Aims: Piper sarmentosum or locally known as Kaduk, is a tropical herb plant that was investigated for its phenolic content by previous researchers. The present study aimed at the analysis of crude methanolic extract of P. sarmentosum leaves for phenolic compounds identification and its anti-amoebic properties against pathogenic Acanthamoeba castellanii. Methodology and results: Folin-Ciocalteu assay was used to determine P. sarmentosum leaves methanolic extract (PSLME)’s total phenolic content (TPC). The extract was further characterized by using gas chromatography-mass spectrometry (GC-MS), reverse phase-high performance liquid chromatography (RP-HPLC) and liquid chromatography-mass spectrometry (LC-MS) analyses to determine the chemical constituents in methanolic PSLME extract. The cytotoxicity of the extract was evaluated through the determination of inhibition concentration for half of cell population (IC50) of pathogenic A. castellanii followed by cell morphological analysis using inverted light and scanning electron microscopies. Acridine-orange/Propidium iodide (AOPI) staining was also conducted to determine the integrity of cell membrane for quantitative analysis. The results demonstrated that the TPC from PSLME was 142.72 mg [GAE]/g with a total of 33 phenolic compounds identified. The IC50 value obtained for A. castellanii was low (74.64 μg/mL) which indicates promising anti-acanthamoebic activity. Microscopy analyses showed that the plant extract caused cells encystment, in which exhibited by distinctive morphological changes on the cells shape and organelle, as well as shortening of acanthopodia. The dual staining and its quantitative analysis prove compromised membrane integrity in the treated amoeba. Conclusion, significance and impact of study This finding provides the evidence that PSLME contains active phenolic compounds contributing to the anti-acanthamoebic activity on pathogenic Acanthamoeba species.
Piperaceae ; Acanthamoeba castellanii--pathogenicity

Acanthamoeba keratitis has been increasing in recent years. Main risk factors are contact lens wear and their cleaning solutions. Most contact lens wearers use multipurpose disinfecting solutions (MPDS) for cleansing and disinfecting microorganisms because of its convenience. We determined amoebicidal effects of MPDS made in Korea and their cytotoxicity on human corneal epithelium cells. Fifteen commercial MPDS (A to O) were tested for their amoebicidal effects on Acanthamoeba castellanii trophozoites and cysts by using a most probable number (MPN) technique. Among them, 7 kinds of MPDS showed little or no amoebicidal effects for 24 hr exposure. Solutions A, B, G, H, L, and O showed positive amoebicidal effects, and solutions M and N killed almost all trophozoites and cysts after 24 hr exposure. However, 50%-N solution showed 56% cytotoxicity on human corneal epithelial cells within 4 hr exposure, and 50%-O solution also showed 62% cytotoxicity on human cells within 4 hr exposure. Solution A did not show any cytotoxicity on human cells. These results revealed that most MPDS made in Korea were ineffective to kill Acanthamoeba. The solutions having amoebicidal activity also showed high levels of cytotoxicity on human corneal epithelial cells. New formulations for improved MPDS that are amoebicidal but safe for host cells are needed to prevent Acanthamoeba keratitis.
Acanthamoeba castellanii* ; Acanthamoeba Keratitis ; Acanthamoeba* ; Epithelial Cells ; Epithelium, Corneal ; Humans ; Korea ; Risk Factors ; Trophozoites

PURPOSE: To evaluate the cysticidal effect of 5 kinds of commercially available contact lens disinfectants against 2 clinical isolates of Acanthamoeba. METHODS: Five kinds of commercially available contact lens disinfectants were soaked with cysts of Acanthamoeba ludgdunesis and castellanii at the concentration of 10(3), 10(4), and 10(5) cells/ml for 1 and 4 or 6 hours. Cysts which were not excysted in 7 days after treatment were recognized to be killed. Morphologic changes were evaluated by electron microscopic observation. RESULTS: Contact lens disinfectants which contain myristamidopropyl dimethylamine (MAPD) showed the best cysticidal effect. These disinfectants demonstrated a cysticidal effect on both Acanthamoeba species of all concentrations in 6-hour treatment. Contact lens disinfectants which contain polyhexamethylene biguanide (PHMB) did not demonstrate cysticidal effect, except for Acanthamoeba castellanii at the concentration of 10(3) cells/ml, in either 4- or 6-hour treatment. Separation of plasma membrane from endocyst and damage of organelles were prominent in cases showing a cysticidal effect. CONCLUSIONS: Contact lens disinfectant which contains MAPD may be helpful in preventing the Acanthamoeba keratitis. A higher concentration of PHMB is required to be effective in preventing Acanthamoeba keratitis.
Acanthamoeba castellanii ; Acanthamoeba Keratitis ; Acanthamoeba* ; Cell Membrane ; Contact Lens Solutions ; Disinfectants ; Organelles

