Detection of Glycogen-Debranching System in Trophozoites of Entamoeba histolytica

ABSTRACT. Homogenates of trophozoites of Entamoeba histolytica were shown to bring about the total degradation of glycogen while purified phosphorylase of the same source alone yielded a limit dextrin as end product. An enzyme system capable of debranching the limit dextrin was obtained from the 40,000 g pellet by extraction in aqueous medium, purified by gel filtration on Fractogel TSK HW-55(F), and separated from phosphorylase by chromatography on Blue Sepharose CL-6B and aminobutyl Agarose. The glycogen-debranching system was purified 540-fold to a state of homogeneity by criterion of disc-gel electrophoresis. The purified enzyme was able to degrade glycogen-limit dextrin in the presence of phosphorylase and exhibited activities of both amylo-1,6-glucosidase (EC 3.2.1.33) and 4- α -glucanotransferase (EC 2.4.1.25). Although amylo-1,6-glucosidase released glucose from a glycogen-phosphorylase limit dextrin, transferase activity moved single glucose residues from the limit dextrin to 4-nitrophenyl- α -glucoside yielding successively 4-nitrophenyl- α -maltoside and 4-nitrophenyl- α -maltotrioside that could be detected by HPLC. Native glycogen-debranching system exhibited a relative molecular mass of M_r= 180,000 ± 10% by gel filtration and gel electrophoresis in both denaturing and nondenaturating conditions.     

Scanning Electron Microscopy of Attached Trophozoites of Pathogenic Entamoeba histolytica1

The surface morphology of Entamoeba histolytica trophozoites of HM 1:IMSS (axenic and monoxenic) and HK9 (axenic) strains cultured on plastic and MDCK cell substrates was examined using scanning electron microscopy (SEM). The conditions for processing trophozoites were determined by comparing the SEM observations with the morphology of living amebas examined by light microscopy. The most frequent surface differentiations in all the amebas observed with SEM were lobopodia. Round cytoplasmic projections were found in approximately half of the axenic amebas. Endocytic stomas and filopodia were more common in monoxenic cultures while the uroid was found in only 2–8% of all examined amebas. The basal surfaces of the trophozoites, involved in both attachment and cytolysis, showed no unusual features, except for the presence of a small number of short filopodia at the outer edge. No differences were found in the morphology of amebas grown on artificial and natural substrates. These observations demonstrate that there are significant quantitative differences in the surface morphology of cultured trophozoites of different strains of E. histolytica and that association with bacteria produces an increase in the relative number of surface specializations of the parasite.     

In vitro Induction of Entamoeba histolytica Cyst-like Structures from Trophozoites

Inhibition of encystment can be conceived as a potentially useful mechanism to block the transmission of Entamoeba histolytica under natural conditions. Unfortunately, amoeba encystment has not been achieved in vitro and drugs inhibiting the formation of cysts are not available. Luminal conditions inducing encystment in vivo are also unknown, but cellular stress such as exposure to reactive oxygen species from immune cells or intestinal microbiota could be involved. A role for certain divalent cations as cofactors of enzymes involved in excystment has also been described. In this study, we show that trophozoite cultures, treated with hydrogen peroxide in the presence of trace amounts of several cations, transform into small-sized spherical and refringent structures that exhibit resistance to different detergents. Ultrastructural analysis under scanning and transmission electron microscopy revealed multinucleated structures (some with four nuclei) with smooth, thick membranes and multiple vacuoles. Staining with calcofluor white, as well as an ELISA binding assay using wheat germ agglutinin, demonstrated the presence of polymers of N-acetylglucosamine (chitin), which is the primary component of the natural cyst walls. Over-expression of glucosamine 6-phosphate isomerase, likely to be the rate-limiting enzyme in the chitin synthesis pathway, was also confirmed by RT-PCR. These results suggest that E. histolytica trophozoites activated encystment pathways when exposed to our treatment.     

Acid Intracellular Vesicles and the Cytolysis of Mammalian Target Cells by Entamoeba histolytica Trophozoites1

ABSTRACT. Entamoeba histolytica kills mammalian target cells in a multi-step sequential process with separate adherence, cytolytic, and phagocytic events. In the studies reported here, we used fluorescein isothiocyanate linked to dextran to label the endocytic vesicles of the HM1 strain of E. histolytica and measure vesicle pH (5.1 ± 0.2 by spectrofluorimetry). Concentrations of NH₄Cl (1.0–10.0 mM) sufficient to increase vesicle pH to °5.7 inhibited amebic killing of target Chinese hamster ovary (CHO) cells as assayed by trypan blue staining, by the release of ³H-thymidine previously incorporated into CHO cell monolayers, and by the release of ¹¹¹indium oxine from radiolabeled CHO cells. Similar effects were also observed with two other weak bases, primaquine and chloroquine (both 50 μM). In contrast, NH₄Cl (10 mM) did not affect either the adherence or phagocytic events, as measured by amebic adherence to CHO cells at 4°C and by the binding and ingestion of ³H-leucine-labeled bacteria. In the presence of NH₄Cl and and the carbohydrate ligand asialofetuin, there was no evidence of intracellular trapping of the amebic galactose-inhibitable lectin; inhibition of adherence by cycloheximide (10 μg/ml for 3 h) suggested rapid turnover of the surface lectin. Prolonged exposure to NH₄Cl for 48 h (which had no effect on amebic protein synthesis) or shorter exposure to cycloheximide (10 μg for 3 h) produced persistent inhibition of cytolysis. These results indicate that an uninterrupted acid pH in intracellular endocytic vesicles is necessary for the cytolysis of target cells by E. histolytica trophozoites.     

