Transcriptional silencing of the gene coding for amoebapore A (AP-A) was observed when trophozoites of Entamoeba histolytica were transfected with a hybrid plasmid construct containing the ap-a gene flanked by the upstream and downstream segments of the original Ehap-a gene. Transfectants were totally devoid of ap-a transcript and AP-A protein. An identical silencing effect was observed upon transfection with a plasmid that contained only the 5 upstream region of ap-a. Removal of the selecting antibiotic enabled the isolation of plasmidless clones, which retained in their progeny the silenced phenotype. E. histolytica cells were able to overexpress ap-a when transfected with a plasmid containing the gene flanked by the 5 and 3 regions of the EhRP-L21 gene. This plasmid, however, could not express ap-a in the retransfected, cloned trophozoites lacking AP-A. This is the first report of gene silencing in E. histolytica, and the mechanism appears to belong to transcriptional gene silencing and not to posttranscriptional gene silencing. This conclusion is based on the following results: (i) silencing was achieved by transfection of homologous 5 flanking sequences (470 bp of the Ehap-a gene), (ii) transcription initiation of Ehap-a was found to be blocked, and (iii) short double-stranded RNA fragments of the ap-a coding and noncoding sequences were not detected. Trophozoites lacking AP-A are nonpathogenic and impaired in their bacteriolytic capability.The amoebapores (AP) are an important virulence factor of Entamoeba histolytica (12,30). Three isoforms of the small (77 amino acid) AP protein exist as mature and potentially active peptides inside distinct cytoplasmic granules of the trophozoite (30-32). The present view of the mode of action of AP is that following lectin-mediated recognition and intimate adherence between the trophozoite and its target cell, the AP molecules are inserted into the membranes of the latter without depending on the interaction with a specific membrane receptor and that antibodies against AP are thus unable to inhibit its toxic effect. To investigate the specific role of AP-A, the most abundant among the three isoforms (38), in the pathogenicity of the parasite, the levels of AP-A expression were modulated by transfection of trophozoites with different hybrid plasmid constructs. Down-regulation (60%) of expression of AP-A by antisense mRNA caused a drastic reduction in amoeba pathogenicity and clearly demonstrated its importance in the parasite's virulence (12). Interestingly, overexpression (fourfold) of AP-A also caused a dramatic reduction in virulence (13). This result has been attributed to an observed spillover of AP-A from the granules into the cytoplasm and a continued release of AP-A by viable trophozoites into the surrounding medium. In an attempt to overcome the problem of the apparent mislocalization of the overexpressed AP-A, we prepared another hybrid plasmid construct in which the Ehap-a gene was inserted into the vector, flanked by its original 5Ј and 3Ј regulatory element...
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.
Transgene homology-dependent silencing of gene expression is a well-recognized phenomenon that has been studied in fungi, plants, and animals (6,10,12,20,27,38). Gene-silencing mechanisms that are based on the recognition of nucleic acid sequence homology have been reported to occur via two diverse strategies: inactivation at the transcriptional level (TGS) or at the posttranscriptional level (PTGS) (26,29). Gene silencing is usually accompanied by epigenetic changes such as DNA methylation, as well as chromatin structure changes via modifications of the amino-terminal tails of core histones by acetylation, methylation, phosphorylation, ADP-ribosylation, ubiquitination, or ␣-sumoylation (33, 35). Several studies have recently shown that the transcriptional activity of some nonlong-terminal-repeat (non-LTR) transposable elements is sensitive to the presence of homologous transgenes, suggesting the involvement of homology-dependent gene-silencing mechanisms in their regulation (21,22,31). It has also been shown that transcriptional activation of retrotransposons can alter the expression of adjacent genes in wheat as well as in mice (23, 41). Primitive amitochondrial eukaryotic organisms such as Giardia and Entamoeba have been shown to harbor non-LTRs which are either long interspersed elements (LINEs) or short interspersed elements (SINEs) (7,25,36). These SINE1-like elements, also termed IE/Ehapt2 (11, 42), are noncoding retroposons that are widely dispersed in the Entamoeba histolytica genome and are abundantly transcribed. Their function is not yet known. Recent findings suggest that LINE-encoded enzymes may play a role in SINE mobilization (25).The starting point of our work was the observation that transfection of E. histolytica trophozoites with a plasmid containing a genomic copy of the amoebapore gene (ap-a), including its upstream and downstream regulating elements, caused, instead of overexpression, a complete suppression of transcription of both the episomal and chromosomal genes (9). Nuclear run-on experiments revealed that gene silencing was at the transcription initiation level (TGS). Furthermore, removal of the plasmid from silenced trophozoites and cultivation of cloned, plasmid-less trophozoites (termed G3) in the presence of inhibitors of DNA methyltransferases such as 5-azacytidine (39) and zebularine or the histone deacetylase inhibitor trichostatin A (32) or butyrate failed to restore the expression of the ap-a gene (9). These observations prompted us to analyze the sequences present in the plasmid construct which triggered the silencing phenomenon. The analysis revealed that the 5Ј flanking segment (473 bp) of the ap-a gene that was used in the plasmid construct included 140 bp of a neighboring SINE1 which is transcribed in the opposite orientation, and these were preceded by a unique T-rich stretch of 48 bp. Our present findings indicate that the transfection of a plasmid containing a truncated SINE1 and the T-rich region upstream of the 5Ј regulating element of the ap-a gene enabled the sup...
SummaryAmoebapores have been proposed to be a major pathogenicity factor of the protozoan parasite Entamoeba histolytica, which is responsible for the killing of target cells. These 77-residue peptides are structural and functional analogues of NK-lysin and granulysin of porcine and human cytotoxic lymphocytes. Inhibition of amoebapore gene expression in amoebae was obtained following transfection with a hybrid plasmid construct (pAP-R2) containing the Neo resistance gene and the gene coding for amoebapore A, including its 58 and 38 untranslated region (UTR) sequences, in reverse orientation under a promoter (g34) taken from one of the E. histolytica ribosomal protein (RP-L21) gene copies. Transfectants of virulent E. histolytica strain HM-1:IMSS, in which the expression of amoebapore was inhibited by , 60%, were signi®cantly less pathogenic. Cytopathic and cytolytic activities of viable trophozoites against mammalian nucleated cells, as well as lysis of red blood cells, were markedly inhibited. Moreover, trophozoite extracts of pAP-R2 transfectant displayed lower pore-forming activity and were less potent in inhibiting bacterial growth compared with controls. Notably, liver abscess formation in hamsters by the pAP-R2 transfectant was substantially impaired. These results demonstrate for the ®rst time that amoebapore is one of the pathogenicity factors by which trophozoites of E. histolytica exert their remarkable cytolytic and tissue destructive activity.
Guinea pig colonic epithelial cells released by treating sections of the colon with solutions containing EDTA, dithiothreitol, and citrate avidly adhered Shigella flexneri bacteria. Separation of the intestinal cells from nonbound bacteria was achieved by differential sedimentation on a Percoll gradient. Adherence of S. flexneri 3a was obtained from E. Romanowska, Po-land. Shigella strains were grown in Luria broth supplemented with 5 mM CaCl2 and 0.2% glucose. Cells were stored at -70°C as described (10). Their virulence was repeatedly tested by the guinea pig eye inoculation technique (21). One of the S. flexneri (lb) strains was used throughout these experiments. E. coli strains were grown in 1% peptone (Difco Laboratories, Detroit, Mich.) 0.5% yeast extract, and 0.5% NaCl.Preparation of radiolabeled bacteria. S. flexneri cells were inoculated in Luria broth supplemented with 0.5% glucose, 5 mM CaCl2, and 0.1 mCi of ["4C]glucose per ml (239 mCi/mmol; The Radiochemical Centre, Amersham, England) and incubated at 37°C overnight. The bacteria were harvested by centrifuga-1110 on August 1, 2020 by guest http://iai.asm.org/ Downloaded from on August 1, 2020 by guest http://iai.asm.org/ Downloaded from
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