• 대한의생명과학회지
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• 제18권3호
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• pp.324-327
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• 2012

Acanthamoeba is a free-living amoeba that causes human infections, and recently the incidence of amoebic keratitis has increased among contact lens wearers. In order to investigate Acanthamoeba contamination of contact lens storage cases, a short survey was performed on 57 contact lens wearers, and Acanthamoeba was found in one contact lens storage case. To diagnose Acanthamoeba, the 18s small subunit ribosomal DNA (18s rDNA) gene was amplified by polymerase chain reaction (PCR), and subsequently, the isolate was identified as A. lugdunensis. This species was originally isolated from a freshwater pool in France, and was reported recently to be a cause of amoebic keratitis. This observation indicates the need for a large survey to investigate the extent of Acanthamoeba contamination, and suggests that contact lens wearers be aware of the importance of hygiene and of the implications of Acanthamoeba infection.

• Parasites, Hosts and Diseases
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• 제54권4호
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• pp.447-453
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• 2016

Acanthamoeba, a free-living amoeba, is widely distributed in the environment, water sources, soil, dust, and air. It can cause keratitis in contact lens wearers with poor hygiene and also fatal granulomatous amebic encephalitis (GAE) in immunocompromised hosts. The aim of this study was to gain some insights into the distribution and genotypes of the potentially pathogenic species of Acanthamoeba present in water sources in north of Iran. Total 43 Acanthamoeba species were isolated from 77 water samples taken from different water sources within the Mazandaran province in Northern Iran (Sari city and suburbs). Isolates were identified based on cyst and trophozoite morphological characteristics as well genetics. PCR fragments corresponding to the small-subunit 18S rRNA gene were sequenced for 20 of 43 positive isolates. The results revealed that 83.3% of sequenced isolates belonged to the T4 genotype and the rest belonged to the T2 genotype. Our results indicated that Acanthamoeba is widely distributed in Sari city. As the incidence in Iran of amoebic keratitis has increased in recent years, the exact estimation of the prevalence of this amoeba and its predominant genotype may play a crucial role in prevention of the disease. Sari city has several rivers, seashores, and natural recreational amenities, which attract visitors during the year. This is the first report of Acanthamoeba genotypes from water sources in Sari city, Mazandaran province of Iran, and the results suggest that more attention is needed to protect the visiting population and immunocompromised individuals.

• 한국동물위생학회지
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• 제42권4호
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• pp.169-175
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• 2019

Naegleria fowleri, a pathogenic free-living amoeba, leads to a fatal infection known as primary amebic meningoencephalitis (PAM) in human and animals. PAM is an acute, fulminant, necrotizing, and hemorrhagic disease that leads to death in approximately seven days. In this study, we investigate the cytotoxicity of target cells and the secreted molecules of N. fowleri under the non-contact condition. The target cell (U87MG cell) treated with N. fowleri lysates showed no morphological changes and no cytotoxicity. By contrast, the U87MG cells co-cultured with N. fowleri trophozoites under the non-contact condition induced morphological changes and reduction in number. When U87MG cells were co-cultured with N. fowleri trophozoites under the non-contact condition for 30 min, 2 hr, and 4 hr, the levels of cytotoxicity of target cells were 32.3, 35.5, and 37.8%, respectively. Particularly, when the ratio of amoeba to target cells is 10 to 1, the level of cytotoxicity of target cells was 49.7% at 30 min. To show the proteins secreted from N. fowleri under the non-contact condition, we carried out 2D electrophoresis and observed 6 major proteins. Finally, these results suggest that the molecules released from N. fowleri under the non-contact condition induce the cell death and this process is an important step in pathogenesis of N. fowleri.

