Effects of Mannose on Pathogenesis of Acanthamoeba castellanii

Acanthamoeba spp. are single-celled protozoan organisms that are widely distributed in the environment. In this study, to understand functional roles of a mannose-binding protein (MBP), Acanthamoeba castellanii was treated with methyl-alpha-D-mannopyranoside (mannose), and adhesion and cytotoxicity of the amoeba were analyzed. In addition, to understand the association of MBP for amoeba phagocytosis, phagocytosis assay was analyzed using non-pathogenic bacterium, Escherichia coli K12. Amoebae treated with mannose for 20 cycles exhibited larger vacuoles occupying the most area of the amoebic cytoplasm in comparison with the control group amoebae and glucose-treated amoebae. Mannose-selected amoebae exhibited lower levels of binding to Chinese hamster ovary (CHO) cells. Exogenous mannose inhibited >50% inhibition of amoebae (control group) binding to CHO cells. Moreover, exogenous mannose inhibited amoebae (i.e., man-treated) binding to CHO cells by <15%. Mannose-selected amoebae exhibited significantly decreased cytotoxicity to CHO cells compared with the control group amoebae, 25.1% vs 92.1%. In phagocytic assay, mannose-selected amoebae exhibited significant decreases in bacterial uptake in comparison with the control group, 0.019% vs 0.03% (P<0.05). Taken together, it is suggested that mannose-selected A. castellanii trophozoites should be severely damaged and do not well interact with a target cell via a lectin of MBP.     

Acanthamoeba castellanii: structural basis of the cytopathic mechanisms

In this study we report observations on the structural mechanisms of the cytopathic effect of Acanthamoeba castellanii trophozoites on cultured MDCK cell monolayers. Co-incubations were carried out for a maximum of 24h. The first evidence of damage to the cell monolayer was detected by measuring the transepithelial resistance of cell monolayers that interacted with the amoebae. At 6h, transepithelial resistance diminished to 51% and amoebae required 5-6h to produce evidence of structural injury at the light microscopy level. Following 12h of incubation, the cell monolayer was severely damaged. After making intimate contact with the surface of target cells, trophozoites detached cells from the substrate, lysed and by means of food-cups ingested the damaged cells. There was no morphological evidence of modifications in MDCK cell membranes, membrane fusion or junction formation between the amoeba and host plasma membrane. The lytic capacity of the amoebas appears to be the result of cytotoxic factors secreted by the amoebae since, when monolayers were incubated with conditioned medium, there was also a decrease in the transepithelial resistance. Besides, mechanical injury produced by the attachment and movement of the trophozoites may contribute to the disruption of the cell monolayer. As in other pathogenic amoebae, the cytopathic action of A. castellanii on the cell monolayers can subjectively be separated into four stages: adhesion, cytolysis, phagocytosis, and intracellular degradation.     

Acanthamoeba castellanii: identification and distribution of actin cytoskeleton

The presence of the cytoskeleton of Acanthamoeba castellanii was observed by means of cryo-electronmicroscopy and immunofluorescence techniques. This structure is formed largely by fibers and networks of actin located mainly in cytoplasmic locomotion structures as lamellipodia and as well as in various endocytic structures. In addition, the comparision between total actin content in whole extracts among different amoebae was made. The molecular weight of actin in A. castellanii was 44 kDa, and 45 kDa for Naegleria fowleri and Entamoeba histolytica.     

Interaction of Escherichia coli K1 and K5 with Acanthamoeba castellanii trophozoites and cysts

