Virulence of Entamoeba histolytica trophozoites. Effects of bacteria, microaerobic conditions, and metronidazole.

The association of axenically grown trophozoites of Entamoeba histolytica strains HK-9 or HM-1:IMSS with various types of gram-negative bacteria for relatively short periods markedly increased their virulence, as evidenced by their ability to destroy monolayers of tissue-cultured cells. Interaction of trophozoites with bacteria that were heat inactivated, glutaraldehyde fixed, or disrupted by sonication, or bacteria treated with inhibitors of protein synthesis, did not augment amebic virulence. Lethally irradiated bacteria, however, retained their stimulative properties and trophozoites that ingested bacteria were protected from the toxic effects of added hydrogen peroxide. An increase in virulent properties of amebae was also found in experiments carried out under microaerobic conditions (5% O2, 10% CO2). The augmentation of amebic virulence due to association with bacteria was specifically blocked by metronidazole, but not by tetracycline or aminoglycosides, and the rate of metronidazole uptake in stimulated trophozoites was two to three times higher. The results obtained suggest that virulence of axenically grown E. histolytica trophozoites may depend to a considerable extent on the cell's reducing power. Both microaerobic conditions and the association with bacteria apparently stimulate the electron transport system of the ameba. Bacteria may function as broad range scavengers for oxidized molecules and metabolites through the contribution of enzymatic systems, components, or products.

Trophozoites of Entamoeba histolytica epigenetically silenced in several genes are virulence-attenuated.

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 (Ehapa) 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 RB9 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.

Inhibition of gene expression in Entamoeba by the transcription of antisense RNA: effect of 5' and 3' regulatory elements.

Down regulation of gene expression by antisense RNA is one of the ways to investigate the specific contribution of certain components to the physiology and activities of a cell. A successful inhibition of gene expression in Entamoeba trophozoites was achieved in stable transfectants by using hybrid plasmid constructs containing promotors that produce transcripts which do not bind to polysomes. Different promotors were found to be required for Entamoeba histolytica or Entamoeba dispar. In E. histolytica one of the two copies (g34) of the gene coding for ribosomal protein L21 was previously found to be transcribed but not translated. Inhibition of gene expression was obtained by placing in a transfection vector, the amoebapore A gene, in its antisense orientation, under the control of the g34 promotor. Transfectants of E. histolytica were shown to accumulate antisense transcripts and inhibit amoebapore synthesis. In contrast, transfectants with plasmid constructs in which the amoebapore gene was placed under the control of the gLE3 promotor of RP-L21, which is known to be translated, did not accumulate antisense transcript or inhibit gene expression. Maximal inhibition of amoebapore expression was obtained when the antisense construct also included the 5' and 3' untranslated regions of the amoebapore gene. In E. dispar the opposite situation was found, plasmid constructs containing the promotor regions of the gLE3 copy, which were shown to be poorly translated, were more efficient in inhibiting the synthesis of a 30 kDa surface-specific antigen than a construct with the g34 promotor element.

Inhibition of expression of the lysine-rich 30 kDa surface antigen of Entamoeba dispar by the transcription of its antisense RNA.

The gene coding for the 30 kDa lysine rich surface antigen (Ed-Ag) that is present on membrane surfaces of Entamoeba dispar trophozoites has been characterized. A specific monoclonal antibody MAb 318-28 prepared against this antigen reacts with all E. dispar strains tested, but not with any of the antigens of E. histolytica. In order to understand the function of this antigen, we constructed two plasmids, pEdA-9 and pEdA-Rev, in which the antigen-coding sequence was introduced into the pEhAct-Neo shuttle vector in the direct and opposite orientation, respectively. When E. dispar trophozoites were transfected with pEdA-9, only a slight increase was observed in the expression of the antigen. However, when E. dispar trophozoites were transfected with pEdA-Rev, the expression of the native 30 kDa antigen was significantly inhibited. This inhibition was proportional to the level of resistance of the E. dispar culture to the neomycin derivative G418. Cytopathic assays detected only a slight difference between untransfected, pEdA-9 transfected and pEdA-Rev transfected trophozoites.

