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Naegleria fowleri, a brain-eating amoeba, thrives in lakes and rivers with aquatic vegetation and causes primary amoebic meningoencephalitis (PAM) in humans. Most recently, it has become such a serious problem that N. fowleri was detected in tap water in Houston, USA. Several pathogenic factors are considered very important to destroy target cells in the brain. In particular, the food-cup where N. fowleri antigen-1 (Nfa1) is located, is strongly expressed in pseudopodia involved in the movement of N. fowleri, and is involved in phagocytosis by attaching to target cells. In this article, we reviewed the role of the Nfa1 protein and its associated pathogenicity. The nfa1 gene was cloned by cDNA library immunoscreening using infection serum and immune serum. Nfa1 protein is mainly distributed in pseudopodia important to movement and vacuoles. Moreover, heat shock protein 70, cathepsin-like proteare and Nf-actin are also associated with pseudopodia in which Nfa1 is localized. Interestingly, the amount of the nfa1 gene changed as N. fowleri trophozoites transformed into cysts. Polyclonal antiserum against Nfa1 showed a protective effect against cytotoxicity of approximately 19.7%. Nfa1-specific IgA antibodies prevent N. fowleri trophozoites from adhering to the nasal mucosa, delaying invasion. The nfa1-vaccinated mice showed significantly higher levels of Nfa1-specific antibody. The duration of anti-Nfa1 IgG in the vaccinated mice lasted 12 weeks, strongly suggesting that nfa1 is a significant pathogenic gene and that Nfa1 is a pathogenic protein. Several factors related to pseudopodia and locomotion have been linked to Nfa1. A clearer function of N. fowleri targeting nfa1 with other genes might enable target-based inhibition of N. fowleri pathogenicity. [ABSTRACT FROM AUTHOR]

The article "Retraction Note: Establishment of an Acanthamoeba keratitis mouse model confirmed by amoebic DNA amplification" from Scientific Reports, published on February 18, 2021, has been retracted due to concerns about image duplication and rotation in Figure 5. The Editors lost confidence in the results and conclusions of the article as the Authors did not provide satisfactory responses to the issues raised. The retraction was reported by multiple authors involved in the study. [Extracted from the article]

Naegleria fowleri, a free-living amoeba that is found in diverse environmental habitats, can cause a type of fulminating hemorrhagic meningoencephalitis, primary amoebic meningoencephalitis (PAM), in humans. The pathogenesis of PAM is not fully understood, but it is likely to be primarily caused by disruption of the host's nervous system via a direct phagocytic mechanism by the amoeba. Naegleria fowleri trophozoites are known to secrete diverse proteins that may indirectly contribute to the pathogenic function of the amoeba, but this factor is not clearly understood. In this study, we analyzed the inflammatory responses in BV-2 microglial cells induced by excretory and secretory proteins of N. fowleri (NfESP). Treatment of BV-2 cells with NfESP induced the expression of various cytokines and chemokines, including the proinflammatory cytokines IL-1α and TNF-α. NfESP-induced IL-1α and TNF-α expression in BV-2 cells were regulated by p38, JNK, and ERK MAPKs. NfESP-induced IL-1α and TNF-α production in BV-2 cells were effectively downregulated by inhibition of NF-kB and AP-1. These results collectively suggest that NfESP stimulates BV-2 cells to release IL-1α and TNF-α via NF-kB- and AP-1-dependent MAPK signaling pathways. The released cytokines may contribute to inflammatory responses in microglia and other cell types in the brain during N. fowleri infection. [ABSTRACT FROM AUTHOR]

Free‐living amoeba, Naegleria fowleri, destroys target cells through contact‐dependent mechanisms, such as phagocytosis and/or trogocytosis. A previous experiment showed that the nf‐actin gene consisted of 1.2 kbp, produced a 50.1 kDa recombinant protein (Nf‐actin), and was localized on the cytoskeleton, pseudopodia and amoebastome. In this study, cellular characterization of the nf‐actin gene concerned with contact‐dependent mechanisms in N fowleri was performed. The nf‐actin gene was amplified from a gene‐cloned vector, pEXQP5‐T7/NT TOPO. The nf‐actin gene was introduced into the Ubi‐pEGFP‐C2 vector, and Ubi‐pEGFP‐C2/nf‐actin was transfected into N fowleri trophozoites. Strong GFP fluorescence was detected in N fowleri trophozoites transfected with Ubi‐pEGFP‐C2/nf‐actin. Expression of EGFP‐Nf‐actin protein was detected by Western blot analysis. The nf‐actin‐overexpressing N fowleri showed significantly increased adhesion activity against extracellular matrix components, fibronectin, collagen I and fibrinogen, compared with wild‐type N fowleri. Moreover, nf‐actin‐overexpressing N fowleri showed increased phagocytic activity and cytotoxicity in comparison with wild‐type N fowleri. In summary, the overexpressed nf‐actin gene has an important function in ability to increase cell adhesion, cytotoxicity and phagocytosis by N fowleri. [ABSTRACT FROM AUTHOR]

Plasmodium lactate dehydrogenase (pLDH) is a strong target antigen for the determination of infection with Plasmodium species specifically. However, a more effective antibody is needed because of the low sensitivity of the current antibody in many immunological diagnostic assays. In this study, recombinant Plasmodium vivax LDH (PvLDH) was experimentally constructed and expressed as a native antigen to develop an effective P. vivax-specific monoclonal antibody (mAb). Two mAbs (2CF5 and 1G10) were tested using ELISA and immunofluorescence assays (IFA), as both demonstrated reactivity against pLDH antigen. Of the 2 antibodies, 2CF5 was not able to detect P. falciparum, suggesting that it might possess P. vivax-specificity. The detection limit for a pair of 2 mAbs-linked sandwich ELISA was 31.3 ng/ml of the recombinant antigen. The P. vivax-specific performance of mAbs-linked ELISA was confirmed by in vitro-cultured P. falciparum and P. vivax-infected patient blood samples. In conclusion, the 2 new antibodies possessed the potential to detect P. vivax and will be useful in immunoassay.

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.

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 (51)Cr 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.