FIGURE 2.1 Toxoplasma gondii tachyzoite ultrastructure.

(A) Sagittal section of an intravacuolar tachyzoite. A, apicoplast; C, conoid; DG, dense granule; er, endoplasmic reticulum; G, Golgi body; HCN, host-cell nucleus; MN, micronemes; Mi, mitochondria; N, nucleus; nu, nucleolus; PV, parasitophorous vacuole; R, rhoptry; ac, acidocalcisome; tvn, tubo vesicular network. Bar = 1 mm.

(B) Enlargement of the Golgi area, showing the apicoplast, A, surrounded by four membranes (arrows). Bar = 0.5 mm.

FIGURE 2.1 Toxoplasma gondii tachyzoite ultrastructure.

(A) Sagittal section of an intravacuolar tachyzoite. A, apicoplast; C, conoid; DG, dense granule; er, endoplasmic reticulum; G, Golgi body; HCN, host-cell nucleus; MN, micronemes; Mi, mitochondria; N, nucleus; nu, nucleolus; PV, parasitophorous vacuole; R, rhoptry; ac, acidocalcisome; tvn, tubo vesicular network. Bar = 1 mm.

(B) Enlargement of the Golgi area, showing the apicoplast, A, surrounded by four membranes (arrows). Bar = 0.5 mm.

granules), endosymbiontic derived organelles (mitochondrion, apicoplast), eukaryotic universal organelles (nucleus, endoplasmic reticulum, Golgi apparatus, ribosomes), and specific structures (acidocalcisomes), all enclosed by a complex membranous structure termed the pellicle. The cytoskeleton comprises:

1. Two apical rings located beneath the plasma membrane at the apical tip of the parasite.

They are uncharacterized at the molecular level, but both are made of a thin ring of electron-dense material; the upper one is 160 nm, the posterior one 200 nm in diameter (200 nmD).

2. The conoid, which is a hollow truncated cone consisting of fibers wound into a spiral, like a compressed spring, 400 nm in diameter at the base and 250 nm high. It is made of tubulin organized in a unique fashion, consisting of asymmetrical filaments of about 9 protofilaments, very different from typical microtubules (Hu et al., 2002a).

3. Two polar rings that encircle the top of the resting conoid (Figure 2.2A). The outer ring consists of dense material covering the anterior rim of the inner membrane complex (IMC, see below). The inner ring is formed of material which anchors the 22 subpellicular micro-tubules that extend underneath the IMC for approximately two-thirds of the body length (Nichols and Chiappino, 1987). These microtubules are classical 22-nm diameter hollow tubes, comprising 13 protofilaments made of tubulin (Hu et al., 2002a).

4. A pair of adjacent intraconoidal microtubules, extending for a short distance (less than 1 pm) into the apical cytoplasm and ending anteriorly next to an apical vesicle of 40 nm that adheres to the plasma membrane (Hu et al., 2002a).

The pellicle is a distinctive membrane complex that encloses the infectious stages. It consists of an outer unit membrane (plasmalemma) that completely encloses the organism, and an inner layer of two closely applied unit membranes found at a fixed distance (approx 15 nm) from the plasmalemma. The inner membrane complex (IMC) consists of fused plates formed from flattened vesicles derived from the ER-Golgi system (Vivier and Petitprez, 1969). The inner layer is interrupted by circular apertures at the anterior end (outer polar ring), where the conoid protrudes, and at the posterior end. The organization of the IMC has been essentially unravelled by EM freeze fracture (Porchet and Torpier, 1977; Morrissette et al., 1997). It is made of an apical plate, which is a single truncated cone, approximately 1 pm high, on

FIGURE 2.2 Ultrastructural details of bradyzoite and tachyzoite.

(A) Apical area of a bradyzoite showing the two apical rings (a1, a2), and the two polar rings (p1, p2) above and around the conoid. Bar = 0.1 |im.

(B) Upper picture: micropore showing the invagination of the zoite plasmalemma (arrow) through an opening and indentation (arrowhead) of the inner membrane complex (imc). Lower picture: uptake of PV vesicular material through the micropore. Bar = 0.1 | m.

(C) Freeze-fracture image of the pellicle of a tachyzoite (taken from Morrissette et al., 1997). The three successive membranes are shown: Pe, protoplasmic face of the plasmalemma; Em, exoplasmic face of the external layer of the inner membrane complex; Pi, protoplasmic face of the inner layer of the inner membrane complex. Arrowheads point at lines of higher IMP density corresponding to underlying subpellicular microtubules. Bar = 0.1 |im.

which six longitudinal rows of rectangular plates are attached. The rows end at the posterior end of the tachyzoite in triangular plates. The rows can extend straight or be twisted helically. The protoplasmic faces on both sides of the IMC are covered with lines of intramembranous particles (IMPs), with 22 lines of higher density corresponding to the underlying subpellicular microtubules (Figure 2.2B). IMPs have a 32-nm longitudinal periodicity, and are lined approximately 30 nm apart (Morrissette et al., 1997). The organization of IMPs in the apical plate is markedly different from that in the other plates, suggesting a distinct molecular structure in this apical area. An additional organized structure associated with the inner side of the IMC has been described, by negative staining after detergent extraction, as a network of 8-10-nm filaments containing two novel proteins with extended coiled-coiled domains that may play a role in determining cell shape (Mann and Beckers, 2001). The precise correlation between this network and the IMP alignments has not been defined.

