Hanyang Med Rev.  2010 Aug;30(3):204-212. 10.7599/hmr.2010.30.3.204.

The nfa1 Gene Contributed on the Contact-dependent Pathogenic Mechanisms of Naegleria fowleri

• Shin HJ

• KMID: 1460255
• DOI: http://doi.org/10.7599/hmr.2010.30.3.204

Abstract

Free-living Naegleria fowleri is a causal agent of primary amoebic meningoencephalitis in mainly children and young adults. An nfa1 gene, encoding 360 bp of nucleotides, was cloned from a N. fowleri cDNA library by SEREX method. By immunohistochemistry and a confocal microscope, Nfa1 protein was found in amoebic pseudopods, especially in food-cups, when amoeba was in contact with target cells. When an anti-Nfa1 antibody was added to the coculture system, the cytotoxicity of N. fowleri trophozoites onto target cells was decreased, and the severe morphological destruction of rat microglial cells cocultured with N. fowleri trophozoites was reduced. In a tansfection system, an expression vector with an nfa1 gene was successful transfected into nonpathogenic N. gruberi, and transgenic N. gruberi showed the increasing in vitro cytotoxicity. The siRNA decreased the expression levels of nfa1 mRNA and Nfa1 protein in transfected N. fowleri trophozoites. On the immunization of mice with the rNfa1 protein, the protective immunity of host was induced. Thus, mice showed the prolonged mean survival times in PAM-developed mice. In final, the nfa1 gene and Nfa1 protein play an important role in the pathogenesis of N. fowleri infection.

Keyword

Naegleria fowleri; nfa1 gene; Cytotoxicity; Protective immunity; Recombinant protein

MeSH Terms

Amoeba
Animals
Child
Clone Cells
Coculture Techniques
Gene Library
Humans
Immunization
Immunohistochemistry
Meningoencephalitis
Mice
Naegleria
Naegleria fowleri
Nucleotides
Rats
RNA, Messenger
RNA, Small Interfering
Survival Rate
Trophozoites
Young Adult
Nucleotides
RNA, Messenger
RNA, Small Interfering

Figure

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  Fig. 1 Life-cycle of Naegleria fowleri.

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  Fig. 2 Three hypothetical mechanisms for Naegleria fowleri pathogenesis. (A) Contact-dependent mechanism. (B) Contact-independent mechanism. (C) Autophagy mechanism.

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  Fig. 3 SDS-PAGE and western blotting results. A) Lane 1 and 2 show a N. fowleri lysate and a recombinant His-tag fusion Nfa1 protein. Lane 3 and 4 show reacting bands by western blotting using an anti-Nfa1 polyclonal antibody. B) All lanes show band patterns of recombinant His-tag fusion Nfa1 proteins with anti-Nfa1 monoclonal antibody produced from various hybridoma clones. PM, prestained marker.

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  Fig. 4 Confocal microscopic findings of the Nfa1 protein in Naegleria fowleri trophozoites cocultured with CHO cells (red color) for 3 hr (A) and 6 hr (B). Strong fluorescent signals were shown in the food-cups of the N. fowleri trophozoite (×400).

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  Fig. 5 Flow-chart of immunization and infection schedule.

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  Fig. 6 Serum IgG level in mice immunized with the rNfa1 protein. rNfa1, recombinant Nfa1 protein; PBS, phosphate buffered saline.

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  Fig. 7 Survival rate in mice infected intraperitoneally with N. fowleri trophozoites post immunization with the rNfa1 protein.

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