Identification of Protein Arginine Methyltransferase 5 as a Regulator for Encystation of Acanthamoeba

Abstract

Encystation is an essential process for Acanthamoeba survival under nutrient-limiting conditions and exposure to drugs. The expression of several genes has been observed to increase or decrease during encystation. Epigenetic processes involved in regulation of gene expression have been shown to play a role in several pathogenic parasites. In the present study, we identified the protein arginine methyltransferase 5 (PRMT5), a known epigenetic regulator, in Acanthamoeba castellanii. PRMT5 of A. castellanii (AcPRMT5) contained domains found in S-adenosylmethionine-dependent methyltransferases and in PRMT5 arginine-N-methyltransferase. Expression levels of AcPRMT5 were increased during encystation of A. castellanii. The EGFP-PRMT5 fusion protein was mainly localized in the nucleus of trophozoites. A. castellanii transfected with siRNA designed against AcPRMT5 failed to form mature cysts. The findings of this study lead to a better understanding of epigenetic mechanisms behind the regulation of encystation in cyst-forming pathogenic protozoa.

Keywords: Acanthamoeba; cellulose synthase; encystation; endocyst.

Fig. 1.

Amino acid sequence alignment of Acanthamoeba castellanii PRMT5 with PRMT5 of Plasmodium reichenowi, Plasmodium falciparum, Dictyostelium discoideum, Entamoeba histolytica, Trichomonas vaginalis, and Trypanosoma brucei. Sequences characteristic of methyltransferase motifs I, post I, II, and III are underlined.

Fig. 2.

mRNA expression of AcPRMT5 using real-time PCR analysis. AcPRMT5 showed high expression 72 hr after encystation induction. Values indicate the mean (±SD) of 3 experiments. ^**Means are significantly different one another (Student’s t-test, P<0.01).

Fig. 3.

Stable transfection of pGAPDHgPRMT5 for intracellular localization. (A) EGFP-PRMT5 fusion protein of A. castellanii was mainly localized in the nucleus of trophozoites. (B) After encystation, EGFP-PRMT5 fusion protein was localized in the nucleus of cysts. Localization of AcPRMT5 in the nucleus was confirmed by DAPI staining.

Fig. 4.

Transfection of A. castellanii with siRNA designed against PRMT5. (A) PRMT5 showed a high expression level during encystation (), but was down-regulated in PRMT5-siRNA transfected cells (). (B) This inhibition by PRMT5-siRNA was found to reduce encystation ratios. Values indicate the mean (±SD) of 3 experiments. ^**Means are significantly different one another (Student’s t-test, P<0.01).