• Parasites, Hosts and Diseases
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• v.56 no.1
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• pp.1-9
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• 2018

Giardia lamblia, an anaerobic, amitochondriate protozoan parasite causes parasitic infection giardiasis in children and young adults. It produces pyruvate, a major metabolic product for its fermentative metabolism. The current study was undertaken to explore the effects of pyruvate as a physiological antioxidant during oxidative stress in Giardia by cysteine-ascorbate deprivation and further investigation upon the hypothesis that oxidative stress due to metabolism was the reason behind the cytotoxicity. We have estimated intracellular reactive oxygen species generation due to cysteine-ascorbate deprivation in Giardia. In the present study, we have examined the effects of extracellular addition of pyruvate, during oxidative stress generated from cysteine-ascorbate deprivation in culture media on DNA damage in Giardia. The intracellular pyruvate concentrations at several time points were measured in the trophozoites during stress. Trophozoites viability under cysteine-ascorbate deprived (CAD) medium in presence and absence of extracellular pyruvate has also been measured. The exogenous addition of a physiologically relevant concentration of pyruvate to trophozoites suspension was shown to attenuate the rate of ROS generation. We have demonstrated that Giardia protects itself from destructive consequences of ROS by maintaining the intracellular pyruvate concentration. Pyruvate recovers Giardia trophozoites from oxidative stress by decreasing the number of DNA breaks that might favor DNA repair.

• Parasites, Hosts and Diseases
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• v.49 no.4
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• pp.349-356
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• 2011

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.

• Parasites, Hosts and Diseases
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• v.42 no.1
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• pp.35-40
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• 2004

The nfa 1 gene was cloned from a cDNA library of pathogenic Naegleria fowleri by immunoscreening; it consisted of 360 bp and produced a 13.1 kDa recombinant protein (rNfa1) that showed the pseudopodia-specific localization by immunocytochemistry in the previous study. Based on the idea that the pseudopodia-specific Nfa1 protein mentioned above seems to be involved in the pathogenicity of N. fowleri, we observed the effect of an anti-Nfa1 antibody on the proliferation of N. fowleri trophozoites and the cytotoxicity of N. fowleri trophozoites on the target cells. The proliferation of N. fowleri trophozoites was inhibited after being treated with an anti-Nfa1 polycional antibody in a dose-dependent manner for 48 hrs. By a light microscope, CHO cells co-cultured with N. fowleri trophozoites (group I) for 48 hrs showed severe morphological destruction. On the contrary, CHO cells co-cultured with N. fowleri trophozoites and anti-Nfa1 polyclonal antibody (1:100 dilution) (group II) showed less destruction. In the LDH release assay results, group I showed 50.6% cytotoxicity, and group II showed 39.3%. Consequently, addition of an anti-Nfa1 polyclonal antibody produced a decreasing effect of in vitro cytotoxicity of N. fowleri in a dose-dependent manner.

• Parasites, Hosts and Diseases
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• v.46 no.4
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• pp.217-221
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• 2008

Free-living Naegleria fowleri leads to a fatal infection known as primary amebic meningoencephalitis in humans. Previously, the target cell death could be induced by phagocytic activity of N. fowleri as a contact-dependent mechanism. However, in this study we investigated the target cell death under a non-contact system using a tissue-culture insert. The human microglial cells, U87MG cells, co-cultured with N. fowleri trophozoites for 30 min in a non-contact system showed morphological changes such as the cell membrane destruction and a reduction in the number. By fluorescence-activated cell sorter (FACS) analysis, U87MG cells co-cultured with N. fowleri trophozoites in a non-contact system showed a significant increase of apoptotic cells (16%) in comparison with that of the control or N. fowleri lysate. When U87MG cells were co-cultured with N. fowleri trophozoites in a non-contact system for 30 min, 2 hr, and 4 hr, the cytotoxicity of amebae against target cells was 40.5, 44.2, and 45.6%, respectively. By contrast, the cytotoxicity of non-pathogenic N. gruberi trophozoites was 10.2, 12.4, and 13.2%, respectively. These results suggest that the molecules released from N. fowleri in a contact-independent manner as well as phagocytosis in a contact-dependent manner may induce the host cell death.

• Parasites, Hosts and Diseases
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• v.23 no.2
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• pp.293-299
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• 1985

