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.

Figure 1. Changes in the Transcription of Other Amoebapore-Like Genes in G3 Trophozoites as Determined by RT-PCR

(A) Total RNA was prepared from freshly harvested HM1-IMSS and G3 trophozoites. The RNA was treated with RNase-free DNase, and reversed transcribed using oligo dT-adaptor primer (Table 1, primer I). PCR was then performed using a sense primer from the gene of interest (Table 1) and the adaptor primer (Table 1, primer II) as antisense for all the genes. Lanes: 1, EhRPL21; 2, Ehap-a; 3, Ehap-b; 4, Ehap-c; 5, SAPLIP 1; 6, SAPLIP 5; 7, SAPLIP 14. (B) Sequence comparisons between the three genes that were silenced: I, Ehap-a; II, Ehap-b; III, SAPLIP 1.

Figure 2. Silencing of the Ehlgl1 Gene

(A) Diagrams of the Ehlgl1-containing plasmids pB33 and pAY. The 473 bp Ehap-a promoter, SINE and T-rich (Tr) regions are marked. Plasmid pAY includes 44 bp of the signal peptide (sp) of the Ehap-a gene. Both plasmids contain the ORF of Ehlgl1 gene and the 3′ regulatory sequence from the Ehactin gene. (B) Northern blot analysis of amoebic RNA extracts. Lanes: 1, untransfected HM1:IMSS; 2, HM-1:IMSS transfected with plasmid pB33; 3, HM-1:IMSS transfected with pAY; 4, untransfected clone G3; 5, clone G3 transfected with pB33; 6, clone G3 transfected with pAY. Blots were probed as indicated. (C) Western blot analysis of protein lysates separated on SDS-PAGE (12%) showing the blot that reacted with the polyclonal antibodies against the Lgl1 protein. Lanes 1–6 are as (B). Lane 7 contains lysate from the plasmidless RBV trophozoites (see Results).

Figure 3. Identification of the Source of Ehlgl1 Transcripts

(A) Diagrams of the location of the two different sense primers used to differentiate between the two types of Ehlgl1 transcripts (genomic and plasmid-derived). (B) RT-PCR analysis of different transfectants. Isolated RNA was subjected to reverse transcription and PCR for several types of transcripts: genomic Ehlgl1 (Table 1, primers XI + XIII), Ehlgl1 derived from plasmid (primers X + XIII), Ehap-a (primers II + III), and EhRP-L21 (primers II + IX). Lanes: 1, HM-1:IMSS transfected with pB33; 2, HM-1:IMSS transfected with pAY; 3, G3 transfected with pB33; 4, G3 transfected with pAY; 5, untransfected HM-1:IMSS; 6, untransfected G3.

Figure 4. Induction of Capping of the Gal/GalNAc-Lectin to the Uroid Region of the Trophozoites

Confocal microscopy of trophozoites; left photomicrographs, trophozoites incubated at 4 °C; right photomicrographs, trophozoites were incubated at 37 °C for 20 min to observe the induction of capping using two monoclonal antibodies against the heavy subunit of the Gal-lectin [35]. Each image shows the fluorescent Gal-lectin superimposed on a Nomarsky section. Samples are HM-1:IMSS, G3, and RBV (the plasmidless strain silenced in Ehlgl1 and Ehap-a).

Figure 5. Silencing of Cysteine Proteinase 5 (EhCP5)

(A) Diagram of the pAP-CP5 construct. (B) Northern blot analysis. Lanes: 1, RNA from untransfected HM1:IMSS; 2, RNA from untransfected G3; 3, RNA from G3 transfected with pAP-CP5 (probes used are as indicated).

Figure 6. In Vivo Labeling of Cysteine Proteinases

The different trophozoite cultures were grown for 18 h with the radiolabeled cysteine proteinase inhibitor Fmoc-[I¹²⁵]Tyr-Ala-diazomethylketone (10 μg/ml, 10 μCi/ml), harvested, washed, and lysed [49]. Samples of each cell lysate (25 μg) were separated on a 12% acrylamide reducing gel and exposed to X-ray film to reveal the different cysteine proteinase bands. Samples in the lanes on the left radiograph are: 1, untransfected HM-1:IMSS; 2, untransfected G3; 3, G3 transfected with pAP-CP5 and grown with 50 μg/ml of G418; 4, RB8 trophozoites after removal of the plasmid (see Results); 5, HM-1:IMSS grown with a lower specific activity inhibitor (0.05 μCi/μg). Samples on the right radiograph demonstrate the CP-5 band location in HM-1:IMSS and its absence from the E. dispar culture [50].

Figure 7. Determination of Transcripts of the Three Different Ehlgl Genes by RT PCR

Total RNA was prepared from freshly harvested G3 and RBV trophozoites. PCR was performed using antisense conserved primer for Ehlgl1, Ehlgl2, and Ehlgl3 (Table 1, primer XV) and a specific sense primer for each of the genes as indicated. Lanes: 1, primer specific for Ehlgl1 (Table 1, primer XI); 2, primer specific for Ehlgl2 (Table 1, primer XVI); 3, primer specific for Ehlgl3 (Table 1, primer XVII); 4, primers specific for EhRPL21 (Table 1 primers II + IX).

Figure 8. Additional Plasmid Constructs Tested for Their Silencing Capabilities

(A) Diagrams of plasmid pSG in which the truncated SINE 1 and the T-rich sequences were ligated upstream to the Ehlgl1 promoter and ORF regions. Plasmid pP10 has the SINE1 sequences removed from the Ehap-a promoter region. Plasmid pTL contains a truncated segment of the Ehlgl1 gene without the 3′ regulatory region. Plasmid constructs can be compared to plasmid pB33 in Figure 2. (B) Northern blots of RNA extracts of trophozoite transfectants. Probes used as indicated. Lanes: 1, untransfected HM-1:IMSS; 2, HM-1:IMSS transfected with pSG; 3, HM-1:IMSS transfected with pP10; 4, G3; 5, G3 transfected with pSG; 6, G3 transfected with pP10. (C) Dot blot of RNA extracts from G3 trophozoites transfected with plasmid pTL and hybridized with probes for either EhRP-L21 or Ehlgl1.