Polyphenol oxidase produced during encystation of Acanthamoeba castellanii

Acanthamoeba castellanii has a phenol oxidase activity that is believed to be a laccase. Enzyme activity was found in the outer cyst wall, in the cytoplasm of encysting amoebae and in the encystment medium. Encystment procedures were modified to promote an increase in the amount of soluble enzyme secreted during encystation. Acanthamoeba polyphenol oxidase has a pH optimum of 6.0 and a Km value of 0.21 mM with dihydroxyphenylalanine. The enzyme does not oxidize tyrosine, and it is inhibited by chloride but not by inhibitors of peroxidase. Its synthesis coincides with encystation, and known inhibitors of polyphenol oxidase prevent encystation. Polyphenol oxidase may have a role in making the cyst resistant to mechanical and chemical breakdown.     

Essential Role for an M17 Leucine Aminopeptidase in Encystation of Acanthamoeba castellanii

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 starvation, low temperatures, and exposure to biocides. During encystation, a massive turnover of intracellular components occurs, and a large number of organelles and proteins are degraded by proteases. Previous studies with specific protease inhibitors have shown that cysteine and serine proteases are involved in encystation of Acanthamoeba, but little is known about the role of metalloproteases in this process. Here, we have biochemically characterized an M17 leucine aminopeptidase of Acanthamoeba castellanii (AcLAP) and analyzed its functional involvement in encystation of the parasite. Recombinant AcLAP shared biochemical properties such as optimal pH, requirement of divalent metal ions for activity, substrate specificity for Leu, and inhibition profile by aminopeptidase inhibitors and metal chelators with other characterized M17 family LAPs. AcLAP was highly expressed at a late stage of encystation and mainly localized in the cytoplasm of A. castellanii. Knockdown of AcLAP using small interfering RNA induced a decrease of LAP activity during encystation, a reduction of mature cyst formation, and the formation of abnormal cyst walls. In summary, these results indicate that AcLAP is a typical M17 family enzyme that plays an essential role during encystation of Acanthamoeba.     

The cyclic AMP phosphodiesterase RegA critically regulates encystation in social and pathogenic amoebas

Amoebas survive environmental stress by differentiating into encapsulated cysts. As cysts, pathogenic amoebas resist antibiotics, which particularly counteracts treatment of vision-destroying Acanthamoeba keratitis. Limited genetic tractability of amoeba pathogens has left their encystation mechanisms unexplored. The social amoeba Dictyostelium discoideum forms spores in multicellular fruiting bodies to survive starvation, while other dictyostelids, such as Polysphondylium pallidum can additionally encyst as single cells. Sporulation is induced by cAMP acting on PKA, with the cAMP phosphodiesterase RegA critically regulating cAMP levels. We show here that RegA is deeply conserved in social and pathogenic amoebas and that deletion of the RegA gene in P. pallidum causes precocious encystation and prevents cyst germination. We heterologously expressed and characterized Acanthamoeba RegA and performed a compound screen to identify RegA inhibitors. Two effective inhibitors increased cAMP levels and triggered Acanthamoeba encystation. Our results show that RegA critically regulates Amoebozoan encystation and that components of the cAMP signalling pathway could be effective targets for therapeutic intervention with encystation.     

Analysis of cellulase and polyphenol oxidase production by southern pine beetle associated fungi

In this study, the production of extracellular enzymes by fungi associated with southern pine beetle was investigated for the first time. Cellulase and polyphenol oxidase production were analyzed for three beetle associated fungi. Only the mutualistic symbiont Entomocorticium sp. A was found to produce cellulases and polyphenol oxidase. In time course analyses of cellulase production in batch cultures, Entomocorticium sp. A showed maximum activity of 0.109 U/ml and 0.141 U/ml for total cellulase and endoglucanase activity respectively. Polyphenol oxidase production was simultaneous with fungal growth. Characterization of polyphenol oxidase by activity staining suggests that the enzyme is a tyrosinase/catechol oxidase. Enzyme assays in the presence of polyphenol oxidase inhibitors support the results of the activity staining.     

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.     

