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

Ho-Joon Shin

doi:10.7599/hmr.2010.30.3.204

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

Review Article

Hanyang Medical Reviews

Hanyang Medical Reviews 2010; 30(3): 204-212.

Published online: 31 August 2010

DOI: https://doi.org/10.7599/hmr.2010.30.3.204

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

Ho-Joon Shin, Ph.D.

Department of Microbiology, and Department of Molecular Science and Technology, Ajou University School of Medicine, Suwon, Korea.

Corresponding author (hjshin@ajou.ac.kr)

Received 8 June 2010 Accepted 29 June 2010

(open-access):

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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.

Keywords: Naegleria fowleri, nfa1 gene, Cytotoxicity, Protective immunity, Recombinant protein

Figures and Tables

Fig. 1

Life-cycle of Naegleria fowleri.

Fig. 2

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

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.

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).

Fig. 5

Flow-chart of immunization and infection schedule.

Fig. 6

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

Fig. 7

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

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