Proteinase Activity of Naegleria spp. with Special Reference to their Pathogenicity

Seo, Jang-Hoon,Park, Hyun,Soh, Chin-Thack,Im, Kyung-il INSTITUTE OF TROPICAL MEDICINE YONSEI UNIVERSITY 1993 YONSEI REPORTS ON TROPICAL MEDICINE Vol.24 No.1

자유생활아메바의 병원성과 단백분해효소 활성도의 상관성을 밝히고자 병원성인 Naegleria fowleri와 비병원성인 N. gruberi 영양형의 용해물과 배양액으로부터 단백분해효소 활성도를 관찰하였다. 또한 Chinese hamster ovary(CHO) 세포를 사용하여 51Cr 방출 검사법과 주사 전자현미경법으로 세포독성 물질을 평가(assay)하였고 단백분해효소의 생화학적 성질을 규명한 결과 다음과 같은 결과를 얻었다. Naegleria 영양형을 마우스에 접종시킨 후 사망율을 관찰한 결과 N. fowleri를 접종시킨 마우스에서 사망율이 75% 이었으나, N. bruberi는 사망율이 0%로 병원성이 없음을 확인하였다. 여러가지 단백분해효소 억제제들과 기질을 사용하여 단백분해효소의 활성을 측정한 결과 gelatin을 기질로한 전기영동과 azocasein을 기질로한 활성부위 잔기 억제실험에서 N. fowleri와 N. gruberi의 단백분해효소는 서로 상이하였다. N. fowleri의 용해물과 배양액을 이용하여 CHO 세포에 대한 세포독성을 관찰한 결과 그 용해물과 배양액 모두 단백질 농도의 증가에 따라 세포독성이 증가하였으나 N. gruberi의 용해물과 배양액에서 세포독성이 거의 없었다. N. fowleri의 용해물과 배양액의 단백분해효소는 Rf값을 서로 달리하는 효소분획으로 되어있음이 gelatin을 기질로한 SDS-PAGE에서 관찰되었으며, Rf 0.87의 단백분해효소 분획을 용액물과 배양액에서 공통적으로 갖고 있었다. 이 N. fowleri 배양액의 정제는 초원심분리, ammonium sulfate 분별침전, Spectra/Gel AcA 44 column chromatography를 이용하여 배양액내 단백분해효소가 23.6배로 정제되었고, 회수율은 19.9%였다. Gelatin을 기질로한 SDS-PAGE를 한 결과 Rf값이 0.87이었으며, 분자량을 SDS-PAGe로 측정한 결과 17,500 dalton이었다. N.fowleri의 배양액에서 부분정제한 분획은 여러 단백분해효소 활성부위 잔기 억제제들과 기질을 사용하여 단백분해효소 활성이 측정되었고, 단백분해효소의 활성을 억제하였던 leupeptin, antipain, pepstatin에 의해 세포독성도 억제되었다. 이로써 cysteine과 aspartic acid의 잔기가 활성에 관여함을 알 수 있었다. 부분정제한 배양액내 효소분획을 CHO 세포에 처리한 후 주사 전자현미경으로 관찰한 결과 처리 30분후 표적세포의 융모막(microvilli)이 점차 감소하였고 세포막에 수포와 구멍이 형성되었으며, 3시간 후에는 세포막이 일부가 파괴되었다. 실험성적을 종합하면 N. fowleri 배양액에서 단백분해효소의 활성이 측정되었고, 세포독성을 나타내는 물질을 부분정제 할 수 있었다. It is suspected that the proteinases of parasites may be closely related to their pathogenicity and cytotoxicity with regard to the host-parasite relationship. The aim of the present study was to elucidate whether proteinases from the lysates or secretory materials of pathogenic Naegleria fowleri and nonpathogenic N. gruberi are related to their cytotoxic activities. Mice inoculated with 1×105 N.fowleri trophozoites showed a cumulative mortality rate of 75% during observation, while no mice died in a group inoculated with the same amount of trophozoites of nonpathogenic N. gruberi. Utilizing a Chinese hamster ovary (CHO) cell line incubated with radioactive 51Cr (51Cr-labeled CHO cells), cytotoxicities of free-living amoebae were assayed. Scanning electron microscopic observations of target CHO cells affected with a purified cytotoxic substance of N. fowleri were also carried out. To determine the types and electrophoretic banding patterns of proteinases, the effect of various proteinase inhibitors on cell activity was examined, and it was found that enzymes from N. fowleri showed different patterns than N. gruberi. Cytotoxic activities, as determined by the release of 51Cr from CHO cells, were significantly detected in the lysate and cultured media of N. fowleri, but very little cytotoxic activity was observed in the lysate and cultured media of N. gruberi. The rate of flow(Rf) values for the electrophoretic banding patterns varied between the proteinase of the lysate and the cultured media of N. fowleri, but a Rf value of 0.87 was common in both bands. Purification of proteinase in the cultured media of N. fowleri was performed by centrifugation, ammonium sulfate fractionation, and spectra/Gel AcA 44 column chromatography. Purification was achieved up to a 23.6-fold increase in comparison with the raw materials of the intact N. fowleri cultured media and yielded about 19.9% of the total amount. The partially purified cytotoxic substance determined by SDS-PAGE had a molecular weight of 17,500 daltons and an Rf value of 0.87 was determined by the gelatin containing SDS-PAGE. Enzymatic activities in the N. fowleri cultured media were examined using various proteinase inhibitors and substrates, and it was confirmed that cysteine and aspartic acid residues were responsible for the cytotoxic activity. The scanning electron microscopic findings of the CHO cells treated with a partially purified cytotoxic substance of N. fowleri and observed for 30 minutes up to 180 minutes, showed deterioration of the microvilli over the entire surface of the cell, plasma membrane blebbing and holes in the plasma membrane. From the above findings of the biochemical and physical properties of the cytotoxic substance from N. fowleri cultured media, it is suggested that the cysteine proteinase secreted from N. fowleri may damage the cell membrane of target cells.