Acanthamoeba castellanii: Proteins involved in actin dynamics, glycolysis, and proteolysis are regulated during encystation

Acanthamoeba castellanii is a pathogenic free-living amoeba. Cyst forms are particularly important in their pathogenicity, as they are more resistant to treatments and might protect pathogenic intracellular bacteria. However, encystation is poorly understood at the molecular level and global changes at the protein level have not been completely described. In this study, we performed two-dimensional gel electrophoresis to compare protein expression in trophozoite and cyst forms. Four proteins, specifically expressed in trophozoites, and four proteins, specifically expressed in cysts, were identified. Two proteins, enolase and fructose bisphosphate aldolase, are involved in the glycolytic pathway. Three proteins are likely actin-binding proteins, which is consistent with the dramatic morphological modifications of the cells during encystation. One protein belongs to the serine protease family and has been already linked to encystation in A. castellanii. In conclusion, this study found that the proteins whose expression was modified during encystation were likely involved in actin dynamics, glycolysis, and proteolysis.     

Rapid loss of ornithine decarboxylase activity during encystation of Entamoeba invadens

Abstract Stimulation of encystation of Entamoeba invadens by incubation of trophozoites under glucose-limiting conditions brought about a dramatic fall of ornithine decarboxylase activity, a key enzyme in polyamine biosynthesis. Levels of enzyme specific activity after 24 and 48 h of encystation represented only 11% and 1.3%, respectively, of those detected at the start of incubation. Induction of encystation in the presence of exogenously added polyamines resulted in a marked reduction in cyst formation. Thus, after 72 h of incubation, 1.0 mM putrescine, 1.0 mM spermidine or 0.5 mM spermine reduced encystation by 48 to 56%. Inhibition was enhanced to 70–73% in response to a two-fold increase in the concentration of either putrescine or spermine. Our results indicate that polyamine biosynthesis from ornithine is rapidly turned off at the onset of encystation.     

Down-Regulation of Cellulose Synthase Inhibits the Formation of Endocysts in Acanthamoeba

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.     

Characterization of a serine proteinase mediating encystation of Acanthamoeba

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.     

Tea Leaf Polyphenol Oxidase

A remarkable increase in polyphenol oxidase activity was observed in tea leaves after plucking. When the tea leaves treated with the specific inhibitors for the protein biosynthesis such as blasticidin S and puromycin A by the absorption through stem or by the vacuum infiltration, the increase in the enzyme activity was severely prevented. Therefore, it was thought that these antibiotics inhibited the activation of polyphenol oxidase. In addition, CM-cellulose column chromatogram of the enzyme protein of the withered leaves was different from that of the fresh leaves. It was also considered that new protein containing the enzyme activity developed in the withered leaves.

These results suggest that the synthesis of the enzyme protein occurred in tea leaves after plucking.     

Glucosylceramide Transferase Activity Is Critical for Encystation and Viable Cyst Production by an Intestinal Protozoan,Giardia lamblia

The production of viable cysts by Giardia is essential for its survival in the environment and for spreading the infection via contaminated food and water. The hallmark of cyst production (also known as encystation) is the biogenesis of encystation-specific vesicles (ESVs) that transport cyst wall proteins to the plasma membrane of the trophozoite before laying down the protective cyst wall. However, the molecules that regulate ESV biogenesis and maintain cyst viability have never before been identified. Here, we report that giardial glucosylceramide transferase-1 (gGlcT1), an enzyme of sphingolipid biosynthesis, plays a key role in ESV biogenesis and maintaining cyst viability. We find that overexpression of this enzyme induced the formation of aggregated/enlarged ESVs and generated clustered cysts with reduced viability. The silencing of gGlcT1 synthesis by antisense morpholino oligonucleotide abolished ESV production and generated mostly nonviable cysts. Interestingly, when gGlcT1-overexpressed Giardia was transfected with anti-gGlcT1 morpholino, the enzyme activity, vesicle biogenesis, and cyst viability returned to normal, suggesting that the regulated expression of gGlcT1 is important for encystation and viable cyst production. Furthermore, the overexpression of gGlcT1 increased the influx of membrane lipids and fatty acids without altering the fluidity of plasma membranes, indicating that the expression of gGlcT1 activity is linked to lipid internalization and maintaining the overall lipid balance in this parasite. Taken together, our results suggest that gGlcT1 is a key player of ESV biogenesis and cyst viability and therefore could be targeted for developing new anti-giardial therapies.     

Differentially expressed genes of Acanthamoeba castellanii during encystation

To examine the expressed gene profile during encystation of Acanthamoeba castellanii Castellani, we used differentially expressed gene (DGE) screening by RT-PCR with 20 sets of random primers. From this analysis, we found that approximately 16 genes showed upregulation during encystation. We chose 6 genes, which had relatively higher expression levels, for further investigation. Based on homology search in database, DEG2 showed 55% of similarity with xylose isomerase, DEG9 showed 37% of similarity with Na P-type ATPase, and DEG14 showed 77% of similarity with subtilisin-like serine proteinase. DEG3 and DEG26 were identified as hypothetical proteins and DEG25 exhibited no significant similarity to any known protein. Encystation of Acanthamoeba has been suggested to be a process to resist adverse environmental or nutritional conditions. Further characterization studies of these genes may provide us with more information on the encystation mechanism of Acanthamoeba.     

