Acanthamoeba castellanii Castellani was obtained from the American Type Culture Collection (ATCC 30011). Trophozoites of A. castellanii were axenically cultured in PYG medium (2% proteose peptone, 0.1% yeast extract, and 100 mM glucose) at 25°C in an incubator. Encystation of A. castellanii was induced in an encystation media (0.1 M KCl, 0.008 M MgSO₄, 0.0004 M CaCl₂, and 0.02 M 2-amino-2-methyl-1,3-propanediol (pH 9.00)) for 3 days.

Acanthamoeba trophozoites were treated with 10 μM of 5-azacytidine (Sigma-Aldrich, St. Louis, Missouri, USA) and incubated in PYG media or encystment media for 3 days. Mature cysts were counted under a light microscope after treating them with 0.5% SDS. Encystation ratios were calculated. Data are expressed as means±SD of the means from 3 independent experiments. Statistical significance was analyzed using unpaired Student’s t-test. A P-value of less than 0.05 was considered statistically significant.

Genomic DNA of Acanthamoeba trophozoite was purified using QIAamp genomic DNA kits (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Promoter sequences were obtained using GenomeWalker kit (BD Biosciences Clontech, Palo Alto, California, USA), following the protocol recommended by the manufacturer. Briefly, the first step was the construction of pools of adaptor-ligated genomic DNA fragments using 1 of 4 restriction enzymes (EcoRV, DraI, PvuII, or SspI). The first PCR amplification was performed with outer adaptor primer (AP1) provided in the kit and cysteine proteinasegene-specific primer (CP-GSP 1) (Table 1). Nested GSP (CP-GSP 2) (Table 1) and nested adaptor primer (AP2) were used in sequential PCR to amplify the promoter. For primary PCR, the following cycling parameters were used: 7 cycles of 25 sec at 94°C and 4 min at 72°C followed by 32 cycles of 25 sec at 94°C and 4 min at 67°C with a final extension at 67°C for an additional 4 min. Primary PCR products were diluted 1:50 and 1 μl of the diluted primary PCR product was used as template for secondary amplification. Cycling parameters for the secondary PCR were the same as those used for the primary PCR except that the first and second steps consisted of 5 and 22 cycles, respectively. A single band of ~3,000 bp was obtained after PCR amplification. It was cloned into pGEM-T easy vector system (Promega, Madison, Wisconsin, USA). Two clones were picked for plasmid DNA purification for sequencing analysis.

Promoter CpG islands and bisulfite sequencing PCR primers were designed based on Methprimer site (Table 1). The primers were designed that they didn’t contain any CpGs in order to facilitate their binding to both methylated and unmethylated sequences. Genomic DNA of Acanthamoeba trophozoite and cyst were prepared with QIAamp genomic DNA kits (Qiagen), and Bisulfite kit (Qiagen) was used for bisulfite sequencing PCR. Bisulfite conversion and subsequent purification were performed according to the respective protocols. Seven PCR products (F1–7) were cloned into the pGEM-T easy vector system, and each ten clones were picked for sequencing analysis.

In order to identify the effect of 5-azacytidine on growth and encystation of Acanthamoeba, trophozoites were cultured with 10 μM 5-azacytidine for 3 days. Results showed that 5-azacytidine had limited effect on the growth of Acanthamoeba compared to untreated control (Fig. 1A, B, E). However, when trophozoites were transferred to encystment media, 5-azacytidine reduced the encystation ratio (Fig. 1C, D, F). Several trophozoites were observed (arrows) after induction into encystment (Fig. 1D). The number of mature cysts was significantly reduced in 5-azacytidine treated cells (52.2%) compared to that in control cells (80.5%) (Fig. 1F). These data indicated that, although 5-azacytidine did not significantly affect Acanthamoeba growth, it reduced encystation ratio.

To identify the promoter region of cyst specific cysteine proteinase (CSCP) of Acanthamoeba, a 2,878 bp segment at the 5′-flaking region of CSCP was amplified by PCR using specific primers (Table 1; Fig. 2). CG dinucleotides in the promoter region were underlined. Scanning for CpG islands in the submitted sequence revealed 3 CpG islands in Methprimer (Fig. 3A). The 3 CpG island regions were at least 200 bp with a GC percentage greater than 50%. The observed-to-expected CpG ratio was greater than 60%. The 3 CpG islands were divided into 7 fragments for bisulfite sequencing PCR (Fig. 3B). A total of 14 bisulfite sequencing PCR primers were then designed according to CpG islands (Table 1).

To determine the effect of promoter methylation on gene expression, we performed bisulfite sequencing for encystation mediating CSCP in Acanthamoeba trophozoite and cyst. In trophozoite, 8.2%, 4.2%, and 3.6% methylation were observed in CpG island 1, CpG island 2, and CpG island 3, respectively. At cyst stage, 7.3%, 5.8%, and 3.6% methylation were observed in CpG island 1, CpG island 2, and CpG island 3, respectively. Total promoter methylation status of CSCP in trophozoite was approximately equal to that in cyst. However, CpG island 1 of trophozoite showed higher methylation (8.2%) than that of cyst (7.3%) (Fig. 4). The length of CpG island 1 was 1,633 bp. There were 148 CpG sites.