Antisense inhibition of expression of cysteine proteinases does not affect <i>Entamoeba histolytica</i> cytopathic or haemolytic activity but inhibits phagocytosis

Ankri, Serge; Stolarsky, Tamara; Mirelman, David

doi:10.1046/j.1365-2958.1998.00837.x

Cited by 135 publications

(124 citation statements)

References 32 publications

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“…Cysteine protease activity is necessary for the trophozoites to traverse a colonic mucus barrier before cell cytolysis (8,16). In addition, the proteases are necessary for the parasite to disrupt the epithelial cell monolayers in vitro (15), to disseminate through host tissue (26), and to cause liver abscesses (27,28). The mechanism by which the parasite disrupts and penetrates the mucin polymeric network was previously unknown, but recent evidence suggests that the amoeba disrupts the MUC2 polymers (8).…”

Section: Discussionmentioning

confidence: 99%

Entamoeba histolytica cysteine proteases cleave the MUC2 mucin in its C-terminal domain and dissolve the protective colonic mucus gel

Lidell

Moncada

Chadee

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

237

186

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In order for the protozoan parasite Entamoeba histolytica (E.h.) to cause invasive intestinal and extraintestinal infection, which leads to significant morbidity and mortality, it must disrupt the protective mucus layer by a previously unknown mechanism. We hypothesized that cysteine proteases secreted from the amoeba disrupt the mucin polymeric network, thereby overcoming the protective mucus barrier. The MUC2 mucin is the major structural component of the colonic mucus gel. Heavily O-glycosylated and protease-resistant mucin domains characterize gel-forming mucins. Their N-and C-terminal cysteine-rich domains are involved in mucin polymerization, and these domains are likely to be targeted by proteases because they are less glycosylated, thereby exposing their peptide chains. By treating recombinant cysteine-rich domains of MUC2 with proteases from E.h. trophozoites, we showed that the C-terminal domain was specifically targeted at two sites by cysteine proteases, whereas the N-terminal domain was resistant to proteolysis. The major cleavage site is predicted to depolymerize the MUC2 polymers, thereby disrupting the protective mucus gel. The ability of the cysteine proteases to dissolve mucus gels was confirmed by treating mucins from a MUC2-producing cell line with amoeba proteases. These findings suggest a major role for E.h. cysteine proteases in overcoming the protective mucus barrier in the pathogenesis of invasive amoebiasis. In this report, we identify a specific cleavage mechanism used by an enteric pathogen to disrupt the polymeric nature of the mucin gel.colon ͉ parasite

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Section: Discussionmentioning

confidence: 99%

Entamoeba histolytica cysteine proteases cleave the MUC2 mucin in its C-terminal domain and dissolve the protective colonic mucus gel

Lidell

Moncada

Chadee

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

237

186

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“…This fragment was then inserted into plasmid pSA8 (17) replacing the antisense ehcp5 insert to create pZZ1. 1 ϫ 10 7 E. histolytica HM1:1MSS trophozoites in log phase growth were transfected with 100 ng of plasmid pZZ1, pSA8 (antisense construct against E. histolytica cysteine proteinase 5), or the parent pNeo-Act plasmid without an insert (17). Selection was performed by adding 10 g/ml G418 to TYI-S33 media beginning 48 h after transfection and gradually increasing G418 concentration to 50 g/ml over 2 weeks' time.…”

Section: Methodsmentioning

confidence: 99%

“…Amebic lysates derived from pZZ1-transfected trophozoites had 30% of the ADH activity of lysates derived from equivalent numbers of wild type HM1:IMSS trophozoites or trophozoites transformed with plasmid pSA8 (which contains an antisense transcript to the E. histolytica cysteine proteinase 5 gene) (Table I) (17). Using immunoblotting with antiserum raised against EhADH2, we found that EhADH2 was present in lower amounts (83% reduction by scanning densitometry of the autoradiograph bands) in SDS-PAGE-separated lysates from pZZ1-transfected E. histolytica compared with lysates from pSA8-transfected E. histolytica (Fig.…”

Section: Episomal Expression Of An Antisense Rna To the Ehadh2mentioning

confidence: 99%

The Bifunctional Entamoeba histolytica Alcohol Dehydrogenase 2 (EhADH2) Protein Is Necessary for Amebic Growth and Survival and Requires an Intact C-terminal Domain for Both Alcohol Dehydrogenase and Acetaldehyde Dehydrogenase Activity

