[show abstract][hide abstract] ABSTRACT: Autophagy-related protein 8 (Atg8) is an essential component of autophagy formation and encystment of cyst-forming parasites, and some protozoa, such as, Acanthamoeba, Entamoeba, and Dictyostelium, have been reported to possess a type of Atg8. In this study, an isoform of Atg8 was identified and characterized in Acanthamoeba castellanii (AcAtg8b). AcAtg8b protein was found to encode 132 amino acids and to be longer than AcAtg8 protein, which encoded 117 amino acids. Real-time PCR analysis showed high expression levels of AcAtg8b and AcAtg8 during encystation. Fluorescence microscopy demonstrated that AcAtg8b is involved in the formation of the autophagosomal membrane. Chemically synthesized siRNA against AcAtg8b reduced the encystation efficiency of Acanthamoeba, confirming that AcAtg8b, like AcAtg8, is an essential component of cyst formation in Acanthamoeba. Our findings suggest that Acanthamoeba has doubled the number of Atg8 gene copies to ensure the successful encystation for survival when 1 copy is lost. These 2 types of Atg8 identified in Acanthamoeba provide important information regarding autophagy formation, encystation mechanism, and survival of primitive, cyst-forming protozoan parasites.
The Korean Journal of Parasitology 10/2013; 51(5):497-502. · 0.88 Impact Factor
[show abstract][hide abstract] ABSTRACT: Chloroquine has been used massively for vivax malaria prophylaxis and treatment in the Republic of Korea (ROK) military personnel from 1997. Although prophylaxis is generally regarded as successful among ROK military, prophylaxis failure has been repeatedly reported. Before the prophylaxis program was started on July 4th 2011, which was completed on October 16th 2011, by the ROK military, more than 60% of malaria cases were attributed to new infection or long-latency relapse. During the prophylaxis program, the authors re-examined the efficiency of chloroquine chemoprophylaxis in ROK military during the last six months of 2011 by measuring compliance and whole blood chloroquine levels in 41 malaria patients immediately before instituting antimalarial therapy between July and December. Three patients (7.3%) showed good compliance, and had whole blood total chloroquine levels above the minimally inhibitory concentration (100 ng/mL). However, 28 (69.3%) of these 41 patients when admitted to hospital showed poor or no compliance with prophylaxis; 4 of the 28 (14.3%) were stationed outside the mass prophylaxis region, and 5 (17.9%) subjects were infected after the prophylaxis program had finished. These findings indicate that the current malaria control program should be carefully reconsidered, in terms of, individual instruction, current chemoprophylaxis program regimens, and schedules to improve the efficacy of prophylaxis in the ROK military.
Parasitology International 07/2013; · 2.30 Impact Factor
[show abstract][hide abstract] ABSTRACT: Amoebic keratitis (AK) caused by Acanthamoeba is one of the most serious corneal infections. AK is frequently misdiagnosed initially as viral, bacterial, or fungal keratitis, thus ensuring treatment delays. Accordingly, the early detection of Acanthamoeba would contribute significantly to disease management and selection of an appropriate anti-amoebic therapy. Recently, the loop-mediated isothermal amplification (LAMP) method has been applied to the clinical diagnosis of a range of infectious diseases. Here, we describe a rapid and efficient LAMP-based method targeting Acanthamoeba 18S rDNA gene for the detection of Acanthamoeba using clinical ocular specimens in the diagnosis of AK. Acanthamoeba LAMP assays detected 11 different strains including all AK-associated species. The copy number detection limit for a positive signal was 10 DNA copies of 18S rDNA per reaction. No cross-reactivity with the DNA of fungi or other protozoa was observed. The sensitivity of LAMP assay was higher than those of Nelson primer PCR and JDP primer PCR. In the present study, LAMP assay based on directly heat-treated samples was found to be as efficient at detecting Acanthamoeba as DNA extracted using a commercial kit, whereas PCR was only effective when commercial kit-extracted DNA was used. This study showed that the devised Acanthamoeba LAMP assay could be used to diagnose AK in a simple, sensitive, and specific manner.
