Hae-Jin Sohn

The Nf-actin gene is an important factor for food-cup formation and cytotoxicity of pathogenic Naegleria fowleri

Naegleria fowleri destroys target cells by trogocytosis, a phagocytosis mechanism, and a process of piecemeal ingestion of target cells by food-cups. Phagocytosis is an actin-dependent process that involves polymerization of monomeric G-actin into filamentous F-actin. However, despite the numerous studies concerning phagocytosis, its role in the N. fowleri food-cup formation related with trogocytosis has been poorly reported. In this study, we cloned and characterized an Nf-actin gene to elucidate the role of Nf-actin gene in N. fowleri pathogenesis. The Nf-actin gene is composed of 1,128-bp and produced a 54.1-kDa recombinant protein (Nf-actin). The sequence identity was 82% with nonpathogenic Naegleria gruberi but has no sequence identity with other mammals or human actin gene. Anti-Nf-actin polyclonal antibody was produced in BALB/c mice immunized with recombinant Nf-actin. The Nf-actin was localized on the cytoplasm, pseudopodia, and especially, food-cup structure (amoebastome) in N. fowleri trophozoites using immunofluorescence assay. When N. fowleri co-cultured with Chinese hamster ovary cells, Nf-actin was observed to localize around on phagocytic food-cups. We also observed that N. fowleri treated with cytochalasin D as actin polymerization inhibitor or transfected with antisense oligomer of Nf-actin gene had shown the reduced ability of food-cup formation and in vitro cytotoxicity. Finally, it suggests that Nf-actin plays an important role in phagocytic activity of pathogenic N. fowleri.

IL-1β and IL-6 activate inflammatory responses of astrocytes against Naegleria fowleri infection via the modulation of MAPKs and AP-1

Naegleria fowleri, a free‐living amoeba, has been found in diverse habitats throughout the world. It causes primary amoebic meningoencephalitis in children and young adults. The amoeba attaches to nasal mucosa, migrates along olfactory nerves and enters the brain. Astrocytes are involved in the defence against infection and produce inflammatory responses. In this study, we focus on the mechanism of immune responses in astrocytes. We showed, using RNase protection assay, RT‐PCR and ELISA in an in vitro culture system, that N. fowleri lysates induce interleukin‐1beta (IL‐1β) and IL‐6 expression of astrocytes. In addition, cytokine levels of astrocytes gradually decreased due to extracellular signal‐regulated kinase (ERK), c‐Jun N‐terminal kinase (JNK) and p38 inhibitors. To determine the transcription factor, we used transcription inhibitor (AP‐1 inhibitor), which downregulated IL‐1β and IL‐6 expression. These results show that AP‐1 is related to IL‐1β and IL‐6 production. N. fowleri‐mediated IL‐1β and IL‐6 expression requires ERK, JNK and p38 mitogen‐activated protein kinases (MAPKs) activation in astrocytes. These findings show that N. fowleri‐stimulated astrocytes in an in vitro culture system lead to AP‐1 activation and the subsequent expressions of IL‐1β and IL‐6, which are dependent on ERK, JNK and p38 MAPKs activation. These results may imply that proinflammatory cytokines have important roles in inflammatory responses to N. fowleri infection.

Vaccination with Lentiviral Vector Expressing the nfa1 Gene Confers a Protective Immune Response to Mice Infected with Naegleria fowleri

ABSTRACT Naegleria fowleri, a pathogenic free-living amoeba, causes fatal primary amoebic meningoencephalitis (PAM) in humans and animals. The nfa1 gene (360 bp), cloned from a cDNA library of N. fowleri, produces a 13.1-kDa recombinant protein which is located on pseudopodia, particularly the food cup structure. The nfa1 gene plays an important role in the pathogenesis of N. fowleri infection. To examine the effect of nfa1 DNA vaccination against N. fowleri infection, we constructed a lentiviral vector (pCDH) expressing the nfa1 gene. For the in vivo mouse study, BALB/c mice were intranasally vaccinated with viral particles of a viral vector expressing the nfa1 gene. To evaluate the effect of vaccination and immune responses of mice, we analyzed the IgG levels (IgG, IgG1, and IgG2a), cytokine induction (interleukin-4 [IL-4] and gamma interferon [IFN-γ]), and survival rates of mice that developed PAM. The levels of both IgG and IgG subclasses (IgG1 and IgG2a) in vaccinated mice were significantly increased. The cytokine analysis showed that vaccinated mice exhibited greater IL-4 and IFN-γ production than the other control groups, suggesting a Th1/Th2 mixed-type immune response. In vaccinated mice, high levels of Nfa1-specific IgG antibodies continued until 12 weeks postvaccination. The mice vaccinated with viral vector expressing the nfa1 gene also exhibited significantly higher survival rates (90%) after challenge with N. fowleri trophozoites. Finally, the nfa1 vaccination effectively induced protective immunity by humoral and cellular immune responses in N. fowleri-infected mice. These results suggest that DNA vaccination using a viral vector may be a potential tool against N. fowleri infection.

