Transcriptional Silencing of Multiple Genes in Trophozoites of Entamoeba histolytica

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.

Entamoeba mitosomes play an important role in encystation by association with cholesteryl sulfate synthesis

“…Each of the 10 SULT genes was knocked down in separate E. histolytica transformants by antisense small RNA-mediated transcriptional gene silencing (5,13), and these transformants were designated "SULT1-10gs." Quantitative RT-PCR (qRT-PCR) verified a 92.8-99.9% reduction of the steady-state transcript level of each target gene (Fig.…”

“…Plasmids for gene silencing were constructed by PCR amplification of 380-to 450-bp fragments corresponding to the 5′ end of the ORF of E. histolytica SULT genes with appropriate primer sets (Table S4) (5,13). Amplicons then were digested with StuI and SacI and were inserted into the corresponding sites of the psAP-2-gunma plasmid (5).…”

tRNA Gene Sequences Are Required for Transcriptional Silencing in Entamoeba histolytica

“…Whether SINE can indeed mechanistically underlie silencing is under debate. Recently, trans inactivation of further genes in E. histolytica was achieved but only in cells in which AP-A was already silenced (4,6,32). Here, we report for the first time that AP-A expression can be reconstituted in AP-A-deficient amoebae, opening a way toward silencing of genes of interest without additional AP-A deficiencies.…”

“…So far, various attempts to identify strain-specific differences have been made. These include PCR-based strain typing and sequencing of the AP-A locus and the tRNA array as well as microarray analyses using panels of selected amoeba genes (4,22,38) Future comparative genome sequencing may help to decipher the molecular differences between the strains, thus providing further insights into the mechanism of gene silencing in E. histolytica.…”

“…This is in contrast to a study in which reconstitution of AP-A expression could not be achieved by episomal transfection (5), likely due to differences in the transfection vectors. Irrespectively, reexpression of genes by episomal transfection is a crucial advancement toward more sophisticated investigations of amoeba protein function, cell biology, and pathogenicity, as targeted silencing of genes of interest currently can be achieved only in amoebae in which AP-A is additionally silenced (4,6,22). However, AP-A-deficient cells are highly impaired in pathogenicity and therefore not suitable for the investigation of other pathogenicity factors.…”