Analysis of Antigenic Specificities of Naegleria fowleri Using EITB

Shin,Ho-Joon,Im,Kyung-il INSTITUTE OF TROPICAL MEDICINE YONSEI UNIVERSITY 1992 YONSEI REPORTS ON TROPICAL MEDICINE Vol.23 No.1

Naegleria fowleri는 자연환경 속에서 자유생활을 하는 아메바로써 실험동물과 인체에서 치명적인 원발성 아메바성 수막뇌염을 일으킨다. 이런 원충들을 포함하여 조직에 기생하는 기생충들의 감염에 대한 진단에는 혈청학적 방법들이 많이 이용되고 있는데, 특히 면역요소는 전기영동 이적법(EITB)은 기생충의 각 항원의 분획에 대한 여러 혈청의 반응을 육안적으로 판별 가능하게 하여 특히 항원대 및 공통 항원대 등을 구별할 수 있게 한 방법이다. 이에 본 연구는 마우스에 N. fowleri로 면역시킨 혈청과 N. fowleri로 감염시킨 혈청을 이용하여 N. fowleri에서 추출한 조항원의 특성을 면역효소 전기영동 이적법으로 관찰하여 다음과 같은 결과를 얻었다. N. fowleri의 lysate의 단백질 분획은 9개 즉, 97, 81, 66, 53, 40, 39, 28, 25 및 23 kDa의 분자량을 가진 분획들의 주요 단백질 분획이었다. 면역혈청을 반응시킨 겨로가, 4개의 항원대 즉, 53, 40, 39 및 25 kDa에서 반응대가 관찰되었으며, 감염혈청 (3일, 7일, 10일)에서는 2개의 항원대 즉, 53 및 25 kDa에서 반응대가 관찰되었다. 21일째 혈청에서는 53 및 25 kDa 반응대와 더불어 35 및 31 kDa의 항원대가 더 관찰되었다. 한편, 감염이나 면역되지 않은 대조군의 마우스의 혈청도 53 kDa의 항원대에 반응하여 53 kDa의 단백질은 비특이성 항원대로 생각되었다. 결과적으로 면역혈청에서는 23 kDa의 항원대, 감염혈청에서는 25 kDa의 항원대가 중요한 단백질 분획으로 관찰되었다. Naegleria fowleri causes the fatal primary amoebic meningoencephalitis in the experimental animals and human beings. In the diagnosis of the tissue-invading parasites in cluding this amoeba, the immunological and serological tests have been used. It is important to observe their antigenic specificities. This study was performed to analyze the antigenicity of N. fowleri using immunized an infected mouse sera by EITB. By SDS-PAGE, at least nine bands, 97, 81, 66, 53, 40, 39, 28, 25 and 23 kDa, were noticed as major protein bands in the lysate of N. fowleri. As a result of EITB, the immunized serum was reacted with major four bands of N. fowleri angigens, 53, 40, 39 and 23 kDa. By EITB using infected sera, 53 and 25 kDa bands were major protein bands. In conclusion, 23 and 25 kDa antigens were identified to be specific for immunization and infection, respectively.