Espinosa

Yan²,

Zhang³

et al. 2001

Journal of Biological Chemistry

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The intestinal protozoan pathogen Entamoeba histolytica lacks mitochondria and derives energy from the fermentation of glucose to ethanol with pyruvate, acetyl enzyme Co-A, and acetaldehyde as intermediates. A key enzyme in this pathway may be the 97-kDa bifunctional E. histolytica alcohol dehydrogenase 2 (EhADH2), which possesses both alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase activity (ALDH). EhADH2 appears to be a fusion protein, with separate N-terminal ALDH and C-terminal ADH domains. Here, we demonstrate that EhADH2 expression is required for E. histolytica growth and survival. We find that a mutant EhADH2 enzyme containing the C-terminal 453 amino acids of EhADH2 has ADH activity but lacks ALDH activity. However, a mutant consisting of the N-terminal half of EhADH2 possessed no ADH or ALDH activity. Alteration of a single histidine to arginine in the putative active site of the ADH domain eliminates both ADH and ALDH activity, and this mutant EhADH2 can serve as a dominant negative, eliminating both ADH and ALDH activity when co-expressed with wild-type EhADH2 in Escherichia coli. These data indicate that EhADH2 enzyme is required for E. histolytica growth and survival and that the C-terminal ADH domain of the enzyme functions as a separate entity. However, ALDH activity requires residues in both the N-and C-terminal halves of the molecule.The anaerobic intestinal protozoan parasite Entamoeba histolytica converts pyruvate to ethanol in its fermentation pathway (1). The last two steps of this pathway are the conversion of acetyl-CoA to acetaldehyde followed by the reduction of acetaldehyde to ethanol (1). E. histolytica possesses at least three enzymes with alcohol dehydrogenase (ADH) 1 activity: a NADP-dependent ADH (EhADH1); a 97-kDa NAD(ϩ)-dependent and Fe 2ϩ -dependent bifunctional enzyme with both ADH and acetaldehyde dehydrogenase (ALDH) activities (EhADH2, also known as EhADHE); and a 43-kDa NADP-dependent ADH with some sequence homology to class III microbial alcohol dehydrogenases (EhADH3) (2-5). There are at least two enzymes with ALDH activity, the EhADH2 enzyme and a NADPdependent ALDH, EhALDH1 (2, 5, 6). Given the presence of multiple ADH and ALDH enzymes in E. histolytica, an important question is whether any of these enzymes are essential for E. histolytica growth and survival and thus potential targets for anti-amebic therapy.The EhADH2 enzyme is part of a newly described family of multifunctional enzymes found in Gram-negative and Grampositive bacteria and the intestinal protozoan parasite Giardia lamblia (2, 7-12). EhADH2 and other members of the family appear to be composed of separate C-terminal ADH and Nterminal ALDH domains linked together to create a fusion enzyme (8). The EhADH2 enzyme utilizes NAD and Fe 2ϩ as co-factors and does not demonstrate homology with the zincdependent ADH enzymes (13). Regions of EhADH2 that could be involved in iron binding and NAD binding have been identified, but a requirement for specific residues in enzymatic activity has not been demo...

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“…Although targeted disruption of selected E. histolytica genes has not yet been achieved, stable episomal expression of foreign DNA is possible in amoebae by maintaining continuous selective pressure [Hamann et al 1995, Vines et al 1995. This has enabled the investigators to target specific molecules in E. histolytica by the episomal expression of antisense mRNA or of genes encoding dominant negative mutants [Ankri et al 1998]. The antisense approach has been applied to E. histolytica cysteine proteinases and episomal expression of an antisense RNA to ehcp5 could reduce total amoebic proteinase activity by 80-90% [Ankri et al 1998].…”

Section: Mechanisms Of Hepatocellular Dysfunction and Regeneration: Ementioning

confidence: 99%

“…This has enabled the investigators to target specific molecules in E. histolytica by the episomal expression of antisense mRNA or of genes encoding dominant negative mutants [Ankri et al 1998]. The antisense approach has been applied to E. histolytica cysteine proteinases and episomal expression of an antisense RNA to ehcp5 could reduce total amoebic proteinase activity by 80-90% [Ankri et al 1998]. To assess the role of E. histolytica cysteine proteinases in amoebiasis, in earlier study, human xenografts in SCID-HU-INT mice were infected with amoebic trophozoites expressing the ehcp5 antisense RNA(proteinase-deficient amoebae) or amoebic trophozoites containing the same plasmid without the antisense insert (the control group) [Zhang et al 2000].…”

Section: Mechanisms Of Hepatocellular Dysfunction and Regeneration: Ementioning

confidence: 99%

Mechanisms of Hepatocellular Dysfunction and Regeneration: Enzyme Inhibition by Nitroimidazole and Human Liver Regeneration

Sharma¹

2012

Enzyme Inhibition and Bioapplications

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Antisense inhibition of expression of cysteine proteinases does not affect Entamoeba histolytica cytopathic or haemolytic activity but inhibits phagocytosis

Cited by 135 publications

References 32 publications

Entamoeba histolytica cysteine proteases cleave the MUC2 mucin in its C-terminal domain and dissolve the protective colonic mucus gel

Entamoeba histolytica cysteine proteases cleave the MUC2 mucin in its C-terminal domain and dissolve the protective colonic mucus gel

The Bifunctional Entamoeba histolytica Alcohol Dehydrogenase 2 (EhADH2) Protein Is Necessary for Amebic Growth and Survival and Requires an Intact C-terminal Domain for Both Alcohol Dehydrogenase and Acetaldehyde Dehydrogenase Activity

Mechanisms of Hepatocellular Dysfunction and Regeneration: Enzyme Inhibition by Nitroimidazole and Human Liver Regeneration

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