Rivka Bracha

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.

An ex-vivo human intestinal model to study Entamoeba histolytica pathogenesis.

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 hosts pro-inflammatory cytokine secretion.

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.

Entamoeba histolytica: Effect of growth conditions and bacterial associates on isoenzyme patterns and virulence

In xenic culture, isolates of Entamoeba histolytica from asymptomatic carriers are characterized, with rare exception, by possession of a nonpathogenic zymodeme. During the process of axenizing such an isolate, strain CDC:0784:4, a change in the pattern of the isoenzymes from nonpathogenic zymodeme I to pathogenic zymodeme II was observed 40 days after the amebae had been transferred from a medium for xenic cultivation to one used for axenic cultivation, but before axenization of the amebae had actually occurred. Axenization was accomplished by feeding the amebae lethally irradiated bacteria while suppressing and finally eradicating with antibiotics the bacterial flora accompanying the amebae in the original xenic culture. The change in zymodeme was accompanied by a change in virulence as evidenced by the ability of the amebae to produce hepatic abscesses in hamsters and to destroy monolayers of tissue culture cells. Two explanations are offered for the observed changes in zymodeme and virulence: a zymodeme is not a stable inherent property of the ameba. Alternatively, the original isolate consisted of two zymodeme populations and the conditions of growth selected for one or the other of the populations. In either case, our results suggest that the finding of a particular zymodeme in a culture of E. histolytica isolated from an asymptomatic carrier of the parasite cannot be used to predict a clinical condition or serve as a basis for the recommendation of therapy.

Expression of Amoebapores Is Required for Full Expression of Entamoeba histolytica Virulence in Amebic Liver Abscess but Is Not Necessary for the Induction of Inflammation or Tissue Damage in Amebic Colitis

ABSTRACT 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.

Downregulation of an Entamoeba histolytica Rhomboid Protease Reveals Roles in Regulating Parasite Adhesion and Phagocytosis

ABSTRACT 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.

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.

A virulence attenuated amoebapore-less mutant of Entamoeba histolytica and its interaction with host cells

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.

STUDIES ON THE ELONGATION OF BACTERIAL CELL WALL PEPTIDOGLYCAN AND ITS INHIBITION BY PENICILLIN

Biosynthesis of bacterial cell wall peptidoglycan is a highly complex process, in which three distinct stages have been recognized: 1. synthesis of the two sugar nucleotides that serve as the low-molecular-weight precursors of the peptidoglycan, 2. conversion of the water-soluble nucleotides into lipid-soluble bactoprenol derivatives, and their subsequent polymerization, via a transglycosylation reaction to form linear peptidoglycan strands, and 3. cross-linking of the linear peptidoglycan strands by a transpeptidation reaction to form a three-dimensional polysaccharide-peptide network. The transpeptidase that catalyzes the latter reaction is the focus of intense interest, because it is sensitive to inhibition by very low concentrations of penicillin and is believed to be the “killing site” of the antibiotic.3, Ample evidence for the reactions involved in the first two steps of peptidoglycan biosynthesis has been presented.l. Comparatively little is known about the third step, however, and virtually no information is available on the reactions that lead to cell wall growth and to the attachment of newly synthesized peptidoglycan to the preexisting wall. We have recently described a novel system for the study of peptidoglycan biosynthesis, in which crude cell walls are used as the enzyme source.5Our system has a distinct advantage over the membrane preparations used earlier for this purpose,r-o because it catalyzes not only polymerization of the sugar nucleotide precursors but also attaches the newly synthesized peptidoglycan to the preexisting one on the cell wall. Apparently, such an attachment does occur, because the enzymes involved, which are membrane bound, are intimately associated with the growing region of the preexisting peptidoglycan.Iu With this system, we demonstrated for the first time the occurrence of the transpeptidation reaction in Staphylococcus aureus and in Micrococcus luteus (Zysodeikticus) . 5 9 With crude cell wall preparations from these organisms, incorporation of the nucleotide precursors UDP-N-acety1-D-ghcosamine ( UDP-GlcNAc ) and UDP-N-acetylmuramyl-L-Ala-D-iso-Glu-L-Lys-D-Ala-~Ala ( UDP-MurNAc-pentapeptide) into the preformed peptidoglycan was accompanied by the release of C-terminal D-alanine from the MurNAc-pentapeptide, and this release was completely inhibited by very low concentrations of penicillin. Rather unexpectedly, penicillin also inhibited markedly, though incompletely, the incorporation of GlcNAc and of MurNAc-pentapeptide into the preexisting peptidoglycan. In all these experiments, the peptidoglycan was isolated subsequent to incubation and purified by extensive washing and heating with sodium dodecyl sulfate (SDS, 1 % in buffer). The radioactive compounds