비접촉 조건에서의 Naegleria fowleri에 의한 표적세포의 세포독성

강창근,Il-Hwa Hong,Jong-Hyun Kim 한국동물위생학회 2019 韓國家畜衛生學會誌 Vol.42 No.4

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 hemor-rhagic disease that leads to death in approximately seven days. In this study, we investigate the cytotox-icity 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 cytotox-icity 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 un-der the non-contact condition induce the cell death and this process is an important step in pathogenesis of N. fowleri.

면역시킨 마우스 흉선 및 비장세포에 대한 병원성 Naegleria의 세포병변 효과

안명희,류재숙,민득영 한양대학교 의과대학 1991 한양의대 학술지 Vol.11 No.1

Naegleria fowleri is a causative agent of primary amoebic meningoencephalitis (PAM) in man and experimental animal. We investigated the interaction of immunized mouse thymocyte and splenocyte with N. fowleri trophozoites in vitro. BALb/c mouse was immunized with 150∼200×10⁴/ml N. fowleri trophozoites, intraperitoneally, once a week, for two times. Thymus and spleen cells from immunized or normal mouse cocultured with N.fowleri in 10% FCS+MEM, 37℃, 55%-CO₂incubator and cell: amoeba ratio was 200∼400:1. Thymus or spleen cells and N. fowleri were observed at 24, 48, 72 hrs after incubation by inverted microscope. Spleen cells from immunized mice showed moderate lysis aftre 72 hrs with round amoeba, but other group cells were completely lysed at 48 hrs after incubation with N. fowleri. It may be secreted a certain cytolytic amterial from N. fowleri because of cell lysis occured abruptly after 24 hrs culture. And amoebae showed better condition i.e., pseudopodaand vacuoles, after phagocytosis of lytic cell material. On the other hand, nonadherent cells from immunized spleen were completely lysed at 48 hrs with N. fowleri conculture and immunized thymus cell with aditive activated mactophage showed prolonged cell survival. And so, cooperative reaction of immunized T-lympocyte and macrophage are important to supperss N. fowleri in vitro.

Naegleria fowleri Induces Jurkat T Cell Death via O-deGlcNAcylation

Young Ah Lee,Kyeong Ah Kim,Myeong Heon Shin 대한기생충학열대의학회 2021 The Korean Journal of Parasitology Vol.59 No.5

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.

Analysis of Blastogenesis of Lymphocytes and their Subpopulations in Mice Infected with Naegleria fowleri

Shin, Ho-Joon,Im, Kyung-il,Kim, Yoon-Jin INSTITUTE OF TROPICAL MEDICINE YONSEI UNIVERSITY 1993 YONSEI REPORTS ON TROPICAL MEDICINE Vol.24 No.1