This study is to verify the protective ability against experimental Naegleria meningoencephalitis by immunization with Naegleria fowleri in mice. Naegleria fewleri, strain 0359, and Naegleria gruberi, strain EGB, were used in this study, and cultured in CGVS medium akenically. Inbred BALB/C mice, weighing about 20g, were immunized by three intraperitoneal injection of $1{\times}10^6$ N. fowleri trophozoites at the interval of one week. This N. fowleri trophozoites antigen was fixed with 5% formaldehyde. N. fowleri trophozoites from culture were homogenized with soiicator at $4^{\circ}C$ as monitored by phase contrast microscopy, and their membrane and cell content preparations were made for the immunization of mice. Their inoculation dose in volume was equivalent to the $1{\times}10^6$ trophozoites in each injection for immunization. And N. gruberi trophosoites, whieh was fixed with 5% formaldehyde, were also used for immunisation. Mice were inoculated intranasally with $5{\times}10^4$ N. fowleri trophozoites in a 511 suspension under anesthesia by as intraperitoneal injection of about 1 mg secobarbiturate. Nervousness, rotation or sluggish behaviour were observed in the mice which were infected with N. fewleri. Necrotic lesion was demonstrated in the anterior portion of brain, especially in the olfactory lobe. The inflammatory cell infiltration with numerous H. fowleri trophozoites was noticed. This pathological changes were more extensive in the control than in the experimental groups. Mice were dead due to experimental primary amoebic meningoencephalitis that developed between 8 days and 23 days after inoculation. Mortality rate of the mice was low in the immunized experimental group. Mean survival time, which is the survival duration of mice from the infection to death, was prolonged significantly in the immunized mice except in the mice immunized with JV, fowleri membrane. Even in the mice immunized with N. gruberi, survival time was delayed. In summary, the effectiveness of immunization is demonstrated in terms of protective immunity against Naegleria meningoencephalitis in mice.

• Parasites, Hosts and Diseases
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• v.55 no.2
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• pp.115-120
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• 2017

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.

• Parasites, Hosts and Diseases
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• v.49 no.4
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• pp.341-347
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• 2011

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.

• Parasites, Hosts and Diseases
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• v.57 no.3
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• pp.217-223
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• 2019

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 $IL-1{\alpha}$, IL-6, IL-8, and CXCL1 in HCECs. Secreted levels of $IL-1{\alpha}$, 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.

• Parasites, Hosts and Diseases
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• v.31 no.3
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• pp.249-258
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• 1993

Naegleria fowleri is the cause of primary amoebic meningoencephalitis in man, IL-2 levels after stimulation of T lymphocytes by PHA or N.fowleri lysates. the amounts of T lymphocyte subsets and the blastogenic responses of T lymphocytes in mice after Infected with pathogenic N. fowleri were studied comparing between two study groups, one $1{\;}{\times}{\;}10^4$ trophozoites inoculated mice and the other $1{\;}{\times}{\;}10^5$ trophozoites inoculated mice. All experimental samples were obtained on the day 7, 14 and 24 after inoculation. The mice inoculated with $1{\;}{\times}{\;}10^4$ trophozoites showed a 14.3% mortality rate, and 72.2% in the mice inoculated with $1{\;}{\times}{\;}10^5$ trophozoites. The IL-2 levels on day 14 of two experimental groups were significantly decreased as compared with the control group. Thy 1.2+T cells in the total spleen Iymphocytes of $1{\;}{\times}{\;}10^5$ trophozoites inoculated group on day 7 were significantly increased compared with the control group. There was no significant difference between $1{\;}{\times}{\;}10^4$ trophozoites inoculated group and the control group. $L3T4^{+}{\;}T$ cells and $Ly2^{+}$ T cells in the total spleen Iymphocytes of $1{\;}{\times}{\;}10^5$ trophozoites inoculated group on day 7 were sigrlificantly increased compared with the control group. The DNA S fraction of T cells in the spleen of $1{\;}{\times}{\;}10^5$ trophozoites inoculated group was significantly increased on day 7. The amount of S fractions of DNA were sequentially decreased on day 14 and 24 but they were also signiacantly increased compared with the control group. The results obtained in the experiments indicats that cell mediated immunity after N.fowleri infection acts on very important host's protection immunity around the 7th day after infection. IL-2 level was much suppressed on day 14 which resulted from the exhaustion of host immune response. It was observed that the level of IL-2 production ability and the amounts of T lymphocytes subsets and the blastogenic responses of T lymphocytes were not well correlated during the observation period.

• Parasites, Hosts and Diseases
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• v.54 no.4
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• pp.461-469
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• 2016

Giardia lamblia is a protozoan that causes diarrheal diseases in humans. Cytoskeletal structures of Giardia trophozoites must be finely reorganized during cell division. To identify Giardia proteins which interact with microtubules (MTs), Giardia lysates were incubated with in vitro-polymerized MTs and then precipitated by ultracentifugation. A hypothetical protein (GL50803_8405) was identified in the precipitated fraction with polymerized MTs and was named GlMBP1 (G. lamblia microtubule-binding protein 1). Interaction of GlMBP1 with MTs was confirmed by MT binding assays using recombinant GlMBP1 (rGlMBP1). In vivo expression of GlMBP1 was shown by a real-time PCR and western blot analysis using anti-rGlMBP1 antibodies. Transgenic G. lamblia trophozoites were constructed by integrating a chimeric gene encoding hemagglutinin (HA)-tagged GlMBP1 into a Giardia chromosome. Immunofluorescence assays of this transgenic G. lamblia, using anti-HA antibodies, revealed that GlMBP1 mainly localized at the basal bodies, axonemes, and median bodies of G. lamblia trophozoites. This result indicates that GlMBP1 is a component of the G. lamblia cytoskeleton.