Lipophosphoglycan Is Present in Distinctly Different Form in Different Entamoeba histolytica Strains and Absent in Entamoeba moshkovskii and Entamoeba invadens1

ABSTRACT. Lipophosphoglycan has recently been demonstrated on the cell surface of Entamoeba histolytica strain HM-1:IMSS. A monoclonal antibody against this molecule had failed to react with some other strains of E. histolytica, including the strain Rahman. To determine if a structurally distinct lipophosphoglycan existed in Rahman, [³H]galactose-labeled glycoconjugates were electrophoresed through sodium dodecyl sulfate polyacrylamide gel electrophoresis. The electrophoretic pattern in Rahman was very different compared to that obtained with strains HM-1:IMSS and 200:NIH. A number of experiments including sensitivity to mild acid, nitrous acid and phosphoinositol-specific phospholipase C suggest that the Rahman glycoconjugate is indeed a lipophosphogylcan-like molecule but distinctly different from that of HM-1:IMSS. Mild acid-treated glycoconjugates from Rahman and HM-1:IMSS revealed the presence of neutral trisaccharides and monosaccharides in Rahman but not in HM-1:IMSS. Human immune sera from amoebiasis patients and a polyclonal antibody against HM-1:IMSS liphophosphoglycan both recognized Rahman glycoconjugate. Thus, while lipophosphoglycan molecules from the two strains share common epitopes, they are clearly distinct from each other. Molecules bearing resemblance to lipophosphoglycan could not be detected in other Entamoeba species, namely Entamoeba invadens and Entamoeba moshkovskii.     

The Ultrastructure of Trophozoites of Entamoeba histolytica with Particular Reference to Spherical Arrangements of Osmiophilic Cylindrical Bodies

SYNOPSIS. Trophozoites of Entamoeba histolytica were cultured under xenic, monoxenic and axenic conditions. Some of the monoxenically cultured trophozoites were grown in the presence of Bacteroides symbiosus and others in the presence of Trypanosoma cruzi (Mexican strain). Other trophozoites were obtained from experimentally produced amebic liver abscesses in hamsters. The trophozoites were centrifuged and prepared for study by electron microscopy. Acid phosphatase activity in the parasites was determined by cytochemical reactions.
The various elements of the trophozoites were described and special attention was given to the spherical arrangements of electrons-dense cylindrical units surrounding finely granular material. Their presence was independent of the strain studied and of the nutrient elements in the culture medium. The cylindrical units probably arise from endoplasmic reticulum elements and their possible function in the digestive processes and aggression mechanisms of the trophozoites is discussed.
Acid phosphatase activity was found in round non-branching intranuclear bodies in trophozoites cultured in various media. Whether these bodies represent part of the lysosomal system of the parasite is unknown.
In preliminary work on the action of some amebicidal drugs, a special arrangement of cytomembranes morphologically similar to a Golgi complex was frequently seen in trophozoites of E. histolytica.     

Maintenance of integrity, viability, and adhesion of Entamoeba histolytica trophozoites in different incubation media

We have determined the integrity, viability and adhesion of Entamoeba histolytica HK9 and HM1 trophozoites during their incubation in two basal culture media (TP and TYI) and three saline media ("maintenance medium" MM-1 and two others buffered with HEPES). In basal culture media, more than 70% of the trophozoites maintained their integrity and adhesion to human red blood cells (RBC) for up to 4 h, and the proportion of those excluding Trypan blue decreased slowly after 2 h. In saline media, the number of ameba-RBC complexes reached a maximum after 20-30 min and then decreased rapidly (and fastest in MM-1), less than 10% of the amebae were intact after 3-4 h, and dye exclusion fell abruptly from the start of incubation. The number of ameba-RBC complexes formed and the rate of adhesion were highest in basal TP medium. Normal nonvacuolated refringent (NVR) trophozoites deteriorated progressively in all media--although much faster in the saline ones--to vacuolated refringent (VR), nonrefringent, and disrupted. Trypan blue was excluded by all NVR and a fraction of the VR trophozoites. Horse serum helped to maintain ameba integrity and viability, but inhibited adhesion in a concentration-dependent manner. We conclude that E. histolytica trophozoite integrity and adhesion are adequately preserved and should be characterized only in basal culture media, that refringence without vacuolization is a more stringent characteristic of ameba quality than Trypan blue exclusion, and that some serum component inhibits ameba adhesion.     

Introns of Entamoeba histolytica and Entamoeba dispar.

