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 castellanii has ubiquitous distribution and causes primary acanthamoebic keratitis (AK). AK is a common disease in contact lens wearers and results in permanent visual impairment or blindness. In this study, we observed the cytopathic effect, in vitro cytotoxicity, and secretion pattern of cytokines in human corneal epithelial cells (HCECs) induced by A. castellanii trophozoites and/or cysts. Morphological observation revealed that panked dendritic HCECs co-cultured with amoeba cysts had changed into round shape and gradually died. Such changes were more severe in co-culture with cyst than those of co-cultivation with trophozoites. In vitro cytotoxicity assay revealed the highest cytotoxicity to HCECs in the co-culture system with amoeba cysts. A. castellanii induced the expression of IL-1α, IL-6, IL-8, and CXCL1 in HCECs. Secreted levels of IL-1α, IL-6, and IL-8 in HCECs co-cultured with both trophozoites and cysts were increased at an early incubation time (3 and 6 hr). These results suggested that cytopathic changes and pro-inflammatory cytokines release of HCECs in response to A. castellanii, especially amoebic cysts, are an important mechanism for AK development.
Acanthamoeba castellanii ; Acanthamoeba ; Amoeba ; Blindness ; Coculture Techniques ; Cytokines ; Epithelial Cells ; Humans ; In Vitro Techniques ; Interleukin-6 ; Interleukin-8 ; Keratitis ; Trophozoites ; Vision Disorders

Acanthamoeba spp. are free-living protozoa that are opportunistic pathogens for humans. Cysteine proteases of Acanthamoeba have been partially characterized, but their biochemical and functional properties are not clearly understood yet. In this study, we isolated a gene encoding cysteine protease of A. castellanii (AcCP) and its biochemical and functional properties were analyzed. Sequence analysis of AcCP suggests that this enzyme is a typical cathepsin L family cysteine protease, which shares similar structural characteristics with other cathepsin L-like enzymes. The recombinant AcCP showed enzymatic activity in acidic conditions with an optimum at pH 4.0. The recombinant enzyme effectively hydrolyzed human proteins including hemoglobin, albumin, immunoglobuins A and G, and fibronectin at acidic pH. AcCP mainly localized in lysosomal compartment and its expression was observed in both trophozoites and cysts. AcCP was also identified in cultured medium of A. castellanii. Considering to lysosomal localization, secretion or release by trophozoites and continuous expression in trophozoites and cysts, the enzyme could be a multifunctional enzyme that plays important biological functions for nutrition, development and pathogenicity of A. castellanii. These results also imply that AcCP can be a promising target for development of chemotherapeutic drug for Acanthamoeba infections.
Acanthamoeba castellanii ; Acanthamoeba ; Cathepsin L ; Cathepsins ; Cysteine Proteases ; Cysteine ; Fibronectins ; Genes, vif ; Humans ; Hydrogen-Ion Concentration ; Lysosomes ; Sequence Analysis ; Trophozoites ; Virulence

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 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

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

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

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

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

Acanthamoeba cysts are resistant to unfavorable physiological conditions and various disinfectants. Acanthamoeba cysts have 2 walls containing various sugar moieties, and in particular, one third of the inner wall is composed of cellulose. In this study, it has been shown that down-regulation of cellulose synthase by small interfering RNA (siRNA) significantly inhibits the formation of mature Acanthamoeba castellanii cysts. Calcofluor white staining and transmission electron microscopy revealed that siRNA transfected amoeba failed to form an inner wall during encystation and thus are likely to be more vulnerable. In addition, the expression of xylose isomerase, which is involved in cyst wall formation, was not altered in cellulose synthase down-regulated amoeba, indicating that cellulose synthase is a crucial factor for inner wall formation by Acanthamoeba during encystation.
Acanthamoeba castellanii/*enzymology/genetics/metabolism ; Aldose-Ketose Isomerases/*biosynthesis ; Amebiasis/*pathology ; Benzenesulfonates ; Cell Wall/chemistry/genetics/*metabolism ; Cellulose/biosynthesis ; Down-Regulation ; Encephalitis/parasitology ; Glucosyltransferases/*biosynthesis/genetics ; Keratitis/parasitology ; Microscopy, Electron, Transmission ; RNA Interference ; RNA, Small Interfering