The Korean Journal of Parasitology 06/2013; 51(3):269-77. · 0.88 Impact Factor
[show abstract][hide abstract] ABSTRACT: The encystation of Acanthamoeba leads to the formation of resilient cysts from vegetative trophozoites. This process is essential for parasite survival under unfavorable conditions, such as, those associated with starvation, low temperatures, and biocides. Furthermore, cysteine proteases have been implicated in the massive turnover of intracellular components required for encystation. Thus, strict modulation of the activities of cysteine proteases is required to protect Acanthamoeba from intracellular damage. However, mechanisms underlying the control of protease activity during encystation have not been established in Acanthamoeba. In the present study, we identified and characterized Acanthamoeba cysteine protease inhibitor (AcStefin), which was found to be highly expressed during encystation and to be associated with lysosomes by fluorescence microscopy. Recombinant AcStefin inhibited various cysteine proteases, including human cathepsin B, human cathepsin L, and papain. Transfection with small interfering RNA against AcStefin increased cysteine protease activity during encystation, and resulted in incomplete cyst formation, reduced excystation efficiency, and a significant reduction in cytoplasmic area. Taken together, these results indicate that AcStefin is involved in the modulation of cysteine proteases and that it plays an essential role during the encystation of Acanthamoeba.
[show abstract][hide abstract] ABSTRACT: The mature cyst of Acanthamoeba is highly resistant to various antibiotics and therapeutic agents. Cyst wall of Acanthamoeba are composed of cellulose, acid-resistant proteins, lipids, and unidentified materials. Because cellulose is one of the primary components of the inner cyst wall, cellulose synthesis is essential to the process of cyst formation in Acanthamoeba. In this study, we hypothesized the key and short-step process in synthesis of cellulose from glycogen in encysting Acanthamoeba castellanii, and confirmed it by comparing the expression pattern of enzymes involving glycogenolysis and cellulose synthesis. The genes of 3 enzymes, glycogen phosphorylase, UDP-glucose pyrophosphorylase, and cellulose synthase, which are involved in the cellulose synthesis, were expressed high at the 1st and 2nd day of encystation. However, the phosphoglucomutase that facilitates the interconversion of glucose 1-phosphate and glucose 6-phosphate expressed low during encystation. This report identified the short-cut pathway of cellulose synthesis required for construction of the cyst wall during the encystation process in Acanthamoeba. This study provides important information to understand cyst wall formation in encysting Acanthamoeba.
The Korean Journal of Parasitology 12/2012; 50(4):361-4. · 0.88 Impact Factor
[show abstract][hide abstract] ABSTRACT: Using the microarray to identify encystation mediating factors, significantly higher expression of a cysteine protease gene was observed in cysts, compared with trophozoites. Results of real-time PCR analysis also showed a magnificent increase of cysteine protease levels during encystation of Acanthamoeba. We named the gene cyst specific cysteine protease (cscp) of Acanthamoeba. The purified recombinant protein of CSCP showed activities of papain and cathepsin B against the substrates. During encystation, EGFP fused CSCP showed colocalization with LysoTracker, an autophagosome marker, in transiently transfected amoeba. Amoeba transfected with siRNA against cscp was unable to form mature cysts. Undigested mitochondria in vacuole like structures were observed in cscp siRNA treated cells by transmission electron microscopy. These results provide evidence of the important role of CSCP in autophagosomal degradation of cell constituents, particularly mitochondria, during encystation of Acanthamoeba.
Molecular and Biochemical Parasitology 08/2012; 185(2):121-6. · 2.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: Protein kinase C (PKC) is involved in receptor desensitization, membrane biogenesis, transcription regulation, immune response mediation, and cell growth regulation. Results of ESTs analysis and microarray analysis of Acanthamoeba cysts revealed high expression of PKC during encystation of Acanthamoeba. PKC inhibitor, chelerythrine chloride, inhibited cyst maturation of Acanthamoeba. Through domain search analysis, we found 27 types of PKC genes from Acanthamoeba ESTs (AcPKC1-AcPKC27), all of which were defined as atypical PKC isoforms. Results of fluorescence microscopic analysis showed the localization of AcPKC in cell membranes or nuclear membranes during encystation. siRNA against AcPKC reduced the encystation efficiency of Acanthamoeba. These AcPKC may be involved in several signal transduction pathways, especially during encystation of Acanthamoeba.