The genome of Entamoeba histolytica is considered to possess very few intervening sequences (introns), as only 5 intron-containing genes from this protozoan parasite have been reported so far. However, while sequencing a number of genomic contigs as well as three independent genes coding for ribosomal protein L27a, we have identified 9 additional intron-containing genes of E. histolytica and the closely related species Entamoeba dispar, indicating that introns are more common in these organisms than previously suggested. The various amoeba introns are relatively short comprising between 46 and 115 nucleotides only and have a higher AT-content compared to the corresponding exon sequences. In contrast to higher eukaryotes, amoeba introns do not contain a well-conserved branch point consensus, and have extended donor and acceptor splice sites of the sequences G     

Differentiation of Entamoeba histolytica and Entamoeba dispar infections

In this article, Terry jackson and Jonathan Ravdin briefly review the latest information on monoclonal antibody-based ELISAs that use antigen capture as a tool in the differential detection of human infection with Entamoeba histolytica and E. dispar. Current technology of culture and isoenzyme analysis is not widely available, is cumbersome and too time-consuming. A further potential benefit of antigen detection tests is that they can be used to monitor the efficacy of therapy; this is a shortcoming of serological tests owing to the persistence of the antibody response after successful treatment.     

Molecular Identification and Prevalence of Entamoeba histolytica,Entamoeba dispar and Entamoeba moshkovskii in Erbil City,Northern Iraq

The present study was conducted to evaluate the infection rates of Entamoeba histolytica, Entamoeba dispar, and Entamoeba moshkovskii among asymptomatic individuals in Erbil City, northern Iraq. The research intent was to discover whether pathogenic or nonpathogenic species cause a high rate of symptomless Entamoeba infections. Stool samples were microscopically examined, and the 18S-rRNA gene was targeted utilizing the nested PCR technique in the positive specimens. Initial results based on morphological features showed that the Entamoeba prevalence rate was 7.4%. Significantly higher rates of infections were seen in females than in males and in low-income people than in moderate-income people. The incidence rates among the asymptomatic individuals, as determined by molecular analysis, were as follows: E. histolytica – 6%, E. dispar – 4.3%, and E. moshkovskii – 0.3%. Of all the Entamoeba positive samples, a single infection with E. histolytica was identified in 41.4% samples; the single infection with E. dispar in 18.6% samples, 35.7% samples had mixed infections with two Entamoeba species, and 4.3% had mixed infections with three species. The current study concluded that 7.4% of healthy people, who live in the endemic area under investigation, carry Entamoeba species asymptomatically. Additionally, the majority of asymptomatic Entamoeba infections were caused by the pathogenic E. histolytica (81.4%) compared to E. dispar (58.6%), and E. moshkovskii with the lowest rate of infection. Single and co-infections with E. histolytica and E. dispar were noted. E. moshkovskii, which was identified for the first time in the region, was only seen in mixed infections.Key words: Entamoeba histolytica, Entamoeba dispar, Entamoeba moshkovskii, epidemiology, asymptomatic infections     

Aerobic and anaerobic metabolism in Entamoeba histolytica

Respiration by Entamoeba histolylica is confirmed. A doubling of the rate of oxygen uptake was observed upon the addition of d-glucose to cells in which the glycogen reserve had been partially depleted. In cells metabolizing endogenous substrates the rate of oxygen uptake was not influenced by sodium cyanide or sodium succinate. It was slightly depressed when d-mannose was the added sugar. The end products, CO₂, ethanol, and acetate accounted for essentially all of the glucose carbon utilized in both aerobic and anaerobic experiments. The radioactivity from uniformly labelled ¹⁴C-glucose was found in these products. Three times as much ethanol as acetate was produced in the anaerobic experiments and in the aerobic experiments this ratio was approximately reversed.     

Cell signalling and motility in Entamoeba histolytica

Entamoeba histolytica crawls as a polarized cell following external stimuli, with the translocation of signals modifying extracellular matrix interactions and the amoeba cytoskeleton. Nancy Guillén here describes how the gliding of E. histolytica cells requires the activity of the actomyosin complex, and how actomyosin functions related to motility are necessary for pathogenesis and for amoebal escape from the host immune response.     

Differential expression of surface glycoconjugates on Entamoeba histolytica and Entamoeba dispar

The human large intestine can harbor two morphologically similar amoebae; the invasive Entamoeba histolytica and the non-invasive Entamoeba dispar. Whereas E. histolytica can produce intestinal and extra-intestinal lesions, E. dispar is present in non-symptomatic carriers. Although biochemical, genetic and proteomic studies have identified clear differences between these Entamoebae, it has become clear that several molecules, once assumed to be involved in tissue destruction, exist in both the virulent and the avirulent species. As surface molecules may play a role in invasion and could therefore determine which amoebae are invasive, we analyzed the glycoconjugate composition of E. histolytica and E. dispar using lectins. There was a significant difference between E. histolytica and E. dispar in the expression of glycoconjugates containing d-mannose and N-acetyl-α-d-galactosamine residues, but not between virulent and avirulent strains of E. histolytica. N-glycoconjugates with terminal α (1–3)-linked mannose residues participate in the adhesion and subsequent cytotoxicity of E. histolytica to cultured hamster hepatocytes. One of them probably is the Gal/GalNAc lectin.