A modified Tanabe-Chiba medium for detection of Entamoeba histolytica

Tanabe-Chiba's (T-C) medium, which has been routinely used for detection of Entamoeba histolytica in feces in Japan, was modified by adding some commercially available enrichments to the liquid part and by eliminating the agar slant. This is tentatively called M medium. M medium and T-C medium were compared for their efficiency for detecting E. histolytica in the feces of monkeys. No significant difference in the detection rate was found between the two media. However the rate of multiplication of the amoebae in M medium was significantly higher than that in T-C medium within the limited time of 24-48 h of cultivation.     

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.     

An ion-channel forming protein produced by Entamoeba histolytica.

We have identified a remarkable ion-channel forming material in virulent strains of Entamoeba histolytica that may be responsible for many of the symptoms associated with amoebic dysentery. A polypeptide that we refer to as amoebapore is shed into the growth media and is also found within the amoeba in a high speed sedimentable fraction. Amoebapore has the distinctive property of spontaneously incorporating into lipid bilayers, liposomes, and cells, leading to progressive and irreversible changes in the ion conductance of the target membranes. Exposure of planar lipid bilayers to amoebapore -containing fractions under voltage clamp conditions results in an almost immediate and progressive incorporation of ion channels which continues in an irreversible manner leading to a fall in membrane impedance of up to five orders of magnitude. The ion-channel conductance is moderately cation-selective, voltage dependent, and displays a unit size of 1.6 +/- 0.2 nanoSiemens in 1 M KCl at -10 mV. In the bilayer, the amoebapore -induced conductance exhibits an in situ sensitivity to protease. Amoebapore is mainly concentrated in a fraction sedimenting at 150 000 g. It is insoluble in Triton X-100 but can be dissociated in an active state in 1% SDS. Under these conditions it has an apparent mol. wt. of 13 000 daltons.     

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.     

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)     

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.     

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.     

Structural changes on Entamoeba histolytica trophozoites after cryopreservation in liquid nitrogen.

Trophozoites of Entamoeba histolytica cultures which had been deep-frozen in the presence of 5% DMSO, along with untreated cells and cells treated with DMSO (5%), were examined for fine-structural changes. After deep-freezing in liquid nitrogen only a few amoebae exhibited normal nuclear and cytoplasmic structure. One frequently observed but unspecific finding pertaining to recovered cells is the separation of the cytoplasm into large vacuolated (coarse-granular) and electron-optically fine-granular (hyaline) zones. The glycogen which normally lies in the cytoplasm is always eluted. In many cases numerous short RNP helices are scattered unevenly in the vesicular plasma, but they are also found in larger masses adjacent to the membranes of still intact and already damaged nuclei. Moderately damaged nuclei have a poorly folded membrane and their chromatin is markedly denatured. More heavily damaged nuclei have a membrane which has partly fibrillated or ruptured and then formed conspicuous folds, where the nuclear membrane has ruptured nucleoplasmic remnants of chromatin and button-like bodies appear to pour into the surrounding cytoplasm. The final destruction of the cell is marked by coalescing autolytic zones, first in the vacuolated and later in the fine-granular cytoplasm. Finally only remnants of the nuclear membrane and of the membranes of numerous vacuoles remain. It is assumed that most of the changes in the cytoplasm are of a secondary nature and are caused by the early functional disturbance of the nucleus.     

Purine salvage in Entamoeba histolytica

Pulse-labeling of the nucleotide pool in Entamoeba histolytica with radioactive precursors, and subsequent high performance liquid chromatographic (HPLC) analysis of the radiolabeled nucleotides, indicate that E. histolytica is incapable of de novo synthesis of purine nucleotides. Hypoxanthine, inosine and xanthine could not be converted to nucleotides in E. histolytica, which suggests the absence of interconversion between adenine nucleotides and guanine nucleotides through formation of IMP. Adenosine was actively incorporated into nucleotides at an initial rate of 130 pmoles per minute per 10(6) trophozoites. Adenine, guanosine and guanine were also incorporated at much lower rates. The rate of adenine incorporation was enhanced by the presence of guanosine; the rate of guanine incorporation was significantly increased by adenosine. These stimulatory effects suggest that the ribose moiety of adenosine or guanosine can be transferred to another purine base to form a new nucleoside, and that the purine nucleosides are the immediate precursors of E. histolytica nucleotides. HPLC results showed that the radiolabel in adenine was exclusively incorporated into adenine nucleotides and that guanine was found only among guanine nucleotides, whereas the radioactivity associated with the ribose moiety of adenosine or guanosine was distributed among both adenine and guanine nucleotides.