[show abstract][hide abstract] ABSTRACT: Autophagy, an evolutionarily conserved protein degradation pathway in eukaryotes, plays essential roles during starvation and cellular differentiation by eliminating unwanted and/or unnecessary cell material including organelles. Autophagy protein 16 (Atg16) is an essential component of the autophagic machinery. The present study identified and characterized an Atg16 homologue (AcAtg16) in Acanthamoeba, an opportunistic pathogen responsible for several distinct diseases in humans. AcAtg16 was highly expressed during encystation and was found to be associated with small or large vesicular structures that partially colocalized with autophagolysosomes. Small interfering RNA against AcAtg16 inhibited autophagosome formation and reduced the encystation efficiency of Acanthamoeba. Moreover, most mitochondria remained undigested in these knockdown cells. Taken together, these results indicate that AcAtg16 is involved in autophagosome formation and plays an essential role in the encystation of Acanthamoeba.
Molecular and Biochemical Parasitology 03/2012; 183(2):158-65. · 2.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: Acanthamoeba infection is difficult to treat because of the resistance property of Acanthamoeba cyst against the host immune system, diverse antibiotics, and therapeutic agents. To identify encystation mediating factors of Acanthamoeba, we compared the transcription profile between cysts and trophozoites using microarray analysis. The DNA chip was composed of 12,544 genes based on expressed sequence tag (EST) from an Acanthamoeba ESTs database (DB) constructed in our laboratory, genetic information of Acanthamoeba from TBest DB, and all of Acanthamoeba related genes registered in the NCBI. Microarray analysis indicated that 701 genes showed higher expression than 2 folds in cysts than in trophozoites, and 859 genes were less expressed in cysts than in trophozoites. The results of real-time PCR analysis of randomly selected 9 genes of which expression was increased during cyst formation were coincided well with the microarray results. Eukaryotic orthologous groups (KOG) analysis showed an increment in T article (signal transduction mechanisms) and O article (posttranslational modification, protein turnover, and chaperones) whereas significant decrement of C article (energy production and conversion) during cyst formation. Especially, cystein proteinases showed high expression changes (282 folds) with significant increases in real-time PCR, suggesting a pivotal role of this proteinase in the cyst formation of Acanthamoeba. The present study provides important clues for the identification and characterization of encystation mediating factors of Acanthamoeba.
The Korean Journal of Parasitology 12/2011; 49(4):341-7. · 0.88 Impact Factor
[show abstract][hide abstract] ABSTRACT: Rodent malaria parasites, such as Plasmodium berghei, are practical and useful model organisms for human malaria research because of their analogies to the human malaria in terms of structure, physiology, and life cycle. Exploiting the available genetic sequence information, we constructed a cDNA library from the erythrocytic stages of P. berghei and analyzed the expressed sequence tag (EST). A total of 10,040 ESTs were generated and assembled into 2,462 clusters. These EST clusters were compared against public protein databases and 48 putative new transcripts, most of which were hypothetical proteins with unknown function, were identified. Genes encoding ribosomal or membrane proteins and purine nucleotide phosphorylases were highly abundant clusters in P. berghei. Protein domain analyses and the Gene Ontology functional categorization revealed translation/protein folding, metabolism, protein degradation, and multiple family of variant antigens to be mainly prevalent. The presently-collected ESTs and its bioinformatic analysis will be useful resources to identify for drug target and vaccine candidates and validate gene predictions of P. berghei.
The Korean Journal of Parasitology 09/2011; 49(3):221-8. · 0.88 Impact Factor
[show abstract][hide abstract] ABSTRACT: Autophagy is a catabolic process involved in the degradation of a cell's own components for cell growth, development, homeostasis, and the recycling of cellular products. Autophagosome is an essential component in the protozoan parasite during differentiation and encystation. The present study identified and characterized autophagy-related protein (Atg) 3, a member of Atg8 conjugation system, in Acanthamoeba castellanii (AcAtg3). AcAtg3 encoding a 304 amino acid protein showed high similarity with the catalytic cysteine site of other E2 like enzymes of ubiquitin system. Predicted 3D structure of AcAtg3 revealed a hammer-like shape, which is the characteristic structure of E2-like enzymes. The expression level of AcAtg3 did not increase during encystation. However, the formation of mature cysts was significantly reduced in Atg3-siRNA transfected cells in which the production of Atg8-phosphatidylethanolamine conjugate was inhibited. Fluorescent microscopic analysis revealed that dispersed AcAtg3-EGFP fusion protein gathered around autophagosomal membranes during encystation. These results provide important information for understanding autophagic machinery through the lipidation reaction mediated by Atg3 in Acanthamoeba.
