Yael Nuchamowitz

Transcriptional Silencing of an Amoebapore Gene in Entamoeba 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.

Antisense inhibition of amoebapore expression in Entamoeba histolytica causes a decrease in amoebic virulence

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 ~ 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.

Transcriptional Silencing of Multiple Genes in Trophozoites of Entamoeba histolytica

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.

Involvement of a short interspersed element in epigenetic transcriptional silencing of the amoebapore gene in Entamoeba histolytica.

ABSTRACT 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 (∼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.

Identification of Significant Variation in the Composition of Lipophosphoglycan‐like Molecules of E. histolytica and E. dispar

The lipophosphoglycan‐like (LPG‐like) molecules of E. histolytica virulent strains are clearly distinct from those of the avirulent E. histolytica and E. dispar strains. Abundant ‘LPG’ levels are apparently limited to virulent strains, while lipophosphopeptidoglycans (‘LPPGs) are common to both virulent and avirulent strains of E. histolytica and E. dispar. It is therefore conceivable that ‘LPPG’ performs a function that is essential to survival within the host, while the ‘LPG’ performs a more specific function related to virulence.

Transcriptional Gene Silencing Reveals Two Distinct Groups of Entamoeba histolytica Gal/GalNAc-Lectin Light Subunits

ABSTRACT 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.

Biosynthesis of peptidoglycan in Pseudomonas aeruginosa. 1. The incorporation of peptidoglycan into the cell wall.

Ether-treated cells of Pseudomonas aeruginosa catalyze the formation of crosslinked peptidoglycan from the two nucleotide precursors uridinediphospho-N-acetylglucosamine and uridinediphospho-N-acetylmuramyl-L-alanyl-D-gamma-glutamyl-meso-diaminopimelyl-D-alanyl-D-alanine. The main enzymatic reactions of biosynthesis were similar to those found in Escherichia coli. Part of the reaction products were soluble in 4% sodium dodecylsulfate whereas the other part was covalently bound to the preexisting cell wall peptidoglycan sacculus. The incorporation into cell wall is carried out by a transpeptidation reaction in which the nascent peptidoglycan functions mainly as the donor and the preexisting one as acceptor. The detergent-soluble peptidoglycan is composed of partially crosslinked peptidoglycan strands as well as low-molecular-weight peptidoglycan fragments. Pulse-chase biosynthesis experiments show that the detergent-soluble peptidoglycan is an intermediate that eventually becomes covalently bound to the wall. The DD-carboxypeptidase activity of P. aeruginosa is membrane-bound and does not hydrolyse C-terminal D-alanine residues from the L-lysine-containing nucleotide-precursor analogue. An LD-carboxypeptidase was also detected in P. aeruginosa.

A homologue of the cysteine proteinase gene (ACP1 or Eh-CPp3) of pathogenic Entamoeba histolytica is present in non-pathogenic E. dispar strains

One of the three cysteine proteinase genes, ACP1 (or CP 3), has been reported to be missing in non-pathogenic strains of Entamoeba histolytica (or Entamoeba dispar as recently labeled). Unexpectedly, a gene fragment very similar in its sequence (95% homology) to ACP1 of pathogenic strains was obtained by use of the polymerase chain reaction from genomic DNA and cDNA of various cloned non-pathogenic strains as well as in 23 clinical isolates from asymptomatic carriers. The finding of the ACP1 homologue in non-pathogenic or E. dispar strains rules out the proposed use of its absence for diagnostic purposes.

Amoebapore is an important virulence factor ofEntamoeba histolytica

We have previously demonstrated that inhibition of expression of amoebapore A (AP-A) by antisense RNA caused a marked decrease in the virulence of the parasite. A four-fold over-expression of AP-A was obtained with plasmid (pA7) which has theap-a gene under the control of geneEhgLE-3-RP-L21. The virulence of the transfected trophozoites, however, was also decreased. Excess of AP-A protein was found in the cytosol and a significant amount was released into the surrounding media. Transfection of the parasite with a plasmid (psAP-1) in which theap-a gene was introduced with its own regulatory sequences, caused a total suppression of the transcription and translation of both the genomic and episomalap-a genes. The silenced transfectant was not virulent at all. These results demonstrate that important factors need to be expressed at the correct cellular location and that the parasite has additional internal control mechanisms such as transcriptional gene silencing which can prevent excess amounts of gene expression.

Cell Surface Molecules of Pathogenic and Nonpathogenic Entamoeba Histolytica and their Relation to Virulence

In recent years it has become increasingly clear that human infections with Entamoeba histolytica are caused by two types of morphologically indistinguishable trophozoites (McKerrow, 1992; Mirelman, 1992). Symptomatic disease is always associated with the pathogenic type (P), whereas the nonpathogenic (NP) ameba or Entamoeba dispar as many prefer to name it, usually behaves more as a commensal that does not cause symptoms. The correlation, however, between the clinical picture of the patient and the identity of the type of parasite with which he is infected, does not always fit. This complication may be due to the fact that both P and NP types of amebae can sometimes be present in the same stool, as has been recently reported for quite a number of cases (Acuna-Soto, et al. 1993; Walderich et al. 1995).