A mouse model for Entamoeba histolytica infection

2 strains of Entamoeba histolytica, SAW 760 and SAW 408 (non-invasive zymodeme IX and invasive zymodeme II respectively, Sargeaunt & Williams 1979) were administered intra-intestinally to 15 inbred and 3 outbred strains of mouse. All were known to be free of the mouse amoeba E. muris. During the investigation, all animals were maintained in plastic film isolator units, under gnotobiotic conditions. Both zymodemes were found to be capable of infecting all 18 strains of mice for varying periods of time and both were capable of invading the gut mucosa in genetically susceptible strains. Mortality was high in some of the immunologically deprived strains, particularly with the more invasive strain of amoeba. 4 mouse strains: DBA/2, MRL, NZB, C57BL/6 bg, were found to be particularly good hosts to SAW 760.     

A serum-free, partly defined medium, PDM-805, for axenic cultivation of Entamoeba histolytica Schaudinn, 1903 and other Entamoeba

We describe the first serum-free, partly defined medium (PDM-805) for cultivating the human enteric pathogen, Entamoeba histolytica, and the reptilian amebae E. barreti, E. invadens, and E. terrapinae. PDM-805 was developed by the stepwise replacement of yeast extract, bovine serum, and a casein peptone digest in TYI-S-33, a medium widely used for the axenic cultivation of these parasites. The defined components include amino acids, carbohydrates, B vitamins, ascorbic acid, tocopherol, thioctic acid, nucleic acid precursors, trace metals, and phosphate buffers. The undefined components include a highly purified bovine serum albumin, a lipoprotein-cholesterol solution from bovine serum, and a dialyzable, autoclavable, water-soluble growth factor(s) having a molecular weight of less than 3,500 prepared from casein peptone. To date, studies on the growth requirements of E. histolytica, strain 200:NIH, show the following are essential for sustained multiplication of this ameba: iron, glucose, biotin, folic acid, niacinamide, pantothenate, pyridoxal, riboflavin, thiamine, cysteine, an ammonium moiety (in addition to that present in cysteine), bovine serum albumin, lipoprotein-cholesterol, and casein peptone dialysate.     

Chemotaxis by Entamoeba histolytica

A micropore membrane procedure to assay taxis by Entamoeba histolytica is described and the results of studies of responses to a variety of soluble substances, bacteria, an rat colon washings using this procedure are reported. Trophozoites migrated in blind well chambers through 8-micron pore size polycarbonate membranes but not nitrocellulose membranes up to 12 micron pore size. Amoebae were attracted toward fresh axenic culture medium (TYI-S), an enzymatic hydrolysate of casein (Trypticase), and a partially purified preparation of N-acetylneuraminic acid from egg mucin, but not purified N-acetylneuraminate or a variety of other low molecular weight metabolites. The response was verified as chemotaxis by checkerboard analysis. Amoebae migrated most dramatically toward suspensions of all of seven bacterial species tested, including motile and non-motile, gram-negative and gram-positive rods and cocci. This response was diminished when the bacteria concentration gradient was eliminated. The response to bacteria culture filtrates was less than 10% of that to bacterial suspensions. A response to clarified washings from the rat colon was detected; this was diminished but not eliminated by filter sterilization of the washings. We concluded that some soluble molecules, possibly of intermediate molecular size, whole bacteria, and both soluble and particulate components of the rat colon provide tactic stimuli for E. histolytica. Scanning electron micrographs of trophozoites migrating towards attractants through membranes showed narrow, extended pseudopodia entering the membrane pores, and enlarging spheres exiting as the cells proceeded through.     

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     

Nucleic acids of Entamoeba histolytica

This review will concentrate on certain aspects of the nucleic acids of Entamoeba histolytica. Utilization and synthesis of purines and pyrimidines will initially be briefly discussed, e.g. salvage vs. de novo pathways, uptake studies and recognition of at least 4 transport loci. Data will be presented which show that the distribution and synthesis of RNA (to a lesser extent DNA) in the nucleus is basically the opposite one finds in other eukaryotes, viz. most RNA (ribosomal?) is synthesized (or accumulates) in the peripheral chromatin (functional equivalent of nucleolus?). The DNA is distributed and synthesized primarily throughout the nucleus. It is usually so dispersed that it will not stain with e.g. the standard Feulgen technique, unless the DNA condenses around the endosome (not a nucleolar equivalent) prior to nuclear division. Isolation of rRNA was difficult due, in part, to potent and difficult to inhibit RNase(s), some of which are apparently intimately bound to ribosomal subunits. The 25S (1.3 kDa), 17S (0.8 kDa) and 5S rRNA were recovered after isolation with a high salt SDS-DEP technique. This is the only procedure which enables us to obtain high yields of 25S rRNA; guanidine or guanidinium which permits isolation of intact functional mRNA results in isolation of small amounts of 25S RNA relative to 17S RNA. The 25S RNA is "nicked" (apparently during nuclear processing) and dissociates readily into 17S (0.7 kDa) and 16S (0.6 kDa) species, and a more rigidly bound 5.8S species. A small amount of "unnicked" 25S RNA was recovered with guanidine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Secretory hydrolases of Entamoeba histolytica