Isolation and partial characterization of the hexokinase isoenzymes from pathogenic and non-pathogenic strains of Entamoeba histolytica.

Isoenzyme electrophoretic patterns (zymodemes) are increasingly used to distinguish between pathogenic and non-pathogenic strains of Entamoeba histolytica. Isolates of E. histolytica from asymptomatic and symptomatic cases have been shown to differ in the electrophoretic mobility of their hexokinase and phosphoglucomutase isoenzymes. The hexokinase isoenzymes from a non-pathogenic strain and from a pathogenic strain of E. histolytica were purified by fast protein liquid chromatography in several steps, which included a separation by size, chromatofocusing, and anion exchange chromatography. The isoenzymes differed in their isoelectric points, which ranged from pH 4.8-5.4, but had very similar kinetic properties and almost identical apparent molecular weights (48,000) in sodium dodecyl sulfate polyacrylamide gels, as well as on gel filtration columns. Comparison of tryptic peptide analysis of each of the isoenzymes indicated considerable homology between the non-pathogenic and pathogenic forms. Antibodies produced against each of the two pathogenic hexokinase isoenzymes inhibited their enzymatic activity. The antibodies also inhibited the activity of the isoenzymes of the non-pathogenic strain. Our findings suggest that the isoenzymes have structural similarities, and that the pathogenic ones differ from the non-pathogenic ones in their electromobility due to post-translational modifications.

Cloning and characterization of an unusual elongation factor-1 alpha cDNA from Entamoeba histolytica.

The coding sequence deduced from two overlapping cDNA inserts obtained from a pathogenic strain of Entamoeba histolytica revealed a striking homology (greater than 85%) with elongation factor EF-1 alpha from Saccharomyces cerevisiae and Artemia salina. The deduced amino acid sequence predicted a size of 49 kDa, and antibodies raised against the S. cerevisiae EF-1 alpha cross-reacted with an amoebic protein of similar size (45-47 kDa). Sequence analysis of the cDNA revealed that the 5' untranslated region contained a stretch of 190 nucleotides which was perfectly complementary to a segment of the 3' terminal coding region situated 1015 bases downstream of the methionine initiation codon. Electron microscopy of self-renatured cDNA confirmed the potential of such molecules to form a stem-loop secondary structure. The presence of the complementary sequences was confirmed at the genomic level by sequence analysis of polymerase chain reaction-amplified segments which span both the 3' and 5' terminal complementary regions. Comparison of the deduced amino acid sequence of E. histolytica EF-1 alpha with Ef-Tu from Escherichia coli and EF-1 alpha from different sources, suggested that the major functional domains of the protein are located within the loop structure.

Intraocular lens in a fighter aircraft pilot.

A pseudophakic pilot of the Israeli air force flying an F-15 (Eagle) aircraft was followed up for three years. He experienced about 100 flying hours, 5% of the time under high g stress. The intraocular lens did not dislocate and no complications were observed. It seems that flying high performance fighter aircraft is not contraindicated in pseudophakic pilots.

Intraocular lens implantation in an eye with Behçet's uveitis.

A case of intraocular lens implantation in a patient with Behçet's uveitis is presented. During a 12-month follow-up the uveitis did not flare up and no other complication developed.

Cloning and partial characterization of an antigen detected on membrane surfaces of non-pathogenic strains of Entamoeba histolytica.

A novel and unique mAb (318-28) which specifically interacts with a 60 kDa antigen that is found only on the surfaces of non-pathogenic (NP) strains of E. histolytica has been recently characterized. The antigen appears to be present also on (NP) cyst forms of amebas but was not detected on any of the various (P)-strains tested. It was also not found on other Entamoeba species such as Moshkovskii, Laredo, Huff, Coli, Gingivalis, or Invadens. Clinical trails for the differentiation of (P) and (NP) amebas directly from stools using this mAb are in progress. Cloning of the gene encoding for the (NP)-specific antigen was achieved after screening with mAb 318-28 a lambda gt11 expression library of NP strain SAW 1734R. No sequence homology to any known protein was found in data bases.