자연환경속에서 자유생활을 하는 N. fowleri를 마우스의 비강내로 감염시켜 실험적 수막뇌염을 일으킴에 있어서 감염량을 달리하여, 감염후 경과 기간별로 마우스의 비장세포에서 T 및 B 림프구를 분리하여 각각의 아형들에 대한 단세포군 항체를 처리하고, flow cytometry를 이용하여 T 및 B 림프구 아형들의 변동 정도를 관찰하고자 하였다. 또한, 비장세포 T 림프구의 아세포화 정도를 DNA 염색방법으로 측정하여 감염정도에 따른 실험성적을 비교하고자 하였다. Thy-1.2+ T 세포수는 중감염군(1×105개 감염, 사망율 70%)에서 7일째에 27.9%로 대조군의 23.5% 보다 증가하였다. 경감염군(1×10⁴개 감염, 사망율 15%)에서는 다소 증가하는 듯 하였으나 통계학적 유의성이 없었다. Ly-2+ T 세포수 및 L3/T4+ T 세포수는 중감염군에서만 7일째에 각각 10.9% 및 17.5%로 대조군의 9.1% 및 14.2% 보다 증가되었으며, LR-1+ B 세포수는 실험군에서 10일째에 대조군과 비교하여 증가하는 듯 하였으나 통계학적 유의성이 없었다. DNA 합성정도(S-분획)는 중감염군에서 감염후 7일째 및 10일째에 각각 10.9% 및 7.9%로, 대조군의 4.2% 및 4.0%와 비교할 때 현저히 증가되었다. 또한 경감염군에서도 각각 5.5% 및 6.6%로 대조군의 3.8% 및 3.9% 보다 증가되었다. In this study, the change in blastogenesis and subpopulations of lymphocytes was investigated in mice experimentally infected with Naegleria fowleri and which subsequently developed meningoencephalitis. Lymphocytes separated from the spleen of the mice were treated with monoclonal antibodies aganist T lymphocytes, B lymphocytes and their subsets, and were observed using flow cytometry. The blastogenesis of T cells in mice infected with N. fowleri was also investigated. DNA in the T lymphocytes was stained with propidium and observed of mice infected with 1×105 trophozoites (heavily infected mice, mortality rate 70%) was 27.9%, which was higher than the control group with 23.5% on the 7th day after infection. The number of Thy-15 lymphocytes in mice infected with 1×10⁴ trophozoites(lightly infected mice, mortality rate 15%) tended to increase, but there was no statistically significant difference compared with the control group. The number of Ly-2+ lymphocytes in the heavily infected mice was 10.9%, which was higher than the control group with 9.1% on the 7th day after infection. The number of L3/T4+ lymphocytes in the heavily infected mice was 17.5%, which was higher than control group(14.2%) on the 7th day after infection. The number of LR-1+ lymphocytes in the heavily and lightly infected mice tended to increase, but there was no statistically significant difference compared with the control group. The DNA percentages (S-fraction,%) for T lymphocytes in the heavily infected mice were 10.9 and 7.9 on the 7th and 10th days after infection, respectively, this percentage was significantly higher than that for the control group(4.2 and 4.0 respectively). In lightly infected mice, the DNA amount was 5.5 on the 7th after infection and 6.6 on the 10th day, which was significantly higher than the control group.

Production and characterization of monoclonal antibodies against cathepsin B and cathepsin B-Like proteins of <i>Naegleria fowleri</i>

Seong, Gi-Sang,Sohn, Hae-Jin,Kang, Heekyoung,Seo, Ga-Eun,Kim, Jong-Hyun,Shin, Ho-Joon Elsevier 2017 Experimental parasitology Vol.183 No.-

<P>Naegleria fowleri causes fatal primary amoebic meningoencephalitis (PAM) in humans and experimental animals. In previous studies, cathepsin B (nfcpb) and cathepsin B-like (nfcpb-L) genes of N. fowleri were cloned, and it was suggested that refolding rNfCPB and rNfCPB-L proteins could play important roles in host tissue invasion, immune response evasion and nutrient uptake. In this study, we produced anti-NfCPB and anti-NfCPB-L monoclonal antibodies (McAb) using a cell fusion technique, and observed their immunological characteristics. Seven hybridoma cells secreting rNfCPB McAbs and three hybridoma cells secreting rNfCPB-L McAbs were produced. Among these, 2C9 (monoclone for rNfCPB) and 1C8 (monoclone for rNfCPB-L) McAb showed high antibody titres and were finally selected for use. As determined by western blotting, 2C9 McAb bound to N. fowleri lysates, specifically the rNfCPB protein, which had bands of 28 kDa and 38.4 kDa. 1C8 McAb reacted with N. fowleri lysates, specifically the rNfCPB-L protein, which had bands of 24 kDa and 34 kDa. 2C9 and 1C8 monoclonal antibodies did not bind to lysates of other amoebae, such as N. gruberi, Acanthamoeba castellanii and A. polyphaga in western blot analyses. Immuno-cytochemistry analysis detected NfCPB and NfCPB-L proteins in the cytoplasm of N. fowleri trophozoites, particularly in the pseudopodia and food-cup. These results suggest that monoclonal antibodies produced against rNfCPB and rNfCPB-L proteins may be useful for further immunological study of PAM. (c) 2017 Elsevier Inc. All rights reserved.</P>

