Production of Nfa1-specific monoclonal antibodies that influences the in vitro cytotoxicity of Naegleria fowleri trophozoites on microglial cells

Abstract

Naegleria fowleri, agent of fatal primary amoebic meningoencephalitis, appears to induce cytotoxicity mechanically through its contact with the cell. The nfa1 gene cloned from a cDNA library of pathogenic N. fowleri by immunoscreening consists of 360 bp and expresses a 13.1-kDa recombinant protein (rNfa1) that demonstrated localization in the pseudopodia when examined using immunocytochemistry. To study the mechanisms involved in N. fowleri cytotoxicity, we developed a large volume of rNfa1-specific monoclonal antibody (McAb) against a 17-kDa His-tag fusion rNfa1 protein using a cell fusion technique. We established eight McAb-producing hybridoma cells. The antibodies were all immunoglobulin G2b and reacted strongly with a 17-kDa band representing the rNfa1 fusion protein in Western blotting, demonstrating immunoreactivity to the Nfa1 protein in pseudopodia (especially in the food cups) of N. fowleri trophozoites. A 51 Cr-release assay indicated N. fowleri cytotoxicity by demonstrating that it eliminated 37.8, 60.6, and 98.8% of the target (microglial) cells 6, 12, and 24 h after coincubation, respectively. When an anti-Nfa1 McAb was added to the coculture system, N. fowleri cytotoxicity decreased to 29.8, 44.1, and 66.3%, respectively.

Naegleria fowleri after 50 years: is it a neglected pathogen?

“…Recently, Nf-actin was associated with phagocytosis (Lee et al, 2007). This protein was localized mainly in phagosome-cup structures (amoebostomes) and in the cytoplasm and pseudopodia during phagocytosis (Sohn et al, 2010).…”

Vaccination with Lentiviral Vector Expressing thenfa1Gene Confers a Protective Immune Response to Mice Infected with Naegleria fowleri

“…Transformed and nontransformed BL21(DE3)pLysS E. coli cell extracts were analyzed by SDS-PAGE, and the presence of the expressed gene product (a His tag fusion rNfa1 protein) was confirmed by Western blotting using sera from both immunized and infected mice. The rNfa1 protein was purified by metal affinity chromatography using a nickel-nitrilotriacetic acid (Ni-NTA) agarose column (Qiagen, Hilden, Germany), as described previously (28). Elution was carried out using imidazole buffer (5 M urea, 20 mM Na 2 HPO 4 , 5 M NaCl, and 500 mM imidazole).…”

Development of a high- versus low-pathogenicity model of the free-living amoeba Naegleria fowleri

“…Several proteins, such as Nf-actin (Jung et al, 2009;Lee et al, 2007;Sohn et al, 2010), pore-forming naegleriapores A and B (Herbst et al, 2002(Herbst et al, , 2004, cysteine protease (Aldape et al, 1994) and virulence-related protein (Hu et al, 1991(Hu et al, , 1992, have been implicated in Naegleria pathogenesis, but the exact roles of these proteins are not yet well understood. The present study enables modulation of the in vivo pathogenicity of N. fowleri by using different compositions of axenic growth media.…”

“…In order to assess transcriptional differences between N. fowleri trophozoites grown in the three different media, a quantitative real-time RT-PCR using primers against potential pathogenicity factors (Aldape et al, 1994;Herbst et al, 2002Herbst et al, , 2004Hu et al, 1991Hu et al, , 1992Jung et al, 2009;Lee et al, 2007;Sohn et al, 2010) was performed. The primers used for the PCR are listed in Table 1.…”

“…Another protein involved in N. fowleri-induced cell destruction is Nfactin, which is specifically located around phagocytic foodcups and therefore plays an important role in phagocytic activity (Sohn et al, 2010). Studies using mAbs against Nfactin ( Lee et al, 2007) and gene silencing by small interfering RNAs targeting the Nf-actin gene (Jung et al, 2009) also suggested that Nf-actin is involved in in vitro cytotoxicity. In addition to Nf-actin, it has been reported that N. fowleri secretes proteases such as a cysteine protease, which catalyses the in vitro degradation of extracellular matrix proteins and exerts a cytotoxic potential against mammalian cells (Aldape et al, 1994).…”