This study was performed to investigate the biochemical properties of Acanthamoeba proteinase, its role in the pathogenesis of Acanthamoeba keratitis and the therapeutic effect of the homogenate of amniotic membrane as a proteinase inhibitors. Acanthamoeba castellanii isolated from the keratitis patient was cultured in PYG medium, in which the excretory and secretory products were analysed. The secretory proteinases of A. castellanii wre identified using in vitro azocasein assay, activity-PAGE, and various protein substrate degradation assays, and one of them was purified and characterized. The pruified secretory proteinase was a kind of serine proteinase. Its molecular weight was 105 kDa and optimal pH was 8.5. It was able to degrade the various protein substrates such as fibronectin, IgA, IgG, fibrinogen. The various proteinase ingibitors and the amniotic membrane homogenates were tested in vitro against the purified seirne proteinase. The amniotic membrane homegenates markedly showed the inhibitory effect against the enzyme activity and this inhibitory effect was also revealed in animal study. In vivo study, this purified proteinase was infected into 14 pigmented rabbit corneas, pretreated with steroids. The corneal lesions induced by both of the purified proteinase and A. castellanii, showed similar clinical findings each other, in which the stromal infiltration and opacity with epithelial defect was revealed. These corneal lesions were significantly inhibited without any side effects of the amniotic membrane homogenates. Conclusively, Acanthamoeba proteinase was closely associated with the pathogenesis of Acanthamoeba keratitis. This study provides a successful animal model of Acanthamoeba keratitis using pigmented rabbit. And the fact that Acanthamoeba-induced corneal lesions were inhibited by the amniotic membrane homogenate, suggested that the amniotic membrane homogenate have the ability of the serine protinase inhibition further investigative studies are also necessary.
Acanthamoeba castellanii ; Acanthamoeba Keratitis* ; Acanthamoeba* ; Amnion* ; Animals ; Cornea ; Fibrinogen ; Fibronectins ; Humans ; Hydrogen-Ion Concentration ; Immunoglobulin A ; Immunoglobulin G ; Keratitis ; Models, Animal ; Molecular Weight ; Peptide Hydrolases ; Serine ; Serine Proteases ; Steroids

Pathogenic Acanthamoeba spp. cause granulomatous amoebic encephalitis and keratitis. Acanthamoeba keratitis (AK) is a rare but serious ocular infection that can result in permanent visual impairment or blindness. However, pathogenic factors of AK remain unclear and treatment for AK is arduous. Expression levels of proteins secreted into extracellular space were compared between A. castellanii pathogenic (ACP) and non-pathogenic strains. Two-dimensional polyacrylamide gel electrophoresis revealed 123 differentially expressed proteins, including 34 increased proteins, 7 qualitative increased proteins, 65 decreased proteins, and 17 qualitative decreased proteins in ACP strain. Twenty protein spots with greater than 5-fold increase in ACP strain were analyzed by liquid chromatography triple quadrupole mass spectrometry. These proteins showed similarity each to inosine-uridine preferring nucleoside hydrolase, carboxylesterase, oxygen-dependent choline dehydrogenase, periplasmic-binding protein proteinases and hypothetical proteins. These proteins expressed higher in ACP may provide some information to understand pathogenicity of Acanthamoeba.
Acanthamoeba castellanii ; Acanthamoeba Keratitis ; Acanthamoeba ; Blindness ; Carboxylesterase ; Choline Dehydrogenase ; Chromatography, Liquid ; Electrophoresis, Polyacrylamide Gel ; Encephalitis ; Extracellular Space ; Eye Infections ; Keratitis ; Mass Spectrometry ; Peptide Hydrolases ; Virulence ; Vision Disorders

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.
Acanthamoeba castellanii ; Acanthamoeba* ; Epigenesis, Genetic ; Epigenomics ; Gene Expression Regulation ; Methyltransferases ; Parasites ; Protein-Arginine N-Methyltransferases* ; RNA, Small Interfering ; Trophozoites

Protein arginine methyltransferase (PRMT) is an important epigenetic regulator in eukaryotic cells. During encystation, an essential process for Acanthamoeba survival, the expression of a lot of genes involved in the encystation process has to be regulated in order to be induced or inhibited. However, the regulation mechanism of these genes is yet unknown. In this study, the full-length 1,059 bp cDNA sequence of Acanthamoeba castellanii PRMT1 (AcPRMT1) was cloned for the first time. The AcPRMT1 protein comprised of 352 amino acids with a SAM-dependent methyltransferase PRMT-type domain. The expression level of AcPRMT1 was highly increased during encystation of A. castellanii. The EGFP-AcPRMT1 fusion protein was distributed over the cytoplasm, but it was mainly localized in the nucleus of Acanthamoeba. Knock down of AcPRMT1 by synthetic siRNA with a complementary sequence failed to form mature cysts. These findings suggested that AcPRMT1 plays a critical role in the regulation of encystation of A. castellanii. The target gene of AcPRMT1 regulation and the detailed mechanisms need to be investigated by further studies.
Acanthamoeba castellanii* ; Acanthamoeba* ; Amino Acids ; Clone Cells ; Cytoplasm ; DNA, Complementary ; Epigenomics ; Eukaryotic Cells ; Protein-Arginine N-Methyltransferases* ; RNA, Small Interfering

