev, exoplasmic face) covering the site of rhoptry exocytosis in the tachyzoite plasmalemma (Pe, protoplasmic face). Arrow lower right represents angle of shadowing. Bar = 0.2 pm.

FIGURE 2.3 Host-cell invasion in vitro.

(A) Serial section though the apical area of a tachyzoite at an early stage of Hela cell invasion. The moving junction is covering the apex of the tachyzoite (arrow); an empty rhoptry (eR) has exocytosed its contents in the neighboring host-cell cytoplasm as small vesicles (v). Bar = 0.1 pm.

(B) Freeze-fracture image of the apical area of an invading tachyzoite at a stage corresponding to Figure 2.3A. The typical structure of the moving junction in the protoplasmic face (Pv) of the host-cell plasmalemma (which will turn into the parasitophorous vacuole membrane) is visible (arrows) below the parasite apical exoplasmic plasmalemmal face (Ee). HC, host cell. Arrow lower right represents angle of shadowing. Bar = 0.1 pm.

(C) Freeze-fracture image of the apex of an invading tachyzoite at a similar stage of invasion as Figure 2.3B, but corresponding to the complementary fracture faces, showing the pit (arrow) in the parasitophorous vacuole membrane (Ev, exoplasmic face) covering the site of rhoptry exocytosis in the tachyzoite plasmalemma (Pe, protoplasmic face). Arrow lower right represents angle of shadowing. Bar = 0.2 pm.

FIGURE 2.4 Host-cell invasion in vitro.

(A) Freeze fracture of an invading tachyzoite showing the parasitophorous membrane (Ev), a clump of membrane whorls that may correspond to material exocytosed from the rhop-tries (asterisk), and the plasmalemma of the tachyzoite (Pe). HC, host cell. Arrow lower left represents angle of shadowing. Bar = 0.5 pm.

(B) Section through an invading tachyzoite showing the moving junction (arrows) and the continuity and the difference in electron density between the host-cell plasmalemma (HCM) and the parasitophorous vacuole membrane (PVM). N, nucleus. Bar = 0.2 pm.

the microneme protein AMA1 (Alexander et al., 2005; Lebrun et al., 2005).

Microneme exocytosis has never been clearly visualized, although it is thought to occur during both gliding motility and invasion; what has been shown is the accumulation of alignment of small, clear vesicles inside the conoid in conditions of chemically triggered microneme exocytosis, as though these dense, rod-like organelles gave rise to these small vesicles before or after exocytosis (Carruthers and Sibley, 1999). The docking site for microneme exocytosis is not known.

Rhoptry exocytosis is easily documented upon invasion, as an apical opening in continuity with the parasite plasma membrane, facing the developing parasitophorous vacuole membrane (PVM) (Nichols et al., 1983). Freeze fracture shows an open pit in the PVM at that location, suggesting continuity between rhoptry contents and PVM or even host-cell cytoplasm (Figure 2.3A). The role of the apical vesicle and that of the apical rosette of intramembranous particles located at the rhoptry exocytosis site has never been elucidated, but the rhoptries open precisely at this location and the IMP rosette disappears, just as reported for trichocyst exocytosis in Paramecium sp. (Beisson et al., 1976).

At very early stages of invasion, when the moving junction forms, small vesicles can be seen budding from the developing vacuole or laying in the host-cell cytoplasm (Figures 2.3A, 2.4A). At this stage, empty rhoptries are already observed. Therefore, these vesicles correspond to the physiological counterpart of the evacuoles, which are the product of frustrated rhoptry exocytosis in the host-cell cytoplasm when invasion is blocked by cytochalasin D (Hakansson et al., 2001).

The membrane of the developing vacuole is completely devoid of intramembranous particles (Figure 2.4A) (Dubremetz, et al., 1993), reflecting the selective exclusion of the intramembranous host-cell proteins at the moving junction. However, it will acquire IMPs during the first hour of development (Porchet-Hennere and Torpier, 1983), likely due to parasite contribution, especially from dense-granule protein translocation in the PVM (Dubremetz et al., 1993). Progression of the moving junction along the zoite is sometimes (but not always) correlated with parasite constriction.

2.2.4 Parasitophorous vacuole, intracellular development

Within minutes after closure of the parasitophorous vacuole, the posterior part of the parasite invaginates and the tubulo-vesicular network (TVN) starts developing in this invagination

FIGURE 2.4 Host-cell invasion in vitro.

