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The human intestinal parasite Entamoeba histolytica causes amoebic colitis and amoebic liver abscesses. Three classes of amoebic molecules have been identified as the major virulence factors, the Gal/GalNAc inhibitable lectin that mediates adherence to mammalian cells, the amoebapores which cause the formation of membrane ion channels in the target cells and the cysteine proteinases which degrade the matrix proteins, the intestinal mucus and secretory IgA. Transcriptional silencing of the amoebapore (Ehap-a) gene occurred after transfection of trophozoites with a plasmid containing a segment of the 5’ upstream region of the gene. Transcriptional silencing of the Ehap-a gene continued even after the removal of the plasmid and the cloned amoebae were termed G3. Transfection of G3 trophozoites with a plasmid construct containing the cysteine proteinase (EhCP-5) gene and the light subunit of the Gal- lectin (Ehlgl1) gene, each under the 5’ upstream sequences of the amoebapore gene, caused the simultaneous epigenetic silencing of expression of these two genes. The resulting trophozoites, termed RB-9, were cured from the plasmid and they do not express the three types of virulent genes. The RB-9 amoeba are virulence attenuated and are incapable of killing mammalian cells, they can not induce the formation of liver abscesses and they do not cause ulcerations in the cecum of experimental animals. The gene-silenced amoebae express the same surface antigens which are present in virulent strains and following intra peritoneal inoculation of live trophozoites into hamsters they evoked a protective immune response. Further studies are needed to find out if RB-9 trophozoites could be used for vaccination against amoebaisis.

Entamoeba histolytica is a deep-branching eukaryotic pathogen. Rhomboid proteases are intramembrane serine proteases, which cleave transmembrane proteins in, or in close proximity to, their transmembrane domain. We have previously shown that E. histolytica contains a single functional rhomboid protease (EhROM1) and has unique substrate specificity. EhROM1 is present on the trophozoite surface and relocalizes to internal vesicles during erythrophagocytosis and to the base of the cap during surface receptor capping. In order to further examine the biological function of EhROM1 we downregulated EhROM1 expression by >95% by utilizing the epigenetic silencing mechanism of the G3 parasite strain. Despite the observation that EhROM1 relocalized to the cap during surface receptor capping, EhROM1 knockdown [ROM(KD)] parasites had no gross changes in cap formation or complement resistance. However, ROM(KD) parasites demonstrated decreased host cell adhesion, a result recapitulated by treatment of wild-type parasites with DCI, a serine protease inhibitor with activity against rhomboid proteases. The reduced adhesion phenotype of ROM(KD) parasites was noted exclusively with healthy cells, and not with apoptotic cells. Additionally, ROM(KD) parasites had decreased phagocytic ability with reduced ingestion of healthy cells, apoptotic cells, and rice starch. Decreased phagocytic ability is thus independent of the reduced adhesion phenotype, since phagocytosis of apoptotic cells was reduced despite normal adhesion levels. The defect in host cell adhesion was not explained by altered expression or localization of the heavy subunit of the Gal/GalNAc surface lectin. These results suggest no significant role of EhROM1 in complement resistance but unexpected roles in parasite adhesion and phagocytosis.

We have previously discovered a unique mechanism of epigenetic transcriptional gene silencing in the Entamoeba histolytica trophozoites of strain HM-1:IMSS that resulted in the persistent downregulation of the amoebapore A (ap-a) gene, and that could be successfully applied to silence other virulence genes (cpA5, lgl1). In order to understand how the silencing is maintained throughout generations, we analysed whether modifications occurred at the chromatin level. Chromatin immunoprecipitation assays were done with antibodies specific to the methylated lysine 4 of E. histolytica histone H3. When the genes were in a transcriptionally silent state, the methylation levels of H3K4 in their coding region were significantly reduced. In contrast, the levels of core histone H3 were consistently higher in the silenced genes. Controlled chromatin digestion with micrococcal nuclease was used to assess changes in nucleosome compaction. We found a significant resistance to digestion in the promoter region of the silenced ap-a and cpA5 genes as compared to the parental strain that expresses those genes. Our data lend further support to the idea that histone modifications and heterochromatin formations are at the basis of the transcriptional silencing of genes in E. histolytica.