The existence of symbiotic relationships between Acanthamoeba and a variety of bacteria is well-documented. However, the ability of Acanthamoeba interacting with host bacterial pathogens has gained particular attention. Here, to understand the interactions of Escherichia coli K1 and E. coli K5 strains with Acanthamoeba castellanii trophozoites and cysts, association assay, invasion assay, survival assay, and the measurement of bacterial numbers from cysts were performed, and nonpathogenic E. coli K12 was also applied. The association ratio of E. coli K1 with A. castellanii was 4.3 cfu per amoeba for 1 hr but E. coli K5 with A. castellanii was 1 cfu per amoeba for 1 hr. By invasion and survival assays, E. coli K5 was recovered less than E. coli K1 but still alive inside A. castellanii. E. coli K1 and K5 survived and multiplied intracellularly in A. castellanii. The survival assay was performed under a favourable condition for 22 hr and 43 hr with the encystment of A. castellanii. Under the favourable condition for the transformation of trophozoites into cysts, E. coli K5 multiplied significantly. Moreover, the pathogenic potential of E. coli K1 from A. castellanii cysts exhibited no changes as compared with E. coli K1 from A. castellanii trophozoites. E. coli K5 was multiplied in A. castellanii trophozoites and survived in A. castellanii cysts. Therefore, this study suggests that E. coli K5 can use A. castellanii as a reservoir host or a vector for the bacterial transmission.     

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.     

Identification of Atg8 Isoform in Encysting Acanthamoeba

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.     

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.     

The effect of pH on the alternative oxidase activity in isolated Acanthamoeba castellanii mitochondria

Mitochondria of Acanthamoeba castellanii possess a cyanide-resistant GMP-stimulated ubiquinol alternative oxidase in addition to the cytochrome pathway. In a previous work it has been observed that an interaction between the two ubiquinol-oxidizing pathways exists in intact A. castellanii mitochondria and that this interaction may be due to a high sensitivity of the alternative oxidase to matrix pH. In this study we have shown that the alternative oxidase activity reveals a pH-dependence with a pH optimum at 6.8 whatever the reducing substrate may be. The GMP stimulation of alternative oxidase is also strongly dependent on pH implicating probably protonation/deprotonation processes at the level of ligand and protein with an optimum pH at 6.8. The ubiquinone redox state-dependence of alternative oxidase activity is modified by pH in such a way that the highest activity for a given ubiquinone redox state is observed at pH 6.8. Thus pH, binding of GMP, and redox state of ubiquinone collaborate to set the activity of the GMP-stimulated alternative oxidase in isolated A. castellanii mitochondria. The high pH sensitivity of the alternative oxidase could link inactivation of the cytochrome pathway proton pumps to activation of the alternative oxidase with acceleration of redox free energy dissipation as a consequence.     

Keratitis by Acanthamoeba triangularis: report of cases and characterization of isolates

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.     

Molecular characterization of Acanthamoeba isolated from amebic keratitis related to orthokeratology lens overnight wear

In an effort to characterize, on the molecular scale, the Acanthamoeba initially isolated from the cornea of an amoebic keratitis patient associated with overnight-wear orthokeratology lens in Korea, we conducted mitochondrial DNA restriction fragment length polymorphism, 18S rDNA sequencing, and drug sensitivity analyses on the isolate (KA/PE1). The patient was treated with polyhexamethylene biguanide, chlorhexidine and oral itraconazole, which resulted in resolution of the patientos ocular inflammation. The majority of the molecular characteristics of the KA/PE1 were determined to be identical, or quite similar, to those of A. castellanii Ma strain, which had been isolated also from amoebic keratitis. The risk of Acanthamoeba keratitis as a potential complication of overnight orthokeratology is briefly discussed.     

Interactions Between the Cytochrome Pathway and the Alternative Oxidase in Isolated Acanthamoeba castellanii Mitochondria

The steady-state activity of the two quinol-oxidizing pathways of Acanthamoeba castellanii mitochondria, the phosphorylating cytochrome pathway (i.e. the benzohydroxamate(BHAM)-resistant respiration in state 3) and the alternative oxidase (i.e. the KCN-resistant respiration), is shown to be fixed by ubiquinone (Q) pool redox state independently of the reducing substrate (succinate or exogenous reduced nicotinamide adenine dinucleotide (NADH)), indicating that the active Q pool is homogenous. For both pathways, activity increases with the Q reduction level (up to 80%). However, the cytochrome pathway respiration partially inhibited (about 50%) by myxothiazol decreases when the Q reduction level increases above 80%. The decrease can be explained by the Q cycle mechanism of complex III. It is also shown that BHAM has an influence on the relationship between the rate of ADP phosphorylation and the Q reduction level when alternative oxidase is active, and that KCN has an influence on the relationship between the alternative oxidase activity and the Q reduction level. These unexpected effects of BHAM and KCN observed at a given Q reduction level are likely due to functional connections between the two pathways activities or to protein–protein interaction.     