The Korean Journal of Parasitology 06/2011; 49(2):103-8. · 0.88 Impact Factor
[show abstract][hide abstract] ABSTRACT: The life cycle of Acanthamoeba consists of two stages, trophozoite and cyst. The cyst form is resistant to almost all antibiotics. By long term cultivation, Acanthamoeba severely attenuated the encysting ability. To determine the changing of gene expression by the long term cultivation, especially focusing an encystation mediating factors, this study compared the ESTs of the fresh strain and the old strain, and trophozoite. Comparison of the KOG (euKaryotic Orthologous Groups) analysis relative to trophozoite revealed higher percentages of cyst ESTs related to G (Carbohydrate transport and metabolism), H (Coenzyme transport and metabolism), I (Lipid transport and metabolism), D (Cell cycle control, cell division, chromosome partitioning), T (signal transduction mechanisms), and O (Posttranslational modification, protein turnover, chaperones). In addition to this result, KOG analysis of fresh strain relative to old strain showed higher percentage of cyst ESTs related to metabolism category and T (signal transduction mechanisms) article. ESTs of the fresh strain revealed more various gene profiles compared to the old strain including encystation mediating factors like autophagy related proteins (Z article) and signal transduction proteins (T article). Twenty seven kinds of protein kinase C (PKC) like genes were detected in cyst or trophozoite ESTs and twenty one of them were highly expressed during encystation. The information of the expressed genes during encystation in only the fresh strain will provide new clues to understanding the encystation mechanism of encysting protozoa including Acanthamoeba.
[show abstract][hide abstract] ABSTRACT: Paragonimus westermani is a trematode parasite that causes inflammatory lung disease as well as systemic infections in carnivorous mammals. The interaction of the parasite with host cells and paired worms is initiated by adhesion and plays an important role in parasite proliferation and differentiation. In this study, we isolated a cDNA encoding a P. westermani fasciclin I domain-containing protein (Pwfas-I). The fasiclin-I domain is suggested to be involved in cell adhesion, migration, and differentiation. Immunohistochemical analysis of P. westermani adult worms with polyclonal anti-Pwfas-I serum revealed immunoreactivity in the egg shells and the cells lining the sub-tegumental layer of adult worm throughout the contact regions of the cyst wall and paired worms. Using cell adhesion and spreading assays, we showed that Pwfas-I supports cell adhesion and spreading. Furthermore, we determined that the alphanubeta5 integrin was a functional receptor for the Pwfas-I. Taken together, these results suggest that Pwfas-I may be functional for the modulation of cell adhesion via binding with alphanubeta5 integrin in the extracellular matrix of Paragonimus.
[show abstract][hide abstract] ABSTRACT: Amino acids derived from hemoglobin are essential to protein synthesis required for growth and development of the Plasmodium vivax malaria parasite. M17 leucine aminopeptidase (LAP) is a cytosolic metallo-exopeptidase that catalyzes the removal of amino acids from the peptide generated in the process of hemoglobin degradation. Inhibitors of the enzyme have shown promise as drugs against Plasmodium infections, implicating aminopeptidases as a novel potential anti-malarial chemotherapy target. In this study, we isolated a cDNA encoding a 68kDa P. vivax LAP (PvLAP). Deduced amino acid sequence of PvLAP exhibited significant sequence homology with LAP from Plasmodium falciparum. Biochemical analysis of the recombinant PvLAP protein produced in Escherichia coli demonstrated preferential substrate specificity for the fluorogenic peptide Leu-7-amido-4-methylcoumarin hydroxide and inhibition by EDTA, 1,10-phenanthroline, and bestatin, which are conserved characteristics of the M17 family of LAP. PvLAP was optimally active at slightly alkaline pH and its activity was dependent on divalent metal ions. Based on the biochemical properties and immunofluorescence localization, PvLAP is concluded to represent a LAP in P. vivax. The enzyme is most likely responsible for the catabolism of host hemoglobin and, hence, represents a potential target of both P. falciparum and P. vivax chemotherapy.
