The human intestinal pathogen Entamoeba histolytica has a number of virulence factors which can cause damage to the host. Transcriptional silencing of the gene coding for one of its major toxic molecules, the amoebapore (Ehap-a), occurred following the transfection of amoebic trophozoites with a plasmid containing the 5' promoter region of Ehap-a as well as a truncated segment of a neighboring, upstream SINE1 element that is transcribed from the opposite strand. Silencing was dependent on the presence of the truncated SINE1 sequences. Small amounts of short (approximately 140 n), ssRNA molecules with homology to SINE1 were detected in the silenced amoeba but no siRNA. The silenced Ehap-a gene domain had a chromatin modification indicating transcriptional inactivation without any DNA methylation. Removal of the plasmid did not restore transcription of Ehap-a. Transcription analysis by microarrays revealed that a number of additional genes were silenced and some were also up-regulated. Transfections of amoeba which already had a silenced Ehap-a, with a plasmid containing a second gene ligated to the 5' upstream region of Ehap-a, enabled the silencing, in-trans, of other genes of choice. The nonvirulent phenotype of the gene-silenced amoeba was demonstrated in various assays and the results suggest that they may have a potential use for vaccination.
Copyright 2008 IUBMB