Purification and characterization of p-coumaroyl-D-glucose hydroxylase of sweet potato (Ipomoea batatas) roots

p-Coumaroyl-D-glucose hydroxylase in sweet potato (Ipomoea batatas Lam.) has been purified to apparent electrophoretic homogeneity using a combination of anion-and cation-exchange, hydrophobic and gel filtration chromatography. The purified enzyme was a monomer with a molecular weight of 33,000 and pI of 8.3. The purified enzyme showed not only hydroxylase activity but also polyphenol oxidase activity. L-Ascorbic acid was the best electron donor for the hydroxylation reaction, which had an optimum pH of 7.0. The enzyme hydroxylated p-coumaroyl-D-glucose, p-coumaric acid, and p-cresol but did not act on o-coumaric acid, m-coumaric acid, 4-hydroxy-3-methoxycinnamic acid, p-hydroxybenzoic acid or L-tyrosine. While the enzyme utilized p-coumaroyl-D-glucose and p-coumaric acid equally at pH 7.0, it hydroxylated only p-coumaroyl-D-glucose at pH 5.5. The enzyme oxidized diphenols such as D,L-(3,4-dihydroxyphenyl) alanine and caffeic acid, but exhibited no clear pH optimum in this reaction characteristic of polyphenol oxidase. Both the hydroxylase and the polyphenol oxidase activities were strongly inhibited by beta-mercaptoethanol, diethyldithiocarbamate, KCN, and p-coumaric acid (in concentrations higher than 5 mM). Ammonium sulfate and sodium chloride activated the hydroxylase activity but not the polyphenol oxidase activity of the enzyme. The enzyme activity and L-ascorbic acid contents changed in a manner suggesting their involvements in chlorogenic acid biosynthesis during incubation of sliced sweet potato root tissues.     

Short-Cut Pathway to Synthesize Cellulose of Encysting Acanthamoeba

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.     

Biochemical response between insects and plants: an investigation of enzyme activity in the digestive system of Leucoptera coffeella (Lepidoptera: Lyonetiidae) and leaves of Coffea arabica (Rubiaceae) after herbivory

Plant defence mechanisms can reduce the digestive enzyme activity of insect pests. The aim of this study was to determine the relationship between the production of proteinase inhibitors, lipoxygenase and polyphenol oxidase activity in Coffea arabica (Catuai IAC 15) plants, and the digestive enzyme activity in the pest Leucoptera coffeella (Lepidoptera: Lyonetiidae) after feeding on the plant. The production of proteinase inhibitors was evaluated with L‐BApNA as a substrate. We studied lipoxygenase activity with linoleic acid and polyphenol oxidase activity with catechol substrates, in coffee plants damaged (T1) and not damaged (T2) by L. coffeella. L. coffeella digestive enzyme activity was verified by trypsin‐like (substrate l ‐BApNA and l ‐TAME), chymotrypsin‐like (BTpNA and ATEE), cysteine proteases (l ‐BApNA) and total protease (azocasein). Proteinase inhibitor production and lipoxygenase and polyphenol oxidase activity in C. arabica increases (P ≤ 0.05) with L. coffeella damage. Our results provide important information that these enzymatic activities may play a role in plant defence processes in C. arabica. Trypsin‐like activity increases, whereas chymotrypsin‐like and cysteine protease activity decrease in the midgut of L. coffeella, which acts as a defence mechanism.     

Enzyme activities and polyphenols related to mesocarp discolouration of avocado fruit

Avocado fruit showing severe symptoms of the mesocarp discolouration disorder exhibited significantly higher extractable activities of soluble polyphenol oxidase and peroxidase, as well as higher levels of total phenols, hydroxycinnamic acids and proanthocyanidins, when compared to healthy fruit. However, l-phenylalanine ammonia-lyase activity was very variable, and no significant differences were observed between healthy and affected fruit. Extraction of healthy, but not severely affected, fruit in the presence of 0.1 % SDS resulted in increased polyphenol oxidase activity reflecting the release of bound and/or latent enzyme. Qualitative differences between healthy and affected fruit included different patterns of polyphenol oxidase multiple forms and different polyphenol profiles. The pattern of polyphenol oxidase multiple forms from SDS-extracted healthy fruit was similar to that from mildly affected fruit not extracted with detergent.