Fig. 2 shows the metabolic pathway of glycogen degradation (arrow pathway) and cellulose synthesis (dot-arrow pathway). In general, breakdown of glycogen into units of glucose occurs through phosphorylitic cleavage by glycogen phosphorylase, phosphoglucomutase, and glucose-6-phosphatase [5]. Synthesis of cellulose from glucose is also a multi-step process involving 4 enzymes; hexokinase, phosphoglucomutase, UDP-glucose pyrophosphorylase, and cellulose synthase [6]. According to the process, it is 7 steps from glycogen to cellulose in Acanthamoeba. Glucose-1-phosphate is a metabolic product of glycogen in glycogenolysis and converts to glucose through glucose-6-phosphate. For cellulose synthesis, glucose-1-phosphate is also an important branch point to generate cellulose from glucose (Fig. 2). Therefore, we hypothesized that the short-cut process of cellular synthesis from glycogen could be mediated by 3 enzymes in Acanthamoeba (Fig. 2, box).

Our previous ESTs analysis study provided partial sequences of glycogen phosphorylase (GenBank no. JX312797) and UDP-glucose pyrophosphorylase (GenBank no. JX312798) [7]. Using sequence information on cellulose synthase from A. castellanii Neff (Tang et al., unpublished), we cloned a partial cDNA of cellulose synthase from A. castellanii Castellani (GenBank no. JX312799). Results of cDNA microarray analysis between cysts and trophozoites indicated a 2.44-fold higher expression of glycogen phosphorylase in cysts than in trophozoites [8]. However, phosphoglucomutase was expressed less in cysts than in trophozoites (2.4-fold) [8]. These results suggested that the conversion of glucose-1-phosphate to glucose-6-phosphate is not essential in glycogen degradation during encystation of Acanthamoeba. Then, it is possible to hypothesize the short process to synthesize cellulose from glycogen through glucose-1-phosphate, an important branch point.

The results of the quantitative real-time PCR analysis of expression of 3 enzymes supported our hypothesis strongly. As shown in Fig. 3, the mRNA level of glycogen phosphorylase showed a gradual increase and reached the maximum (10.3-fold) at the third day after induction of encystation (Fig. 3A). Dictyostelium discoideum, a species of soil-living amoeba, has been reported to have 2 forms of glycogen phosphorylase [9]. The 2 forms of the enzyme may play different roles in the development of Dictyostelium because they have different expression patterns. Glycogen phosphorylase 1 was found to be functional during differentiation of Dictyostelium into spores which have walls containing cellulose [10]. During the time course of development of Dictyostelium, the 'b' form (glycogen phosphorylase 2) showed a decrease, whereas the 'a' form (glycogen phosphorylase 1) showed an increase [11]. The partial glycogen phosphorylase of A. castellanii Castellani showed 65% similarity with glycogen phosphorylase 1 of D. discoideum, and showed high expression during encystation. The mRNA levels of UDP-glucose pyrophosphorylase of Acanthamoeba showed the highest expression (6.1-fold) on the second day after induction of encystation (Fig. 3B). UDP-glucose pyrophosphorylase 2 of D. discoideum was required for the differentiation and development of the amoeba [12]. The partial UDP-glucose pyrophosphorylase of A. castellanii showed 62% similarity with UDP-glucose pyrophosphorylase 2 of D. discoideum. The expression level of cellulose synthase of A. castellanii showed an increase (3.0-fold) on the second day after induction of encystation (Fig. 3C). The partial sequence of cellulose synthase of A. castellanii showed 99% similarity with that of A. castellanii Neff (ACC008015- Tang et al., unpublished) and 64% similarity with that of D. discoideum. These results suggest the involvement of these enzymes in the encystation process of Acanthamoeba.

The development of the acid-insoluble protein containing ectocyst wall and the cellulose containing endocyst wall leads to emergence of resistance to biocides in encysted Acanthamoeba [13]. Cellulose, the primary component of the cyst wall, is also used for diagnosis of Acanthamoeba cysts [14]. This is the first study that we identified 3 enzymes involving cellulose synthesis in Acanthamoeba and confirm the short-cut pathway to synthesize cellulose by analysis of their expression patterns during encystation. Information on cellulose synthesis is important to understand the mechanism of encystation and to diagnose cyst-forming protozoa. This key process of cellulose synthesis may aid in understanding of cyst wall formation in Acanthamoeba.