• Parasites, Hosts and Diseases
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• 제61권4호
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• pp.449-454
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• 2023

Free-living amoebae (FLA) rarely cause human infections but can invoke fatal infections in the central nervous system (CNS). No consensus treatment has been established for FLA infections of the CNS, emphasizing the urgent need to discover or develop safe and effective drugs. Flavonoids, natural compounds from plants and plant-derived products, are known to have antiprotozoan activities against several pathogenic protozoa parasites. The anti-FLA activity of flavonoids has also been proposed, while their antiamoebic activity for FLA needs to be emperically determined. We herein evaluated the antiamoebic activities of 18 flavonoids against Naegleria fowleri and Acanthamoeba species which included A. castellanii and A. polyphaga. These flavonoids showed different profiles of antiamoebic activity against N. fowleri and Acanthamoeba species. Demethoxycurcumin, kaempferol, resveratrol, and silybin (A+B) showed in vitro antiamoebic activity against both N. fowleri and Acanthamoeba species. Apigenin, costunolide, (-)-epicatechin, (-)-epigallocatechin, rosmarinic acid, and (-)-trans-caryophyllene showed selective antiamoebic activity for Acanthamoeba species. Luteolin was more effective for N. fowleri. However, afzelin, berberine, (±)-catechin, chelerythrine, genistein, (+)-pinostrobin, and quercetin did not exhibit antiamoebic activity against the amoeba species. They neither showed selective antiamoebic activity with significant cytotoxicity to C6 glial cells. Our results provide a basis for the anti-FLA activity of flavonoids, which can be applied to develope alternative or supplemental therapeutic agents for FLA infections of the CNS.

• Parasites, Hosts and Diseases
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• 제30권1호
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• pp.33-42
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• 1992

자유생활 아메바인 Naegleria fowleri는 비강을 통해 인체에 들어와 출혈성 수막뇌염을 일으켜 감염된 사람의 대부분을 일주일 이내에 사망하게 하는 원충이다. 이 실험에서는 N. fowleri에 특이한 단세포군 항체를 만들어 이의 특성 및 그 이용 가능성을 알아보고자 하였다. 7 종류의 단세포군 항체(Nf 1, Nf 2, NE 256, Nf 279, Nf 27, Nf 154, Nf 137)를 제조하였는데 각각의 isotope은 IgGl이 두 종류(Nf 27, Nf 154), IgG3가 1 종류(Nf 137), IgA가 4 종류(Nf 1, Nf 2, Nf 256, Nf 279)이었다. 이들 항체를 N. fwoleri와 다른 종류의 아메바를 항원으로 하여 효소표식 면역검사법을 시행하여 Nf 1 및 Nf 256 항체를 제외한 5종류의 항체가 N. fowleri에 특이한 항체임을 확인하였다. 또한 간접 형광항체법을 통하여 NF 256 항체를 제외한 6 종류의 단세포군 항체가 세포막의 일부에 결합하였음을 관찰하였다. Nf 154 항체를 이용한 immunoperoxidase 염색에서도 세포막의 반응을 관찰하였다. 단세포를 항체가 N. fowleri를 응집시키는지 알아보고자 항체를 N. fowleri 영양형과 반응시켰더니 Nf 27 항 체를 제외한 5 종류의 단세포를 항체에 의해 응집이 일어남을 알 수 있었으며 응집된 덩 양형을 보체로 처리한 후 CGVS배지에서 배양하였더니 영양형의 증식이 억제되었다. 또한 2종류의 단세포군 항체(Nf 2, Nf 154)는 조직 세포(Chinese hamster ovary cell: CHO)에 대한 N.fowleri의 세포독성을 저하시켰다. 단세포군 항채와 반응하는 항원의 분자량을 알아보고자 EITB(Enzyme-linked immunoelectrotransfer blot)를 시행하였는데 Nf 279 항체는 25 kDa 및 28 kDa, Nf 154 항체는 43 kDa, N( 137 항체는 29 kDa에 해당되는 분획에서 항원항체반응을 나타내었다. 이상의 성적을 종합하면 N. fowleri에 대한 7종류의 IgG., IgG3 및 IgA 단세포군 항체를 생산하였다. 그중 Nf 256 항체를 제외한 6 종류는 N. fowleri이 세포막 성분중 28 kDa-43 kDa의 항원과 반응하는 특이한 항체임을 관찰할 수 있었다. 또한 이 단세포를 항체들은 영양형을 응집시키며, 시험관 내에서의 증식을 억제시키고 CHO 세포에 대한 N. fowleri의 세포 독성을 저하시키는 성질을 가지고 있음을 알 수 있었다.