The pellicle has an additional adaptation termed the micropore, which is located in the apical half of the cell normally just anterior to the nucleus. The single micropore consists of a circular (approximately 150 nm in diameter) invagination of the plasmalemma through a break in the inner membrane complex. The latter infolds to form an electron-dense collar around the invagination (Figure 2.2C). These structures are present throughout development, and increase in number during endopolygeny and gametogony. They are thought to act as a cytostome-like structure with extensions of the invaginated plasmalemma budding off, resulting in the uptake of material (Figure 2.2C) (Nichols et al., 1994). This process has been clearly shown to be important in the malaria parasite, where the micropore is responsible for the ingestion of the erythrocyte hemoglobin (Aikawa et al., 1966).

Three distinct secretory organelles have been identified, which can vary in numbers and shape between the invasive stages (see below) (Figures 2.1A, 2.2A). First are small, rod-shaped micronemes (250 x 50 nm), located in the most apical area of the parasite, behind the conoid. They are homogeneously electron-dense. Second are the rhoptries, organized as a group of elongated, club-shaped organelles that extend from within the conoid toward the nucleus. They show a long, narrow neck up to 2.5 pm in length, and a sac-like body about 0.25 x 1 pm in the posterior area. The contents are electron-dense except in the widened part, where the structure can be either labyrinthine or electron-dense in appearance depending on the specific stage. The third type, found throughout the cell but mostly in the posterior part of the parasite, are spherical-shaped (0.3-pm diameter) structures with electron-dense contents, which have been termed the dense granules.

Immuno-electron microscopy has played an important role in our understanding of the functions of these organelles. With the development of antibodies to specific proteins, it has been possible to begin to identify proteins specifically located in the different organelles. It has also been possible to identify proteins (MIC proteins) that are only present in the micronemes or proteins located in the dense granules (GRA proteins). Indeed, in the case of the rhoptries it has been possible not just to identify proteins located in the rhoptries, but also to differentiate between those located in the bulbous region (the ROP proteins) and those specific to the neck region (the RON proteins) (Bradley et al., 2005).

The nucleus occupies a central or basal location, depending on the invasive stage (see below). It is often flattened on the upper side, where the Golgi apparatus is located. It contains a central nucleolus and small clumps of electron-dense heterochromatin scattered throughout the nucleoplasm. The nuclear envelope has numerous nuclear pores, and is covered on its external side with ribosomes, except on the upper face, where the Golgi apparatus is located (Figures 2.1A, 2.1B, 2.23C). The nuclear envelope is in continuity with sheets of rough endoplasmic reticulum that extend into the cytoplasm of the tachyzoite.

On the upper surface of the nucleus a layer of clear vesicles of 70 nm diameter, some of which can be seen budding from the nuclear envelope, is topped by three or four flat Golgi cisternae, on top of which numerous vesicle of various contents and size can be observed (Figures 2.1A, 2.1B, 2.23B, 2.23C).

Using certain preparative technique, one or two vesicles of approximately 200 nm containing one or several electron-dense droplets or crystals of various sizes in a clear background are found near the nucleus or in the posterior part of tachyzoites (Figure 2.1A). These have been termed the acido-calcisomes, and the dark contents are believed to be calcium bound to pyrophosphate and polyphosphates (Luo et al., 2005).

Several mitochondrial profiles of 0.5-pm width and various lengths can usually be observed at various locations above and below the nucleus (Figures 2.1A, 2.1B). These represent sections through a single branched and elongated mitochondrion. They show the typical apicomplexan structure, with bulbous cristae.

Above the Golgi is the apicoplast (Figure 2.1B). This organelle, limited by multiple membranes, has been identified morphologically since the early 1960s (Ogino and Yoneda, 1966; Sheffield and Melton, 1968; Vivier and Petitprez, 1969), but was only recently shown to be a typical plastid (Kohler et al., 1997). Since it appears to be a feature of all members of the Apicomplexa, with the exception of Cryptosporidium sp., the term 'apicoplast' was proposed. In the infectious stage it is relatively uniform in shape, up to 500 nm in diameter, bounded by possibly four membranes, and filled with granular and filamentous content in which ribosomes can be observed. The origin of the four membranes is still a matter of debate but could result from a secondary phagocytosis of an alga already containing an endosymbiont (Kohler, et al., 1997). It has recently been proposed that the 4-membrane structure could result from the extensive invagination of the inner membrane of a double membraned organelle but this requires confirmation (Kohler, 2005).

2.2.2 Comparison of the invasive stages

The infectious stages consisting of the tachyzoite, bradyzoite, merozoite, and sporozoite differ from

TABLE 2.1 Summary of the morphological differences between stages of T. gondii

Life-cycle stage Nucleus Micronemes

Rhoptries number


Dense granules

Polysaccharide granules

Tachyzoite Bradyzoite Merozoite Sporozoite

Central Basal Central Basal

Numerous Few


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