Cells of Entamoeba histolytica grown over a period of four days contained NADP+-dependent alcohol dehydrogenase exclusively inside the cells. No activity of this enzyme could be found in the growth medium after harvesting the cells. Under the same conditions, acid phosphatase, beta-N-acetylglucosaminidase, esterase, alpha-glucosidase, and different amylases of the parasite were found both inside the cells and in the medium. The activities present in the cell homogenate and in the medium before and after growth of the amoebas were partially separated by gel filtration on Sephadex G150 and G75, respectively. The comparison of the elution diagrams revealed that NADP+-dependent alcohol dehydrogenase, acid phosphatase, esterase, and amylases occurred as multiple forms inside the cells. These activities, as well as beta-N-acetylglucosaminidase and alpha-glucosidase, were released into the extracellular environment to a different degree. The enzymes originating from the parasite were identified and distinguished from those of the ingredients of the growth medium according to their molecular mass and pH optimum. Furthermore, the amoebic origin of the secreted enzymes was shown on the basis of their inhibition by antibodies prepared against the supernatant fraction of the homogenate.     

Immunopathology of Entamoeba histolytica infections

Entamoeba histolytica infects almost 10% of the world's population and results in about 100 000 deaths annually(1). Relatively little information is available concerning the immune response and the immunopathology elicited by this parasite, probably due in part to the lack of a truly appropriate animal model(2-4). However, there has been some progress - particularly concerning the interaction of this parasite with cells of the immune system(5,6). This review summarizes the salient features of the cellular immune response and immunopathology, largely from in vitro studies and studies using the gerbil model for invasive amoebiasis(7,8). Overall, the results suggest that invasive amoebtasis induces profound immune dysfunction both at the effector level of macrophages and on their accessory cell potential.     

Glucose transport in Entamoeba histolytica

That the uptake of glucose by the parasitic amoeba Entamoeba histolytica occurs by an equilibrative transport system is supported by the following observations. 1. The rate of glucose uptake is several orders of magnitude greater than the uptake by pinocytosis. 2. The uptake of glucose exhibits saturation kinetics, with K(m)=1.6mm and V(max.) ranging from 2 to 5mumol/min per ml of cells at 37 degrees C. 3. The glucose analogues 2-deoxyglucose, 3-O-methylglucose and d-xylose are transported by the glucose system although with much less affinity. Competitive inhibition was observed between pairs of substrates, with K(i) values for any sugar closely coincident with the corresponding K(m). 4. d-Xylose, a sugar not metabolized by the cells, equilibrated with 80% of the amoebal cell water. 5. Cells equilibrated with xylose exhibited countertransport of this sugar against its concentration gradient when another substrate was added to the medium. 6. Blocking of glycolysis by iodoacetate or F(-) has no immediate effect on transport. The presence of a glucose-transport system in E. histolytica contrasts with the situation found in the non-parasitic amoeba, where pinocytosis seems to be the only mechanism of solute uptake.     

Chemotaxis by Entamoeba histolytica1

A micropore membrane procedure to assay taxis by Entamoeba histolytica is described and the results of studies of responses to a variety of soluble substances, bacteria, an rat colon washings using this procedure are reported. Trophozoites migrated in blind well chambers through 8-m?m pore size polycarbonate membranes but not nitrocellulose membranes up to 12 m?m pore size. Amoebae were attracted toward fresh axenic culture medium (TYI-S), an enzymatic hydrolysate of casein (Trypticase), and a partially purified preparation of N-acetylneuraminic acid from egg mucin, but not purified N-acetylneuraminateora variety of other low molecular weight metabolites. The response was verified as chemotaxis by checkerboard analysis. Amoebae migrated most dramatically toward suspensions of all of seven bacterial species tested, including motile and non-motile, gram-negative and gram-positive rods and cocci. This response was diminished when the bacteria concentration gradient was eliminated. The response to bacteria culture filtrates was less than 10% of that to bacterial suspensions. A response to clarified washings from the rat colon was detected; this was diminished but not eliminated by filter sterilization of the washings. We concluded that some soluble molecules, possibly of intermediate molecular size, whole bacteria, and both soluble and paniculate components of the rat colon provide tactic stimuli for E. histolytica. Scanning electron micrographs of trophozoites migrating towards attractants through membranes showed narrow', extended pseudopodia entering the membrane pores, and enlarging spheres exiting as the cells proceeded through.     

Thioredoxin-linked metabolism in Entamoeba histolytica

Entamoeba histolytica, an intestinal protozoan that is the causative agent of amoebiasis, is exposed to elevated amounts of highly toxic reactive oxygen species during tissue invasion. In this work, we report the molecular cloning, from E. histolytica genomic DNA, of the genes ehtrxr and ehtrx41, respectively coding for thioredoxin reductase (EhTRXR) and thioredoxin (EhTRX41). The genes were expressed in Escherichia coli cells, and the corresponding recombinant proteins were purified and characterized. EhTRXR catalyzed the NADPH (Km=4.5 microM)-dependent reduction of 5,5'-dithiobis-(2-nitrobenzoic) acid (Km=1.7 mM), EhTRX41 (Km=3.6 microM), and E. coli TRX (Km=4.6 microM). EhTRXR and EhTRX41 could be assayed as a functional redox pair that, together with peroxiredoxin, mediate the NADPH-dependent reduction of hydrogen peroxide and tert-butyl hydroperoxide. It is proposed that this detoxifying system could be operative in vivo. Results add value to the genome project information and advise reconsideration of key metabolic pathways operating in E. histolytica.