• Parasites, Hosts and Diseases
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• 제59권5호
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• pp.501-505
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• 2021

The pathogenic free-living amoeba Naegleria fowleri causes primary amoebic meningoencephalitis, a fatal infection, by penetrating the nasal mucosa and migrating to the brain via the olfactory nerves. N. fowleri can induce host cell death via lytic necrosis. Similar to phosphorylation, O-linked β-N-acetylglucosamine (O-GlcNAc) glycosylation (O-GlcNAcylation) is involved in various cell-signaling processes, including apoptosis and proliferation, with O-GlcNAc addition and removal regulated by O-GlcNAc transferase and O-GlcNAcase (OGA), respectively. However, the detailed mechanism of host cell death induced by N. fowleri is unknown. In this study, we investigated whether N. fowleri can induce the modulation of O-GlcNAcylated proteins during cell death in Jurkat T cells. Co-incubation with live N. fowleri trophozoites increased DNA fragmentation. In addition, incubation with N. fowleri induced a dramatic reduction in O-GlcNAcylated protein levels in 30 min. Moreover, pretreatment of Jurkat T cells with the OGA inhibitor PUGNAc prevented N. fowleri-induced O-deGlcNAcylation and DNA fragmentation. These results suggest that O-deGlcNAcylation is an important signaling process that occurs during Jurkat T cell death induced by N. fowleri.

• Parasites, Hosts and Diseases
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• 제57권3호
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• pp.291-294
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• 2019

Primary amebic encephalitis (PAM) is a devastating central nervous system infection caused by Naegleria fowleri, a free-living amoeba, which can survive in soil and warm fresh water. Here, a 43-year-old healthy male was exposed to warm freshwater 5 days before the symptom onset. He rapidly developed severe cerebral edema before the diagnosis of PAM and was treated with intravenous conventional amphotericin B while died of terminal cerebral hernia finally. Comparing the patients with PAM who has similar clinical symptoms to those with other common types of meningoencephalitis, this infection is probably curable if treated early and aggressively. PAM should be considered in the differential diagnosis of purulent meningoencephalitis, especially in patients with recent freshwater-related activities during the hot season.

• 대한의생명과학회지
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• 제24권4호
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• pp.435-438
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• 2018

Acanthamoeba culbertsoni is the causative agent of granulomatous amoebic encephalitis (GAE), a condition that predominantly occurs in immunocompromised individuals and which is typically fatal. A mannose-binding protein (MBP) among lectins was shown to have strong A. castellanii pathogenic potential when correlated with major virulence proteins. In this study, protective effects were analyzed using the monoclonal antibody to A. culbertsoni MBP by quantification and were also compared with other free-living amoebae. For the amoebial cytotoxicity to the target cell, amoeba trophozoites were incubated with Chinese hamster ovary (CHO) cells. For the protective effects of antibodies, amoebae were pre-incubated with them for 4 h and then added to the target cells. After 24 h, the supernatants were collected and examined for host cell cytotoxicity by measuring lactate dehydrogenase (LDH) release. The cytotoxicity of A. culbertsoni to the CHO cells showed about 87.4%. When the monoclonal antibody was pre-incubated with A. culbertsoni, the amoebial cytotoxicity was remarkably decreased as shown at LDH release (1.858 absorbance), which was represented with about 49.9%. Taken together, it suggested that the monoclonal antibody against MBP be important to inhibit the cytotoxicity of A. culbertsoni trophozoites to the target cell. The antibody will be applied into an in vivo functional analysis, which would help to develop therapeutics.

