Epigenetic silencing of virulence genes in Entamoeba histolytica

The human intestinal protozoan parasite Entamoeba histolytica produces a number of virulence factors such as a Gal-specific adhesin , cysteine proteinases and a family of small (77 a.a) ampiphatic pore-forming peptides, termed amoebapores . The concerted action of these virulence factors causes intestinal inflamation, kills the mucosal cells and enables the invasion of the parasite . In earlier work we have shown that stable transfection of virulent amoebic trophozoites with a hybrid plasmid construct which contained the 5π upstream region (470 bp) of the amoebapore gene ( Ehap-a) caused, within a few weeks, the complete inhibition of transcription (TGS) and translation of this gene. Subsequent removal of the plasmid resulted in a stable, Ehap-a silenced, plasmidless trophozoite line (G3). Growing of the G3 silenced trophozoites in the presence of 5π Aza-Cytidine or Trichostatin A did not reverse the gene silencing. Neither DNA methylation of the promoter region of the Ehap-a gene nor siRNA molecules belonging to the Ehap-a ORF were detected in RNA extracts of G3 trophozoites.

Sequence analysis of the 470 bp 5π upstream region of the Ehap-a gene revealed the presence of 140 bp of a neighboring short, interspersed repetitive element (SINE1) which is actively transcribed from the opposite strand as evidenced by the transcription of a CAT reporter gene. Total elimination of the SINE1 sequences from the plasmid construct prevented the silencing of the Ehap-a gene . Silencing occurred only if a truncated segment of the SINE element was included in the plasmid. Nuclear RNA extracts of Ehap-a silenced trophozoites were found to have small amounts of non-productive single stranded RNA molecules of approximately 140 n with homology to the 5π end of the SINE1 element. In addition , a loss of methylation at Lysine 4 of Histone H3 was detected by ChIP analysis in the chromatin domain of the Ehap-a gene of G3 silenced trophozoites . A loss of methylation of H3-lysine 4 has been shown in numerous cases as being indicative of a transcription inactivated domain. Comparison between the transcription profiles of G3 silenced trophozoites and the parent strain revealed that two other genes of the poreforming family ( Ehap-b and a Saposin-like) with considerable homology to Ehap-a , had also become transcriptionally silenced. This prompted us to attempt to silence additional genes of choice. Plasmid constructs in which the gene coding for the light subunit of the Gal-lectin ( EhLgl1) was ligated to the 5π upstream region of the Ehap-a gene (Fig 1), were prepared and transfected into the plasmid-less G3 silenced trophozoites. Transfectants showed that gene silencing occurred in both the plasmid and genomic copies of the EhLgl1 and silencing of the EhLgl1 gene, as well as the Ehap-a genes remained in effect even after removal of the plasmid.

Fig 1A. Plasmid constructs used for silencing the expression of the EhLgl1 and EhCp5 genes by transfection of G3 amoeba, which were already silenced in the Ehap-a gene. B. Northern blots of the double silenced amoebae. Tr- T-Rich Region

The expression of two other genes EhLgl2 and EhLgl3, with high homology to EhLgl1 was also found to be suppressed. In contrast, transfectants of the parent strain were shown to over-express EhLgl1 from the plasmid construct. By a similar procedure we have now been able to silence additional genes such as the cysteine proteinase 5 (EhCP5) and EhLim. We are now trying to find out the molecular mechanism by which the genomic copy of the additional gene becomes silenced by attempting to silence genes which catalyse histone modifications.

In our previous studies we had found out that the G3 trophozoites, which lacked the amoebapore protein, have lost their ability to kill mammalian cells and to induce liver abscesses in the hamster model. The G3 trophozoites, nevertheless, were still able to cause intestinal inflamation and colitis in human colon segments engrafted in SCID-mice. The colitis and inflamation were found to be caused by the cysteine proteinases. We are now in the process of investigating whether the trophozoites silenced in both, the amoebapore and cysteine proteinase, are still capable of inducing inflamation. Our novel method to silence additional genes may be useful for the generation of virulence-attenuated trophozoites which may have the potential to serve as a live vaccine.

