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  • Author: Prasert Auewarakul x
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Supathra Tiewcharoen, Jundee Rabablert, Sittiruk Roytrakul, Waravee Wiyawuth, Nat Malainual, Virach Junnu, Kumchol Chaiyo and Prasert Auewarakul

Abstract

Background: Free-living amoeboflagellate, Naegleria fowleri causes acute fulminant primary amoebic meningoencephalitis (PAM). Interaction of N. fowleri with human cells is essential for the cytopathogenic process before phagocytosis and tissue invasion.

Objective: In order to understand the pathogenesis mechanisms of N. fowleri, subtractive cDNA library was used to analyze overall induction in gene expression of N. fowleri during exposure to the human neuroblastoma cells.

Methods: Co-cultivation of N. fowleri and human neuroblastoma SK-N-MC monolayer cultivation was performed. Subtractive cDNA libraries of inoculated N. fowleri at 15, 30, 45, 60, and 120 minutes were constructed. The PCR amplified products were cloned into E. coli. The specific clones were selected and further sequenced. Nucleotide sequences were compared with those deposited in the Genbank using BlastX. Significant probabilities were considered when E-value was less than 10-4. The induction of several gene expressions was validated by real-time RT-PCR.

Results: Extensive changes in gene expression of N. fowleri during the interaction with the human neuroblastoma SK-N-MC in vitro were detected. One hundred twenty clones were obtained. Among these, five clones containing DNA sequence homologue to known genes were identified. These genes included acetyl Co-A synthetase, 18s ribosome RNA, naegleria pore B precursor, isocitrate dehydrogenase, and pyruvate kinase. Real-time quantitative RT-PCR indicated that the expression level of all five genes was up-regulated within 1 hour after exposure. The expression level of acetyl Co-A synthetase increased and reached 7-times significantly greater than that of the control while pyruvate kinase, isocitrate dehydrogenase, naegleria pore B precursor, and 18s ribosome RNA transcripts increased from 2- to 4-fold, respectively.

Conclusions: A defined set of genes in N. fowleri that was differentially transcribed after contacting with the SKN- MC monolayer cells was identified. The transcription profiles unique for amoebic cell may help elucidate the transcriptional framework of N. fowleri pathogenicity and serve as a basis for identifying transcriptional virulence factors.