Molecular cloning and characterization of a cytosolic heat shock protein 70 from Naegleria fowleri

Abstract

A gene encoding a cytosolic heat shock protein 70 from pathogenic Naegleria fowleri (Nf-cHSP70) was identified. The Nf-cHSP70 was 2,062 bp in length with an open reading frame of 1,980 bp encoding 659 amino acid residues. The deduced amino acid sequence of the gene shared high sequence identities with HSP70s from other parasitic organisms and mammals. The characteristic domains, including N-terminal ATPase domain, calmodulin-binding domain, and EE(D)VD motif, found in HSP70s were also well conserved in this gene. The recombinant Nf-cHSP70 protein showed strong antigenicity against the sera from mice experimentally infected with N. fowleri. Immunofluorescence assay showed that Nf-cHSP70 localized in cytosol of the parasite. The results from semi-quantitative RT-PCR and Western blot analyses demonstrated the expression levels of gene transcripts, and its products were significantly increased at high temperature (42 degrees C). The definitive biological roles of Nf-cHSP70 are not clear, but it may protect the parasite under environmental changes especially high temperature.

Cytopathogenesis of Naegleria fowleri Thai strains for cultured human neuroblastoma cells

“…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). In this study, we propose the SK-N-MC cells as a gold standard model due to the high susceptibility to N. fowleri.…”

Heat shock protein 70 of Naegleria fowleri is important factor for proliferation and in vitro cytotoxicity

“…To investigate the role of HSPs in N. fowleri, Song et al (2007) cloned the gene encoding for cytosolic-HSP70 in N. fowleri and concluded that the definitive roles of the NfcHSP70 protein under induction condition are not clear, but it may protect the parasite from environmental temperature changes (Song et al 2007). Nevertheless, there are only very limited information available regarding its function in the parasite.…”

“…We have previously reported that Nf-cHSP70 gene encodes a protein with 660 amino acids residues, producing a molecular mass of 72 kDa. In addition, the recombinant Nf-cHSP70 protein showed strong antigenicity against mice sera, which were infected experimentally with N. fowleri (Song et al 2007) and showed 68% sequence identity to HSP70s in comparison with other organisms.…”

“…Briefly, after protein concentration was measured by the Bradford assay, 10 μg of samples was loaded onto 12% SDS-PAGE gel. For Western blotting, proteins were transferred onto PVDF sheet and reacted with a polyclonal anti Nf-cHSP70 antibody (Song et al 2007) for 1 h. After washing three times with 0.05% PBST, peroxidase-conjugated goat antimouse whole IgG (1:2,000 dilution; Sigma Chemical Co., St. Louis, USA) was added onto each sheet. They were soaked in enhanced chemiluminescence solution (Intron, Daejon, Korea) and exposed to an X-ray film (Konica, Tokyo, Japan).…”

“…After incubation for 5 h post-transfection, 1 ml of complete Nelson's medium was added to each well. HSP70 expression assay KNK 437 treated or transiently transfected N. fowleri trophozoites were collected and assayed for the expression of HSP70 as described previously (Song et al 2007). Briefly, after protein concentration was measured by the Bradford assay, 10 μg of samples was loaded onto 12% SDS-PAGE gel.…”