Cell Biology

T. gondii must invade host cells in order to replicate (Morisaki et al., 1995) and is able to penetrate virtually any cell type (Figure 5.5). Active forward movement of the parasite is an absolute requirement for host cell penetration (Zaman and Colley, 1972). The ability of T. gondii to actively invade host cells is key to its wide host cell range (Dubremetz, 1998). T. gondii also actively exits the host cell to continue the infection (Schwartzman and Saffer, 1992). Active egress from host cells is likely to be important, both in acute disease and in reactivation of dormant bradyzoites.

The basic behavior of T. gondii in its interaction with cells in culture can be observed by light microscopy (Figure 5.2). Saltatory gliding moti-lity of tachyzoites over a solid substrate is followed by rapid penetration of cultured cells of almost any type (Hakansson et al., 1999). The movement of the parasite is in a forward clockwise helical rotation without obvious deformation of the parasite body, although torsion of the parasite external membrane has been noted by scanning electron microscopy (Bonhomme et al., 1992). Anterior-posterior flexing of non-gliding parasites and forward protrusion of the anterior tip of the organism can also be seen (Nichols and O'Connor, 1981).

Rather than depending on a particular cell type to take the parasite up by host-dependent mechanisms, or on specific cellular receptors that would limit its ability to invade, T. gondii is able to penetrate a very wide variety of cell types in a broad range of host species. Invasion has been shown to depend on gliding movement (Dobro-wolski and Sibley, 1997; Hakansson et al., 1999). The motor for gliding is likely to be actin/myosin (Dobrowolski et al., 1997; Morisaki et al., 1995). Recent work has focused on defining the motor proteins that power active invasion. An unusual family of small myosins has been described in T. gondii that may be implicated in gliding, although the mechanism has yet to be established (Heintzelman and Schwartzman, 1997). Phylo-genetic analysis reveals that the three myosins represent a novel, highly divergent class in the myosin superfamily. T. gondii myosin-A (TgM-A) is an unusually small (approximately 93 kDa) myosin that shows a striking departure from typical myosin heavy chain structure, in that it lacks a neck domain between the head and tail, and in the absence of recognizable regulatory sites. The tail domain of TgM-A encompasses only 57 amino acid residues and has a highly basic charge. Two other Toxoplasma myosins, TgM-B and TgM-C, are proteins of 114kDa and 125 kDa, respectively. These two myosins differ only in their distal tail structure. The tails, like that of TgM-A, share no homology to any other myosin tails apart from a highly basic charge. Both TgM-A and TgM-C are membrane-associated and bind actin in the absence, but not in the presence, of ATP (Heintzelman and Schwartzman, 1999). The localization of TgM-A is to the anterior pole of the parasite, with subtle redistribution to apical patches along the cell membrane in extracellular gliding parasites. Two additional T. gondii genes (TgM-D and TgM-E), which strongly resemble TgM-A in size and sequence, have been cloned (Hettmann et al., 2000). In addition to the work done on T. gondii, a small unconventional myosin highly homologous to TgM-A has also been found in Plasmodium falciparum (PfM-A) (Pinder et al., 1998). This myosin is expressed in motile mer-ozoites and sporozoites, but disappears in the intracellular trophozoite stage. This myosin is localized to the parasite cortex, with some concentration at the apical pole, not unlike the distribution of TgM-A. As PfM-A appears to be the dominant or perhaps the only myosin expressed at this stage, it is the best candidate for the molecular motor driving the invasive process in Plasmodium.

At least 17 different classes of unconventional myosins have been identified in Protozoa, plants and animals across the phylogenetic spectrum (Mermall et al., 1998). Phylogenetic analysis reveals that the five myosins so far described in T. gondii, together with the three myosins from Plasmodium, represent at least one, and more likely two, novel, highly divergent classes in the myosin superfamily. As such, they may perform functions not yet documented for the existing myosins, such as powering gliding motility. Although the functions of the parasite myosins have not yet been determined, the distribution of these molecules does strongly support the hypothetical role of these myosins in parasite motility.

