Transcriptional Silencing of Multiple Genes in Trophozoites of Entamoeba histolytica

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Abstract
In a previous work we described the transcriptional silencing of the amoebapore A (AP-A) gene (Ehap-a) of Entamoeba histolytica strain HM-1:IMSS. The silencing occurred following transfection with a plasmid containing a 5′ upstream region (473 bp) of Ehap-a that included a truncated segment (140 bp) of a short interspersed nuclear element (SINE1). Silencing remained in effect even after removal of the plasmid (clone G3). Neither short interfering RNA nor methylated DNA were detected, but the chromatin domain of Ehap-a in the gene-silenced trophozoites was modified. Two other similar genes (Ehap-b and one encoding a Saposin-like protein, SAPLIP 1) also became silenced. In the present work we demonstrate the silencing of a second gene of choice, one that encodes the light subunit of the Gal/GalNAc inhibitable lectin (Ehlgl1) and the other, the cysteine proteinase 5 (EhCP-5). This silencing occurred in G3 trophozoites transfected with a plasmid in which the 473 bp 5′ upstream Ehap-a fragment was directly ligated to the second gene. Transcriptional silencing occurred in both the transgene and the chromosomal gene. SINE1 sequences were essential, as was a direct connection between the Ehap-a upstream region and the beginning of the open reading frame of the second gene. Gene silencing did not occur in strain HM-1:IMSS with any of these plasmid constructs. The trophozoites with two silenced genes were virulence-attenuated as were those of clone G3. In addition, trophozoites not expressing Lgl1 and AP-A proteins had a significantly reduced ability to cap the Gal/GalNAc-lectin to the uroid region when incubated with antibodies against the heavy (170 kDa) subunit of the lectin. Lysates of trophozoites lacking cysteine proteinase 5 and AP-A proteins had 30% less cysteine proteinase activity than those of HM-1:IMSS strain or the G3 clone. Silencing of other genes in G3 amoebae could provide a model to study their various functions. In addition, double gene-silenced, virulence-attenuated trophozoites may be an important tool in vaccine development. The human intestinal parasite Entamoeba histolytica has numerous genes that code for virulence. Silencing the expression of individual genes is useful to determine their roles. In previous work the authors demonstrated the silencing of the gene coding for amoebapore, which is responsible for killing of human cells. They transfected amoebic trophozoites with a plasmid that contained DNA sequences homologous to the promoter region of the amoebapore gene, as well as a portion of a repetitive DNA element (called a short interspersed nuclear element). This construct induced a modification of the chromatin and prevented the expression of the gene. Removal of the plasmid resulted in stable, amoebapore-deficient parasites possessing low virulence. In the present work, Bracha and colleagues show silencing of additional genes following transfection of E. histolytica trophozoites already silenced in amoebapore with a plasmid containing the second gene directly ligated to the upstream region of the amoebapore gene. The DNA sequences that are essential for transferring the silencing from the plasmid to the chromosomal gene copy were identified. Additional virulence genes that the authors irreversibly silenced are those encoding a subunit of a surface lectin that mediates the adherence of the parasite to host cells, and a cysteine proteinase that plays a role in inflammation and invasion of the intestine.