(A) Freeze fracture of an invading tachyzoite showing the parasitophorous membrane (Ev), a clump of membrane whorls that may correspond to material exocytosed from the rhop-tries (asterisk), and the plasmalemma of the tachyzoite (Pe). HC, host cell. Arrow lower left represents angle of shadowing. Bar = 0.5 pm.

(B) Section through an invading tachyzoite showing the moving junction (arrows) and the continuity and the difference in electron density between the host-cell plasmalemma (HCM) and the parasitophorous vacuole membrane (PVM). N, nucleus. Bar = 0.2 pm.

(Sibley et al., 1995). The origin of the TVN is not fully understood: it contains dense granule proteins that are exocytosed from the anterior end of the parasite, this exocytosis beginning before the completion of the invasion process (Dubremetz et al., 1993). The origin of the tubular material itself, which is likely made of phospholipids, has never been elucidated; what is known is that the GRA2 protein is required to organize this network (Mercier et al., 2002), and that these tubules are in direct continuity with the PVM, although these two structures contain distinct dense-granules derived proteins (Cesbron-Delauw, 1994).

Immediately after invasion, host-cell mitochondria and endoplasmic reticulum surround the PV and persist throughout the intracellular development (see Figure 2.8). The rhoptry protein ROP2, exocytosed during invasion, has been shown to anchor the host mitochondria to the PVM (Sinai and Joiner, 2001). The host ER is devoid of ribo-somes on the side facing the vacuole. The distance between these organelles and the PV is highly conserved, and is about 12 nm and 18 nm for mitochondria and ER respectively (Sinai et al., 1997). The PVM-associated mitochondria may look normal but sometimes show morphological changes, with the cristae becoming larger and irregular in shape and the stroma becoming electron-dense.

The parasitophorous vacuole described above is formed by actively invading parasites and is characterized by the absence of the fusion of the host-cell lysosomes, thus protecting the parasite during intracellular development (Jones and Hirsch, 1972; Jones et al., 1972). This probably relates to the exclusion of the host-cell intramembranous proteins from the parasitophorous vacuole membrane during invasion. In contrast, parasites within vacuoles formed by host-cell phagocytosis exhibit lysosome fusion, and the parasites are broken down in typical phagolysosomes (Jones and Hirsch, 1972).

2.2.5 Endodyogeny

The tachyzoite is unique in it ability to undergo indefinite proliferation by a distinctive process termed 'endodyogeny', which involves parasite growth and division to form two daughters.

Despite grossly resembling binary fission, endodyogeny is a highly complex event, related to the structural complexity of the formation of polarized daughters. In contrast with the canonical asexual division mode of most Apicomplexa and even the coccidian stages of T. gondii, the tachyzoite retains the apical complex until the end of the endodyogeny. Although both events occur simultaneously, we will describe mitosis and daughter formation successively.

2.2.5.1 Mitosis

There have been few descriptions of T. gondii mitosis at the ultrastructural level, and what has been observed can be interpreted by comparison with more detailed studies in related Apicomplexa, especially Eimeria spp. (Dubremetz, 1973). One unique feature of apicomplexan mitosis is the retention of an intact nuclear membrane throughout the process of division. Coccidian-type centrioles (150 nm diameter) consist of nine short tubules (100 nm long) centered on a central tubule. Centrosomes or spindle pole bodies are made of two centioles oriented in parallel (Figure 2.5B). Centrosomes are always found associated with centrocones or mitotic spindle poles, usually on the apical side of the nucleus. The earliest stage of mitosis is a transnuclear funnel containing fibrous material, corresponding to an invagination of the nuclear envelope opened on both sides towards the cytoplasm (Figure 2.5A). The mitotic spindle most likely polymerizes in this funnel, which then opens in the nucleoplasm in its middle part, whereas the poles give rise to the centrocones. The centro-cones are at first subspherical invaginations of the nuclear envelope opened towards the centro-somes and through which the spindle micro-tubules extend. The intranuclear spindle is usually very short and transient, and has rarely been described. What occurs most likely is that the kine-tochores are separated immediately after the funnel opening, and assemble on the nucleoplas-mic side of the centrocones. Indeed, in Coccidia, caryokinesis does not depend on mitotic spindle elongation. Centrocones soon become conical evaginations of the nuclear envelope, opened on

0 0

Post a comment