Fig 2 Proposed model for the spread of heterochromatin to the EhLgl1 gene in the G3 amoeba that is already transcriptionally silenced in the Ehap-a gene. shRNA - small heterochromatic ssRNA molecules

Characterization of other genes involved in the modulation of parasite virulence : Comparison by 2D-Gel of lysates from two types of amoeba, one capable of producing liver abscesses in hamsters and the other incapable , revealed a number of spots which differed their migration. Proteomic MS analysis of two of these spots showed that one of them is a Lim-like protein and the other, 20 KDa antigen. The genes coding for these two proteins have been cloned and the 2D gel differences in migration have been identified , the first as a post-translation modification and the other as a deletion of a repeat sequence. The effects silencing of the expression of EhLim and the 20 KDa antigen the G3 amoeba are under investigation.

Selected publications (from 2005)
Anbar, M., Bracha, R., Nuchamowitz, Y., Li, Y., Florentin, A. and Mirelman, D. (2005) Involvement of a SINE element in the epigenetic transcriptional silencing of the amoebapore gene in Entamoeba histolytica. Eukaryotic Cell, 4: 1775-1784

Villalobo, E., Wender, N. and Mirelman,D. (2005) Entamoeba histolytica : molecular characterization of an aldose-1-epimerase (mutarotase ). Exp. Parasitol. 110 : 298-303

Libros-Ziv, P., Villlobo, E. and Mirelman, D. (2005) Entamoeba histolytica : characterization of an N-ethylmaleimide sensitive fusion protein homologue . Exp. Parasitol. 110 : 276-279

Barrios-Ceballos, M.P., Martinez-Gallardo, N.A., Anaya-Velazquez, F., Mirelman, D. and Padilla-Vaca, F. ( 2005) A novel protease from Entamoeba histolytica homologous to members of the S28 serine proteases . Exp. Parasitol. 110, 270-275

Mirelman,D., Anbar, M., Nuchamowitz, Y. and Bracha, R. (2006) Epigenetic silencing of gene expression in Entamoeba histolytica . Arch. Med. Res. 37, 226-233

Moncada, D.M., Keller, K., Ankri, S., Mirelman, D. and Chadee, K. (2006) Antisense inhibition of Entamoeba histolytica cysteine proteinases inhibits colonic mucus degradation . Gastroenterology, 130, 721-730

Weber, C., Guigon, G., Bouchier, C., Frangeul, L., Moreira, S., Sismeiro, O., Gouyette, C., Mirelman, D., Coppee, J.Y. and Guillen, N. ( 2006) Stress by heat shock induces massive down-regulation of genes and allows differential allelic expression of the Gal/GalNAc lectin of Entamoeba histolytica. Eukaryotic Cell , In Press

Ackers J. and Mirelman D. (2006 ) Progress in research on Entamoeba histolytica pathogenesis . Curr. Opinion in Microbiol. 9, In press

Bracha, R., Nuchamowitz, Y. , Anbar, M. and Mirelman, D. (2006) Transcriptional silencing of multiple genes in trophozoites of Entamoeba histolytica. PLoS Pathogens , In press

Tillack, M., Nowak, N., Lotter, H., Bracha, R., Mirelman, D. , Tannich, E., and Bruchahus, I. ( 2006) . Increased expression of the major cysteine proteinases by stable episomal transfection underlines the important role of EhCP5 for the pathogenicity of Entamoeba histolytica. Molec. Biochem. Parasitol. In press

Acknowledgements Besen-Brender Professorial Chair in Microbiology and Parasitology. Research grants were obtained from the Pasteur-Weizman program, from the Drake Family Foundation and from Mr. Henry H. Meyer