The immune response induced by DNA vaccine expressing nfa1 gene against Naegleria fowleri

The pathogenic free-living amoeba, Naegleria fowleri, causes fatal primary amoebic meningoencephalitis in experimental animals and in humans. The nfa1 gene that was cloned from N. fowleri is located on pseudopodia, especially amoebic food cups and plays an important role in the pathogenesis of N. fowleri. In this study, we constructed and characterized retroviral vector and lentiviral vector systems for nfa1 DNA vaccination in mice. We constructed the retroviral vector (pQCXIN) and the lentiviral vector (pCDH) cloned with the egfp-nfa1 gene. The expression of nfa1 gene in Chinese hamster ovary cell and human primary nasal epithelial cell transfected with the pQCXIN/egfp-nfa1 vector or pCDH/egfp-nfa1 vector was observed by fluorescent microscopy and Western blotting analysis. Our viral vector systems effectively delivered the nfa1 gene to the target cells and expressed the Nfa1 protein within the target cells. To evaluate immune responses of nfa1-vaccinated mice, BALB/c mice were intranasally vaccinated with viral particles of each retro- or lentiviral vector expressing nfa1 gene. DNA vaccination using viral vectors expressing nfa1 significantly stimulated the production of Nfa1-specific IgG subclass, as well as IgG levels. In particular, both levels of IgG2a (Th1) and IgG1 (Th2) were significantly increased in mice vaccinated with viral vectors. These results show the nfa1-vaccination induce efficiently Th1 type, as well as Th2 type immune responses. This is the first report to construct viral vector systems and to evaluate immune responses as DNA vaccination in N. fowleri infection. Furthermore, these results suggest that nfal vaccination may be an effective method for treatment of N. fowleri infection.

NLRP3 Inflammasome Activation in THP-1 Target Cells Triggered by Pathogenic Naegleria fowleri

ABSTRACT Naegleria fowleri, known as the brain-eating amoeba, causes acute primary amoebic meningoencephalitis. During swimming and other recreational water activities, N. fowleri trophozoites penetrate the nasal mucosa and invade the olfactory bulbs, resulting in intense inflammatory reactions in the forebrain tissue. To investigate what kinds of inflammasome molecules are expressed in target cells due to N. fowleri infection, human macrophage cells (THP-1 cells) were cocultured with N. fowleri trophozoites in a noncontact system, and consequently, interleukin-1β (IL-1β) production was estimated. Caspase-1 activation and IL-1β production from THP-1 cells by Western blotting and the culture supernatant by enzyme-linked immunosorbent assay analysis were observed at 3 h after cocultivation. In addition, the increased expression of ASC and NLRP3, which make up an inflammasome complex, was also observed at 3 h after cocultivation. To confirm the caspase-1 activation and IL-1β production via the NLRP3 inflammasome in THP-1 cells triggered by N. fowleri trophozoites, THP-1 cells were pretreated with several inhibitors. The inhibition assay showed that CA-074 (a cathepsin B inhibitor), glybenclamide (an NLRP3 molecule inhibitor), and N-benzyloxycarbony-Val-Ala-Asp(O-methyl)-fluoromethylketone (Z-VAD-FMK; a caspase-1 inhibitor) reduced the levels of caspase-1 activation and IL-1β production from THP-1 cells. This study suggests that N. fowleri infection induces the NLRP3 inflammasome, which activates caspase-1 and subsequently produces IL-1β, thus resulting in inflammation.

Naegleria fowleri Lysate Induces Strong Cytopathic Effects and Pro-inflammatory Cytokine Release in Rat Microglial Cells

Naegleria fowleri, a ubiquitous free-living ameba, causes fatal primary amebic meningoencephalitis in humans. N. fowleri trophozoites are known to induce cytopathic changes upon contact with microglial cells, including necrotic and apoptotic cell death and pro-inflammatory cytokine release. In this study, we treated rat microglial cells with amebic lysate to probe contact-independent mechanisms for cytotoxicity, determining through a combination of light microscopy and scanning and transmission electron microscopy whether N. fowleri lysate could effect on both necrosis and apoptosis on microglia in a time- as well as dose-dependent fashion. A 51Cr release assay demonstrated pronounced lysate induction of cytotoxicity (71.5%) toward microglial cells by 24 hr after its addition to cultures. In an assay of pro-inflammatory cytokine release, microglial cells treated with N. fowleri lysate produced TNF-α, IL-6, and IL-1β, though generation of the former 2 cytokines was reduced with time, and that of the last increased throughout the experimental period. In summary, N. fowleri lysate exerted strong cytopathic effects on microglial cells, and elicited pro-inflammatory cytokine release as a primary immune response.