Fig. 5.5 Active egress of T. gondii from host fibroblasts following stimulation of motility by calcium. (A-D) Intracellular rosettes of parasites are demonstrated by differential-interference-contrast microscopy. Following calcium stimulation of parasite movement, the individual tachyzoites penetrate the wall of the parasitophorous vacuole and traverse the host cell cytoplasm on their own power, to exit the cell. A single tachyzoite is seen in panels C and D, showing constriction of the parasite body as it pushes through the host plasma membrane (arrow). Interval between panels, 10 seconds. Bar=10 ^m. Courtesy of Dr Elijah Stommel, Dartmouth Medical School

Fig. 5.5 Active egress of T. gondii from host fibroblasts following stimulation of motility by calcium. (A-D) Intracellular rosettes of parasites are demonstrated by differential-interference-contrast microscopy. Following calcium stimulation of parasite movement, the individual tachyzoites penetrate the wall of the parasitophorous vacuole and traverse the host cell cytoplasm on their own power, to exit the cell. A single tachyzoite is seen in panels C and D, showing constriction of the parasite body as it pushes through the host plasma membrane (arrow). Interval between panels, 10 seconds. Bar=10 ^m. Courtesy of Dr Elijah Stommel, Dartmouth Medical School

T. gondii motility is actin-dependent, but fibrillar actin has been very difficult to demonstrate in the parasite (Dobrowolski and Sibley, 1997). By the use of an agent that polymerizes and stabilizes actin filaments, specific actin processes can be demonstrated at the anterior pole of the parasite and beneath the parasite plasma membrane (Shaw and Tilney, 1999). This localization is consistent with roles for actin in both the probing movement of the anterior tip of the parasite seen during cell penetration and in gliding motility. Gliding motility is seen in bacteria, fungi, algae and many other protists, but the mechanisms responsible for producing movement over a substratum without deformation of the moving organism are as yet unexplained. Understanding the mechanism of gliding locomotion in T. gondii is complicated by the unusual arrangement of membranes of T. gondii zoites. An apparently ordinary plasma membrane surrounds the organism. Two additional unit membranes, positioned immediately subjacent to the plasma membrane, are arranged as side-by-side envelopes, appearing like 'pavement blocks' (Figure 5.6), with cross-sections showing two unit membranes closely apposed (Schwartzman and Saffer, 1992). The cisternae of the inner membrane complex (IMC) are not continuous beneath the entire parasite cell membrane, being absent at the poles of the organism. Beneath the IMC is an array of 22 longitudinal microtubules (Nichols and Chiap-pino, 1987). The function of the inner membrane complex is unknown.

Penetration and establishment of a parasitophorous vacuole requires constitutive and regulated secretion of parasitic factors (Karsten et al., 1998). The parasite has three secretory organelles, the rhoptries and micronemes that secrete their contents at the anterior pole, and dense granules that secrete along the lateral surface and posterior pole of the parasite (Dubremetz et al., 1993). The combined function of these secretory products appears to be modification of the host

Fig. 5.6 Montage of micrographs of a freeze-fractured T. gondii tachyzoite, shadowed by evaporated platinum to show the exterior surface of the organism. The plasma membrane has been largely lost, and the surface is made up of the several layers of the inner membrane complex, which covers the parasite in large 'pavement blocks'. The longitudinal ridges represent the membranes supported from the interior by the microtubule cytoskeleton

Fig. 5.6 Montage of micrographs of a freeze-fractured T. gondii tachyzoite, shadowed by evaporated platinum to show the exterior surface of the organism. The plasma membrane has been largely lost, and the surface is made up of the several layers of the inner membrane complex, which covers the parasite in large 'pavement blocks'. The longitudinal ridges represent the membranes supported from the interior by the microtubule cytoskeleton membrane to provide a vacuole that does not fuse with host compartments (Sibley and Kra-henbuhl, 1988) and that allows the parasite to salvage small molecular weight molecules for its metabolic and synthetic functions (Schwab et al., 1994) (Figure 5.7).

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