Yeonchul Hong

Characterization of a Serine Proteinase Mediating Encystation of Acanthamoeba

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.

Autophagy protein 8 mediating autophagosome in encysting Acanthamoeba

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.

Acanthamoeba castellanii: Gene profile of encystation by ESTs analysis and KOG assignment

The trophozoite of Acanthamoeba transforms into a cyst, the resistant form under harmful environments such as starvation, cold and certain chemicals used in medical treatment. To investigate the factors mediating encystation, ESTs of encystation-induced A. castellanii were analysed and compared to those of trophozoites. Each EST was compared by the predicted proteins from the ESTs, to the cyst and the trophozoite by reciprocal BLAST analysis, KOG assignment, and gene annotation. In addition to the genes previously reported to encystation mediate such as cyst specific protein 21, protein kinase C, proteasome and heat shock protein, several genes like cullin 4, autophage protein 8 and ubiquitin-conjugating enzymes were identified to be related to encystation. Five kinds of proteinase genes were detected in cyst ESTs. The information of the genes expressed during encystation may open the way to further study on differentiation and resistance of cyst-forming pathogenic protozoa.

Degradation of malic acid in wine by immobilized Issatchenkia orientalis cells with oriental oak charcoal and alginate

Aims:  To test degradation of malic acid content in wine by immobilized Issatchenkia orientalis KMBL 5774 cells recently isolated from Korean wine pomace as a malic acid‐degrading yeast.

Immunization of proteins from Toxascaris leonina adult worm inhibits allergic specific Th2 response

Recently, the influence of parasitic infections on the incidence of allergic diseases has become the focus of increased attention. In order to ascertain whether parasite-derived proteins could inhibit the allergic specific Th2 response, we applied excretory-secretory protein (Tl-ES) or total protein (Tl-TP) of the adult worm Toxascaris leonina to asthma model mice prior to or simultaneously with OVA challenge, after which we assessed the OVA-specific Th2 responses. The group subjected to immunization with Tl-ES and Tl-TP (immunized group) evidenced a thinning of the bronchial epithelial and muscle layer, a disruption and shedding of epithelial cells, a reduction in the number of goblet cells, and a reduction in mucus production as compared to the group treated with Tl-ES coupled with OVA challenge (challenge with OVA groups) and the OVA-induced asthma group. The administration of Tl-ES and Tl-TP, regardless of injection time, was shown to inhibit the recruitment of inflammatory cells into the airway, and in particular, macrophages, neutrophils, and lymphocytes were significantly reduced as the result of the parasite proteins. However, the total number of eosinophils was slightly reduced as the result of the administration of parasite proteins. Sensitization and OVA challenge was shown to accelerate the secretion of Th2 cytokines (IL-4 and IL-5) within the lung, but in the immunized groups, those levels were lower. The administration of Tl-TP and OVA challenge group also evidenced a significant reduction in IL-4 levels as compared to the OVA-challenged group. The concentrations of Th2 cytokines in the Tl-ES and OVA challenge group were more similar to those observed in the OVA-challenged group. The concentration of IL-10 and TGF-beta in the lung was decreased substantially in the OVA-only challenge group, but the Tl-TP immunized group exhibited significantly induced IL-10 cytokine. OVA-specific IgG2a, IgG1, and IgE levels in the immunized groups were significantly lower than those detected in the OVA-challenged group. In conclusion, parasite-derived protein is able to inhibit OVA-specific Th2 responses, and in particular, immunization with parasite proteins exerts a more profound protective effect than is seen with the treatment of allergic reactions. The results of our study are encouraging in terms of our further understanding of the molecular basis of immune evasion by nematodes.

Autophagy protein 16-mediated autophagy is required for the encystation of Acanthamoeba castellanii.

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.

M17 leucine aminopeptidase of the human malaria parasite Plasmodium vivax.

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.

Cysteine protease involving in autophagosomal degradation of mitochondria during encystation of Acanthamoeba

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.

Autophagy Inhibitors as a Potential Antiamoebic Treatment for Acanthamoeba Keratitis

ABSTRACT Acanthamoeba cysts are resistant to extreme physical and chemical conditions. Autophagy is an essential pathway for encystation of Acanthamoeba cells. To evaluate the possibility of an autophagic Acanthamoeba encystation mechanism, we evaluated autophagy inhibitors, such as 3-methyladenine (3MA), LY294002, wortmannin, bafilomycin A, and chloroquine. Among these autophagy inhibitors, the use of 3MA and chloroquine showed a significant reduction in the encystation ratio in Acanthamoeba cells. Wortmannin also inhibited the formation of mature cysts, while LY294002 and bafilomycin A did not affect the encystation of Acanthamoeba cells. Transmission electron microscopy revealed that 3MA and wortmannin inhibited autophagy formation and that chloroquine interfered with the formation of autolysosomes. Inhibition of autophagy or autolysosome formation resulted in a significant block in the encystation in Acanthamoeba cells. Clinical treatment with 0.02% polyhexamethylene biguanide (PHMB) showed high cytopathic effects on Acanthamoeba trophozoites and cysts; however, it also revealed high cytopathic effects on human corneal epithelial cells. In this study, we investigated effects of the combination of a low (0.00125%) concentration of PHMB with each of the autophagy inhibitors 3MA, wortmannin, and chloroquine on Acanthamoeba and human corneal epithelial cells. These new combination treatments showed low cytopathic effects on human corneal cells and high cytopathic effects on Acanthamoeba cells. Taken together, these results provide fundamental information for optimizing the treatment of Acanthamoeba keratitis.

Expression levels of encystation mediating factors in fresh strain of Acanthamoeba castellanii cyst ESTs

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.