Role of the Nfa1 Protein in Pathogenic Naegleria fowleri Co-cultured with Target Cells

Abstract

Naegleria fowleri, a free-living amoeba, exists as a virulent pathogen which causes fatal primary amoebic meningoencephalitis (PAME) in experimental animal and humans. Using infected and immune mouse sera, we previously cloned an antigenic gene (called nfa1) from a cDNA library of N. fowleri by immunoscreening. The nfa1 gene had the coding sequence of 360 bp, producing a 13.1kDa recombinant protein (rNfa1). Anti-Nfa1 polyclonal antibody, revealed pseudopodia-specific immunolocalization of Nfa1 protein in a trophozoite of N. fowleri. An anti-Nfa1 antibody showed a neutralizing effect on the cytotoxicity of N. fowleri trophozoites against CHO cells, much like treating an anti-Nfa1 antibody on a cocultivating system. In spite of the wide use of N. fowleri in free-living amoebic pathogenicity, no informations what proteins are involved in the functions of these organisms are yet available. In this study, we observed the role of Nfa1 protein in a cell-contact mechanism of pathogenic N. fowleri cocultured with target cells (CHO cell) by the immunofluorescence assay. Using confocal microscopic findings, the Nfa1 protein located on pseudopodia of N. fowleri trophozoites. The Nfa1 protein in N. fowleri trophozoites co-cultured with CHO cells was located on pseudopodia and in a food-cup formed as a phagocytic structure close contact with target cells. The amount of nfa1 mRNA of N. fowleri was strongly increased at 6 hr post co-culture. Finally, it was elucidated that the Nfa1 protein played an important role in phagocytic activity, a cell-contact mechanism of pathogenic N. fowleri.