Effect of DNA Vaccination with Retroviral Vector Expressing nfa1 Gene in Naegleria fowleri Infection

강창근,kianpour mehrnoosh,홍일화,손혜진,신호준,김종현 경상대학교 농업생명과학연구원 2017 농업생명과학연구 Vol.51 No.6

Naegleria fowleri is pathogenic free-living amoeba leading to primary amoebicmeningoencephalitis(PAM) in human and animals. The nfa1 gene cloned from N. fowleri islocated on food-cup structure called pseudopodia and function an adherence of host targetcells. To evaluate the effect of nfa1 vaccination against N. fowleri infection, we constructedretroviral vector(pQCXIN) expressing nfa1 gene. To determine the effect of vaccination andprotective immunity in in vivo models, we measured the immunoglobulin levels, cytokineinduction, and survival rate in mice infected with N. fowleri. Both levels of IgG and IgGsubclass in DNA vaccinated mice were significantly elevated. The cytokine analysis showthat DNA vaccinated mice induces production of IL-4 and IFN-γcytokines suggesting aTh1/Th2 mixed type immune response. The levels of nfa1 specific IgG antibody weremaintained highly until 12 weeks post-vaccination in vaccinted mice. The nfa1 vaccinatedmice using retroviral vector increased significantly survival rate(60%) after N. fowleri infection. Consequently, the nfa1 vaccination effectively induces protective immunity by upregulation ofimmune response in mice infected with N. fowleri. These results suggest that DNAvaccination using retroviral vector may be proper trial for treatment and prevention of PAM.

Antiamoebic activities of flavonoids against pathogenic free-living amoebae, Naegleria fowleri and Acanthamoeba species

Hương Giang Lê,Tuấn Cường Võ,강정미,Thu Hằng Nguyễn,황병수,오영택,나병국 대한기생충학ㆍ열대의학회 2023 The Korean Journal of Parasitology Vol.61 No.4

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.

<i>Naegleria fowleri</i>: Functional expression of the Nfa1 protein in transfected <i>Naegleria gruberi</i> by promoter modification

Song, Kyoung-Ju,Jeong, Seok-Ryoul,Park, Sun,Kim, Kyongmin,Kwon, Myung-Hee,Im, Kyung-Il,Pak, Jhang Ho,Shin, Ho-Joon Elsevier 2006 Experimental parasitology Vol.112 No.2

<P><B>Abstract</B></P><P>To establish a transient transfection system in a <I>Naegleria</I>, we constructed three <I>nfa1</I>-pEGFP-N1 vectors by the promoter replacement and insertion of a <I>nfa1</I> gene and transfected the DNAs into <I>Naegleria gruberi</I> using a lipid reagent. The transfection efficiency and usefulness of the three modified vectors were estimated by identifying the expressions of the EGFP and Nfa1 protein from <I>N. gruberi</I>. After transfection, the Nfa1 protein was functionally expressed on pseudopodia of <I>N. gruberi</I>. The strong GFP fluorescence was observed in <I>N. gruberi</I> transfected with the actin-<I>nfa1</I>-pEGFP-N1 vector, of which the CMV promoter region in the expression vector was replaced with the actin 5′ UTR region. Additionally, when transgenic <I>N. gruberi</I> trophozoites were co-cultured with CHO target cells, the Nfa1 protein was also located on cytoplasm and pseudopodia, especially on a food cup that was formed in contact with target cells as it shown in pathogenic <I>N. fowleri</I>.</P>