Amoebiasis (a human intestinal infection affecting 50 million people every year) is caused by the protozoan parasite Entamoeba histolytica. To study the molecular mechanisms underlying human colon invasion by E. histolytica, we have set up an ex vivo human colon model to study the early steps in amoebiasis. Using scanning electron microscopy and histological analyses, we have established that E. histolytica caused the removal of the protective mucus coat during the first two hours of incubation, detached the enterocytes, and then penetrated into the lamina propria by following the crypts of Lieberkühn. Significant cell lysis (determined by the release of lactodehydrogenase) and inflammation (marked by the secretion of pro-inflammatory molecules such as interleukin 1 beta, interferon gamma, interleukin 6, interleukin 8 and tumour necrosis factor) were detected after four hours of incubation. Entamoeba dispar (a closely related non-pathogenic amoeba that also colonizes the human colon) was unable to invade colonic mucosa, lyse cells or induce an inflammatory response. We also examined the behaviour of trophozoites in which genes coding for known virulent factors (such as amoebapores, the Gal/GalNAc lectin and the cysteine protease 5 (CP-A5), which have major roles in cell death, adhesion (to target cells or mucus) and mucus degradation, respectively) were silenced, together with the corresponding tissue responses. Our data revealed that the signalling via the heavy chain Hgl2 or via the light chain Lgl1 of the Gal/GalNAc lectin is not essential to penetrate the human colonic mucosa. In addition, our study demonstrates that E. histolytica silenced for CP-A5 does not penetrate the colonic lamina propria and does not induce the host's pro-inflammatory cytokine secretion.

The human intestinal pathogen Entamoeba histolytica has a number of virulence factors which can cause damage to the host. Transcriptional silencing of the gene coding for one of its major toxic molecules, the amoebapore (Ehap-a), occurred following the transfection of amoebic trophozoites with a plasmid containing the 5' promoter region of Ehap-a as well as a truncated segment of a neighboring, upstream SINE1 element that is transcribed from the opposite strand. Silencing was dependent on the presence of the truncated SINE1 sequences. Small amounts of short (approximately 140 n), ssRNA molecules with homology to SINE1 were detected in the silenced amoeba but no siRNA. The silenced Ehap-a gene domain had a chromatin modification indicating transcriptional inactivation without any DNA methylation. Removal of the plasmid did not restore transcription of Ehap-a. Transcription analysis by microarrays revealed that a number of additional genes were silenced and some were also up-regulated. Transfections of amoeba which already had a silenced Ehap-a, with a plasmid containing a second gene ligated to the 5' upstream region of Ehap-a, enabled the silencing, in-trans, of other genes of choice. The nonvirulent phenotype of the gene-silenced amoeba was demonstrated in various assays and the results suggest that they may have a potential use for vaccination., (Copyright 2008 IUBMB)

The Entamoeba histolytica cell surface Gal/GalNAc-inhibitable lectin is a heterodimer between a heavy (170 kDa) subunit linked via a disulfide bond to a light (31 to 35 kDa) subunit. Five light subunit genes with high homology have been identified (Ehlgl1 to -5). We have previously shown that silencing of the expression of Ehlgl1, in the G3 trophozoites which had already been silenced in the amoebapore gene (Ehap-a), also suppressed the transcription of Ehlgl2 and -3 (strain RBV). The total absence of the lgl1 to -3 subunits in the RBV trophozoites affected their ability to cap the surface Gal-lectin molecules to the uroid region. We have now found that in the RBV trophozoites, the lgl4 and -5 subunits (31 kDa) are overexpressed and appear to compensate for the missing lgl1 to -3 in the heterodimer complex. Transcriptional silencing of Ehlgl5 was achieved by transfection of G3 trophozoites with a plasmid containing the open reading frame of Ehlgl5 ligated to the 5' promoter region of the Ehap-a gene. The transfected trophozoites (strain L5) were silenced in Ehlgl5 and the closely related Ehlgl4, while the expression of the larger lgl1 to -3 subunits was upregulated. L5 trophozoites retained their ability to cap the Gal-lectin molecules. Attempts to simultaneously silence all of the Ehlgl genes have failed so far, possibly due to their crucial importance to the Gal-lectin functions. Our ability to silence part of the genes belonging to the same family can serve as a tool to study the relationships and functions of the members of other gene families.