Molecular and Biochemical Parasitology 11/2009; 170(1):45-8. · 2.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: The virulence of Acanthamoeba can be attenuated by long-term in vitro cultivation, and can be recovered by serial mouse-brain passage via intranasal inoculation. Recovery is concomitant with changes in expression of virulence-related genes. To investigate the virulence factors of Acanthamoeba, expressed sequence tags (ESTs) from two kinds of cDNA libraries-long-term in vitro cultivated A. healyi (OLD) and three times mouse-brain passaged A. healyi (MBP)-were compared using reciprocal BLAST analysis, eukaryotic orthologous groups (KOG) assignment, and gene annotation. A total of 938 (OLD) and 1033 (MBP) ESTs were sequenced and resulted in the assembling of 718 OLD and 833 MBP unique sequences. Comparison of the KOG analysis revealed a relatively higher percentage of MBP ESTs in genes related to transcription (K group), amino acid transport and metabolism (E group), coenzyme transport and metabolism (H group), and secondary metabolites biosynthesis, transport and metabolism (Q group). However, a higher percentage of unidentified MBP ESTs (57.9%) than OLD ESTs (28.9%) was evidence of the limited understanding of virulence-related factors of Acanthamoeba. Characterization of the genes expressed during brain passage in mice will be useful in clarifying the pathogenesis of granulomatous amoebic encephalitis by Acanthamoeba.
[show abstract][hide abstract] ABSTRACT: The genus Acanthamoeba can cause severe infections such as granulomatous amebic encephalitis and amebic keratitis in humans. However, little genomic information of Acanthamoeba has been reported. Here, we constructed Acanthamoeba expressed sequence tags (EST) database (Acanthamoeba EST DB) derived from our 4 kinds of Acanthamoeba cDNA library. The Acanthamoeba EST DB contains 3,897 EST generated from amebae under various conditions of long term in vitro culture, mouse brain passage, or encystation, and downloaded data of Acanthamoeba from National Center for Biotechnology Information (NCBI) and Taxonomically Broad EST Database (TBestDB). The almost reported cDNA/genomic sequences of Acanthamoeba provide stand alone BLAST system with nucleotide (BLAST NT) and amino acid (BLAST AA) sequence database. In BLAST results, each gene links for the significant information including sequence data, gene orthology annotations, relevant references, and a BlastX result. This is the first attempt for construction of Acanthamoeba database with genes expressed in diverse conditions. These data were integrated into a database (http://www.amoeba.or.kr).
The Korean Journal of Parasitology 07/2009; 47(2):103-7. · 0.88 Impact Factor
[show abstract][hide abstract] ABSTRACT: Autophagy is an evolutionally conserved protein degradation pathway in eukaryotes. It plays essential roles during starvation, cellular differentiation, cell death, and aging by eliminating unwanted or unnecessary organelles and recycling the components for reuse. ATG8, a member of a novel ubiquitin-like protein family, is an essential component of the autophagic machinery. The present study identified and characterized autophagy protein 8 in Acanthamoeba castellanii an amphizoic amoeba causing granulomatous amoebic encephalitis and amoebic keratitis in humans. Real-time polymerase chain reaction demonstrated that the A. castellanii Atg8 (AcAtg8) gene encoding a 118 amino acid protein was highly expressed during encystation. Fluorescence microscopic analysis following transient transfection of enhanced green fluorescent protein-AcAtg8 revealed small or large vacuolar fluorescent structures in an encysting amoeba. The Atg8 fluorescent structures on the membrane were identified as autophagosomes by co-localization analysis with LysoTracker. Chemically synthesized small interfering RNA against AcAtg8 reduced the encystation efficiency and inhibited autophagosome formation in Acanthamoeba.
