Antisense inhibition of expression of the light subunit (35 kDa) of the Gal/GalNac lectin complex inhibits Entamoeba histolytica virulence.

One of the under-represented genes identified by cDNA representational difference analysis (RDA) between avirulent Entamoeba histolytica strain Rahman and virulent strain HM-1:IMSS was the amoebic light (35 kDa) subunit of the Gal/GalNac lectin complex. This lectin complex, which mediates the adhesion of the parasite to the target cell, also contains a heavy (170 kDa) subunit, which has the carbohydrate-binding domain. Stable transfectants of the virulent strain in which the expression of the 35 kDa subunit was inhibited by antisense RNA were not significantly affected in their adhesion activity to mammalian or bacterial cells but were strongly inhibited in their cytopathic activity, cytotoxic activity and in their ability to induce the formation of liver lesions in hamsters. These findings suggest that the 35 kDa subunit may have a specific function in the pathogenic pathway and provides a new insight into the role of this component of the Gal/GalNac lectin complex in amoebic virulence.

Antisense inhibition of expression of cysteine proteinases affects Entamoeba histolytica-induced formation of liver abscess in hamsters.

Trophozoites of virulent Entamoeba histolytica transfected with the antisense gene encoding cysteine proteinase 5 (CP5) have only 10% of the CP activity but retain their cytopathic activity on mammalian monolayers. In the present study we found that the transfected trophozoites with low levels of CP activity were incapable of inducing the formation of liver lesions in hamsters.

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

Inhibition of most of the expression of the cysteine proteinases of Entamoeba histolytica strain HM-1:IMSS was successfully performed by transcription of ehcp5 antisense RNA using the promoter of ehg34, which encodes a L21 ribosomal protein of E. histolytica. We have generated a stable transfectant in which the overall level of cysteine proteinase activity is strongly reduced ( 90%). This transfectant has a normal growth rate in Diamond's TYI-S-33 medium, a cytopathic and haemolytic activity similar to the control HM-1:IMSS pEhAct-Neo transfectant but with a significantly lower phagocytic activity.

Regulation of expression of ribosomal protein L-21 genes of Entamoeba histolytica and E. dispar is at the post-transcriptional level.

Two genes, EhgLE3 and Ehg34, encoding the ribosomal protein L21 (rp-L21) were identified and characterized from Entamoeba histolytica. Their coding regions are highly conserved, but their flanking regions differ significantly. Analogous genes (EdgLE3 and Edg34) were characterized in E. dispar. The two rp-L21 copies are transcribed at similar levels in the two parasites. However, their relative binding to the polyribosomal complex during active translation is different. In E. histolytica, binding of EhgLE3 transcripts to the polyribosomes is significantly higher in comparison with that of Ehg34 transcripts, whereas in E. dispar the binding pattern is inverse. The importance of each of the rp-L21 flanking regions to gene translation was investigated by constructing hybrid plasmids containing the CAT reporter gene flanked by rp-L21 flanking regions. The plasmids were stably transfected into E. histolytica and E. dispar, and CAT mRNA and enzymatic activity levels were determined. All plasmids promoted transcription of CAT. Yet, in E. histolytica, high levels of CAT activity were observed only when gLE3 upstream regions flanked CAT. In contrast, in E. dispar, high levels of CAT activity were observed when g34 upstream regions flanked CAT. The downstream regions showed no significant effect on CAT translation.

Comparison of use of enzyme-linked immunosorbent assay-based kits and PCR amplification of rRNA genes for simultaneous detection of Entamoeba histolytica and E. dispar.

A comparison of the use of three commercially available enzyme-linked immunosorbent assay-based kits and PCR amplification of rRNA genes to detect and differentiate Entamoeba histolytica from E. dispar was carried out. Only the Techlab kit did not cross-react with E. dispar antigens, but it was 100 times less sensitive than PCR in detection of and differentiation between the two types of Entamoeba.

Specific labeling of cysteine proteinases in pathogenic and nonpathogenic Entamoeba histolytica.

