Transcriptional Silencing of an Amoebapore Gene inEntamoeba histolytica: Molecular Analysis and Effect on Pathogenicity

Abstract

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...

Identification of an Entamoeba histolytica Serine-, Threonine-, and Isoleucine-Rich Protein with Roles in Adhesion and Cytotoxicity

“…Transcriptional gene silencing has been previously documented in E. histolytica. Bracha et al (11) showed that upon transfection with a plasmid containing the amoebapore gene and a portion of the short interspersed nuclear element EhSINE1, both chromosomal and episomal copies of the amoebapore gene were permanently silenced, and this silencing was stable even after removal of the plasmid (2). This permanent and stable epigenetic silencing mechanism is not completely understood, and whether a similar mechanism is functional in E. histolytica Rahman is not currently known.…”

“…The cytotoxicity data are a significant finding, as the relative difficulty in manipulating gene expression in E. histolytica has limited the ability to associate particular genes with pathogenicity. To date, only a few amebic genes have been genetically shown to be involved in cytotoxicity (3,11,16,25,29,43,47).…”

Solution Structure of the Pore-forming Protein of Entamoeba histolytica

“…The protozoon discharges its effector proteins in the acidic environment of phagolysosomes in which engulfed bacteria are situated (40). Persuasive support for the involvement of amoebapores in extracellular killing of host cells comes from the finding that amoebapores are cytolytic toward eukaryotic cells (41,42) and that amoebae lacking the major form amoebapore A are no longer capable of killing mammalian cells or destroying host tissues in vitro (7). For the scenario of the cytolytic reaction, it may be envisaged that after amoeba-target cell contact has been established, amoebapores are discharged from cytoplasmic granules into the confined environment of the contact zone in which a high protein concentration and a relatively low pH may be reached.…”

“…They are capable of lysing a broad spectrum of target cells, including human host cells and bacteria. It has been recently shown that trophozoites of E. histolytica lacking amoebapore A, due to transcriptional silencing of the encoding gene, became avirulent (7), demonstrating that this protein is a key pathogenicity factor of the parasite. All three amoebapore isoforms have been isolated and biochemically characterized, and their primary structure has been elucidated by molecular cloning of the genes of their precursors (8 -10).…”

New insights into host-pathogen interactions duringEntamoeba histolyticaliver infection

“…The resulting disturbance of the endothelial barrier likely facilitates passage of trophozoites from the blood circulation into the hepatic tissue. E. histolytica then divides and induces abscess formation [7], dependent upon the expression of virulence factors like Gal/GalNAc lectin [20], amoebapore A [21], CP-A5 [22] and the highly positively-charged lysine-and glutamic acid-rich protein KERP1 [23].…”