Molecular and Biochemical Parasitology 07/2009; 168(1):43-8. · 2.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: Three Acanthamoeba isolates (KA/E9, KA/E17, and KA/E23) from patients with keratitis were identified as Acanthamoeba triangularis by analysis of their molecular characteristics, a species not previously recognized to be a corneal pathogen. Epidemiologic significance of A. triangularis as a keratopathogen in Korea has been discussed. Morphologic features of Acanthamoeba cysts were examined under a microscope with differential interference contrast (DIC) optics. Mitochondrial DNA (mtDNA) of the ocular isolates KA/E9, KA/E17, and KA/E23 were digested with restriction enzymes, and the restriction patterns were compared with those of reference strains. Complete nuclear 18S and mitochondrial (mt) 16S rDNA sequences were subjected to phylogenetic analysis and species identification. mtDNA RFLP of 3 isolates showed very similar patterns to those of SH621, the type strain of A. triangularis. 16S and 18S rDNA sequence analysis confirmed 3 isolates to be A. triangularis. 18S rDNA sequence differences of the isolates were 1.3% to 1.6% and those of 16S rDNA, 0.4% to 0.9% from A. triangularis SH621. To the best of our knowledge, this is the first report, confirmed by 18S and 16S rDNA sequence analysis, of keratitis caused by A. triangularis of which the type strain was isolated from human feces. Six isolates of A. triangularis had been reported from contaminated contact lens cases in southeastern Korea.
The Korean Journal of Parasitology 10/2008; 46(3):157-64. · 0.88 Impact Factor
[show abstract][hide abstract] ABSTRACT: Members of the genus Acanthamoeba, amphizoic protozoan parasites, are causative agents of granulomatous amoebic encephalitis and amoebic keratitis. Proteinases play a role in various biologic actions in Acanthamoeba, including host tissue destruction, pathogenesis, and digestion of phagocytosed food. Interestingly, we found that encystation of Acanthamoeba was inhibited by the serine proteinase inhibitor phenylmethanesulfonyl fluoride. In this study, we characterize a serine proteinase that is involved in mediating the encystation of Acanthamoeba. This encystation-mediating serine proteinase (EMSP) is shown to be highly expressed during encystation by real-time PCR and Western blot analysis. Chemically synthesized small interfering RNA against EMSP inhibited the expression of EMSP mRNA and significantly reduced the encystation efficiency of Acanthamoeba. An EMSP-enhanced green fluorescent protein fusion protein localized to vesicle-like structures within the amoeba. Using LysoTracker analysis, these vesicular structures were confirmed to be lysosomes. After incubation of the transfected amoeba in encystment media, small fluorescent vesicle-like structures gathered and formed ball-like structures, which were identified as colocalizing with the autophagosome. Taken together, these results indicate that EMSP plays an important role in the differentiation of Acanthamoeba by promoting autolysis.
[show abstract][hide abstract] ABSTRACT: Paragonimus westermani is a tissue-invading trematode parasite that causes inflammatory lung disease as well as systemic infections including cerebral invasion in carnivorous mammals. While aminopeptidases play important roles in trematodes in the catabolism of host hemoglobin, an essential source of nutrient for the parasite, little is known about aminopeptidase in Paragonimus. Presently, we isolated a cDNA encoding a 58 kDa P. westermani leucine aminopeptidase (PwLAP). Deduced amino acid sequence of PwLAP exhibited significant sequence homology with LAP from Schistosoma spp. and Fasciola hepatica. Biochemical analysis of the recombinant PwLAP protein demonstrated preferential substrate specificity for Leu-NHMec and inhibition by EDTA, 1,10-phenanthroline, and bestatin, which are conserved characteristics of the M17 family of leucine aminopeptidase. PwLAP exhibited relatively higher enzyme activity in the presence of Mn2+ compared to Schistosoma mansoni LAP. Based on the biochemical properties and immunohistochemical analysis, PwLAP is concluded to represent a leucine aminopeptidase. The enzyme is most likely responsible for the catabolism of host hemoglobin, and, hence, represents a potential target of Paragonimus chemotherapy.
Parasitology International 10/2008; 57(3):334-41. · 2.30 Impact Factor