Growth of Entamoeba histolytica trophozoites was inhibited by 50% at low concentrations (2.0 micrograms/ml) of the diazopeptidyl inhibitor benzyloxycarbonyl-leucyl-L-tyrosyldiazomethane (Z-L-Leu-L-Tyr-CHN2). Iodination of the tyrosine residue lowered the growth inhibitory efficacy of the diazopeptidyl inhibitor (50% inhibition, approximately 10 micrograms/ml). However, even at this concentration, practically all of the cysteine proteinase activity of the cells was irreversibly inactivated as shown by fluorescence microscopy with the dipeptide substrate L-Arg-L-Arg-4-methoxy-beta-napthylamide or colorimetrically with azocasein as the substrate. Growth of trophozoites of E. histolytica from various strains, including both pathogenic and nonpathogenic zymodemes, was similarly inhibited. The concentration of inhibitor required to inactivate the proteinase activity of nonpathogenic cells was lower. Lysates from trophozoites grown in the presence of sublethal concentrations of 125I-labeled protease inhibitor (10 micrograms/ml) showed as many as eight radioactive bands by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (molecular sizes, 73, 68, 56, 40, 39, 35, 29, and 27 kilodaltons). Two of these bands (molecular sizes, 29 and 27 kilodaltons) could be seen in gels of the cytoplasmic fraction, whereas the high-molecular-size bands were mostly associated with the membrane fraction. The radioactive bands in pathogenic and nonpathogenic strains were very similar with only minor differences. The results obtained show that E. histolytica cells, irrespective of their pathogenicity, possess a number of cysteine proteinases of similar molecular sizes which are vital for cell growth.

Identification of protein kinase C and its potential substrate in Entamoeba histolytica.

1. Protein kinase C (PKC) activity has been identified in various strains of the human parasite, Entamoeba histolytica. 2. An amoebic protein of mol. wt 78,000 was recognized by polyclonal antibodies raised against the 82,000 mol. wt rat brain protein kinase C. 3. A partially purified PKC preparation from E. histolytica phosphorylated histone I in the presence of calcium, phospholipids and diacylglycerol, and specifically bound tritiated phorbol ester at an apparent KD of 9 nM. 4. A relocalization of the amoebic PKC activity from the cytosol to the membrane fraction was observed when trophozoites were actively phagocytising bacteria. Under these conditions, a labelled phosphoprotein of mol. wt 68,000 was identified. 5. Similar to what was found during macrophage activation, a myristoylated mol. wt 68,000 protein was detected in amoebae grown in the absence of bacteria, but not in amoebae which were active in phagocytosis.

Effects of covalently bound silica-nitroimidazole drug particles on Entamoeba histolytica.

Most commonly used antiamoebic drugs are effective in invasive amebiasis, but their response against trophozoites of Entamoeba histolytica, present in the lumen of the human colon, is inadequate. We report the development of an antiamoebic drug carrier that may be effective against luminal infections. Our preparation consists of small silica particles (5-10 microns in diameter) covalently linked to a potent antiamoebic drug, 2-(4-aminophenoxymethyl)-5-nitro-1-methyl imidazole. Silica-drug particles were injected into mice, hamsters, and guinea pigs. We found that trophozoites phagocytosed the particles in vivo and in vitro, followed by rapid cell death due to the released drug. Analysis of mouse serum revealed that no drug was absorbed from the intestine after placement of the drug-containing particles in the intestine. The antiamoebic activity of particles recovered from the intestine was almost fully retained. This novel antiamoebic concept may be useful for luminal therapy for asymptomatic amebiasis and may minimize side effects and frequency of administration.

A stage-specific sialoglycoprotein in encysting cells of Entamoeba invadens.

A novel sialoglycoprotein with an apparent molecular mass of approximately 250 kDa was detected on the surface of cysts of Entamoeba invadens. Sialic acid was identified in this glycoprotein by gas chromatography after methanolysis; N-acetyl- and N-glycolyl neuraminic acid were identified by thin layer chromatography in hydrolysates of partially purified preparations of the 250 kDa glycoprotein as well as in whole cysts. The sialoglycoprotein is stage-specific and could be detected by binding of wheat germ agglutinin and a specific monoclonal antibody (JAM3) only to precysts and mature cysts but not to trophozoites. A 250 kDa protein could be metabolically labeled with [35S]methionine. This, together with the absence of such a glycoprotein in the encystation medium, suggests that the 250 kDa sialoglycoprotein is not an adsorbed serum glycoprotein. Indirect evidence suggests that the parasite may utilize serum components as a source for sialic acid.

Encystation of Entamoeba invadens IP-1 is induced by lowering the osmotic pressure and depletion of nutrients from the medium.