The protozoan Entamoeba histolytica causes intestinal inflammation and liver abscess. Cysteine proteinases (CPs) have been proposed as important virulence factors for amoebiasis. To test the role of the various CPs for amoeba induced pathology, the three major enzymes of the parasite, namely EhCP1, EhCP2 and EhCP5 accounting for about 90% of total proteinase activity, were overexpressed by stable episomal transfection. Total CP activity of recombinant amoebae increased by three- to six-fold depending on the gene transfected. Interestingly, overexpression of the genes for EhCP1 or EhCP2 increased the activity of the corresponding enzyme only, whereas overexpression of the gene for EhCP5 increased the activity of all three enzymes, which is consistent with enzyme-converting activity of EhCP5. Cytopathic activity, measured by in vitro monolayer disruption, was dramatically increased in ehcp5-transfectants (five-fold) but showed only a modest increase in ehcp1- or ehcp2-transfectants (1.5-2-fold). In addition, overexpression of ehcp5 but not of ehcp1 or ehcp2 significantly increased amoebic liver abscess formation in laboratory animals. Moreover, transfection and overexpression of ehcp5 was able to compensate the reduction of in vivo pathogenicity in parasites, which have been silenced for the gene encoding the pore-forming protein amoebapore A. In summary, these results further support the important role of EhCP5 in E. histolytica pathogenicity.

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.

Transcriptional silencing of an amebapore (ap-a) gene occurred in Entamoeba histolytica following the transfection of plasmids containing a DNA segment (473 bp) homologous to the 5' upstream region of the gene. This segment contains the promoter region of the ap-a gene, a T-rich stretch, followed by a truncated SINE1 (short interspersed element) that is transcribed from the opposite strand. The downstream silencing of the ap-a gene did not occur with plasmids containing the entire SINE1 sequence or lacking the entire SINE1 sequences including the T-rich stretch. Such plasmids promoted the overexpression of the ap-a gene. The transcription of the SINE element required both the T-rich stretch as well as sequences from the 5' end of SINE. RNA extracts from gene-silenced cultures showed small amounts of short (approximately 140 nt), single-stranded molecules with homology to SINE1 transcripts but no siRNA. Chromatin immunoprecipitation (ChIP) analysis of silenced G3 trophozoites with an antibody against methylated K4 of histone H3 revealed a demethylation of K4 at the domain of the ap-a gene indicating transcriptional inactivation. These results suggest the involvement of the SINE1 element in triggering the gene silencing and the role of histone modification in its epigenetic maintenance. The avirulent phenotype of the silenced trophozoites was demonstrated in various assays and the results suggest they may have a potential use for vaccination.

Transcriptional silencing of an amoebapore (ap-a) gene occurred in Entamoeba histolytica following the transfection of plasmids containing a DNA segment (473 bp) homologous to the 5' upstream region of the gene (R. Bracha, Y. Nuchamowitz, and D. Mirelman, Eukaryot. Cell 2:295-305, 2003). This segment contains the promoter region of the ap-a gene, a T-rich stretch, followed by a truncated SINE1 (short interspersed element 1) that is transcribed from the antisense strand. Transfection of plasmids containing truncated SINE1 sequences which lack their 3' regulatory elements upstream of the ap-a gene was essential for the downstream silencing of the ap-a gene while transfection with plasmids containing the entire SINE1 sequence or without the T-rich stretch promoted the overexpression of the ap-a gene. Both the T-rich stretch and sequences of the 5' SINE1 were essential for the transcription of SINE1. RNA extracts from gene-silenced cultures showed small amounts of short (approximately 140-nucleotide), single-stranded molecules with homology to SINE1 but no short interfering RNA. Chromatin immunoprecipitation analysis with an antibody against methylated K4 of histone H3 showed a demethylation of K4 at the domain of the ap-a gene, indicating transcriptional inactivation. These results suggest the involvement of SINE1 in triggering the gene silencing and the role of histone modification in its epigenetic maintenance.