Growth,root colonization and nutrient status of Helianthemum sessiliflorum Desf. inoculated with a desert truffle Terfezia boudieri Chatin

This study aims to investigate the effects of inoculation using Terfezia boudieri Chatin ascospores (ectomycorrhizal fungus) on growth, root colonization and nutrient status of Helianthemum sessiliflorum Desf. seedlings grown in pots on two-soil types (gypseous and sandy loam). Mycorrhizal seedlings had significantly increased their height and leaf number compared to non-mycorrhizal ones. Regardless of mycorrhizal inoculation treatments, the plants growing on gypseous soil showed higher growth as compared to sandy loam one. It appears that inoculation with T. boudieri changed root morphology, increasing branching of first-order lateral roots of H. sessiliflorum seedlings. The highest root mycorrhizal colonization was recorded in inoculated seedlings on sandy loam soil (89%) when compared to gypseous one (52%). N, P and K concentrations in mycorrhizal seedlings were significantly improved by fungal inoculation. It can be concluded that inoculation of H. sessiliflorum with T. boudieri increased growth attributes and improved plant nutritional status.     

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.     

Transgenics in groundnut (Arachis hypogaea L.) expressing cry1AcF gene for resistance to Spodoptera litura (F.)

Large number of primary transgenic events were generated in groundnut by an Agrobacterium mediated, in planta transformation method to assess the efficacy of cry1AcF against the Spodoptera litura. The amplification of required size fragment of 750 bp with npt II primers and 901 bp with cry1AcF gene primers confirmed the integration of the gene. The expression of the cry gene was ascertained by ELISA in T₂ generation, and the maximum concentration of cry protein in transgenic plants reached approximately 0.82 μg/g FW. Further, Southern blot analysis of ten T₂ transgenic plants proved that transgene had been integrated in the genome of all the plants and Northern analysis of the same plants demonstrated the active expression of cry1AcF gene. The highest mean % larval mortalities 80.0 and 85.0 with an average mean % larval mortalities 16.25 (n = 369) and 26.0 (n = 80) were recorded in T₁ and T₂ generations, respectively. Segregation analysis of the selected lines in the T₃ generation demonstrated homozygous nature. This clearly proved that though there is considerable improvement in average mean % larval mortality in T₂ generation, the cry1AcF gene was effective against S. litura only to some extent.     

Occurrence,Parasitism, and Pathogenicity of Nematodes Associated with Sycamore (Platanus occidentalis L.)

Ten species of stylet-bearing nematodes were recovered in a survey of sycamore (Platanus occidentalis L. ) stands in Georgia. Helicotylenchus, Xiphinema, and Criconemoides were the genera found most frequently. Populations of Hoplolaimus galeatus, Scutellonema brachyurum, Helicotylenchus dihystera and H. pseudorobustus increased on greenhouse-grown sycamore, but Trichodorus christiei, Xiphinema americanum, Meloidogyne hapla, M. arenaria and M. incognita did not. Hoplolaimus galeatus and S. brachyurum are semi-endoparasites; H. dihystera and H. pseudorobustus are migratory endoparasites. Hoplolaimus galeatus caused extensive root necrosis and marked decrease of fresh weights of seedling roots and tops. Helicotylenchus dihystera and S. brachyurum produced only qualitatively different sparse and unhealthy root growth. Helicotylenchus pseudorobustus caused only a reduction in root surface area.     

L-Dopa production and antioxidant activity in Hybanthus enneaspermus (L.) F. Muell regeneration

Physiology and Molecular Biology of Plants - Hybanthus enneaspermus is an ethanobotanical plant extensively used in Indian traditional medicine. Quick and efficient in vitro mass propagation of...