T gondii displays a uniquely simple pathway for amylopectin synthesis

Using bioinformatic searches, several gene candidates encoding enzymes that are probably involved in amylopectin biosynthesis were identified (Coppin et al., 2005). These putative enzymes can be grouped in two classes:

1. Enzymes that are involved in amylopectin synthesis, such as amylopectin synthase, branching enzymes, UDP-glucose pyrophosphorylase, isoamylase, indirect debranching enzyme, a-1, 4-glucanotransferase, and glycogenin

2. Enzymes for amylopectin degradation, like a-amylase, dikinase or R1 protein, phosphory-lase, and a-glucosidase. Based on the presence of these enzymes, metabolic pathways and enzymes involved in amylopectin synthesis in T. gondii are probably similar to those of starch synthesis in the unicellular green algae Chlamydomonas reinhardtii (Figure 8.2).

Surprisingly, all of these genes are present in the Toxoplasma genome as a unique copy, suggesting that redundant genes are not required for the synthesis of a genuine crystalline amylopectin in this protozoan parasite (Ball and Morell, 2003; Coppin et al., 2005). This is in violation of the current dogma that suggests that redundancy of genes is required to build a crystalline starch in plants. Even in the simplest unicellular picophytoplanktonic green alga, Ostreococcus tauri, there is multiplicity

T. gondii Cyst Bradyzoite Tachyzoite

FIGURE 8.1 Transmission electron micrographs of bradyzoites (Br) within a tissue cyst (Panel A). Note the presence of the cyst wall (CW) and numerous amylopectin granules (AG) in the cytoplasm of the bradyzoites. Panels B and C shows a higher magnification of ultrastructural morphology of bradyzoite and tachyzoite which lacks amylopectin granules. Rh, rhoptry; DG, dense granules; Mi, micronemes and M, mitochondrion; N, nucleus; CW, cyst wall; PV, parasitophorous vacuole.

T. gondii Cyst Bradyzoite Tachyzoite

FIGURE 8.1 Transmission electron micrographs of bradyzoites (Br) within a tissue cyst (Panel A). Note the presence of the cyst wall (CW) and numerous amylopectin granules (AG) in the cytoplasm of the bradyzoites. Panels B and C shows a higher magnification of ultrastructural morphology of bradyzoite and tachyzoite which lacks amylopectin granules. Rh, rhoptry; DG, dense granules; Mi, micronemes and M, mitochondrion; N, nucleus; CW, cyst wall; PV, parasitophorous vacuole.

of genes and redundancy of isoenzymes involved in starch synthesis (Ral et al., 2004).

Only UDP-glucose pyrophosphorylase and UDP-glucose utilizing amylopectin synthase are found in T. gondii. Comparative genomic analyses involving the unicellular red alga Cyanidioschyzon merolae (Matsuzaki et al., 2004), the unicellular green alga Chlamydomonas rein-hardtii, the yeast Saccharomyces cerevisiae, and the bacterium Escherichia coli revealed that both C. merolae and T. gondii contain a UDP-glucose utilizing glycogen (starch) synthase-like sequences and glycogenins. These enzymes are specific for the eukaryote UDP-glucose based pathway. In addition, UDP-glucose utilizing glycogen synthase activity has been detected in the crude extract from T. gondii while only ADP-glucose dependent activity is present in Chlamydomonas lysates (Coppin et al., 2005). T. gondii also contains an indirect debranching enzyme, a bifunctional enzyme that carries both a-1,4-glucanotransferase and amylo-1,6-glucosidase activities in fungi and animals (Figure 8.2).

However, the characteristic most typifying the amylopectin biosynthetic pathway in T. gondii is the presence of genes that are of plant origin. Among the genes that distinguish plant starch metabolism from those of the animal, fungal, and bacterial glycogen pathways are the isoamylase and R1 (glucan water dikinase activity)-like sequences in T. gondii. This suggests that both plant- and animal-like amylopectin biosynthetic pathways are required for the synthesis of crystalline amylopectin in the parasite (Figure 8.2).

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