Trophozoites of Entamoeba invadens IP-1 can be induced to encyst in simple solutions composed of semipermeable constituents (buffer, salts, or sugars) provided that their osmotic pressure is in the range of 60-160 mosmol/kg. Optimal yield of mature cysts was obtained when the osmotic pressure of the medium was 110 mosmol/kg. Encystation could be obtained in the absence of serum although higher yields were obtained in its presence. No difference in the yield of mature cysts was found when either dialyzed or full serum was used. High yields of encystation were obtained (greater than 70%) in the presence of 5% serum in solutions of NaCl, KCl, or MgSO4, suggesting that the mechanism of encystation is not induced via sodium or potassium channels. Cysts were obtained in the presence of 72 mM glucose, indicating that depletion of a carbon source is not the only requirement for encystation. A rapid change in the density of the Entamoeba cells was observed upon transfer of trophozoites (density 1.061-1.073 g/ml) from growth medium to the low osmotic pressure encystation solutions. Within the first 2 min their density decreased (to 1.050 g/ml), but it soon increased, reaching within 30 min a density higher than 1.120 g/ml. As the encystation process continued to completion, the density of the cells gradually decreased, the mature cysts reaching a density of 1.049-1.061 g/ml.

Retinoic acid restores shape-dependent growth control in neoplastic cells cultured on poly(2-hydroxyethyl methacrylate)-coated substrate.

The ability of retinoic acid to modulate cell-shape-dependent growth of untransformed (human skin fibroblasts and mouse embryo Swiss 3T3 fibroblasts) and neoplastic cells (human cervical carcinoma HeLa-S3, osteosarcoma Hs791, and murine melanomas B16-F1, S91-C2 and S91-C154) was examined. The cells were plated on tissue culture dishes coated with increasing concentrations of poly(2-hydroxyethyl methacrylate), poly(HEMA) which cause a gradual decrease in substrate adhesiveness. Untreated cells as well as cells pretreated with 10 microM retinoic acid for 4 days displayed a similar graded series of cell shapes between flat and spherical on these modified substrata, with the exception of HeLa-S3 cells which were rounded and loosely attached even on uncoated plastic dishes. A marked cell-shape-dependent decrease in DNA synthesis was observed in untransformed human skin fibroblasts, Swiss 3T3 fibroblasts and neoplastic human Hs791 cells 20 h following plating of untreated cells on poly(HEMA)-coated substrates of decreasing adhesiveness. Conversely, in B16-F1, HeLa-S3 and S91-C154 cells DNA synthesis was only slightly affected by changes in cell shape. Pretreatment with retinoic acid rendered DNA synthesis in Swiss 3T3, Hs791, B16-F1 and S91-C2 cells much more sensitive to changes in cell shape. In contrast, retinoic acid exerted only marginal effects on the sensitivity of DNA synthesis to changes in cell shape in untransformed human skin fibroblasts, in HeLa-S3 cells and in the retinoic-acid-resistant S91-C154 cells. The results suggest that retinoic acid can restore in certain tumor cells the tight coupling between cell shape and DNA synthesis that exists in untransformed cells.

Isolation and analysis of melanoma cell mutants resistant to the antiproliferative action of retinoic acid.

Retinoic acid inhibits both the anchorage-dependent and the anchorage-independent growth of the murine melanoma S91-C-2 cells. To explore the mechanism of these effects, several mutant cell clones resistant to retinoic acid-induced growth inhibition have been derived from the S91-C-2 cells by exposing them to the mutagen ethyl methane sulfonate and plating in soft agarose in the presence of 1 microM beta-all-trans-retinoic acid. Under such conditions, the nonmutagenized S91-C-2 cells failed to grow; however, 2 X 10(-6) of the mutagenized cells did form colonies. These colonies were isolated, expanded in culture, and recloned in agarose containing retinoic acid. Five cell clones that retained their drug-resistant phenotype after repeated subculture for 3 months, in the absence of retinoic acid, were characterized further. They were found to be 3- to greater than 1000-fold and 100- to greater than 100-fold resistant to retinoic acid-induced inhibition of anchorage-independent and anchorage-dependent growth relative to the wild-type C-2 cells, respectively. The rate of uptake of [3H]-retinoic acid by the resistant cell clones was similar to that of the sensitive C-2 cells, indicating that resistance is not the result of reduced uptake. Analysis of cytoplasmic retinoic acid-binding protein revealed that it is present in the most resistant clones in amounts that are similar to or even greater than those found in the sensitive S91-C-2 cells. These results indicate that resistance is not the result of the absence of the binding protein. The retinoic acid-resistant mutants exhibited cross-resistance to related retinoids such as 13-cis-retinoic acid and all-trans-retinol as well as to the arotinoid p-[(E)-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthyl) propenyl]benzoic acid suggesting that they all share a similar mechanism of action. These resistant mutants may provide a useful system for further studies of the molecular processes through which retinoic acid exerts its antiproliferative effects.