• Journal of Microbiology and Biotechnology
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• 제29권5호
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• pp.713-720
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• 2019

Acanthamoeba castellanii belonging to the T4 genotype may cause a fatal brain infection known as granulomatous amoebic encephalitis, and the vision-threatening eye infection Acanthamoeba keratitis. The aim of this study was to evaluate the antiamoebic effects of three clinically available antidiabetic drugs, Glimepiride, Vildagliptin and Repaglinide, against A. castellanii belonging to the T4 genotype. Furthermore, we attempted to conjugate these drugs with silver nanoparticles (AgNPs) to enhance their antiamoebic effects. Amoebicidal, encystation, excystation, and host cell cytotoxicity assays were performed to unravel any antiacanthamoebic effects. Vildagliptin conjugated silver nanoparticles (Vgt-AgNPs) characterized by spectroscopic techniques and atomic force microscopy were synthesized. All three drugs showed antiamoebic effects against A. castellanii and significantly blocked the encystation. These drugs also showed significant cysticidal effects and reduced host cell cytotoxicity caused by A. castellanii. Moreover, Vildagliptin-coated silver nanoparticles were successfully synthesized and are shown to enhance its antiacanthamoebic potency at significantly reduced concentration. The repurposed application of the tested antidiabetic drugs and their nanoparticles against free-living amoeba such as Acanthamoeba castellanii described here is a novel outcome that holds tremendous potential for future applications against devastating infection.

• Parasites, Hosts and Diseases
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• 제26권2호
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• pp.95-106
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• 1988

아메바의 cytolytic process가 주로 가수분해 효소 환성과 관계 깊다는 연구들이 70년대 후반부터 병원성 Entamoeba sp.와 Naegleria sp.에서 보고되었지만 Acanthamoeba sp.에서는 가수분해 효소 환성에 관한 연구는 찾아볼 수 없다. 본 연구에서는 병원성이 없는 것으로 알려진 Acanthamoeba royreba를 대조로 하여 병원성이 있는 A. culbertsonl이 가수분해 효소 활성도를 측정하고 이들을 비교하고자 하였다. A. culbertsoni를 마우스에 감염시켰더니 15일내에 마우스 모두가 사망하였으며, 평균 생존기간은 8.5일이었고, 아베바성 수막뇌염으로 사망했음을 알 수 있었다. 반면에 A. royreba를 감염시킨 마우스에서는 수막뇌염이 발생되지 않았다. CGV배지에서 두 종(種) 아메바를 배양하여 아메바 추출물 및 배양액에서 가수분해 효소의 환성을 측정한 결과 A. rcyreba에 비해 A. culbertsoni 세포의 추출물 및 배양액에서 현저히 활성도가 높은 가수분해 효소는 acid phosphatase, p-N-acetyl galactosaminidase, β-N-acetyl glucosaminidase, α-mannosidase, neutral proteinase, acid proteinase였다. 표적 세포인 CHO 세포에 혼합한 A. culbertsoni는 대조군에 비해 강한 세포독성을 나타내었고 CHO 세포와의 혼합후 48시간 후 80%, 72시간후 95% 이상의 CHO 세포가 사멸하는 것을 알 수 있었다. 반면에 A. royreba를 혼합하였을 때는 별다른 세포독성을 나타나지 않았다. CHO 세포에 혼합한 A. tulbgrtsoni이 가수분해 효소 활성도를 경과 시간별로 측정하였던 바 CGV 배지에서 배 양시간이 지남에 따라 아메바내보다 그 배양액에서 더 높은 효소 활성도를 나타내었던 acid phosphatase, β-N·acetyl galactosaminidase, β-N·acetyl glucosaminidase, α-mannosidase, acid proteinase는 혼합 120시간 동안 아메바내에서 더 높은 효소 활성도를 나타내었으며, neutral proteinase의 활성도는 경과시간이 지남에 따라 아메바내에서는 일정하였지만 그 배양액인 EBSS용액내에서는 점점 높아짐을 알 수 있었다. 이상의 성적으로 보아 마우스에서 수막뇌염을 일으키는 A. cuzbertsoni는 비 병원성인 A. reyreba와 비교할 때 여러 종류 가수분해 효소들의 활성도에 현저한 차이를 나타내고 있음을 알 수 있었다. 또한 표적세포인 CHO세포에 대한 A. cuzbertsoni의 세포독성은 강함을 알 수 있었고, 이 때 몇 종류 가수분해 효소들의 환성도도 높게 나타났으며, 이러한 소견이 A. culbertsoni의 병원성과 관련이 있을 것으로 추측되었다.