Effects of immunization with the rNfa1 protein on experimental Naegleria fowleri-PAM mice

Free‐living Naegleria fowleri causes primary amoebic meningoencephalitis (PAM) in humans and animals. To examine the effect of immunization with Nfa1 protein on experimental murine PAM because of N. fowleri, BALB/c mice were intra‐peritoneally or intra‐nasally immunized with a recombinant Nfa1 protein. We analysed Nfa1‐specific antibody and cytokine induction, and the mean survival time of infected mice. Mice immunized intra‐peritoneally or intra‐nasally with rNfa1 protein developed specific IgG, IgA and IgE antibodies; the IgG response was dominated by IgG1, followed by IgG2b, IgG2a and IgG3. High levels of the Th1 cytokine, IFN‐γ, and the regulatory cytokine, IL‐10, were also induced. The mean survival time of mice immunized intra‐peritoneally with rNfa1 protein was prolonged compared with controls, (25·0 and 15·5 days, respectively). Similarly, the mean survival time of mice immunized intra‐nasally with rNfa1 protein was 24·7 days, compared with 15·0 days for controls.

Therapeutic effect of rokitamycin in vitro and on experimental meningoencephalitis due to Naegleria fowleri

Inhalation of freshwater containing the free-living amoeba Naegleria fowleri leads to a potentially fatal infection known as primary amoebic meningoencephalitis (PAME). Amphotericin B is the only agent with clinical efficacy in the treatment of PAME in humans, however this drug is often associated with adverse effects on the kidney and other organs. In an attempt to select other useful therapeutic agents for treating PAME, the amoebicidal activities of antibacterial agents including clarithromycin, erythromycin, hygromycin B, neomycin, rokitamycin, roxithromycin and zeocin were examined. Results showed that the growth of amoeba was effectively inhibited by treatment with hygromycin B, rokitamycin and roxithromycin. Notably, when N. fowleri trophozoites were treated with rokitamycin, the minimal inhibitory concentration was 6.25 microg/mL on Day 2. In the treatment of experimental meningoencephalitis due to N. fowleri, survival rates of mice treated with roxithromycin and rokitamycin were 25% and 80%, respectively, over 1 month. The mean time to death for roxithromycin and rokitamycin treatment was 16.2 days and 16.8 days, respectively, compared with 11.2 days for control mice. Finally, rokitamycin showed both in vitro and in vivo therapeutic efficacy against N. fowleri and may be a candidate drug for the treatment of PAME.

Effective PCR-based detection of Naegleria fowleri from cultured sample and PAM-developed mouse.

Increasing numbers of Primary Amoebic Meningoencephalitis (PAM) cases due to Naegleria fowleri are becoming a serious issue in subtropical and tropical countries as a Neglected Tropical Disease (NTD). To establish a rapid and effective diagnostic tool, a PCR-based detection technique was developed based on previous PCR methods. Four kinds of primer pairs, Nfa1, Nae3, Nf-ITS, and Naegl, were employed in the cultured amoebic trophozoites and a mouse with PAM experimentally developed by N. fowleri inoculation (PAM-mouse). For the extraction of genomic DNA from N. fowleri trophozoites (1×10(6)), simple boiling with 10μl of PBS (pH 7.4) at 100°C for 30min was found to be the most rapid and efficient procedure, allowing amplification of 2.5×10(2) trophozoites using the Nfa-1 primer. The primers Nfa1 and Nae3 amplified only N. fowleri DNA, whereas the ITS primer detected N. fowleri and N. gruberi DNA. Using the PAM-mouse brain tissue, the Nfa1 primer was able to amplify the N. fowleri DNA 4 days post infection with 1ng/μl of genomic DNA being detectable. Using the PAM-mouse CSF, amplification of the N. fowleri DNA with the Nae3 primer was possible 5 days post infection showing a better performance than the Nfa1 primer at day 6.

Improvement of a rapid diagnostic application of monoclonal antibodies against avian influenza H7 subtype virus using Europium nanoparticles

The development of a sensitive and rapid diagnostic test is needed for early detection of avian influenza (AI) H7 subtype. In this study, novel monoclonal antibodies (mAbs) against influenza A H7N9 recombinant hemagglutinin (rHA)1 were developed and applied to a Europium nanoparticle–based rapid fluorescent immunochromatographic strip test (FICT) to improve the sensitivity of the rapid diagnostic system. Two antibodies (2F4 and 6D7) exhibited H7 subtype specificity in a dot-FICT assay by optimization of the conjugate and the pH of the lysis buffer. The subtype specificity was confirmed by an immunofluorescence assay and Western blot analysis. The limit of detection of the FICT employing novel mAbs 31 ng/mL for H7N9 rHA1 and 40 hemagglutination units/mL for H7 subtype virus. Sensitivity was improved 25-fold using Europium as confirmed by comparison of colloidal gold-based rapid diagnostic kit using the 2F4 and 6D7 mAbs.