To evaluate the role of heat shock 70 protein (HSP70) in free-living amoeba, a constitutive and inducible heat shock 70 gene of pathogenic Naegleria fowleri has previously been cloned, characterized, and named as NfcHSP70. The Nf-cHSP70 is localized in the cytoplasm, pseudopodia, and phagocytic food-cups. To investigate the role of Nf-cHSP70 in the pathogenicity of N. fowleri, the synthesis of N. fowleri HSP70 was first inhibited with benzylidene lactam compound (KNK437), and Nf-cHSP70 gene was knock-downed with antisense oligomers, which were designed with a start region-specific antisense oligonucleotides (24 oligomers) and modified with phosphorothioate. KNK437 inhibited the induction of N. fowleri HSP70 in a dose-dependent manner. In addition, 300 μM KNK437 reduced the proliferation of N. fowleri to 79.4% of untreated control (100%). Nf-cHSP70 knock-downed N. fowleri with antisense oligomers showed 68.5% reduction of proliferation in comparison with untreated control (100%). The cytotoxicity of N. fowleri against CHO target cells was reduced to 42.1% by KNK437 and 68.6% by antisense oligomers. These results suggest that the cloned Nf-cHSP70 plays an important role in the proliferation and cytotoxicity of pathogenic N. fowleri.

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.