The induction of NAD(P)H: quinone oxidoreductase (QR), glutathione S-transferase (GST), and glutathione (GSH) levels in hepa1c1c7 cells (murine hepatoma) by waxy brown rice cultured with Phellinus igniarius to induce anticarcinogenic enzymes were measured. In addition, the inhibition of polyamines metabolism was tested with the growth of Acanthamoeba castellanii. The result shows that QR, GST activities, and GSH levels of experimental animals were increased much more by feeding the methanol extract of waxy brown rice cultured with Phellinus igniarius than those of the rats received the ethanol of uncultured brown rice. The growth of A. castellanii was inhibited mostly at 40 mg/3 ml concentration of methanol extract of waxy brown rice cultured with P. igniarius. The results suggested that waxy brown rice cultured with P. igniarius possess chemopreventive activity by inducing anticarcinogenic enzymes and inhibiting polyamine metabolism.
Acanthamoeba castellanii ; Animals ; Chemoprevention ; Ethanol ; Glutathione ; Glutathione Transferase ; Metabolism ; Methanol ; Polyamines ; Rats

Encystation mediating cyst specific cysteine proteinase (CSCP) of Acanthamoeba castellanii is expressed remarkably during encystation. However, the molecular mechanism involved in the regulation of CSCP gene expression remains unclear. In this study, we focused on epigenetic regulation of gene expression during encystation of Acanthamoeba. To evaluate methylation as a potential mechanism involved in the regulation of CSCP expression, we first investigated the correlation between promoter methylation status of CSCP gene and its expression. A 2,878 bp of promoter sequence of CSCP gene was amplified by PCR. Three CpG islands (island 1–3) were detected in this sequence using bioinformatics tools. Methylation of CpG island in trophozoites and cysts was measured by bisulfite sequence PCR. CSCP promoter methylation of CpG island 1 (1,633 bp) was found in 8.2% of trophozoites and 7.3% of cysts. Methylation of CpG island 2 (625 bp) was observed in 4.2% of trophozoites and 5.8% of cysts. Methylation of CpG island 3 (367 bp) in trophozoites and cysts was both 3.6%. These results suggest that DNA methylation system is present in CSCP gene expression of Acanthamoeba. In addition, the expression of encystation mediating CSCP is correlated with promoter CpG island 1 hypomethylation.
Acanthamoeba castellanii* ; Acanthamoeba* ; Computational Biology ; CpG Islands ; Cysteine Proteases ; DNA Methylation* ; DNA* ; Epigenomics ; Gene Expression Regulation ; Gene Expression* ; Methylation ; Negotiating ; Polymerase Chain Reaction ; Trophozoites

Pathogenic Naegleria fowleri, Acanthamoeba castellanii, and Acanthamoeba polyphaga, are distributed worldwide. They are causative agents of primary amoebic meningoencephalitis or acanthamoebic keratitis in humans, respectively. Trophozoites encyst in unfavorable environments, such as exhausted food supply and desiccation. Until recently, the method of N. fowleri encystation used solid non-nutrient agar medium supplemented with heat-inactivated Escherichia coli; however, for the amoebic encystment of Acanthamoeba spp., a defined, slightly modified liquid media is used. In this study, in order to generate pure N. fowleri cysts, a liquid encystment medium (buffer 1) modified from Page’s amoeba saline was applied for encystation of N. fowleri. N. fowleri cysts were well induced after 24 hr with the above defined liquid encystment medium (buffer 1). This was confirmed by observation of a high expression of differential mRNA of nfa1 and actin genes in trophozoites. Thus, this liquid medium can replace the earlier non-nutrient agar medium for obtaining pure N. fowleri cysts. In addition, for cyst formation of Acanthamoeba spp., buffer 2 (adjusted to pH 9.0) was the more efficient medium. To summarize, these liquid encystment media may be useful for further studies which require axenic and pure amoebic cysts.
Acanthamoeba ; Acanthamoeba castellanii ; Actins ; Agar ; Amoeba* ; Desiccation ; Escherichia coli ; Food Supply ; Humans ; Hydrogen-Ion Concentration ; Keratitis ; Meningoencephalitis ; Methods ; Naegleria fowleri ; RNA, Messenger ; Trophozoites