Expression of the nfa1 Gene Cloned from Pathogenic Naegleria fowleri in Nonpathogenic N. gruberi Enhances Cytotoxicity against CHO Target Cells In Vitro

Abstract

The pathogenic amoeba Naegleria fowleri has a 360-bp nfa1 gene that encodes the Nfa1 protein (13.1 kDa), which is located in the pseudopodia of the amoeba, and an anti-Nfa1 antibody reduces N. fowleri-induced mammalian-cell cytotoxicity in vitro. In contrast, an anti-Nfa1 antibody cannot detect Nfa1 protein expression in the nonpathogenic amoeba Naegleria gruberi, which also possesses the nfa1 gene. In the present study, the nfa1 gene cloned from pathogenic N. fowleri was transfected into nonpathogenic N. gruberi to determine whether it was related to pathogenicity. The nfa1 gene was initially inserted into a eukaryotic transfection vector, pEGFP-C2, containing a cytomegalovirus promoter and the green fluorescent protein (GFP) gene, and was designed as pEGFP-C2/nfa1UTR (nfa1UTR contains 5 upstream regions, the nfa1 open reading frame, and 3 downstream regions). After transfection, the green fluorescence was observed in the cytoplasm of N. gruberi trophozoites. These transfectants were preserved for more than 9 months after selection. The transfected nfa1 gene was observed by PCR using nfa1-and vector-specific primers in the genomic DNA of N. gruberi transfected with the pEGFP-C2/nfa1UTR vector. In addition, the nfa1 and GFP genes were identified by reverse transcription-PCR in transgenic N. gruberi. The Nfa1 protein expressed in transgenic N. gruberi was identified as a 13.1-kDa band by Western blotting using an anti-Nfa1 antibody. Finally, N. gruberi transfected with the pEGFP-C2/nfa1UTR vector was found to have enhanced cytotoxicity against CHO cells compared with naïve N. gruberi.

Cellular characterization of actin gene concerned with contact‐dependent mechanisms in Naegleria fowleri

“…The contact‐independent mechanism involves secretory molecules, such as serine protease, phospholipase A, neuraminidase and cysteine protease . In contrast, the contact‐dependent mechanism is associated with phagocytic food‐cup structures or amoebastomes activated by Nfa1 protein, heat‐shock protein 70 and Nf‐actin protein …”

Effects of Mannose on Pathogenesis of Acanthamoeba castellanii

“…Bacteria were routinely cultured in Luria-Bertini (LB) medium at 37℃ overnight as previously described [7]. Chinese hamster ovary (CHO) cells were used for A. castellanii adhesion and in vitro cytotoxicity assay [8]. Briefly, CHO cells were cultured as a monolayer in Earle's minimal essential medium (EMEM; Gibco BRL, Grand Island, New York, USA) at 37℃.…”

Cytopathogenesis of Naegleria fowleri Thai strains for cultured human neuroblastoma cells

“…It is reasonable to propose that Siriraj strain, which was isolated from a patient, secreted some biological substances to the cell surface of the infected cells (Fulford and Marciano-Cabral 1986;Yum et al 2002). Previous studies reported that N. fowleri produced Nfa1 protein as the contact mechanism on the cell surface of Chinese Hamster Ovary target cells in coculture (Jeong et al 2005). In addition, cytosolic heat shock protein 70 was a new finding for heat tolerance and may relate to virulence of the amoebae (Song et al 2007).…”

“…Vero cells have also been reported as a suitable model for evaluating the cytopathogenicity of N. fowleri (John and John 1994a, b) even though the cells had been isolated from monkey kidney. Moreover, the CHO target cell (Chinese Hamster ovary cells) has also used for testing the cytotoxicity of the amoebae and the results demonstrated that the amoebae produced the Nfa1 protein during cytopathogenesis (Jeong et al 2005). However, the suitable gold standard model representing human neuron has not yet been found.…”