Entamoeba histolytica trophozoites produce amoebapores, a family of small amphipathic peptides capable of insertion into bacterial or eukaryotic membranes and causing cellular lysis. Recently, E. histolytica trophozoites that are totally deficient in the production of amoebapore-A were created through a gene silencing mechanism (R. Bracha, Y. Nuchamowitz, and D. Mirelman, Eukaryot. Cell 2:295-305, 2003). Here we tested the virulence of amoebapore A(-) trophozoites in models of the two major forms of amebic disease: amebic liver abscess and amebic colitis. We demonstrate that amoebapore expression is required for full virulence in the SCID mouse model of amebic liver abscess, but E. histolytica trophozoites that do not express amoebapore-A can still cause inflammation and tissue damage in infected human colonic xenografts. These data are consistent with the concept that tissue damage may proceed by different mechanisms in amebic liver abscess compared to amebic colitis.

Entamoeba histolytica, the protozoan parasite which causes amoebiasis, is an exclusively human pathogen so developing a vaccine could effectively impact the spread of the disease. Recently we developed a genetically modified avirulent strain, termed G3, from the virulent E. histolytica strain HM-1:IMSS. The new strain lacks the important virulence factor, the amoebapore-A. The objective of our current study was to investigate the avirulence of the attenuated strain as well as to examine the antigenic and immunogenic responses of these trophozoites as potential candidates for a live vaccine. Functional assays were conducted to characterise the virulent behaviour of the G3 strain. This behaviour was compared to the virulent strain HM-1:IMSS and the non-virulent strain Rahman. Western blots were conducted to confirm the lack of amoebapore-A in the E. histolytica G3 strain and to demonstrate that it had no influence on the presence of other virulence factors. Results of these two sets of tests proved the G3 strain to be phenotypically similar to the avirulent Rahman strain while antigenically identical to the virulent HM-1:IMSS, apart from the lack of the amoebapore-A protein. Intraperitoneal immunisation of hamsters with G3 trophozoites compared to sham immunised hamsters resulted in IgG anti-HM-1:IMSS antibodies. The level of humoral response was variable and further testing has to take place before introducing this new strain as a vaccine.

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.

Purpose: To evaluate the outcome of paediatric rhegmatogenous retinal detachment treated by segmental scleral buckling plus an encircling element., Methods: A retrospective review was carried out of 15 consecutive paediatric patients (16 eyes)., Results: Thirteen of the 15 patients were male (87%), 2 (13%) were female. Their average age was 10.9 years (range 6-18 years; median 10 years). Retinal detachment was caused by trauma in 9 of 16 eyes (56%), high myopia in 5 of 16 eyes (31%) and had an unknown cause in 2 of 16 eyes (13%). Diagnosis was delayed by more than 1 month in 10 of 16 eyes (63%). The visual acuity was 6/60 or worse and the macula was detached on presentation in 13 of 16 eyes (81%). Two of 16 eyes (13%) had mild proliferative vitreoretinopathy. Final reattachment was achieved in all cases. Of 11 eyes with a follow-up of 6 months or more, there was improvement in visual acuity in 7 (63%), and a best-corrected visual acuity of 6/20 or better in 5 (46%)., Conclusion: Paediatric rhegmatogenous retinal detachment is characterised by a delay in diagnosis and a high degree of macular involvement on presentation. Anatomical reattachment with segmental scleral buckling plus an encircling element was successful in all eyes, and improvement of visual acuity was achieved in one-half of the eyes which had a follow-up of 6 months or more.

Amoebapores 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 5' and 3' 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 approximately 60%, were significantly 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 first time that amoebapore is one of the pathogenicity factors by which trophozoites of E. histolytica exert their remarkable cytolytic and tissue destructive activity.