Axenic Amoebae Research Articles

Interaction of antibodies with Entamoeba histolytica trophozoites from experimental amebic liver abscess: an immunocytochemical study.

Using immunocytochemical techniques, we studied the interaction of antibodies with Entamoeba histolytica trophozoites present during the development of amebic liver abscess. Hamsters were intrahepatically inoculated with HM1-IMSS axenic amebas and sacrificed at different days post-inoculation. IgG of rabbit anti-E. histolytica and IgG of rabbit anti-IgG of hamster were used, both labeled with peroxidase. With the rabbit anti-E. histolytica, all trophozoites present in hepatic lesions from 1-7 days post-inoculation were highly labeled. The IgG of rabbit anti-IgG of hamster intensively stained only those trophozoites present in lesions from 1-2 days post-inoculation. From day 3, the intensity and number of labeled trophozoites decreased progressively. The results suggest that the interaction between the amebas and the IgG of hamster is non-specific during the first 2 days. The absence of labeling in the chronic stages could be due to changes in the membrane antigens of the parasite or to alterations in the bloodstream around necrosis. Also, the anti-E. histolytica antibodies produced in the serum during the development of the hepatic disease are apparently incapable of reaching and interacting with the trophozoites present on the liver abscess. This can explain in part why antibodies do not have an important role in the defense of the host.

A new method of preservation of axenic amoebae of the genera Naegleria and Acanthamoeba

A new method of preservation of axenic strains of amoebae of the Naegleria and Acanthamoeba genera is described. The trophozoites are frozen in a solution with 2 cryoprotec-tors: fetal calf serum at 90% and dimethylsulphoxide at 10%. For 4 h and with a slow rate of cooling up to − 80 °C, one step is sufficient to reduce the temperature before long-term storage in liquid nitrogen. The ampules are quickly thawed at 40 °C and the medium is changed 4 h later. This technique gives confluent cells in 5 days and is quicker and more reliable than the methods previously described.

Zymodeme alteration of Entamoeba histolytica isolates under varying conditions

Cysts of Entamoeba histolytica obtained from 2 asymptomatic subjects were cloned in vitro and the isoenzyme patterns and virulence of the cultures derived from them were determined. Incubation in Diamond's TYI-S33 medium with Crithidia sp. for 80 d resulted in a change from zymodeme III to zymodeme II, with consistent virulence. Axenization by replacing Crithidia sp. with crithidial lysate had no further effect on the zymodeme but the virulence of the amoebae was attenuated. A reversal to zymodeme III, together with augmentation of virulence, was observed on the 20th day after transfer of axenic amoebae to Diamond's TYSGM-9 medium containing the original bacterial flora. The possibility that the isoenzyme pattern determining the zymodeme and the virulence behave as 2 independent variables in E. histolytica is discussed.

Modulation of a surface antigen of Entamoeba histolytica in response to bacteria

Changes in the cell surface of Entamoeba histolytica, a human intestinal parasite and the causative agent of amebic dysentery, were examined with a monoclonal antibody, 2D7.10, which selectively recognizes carbohydrate epitopes in some axenic amebic strains. While high-level expression of this epitope was observed in axenic amebae, it was either absent or present only in small amounts in xenic amebae. Furthermore, reassociation of the axenic amebae with intestinal flora resulted in loss of the 2D7.10 epitope. Our data suggest that surface antigens of E. histolytica can be modulated in response to bacteria and may provide an explanation for the observed influence of bacteria on amebic virulence.

Synthetic oligonucleotide probes for detection of mercury-resistance genes in environmental freshwater microbial communities in response to pollutants

Mercury-resistance genes were detected byin situ hybridization using new synthetic oligonucleotide probes specific formerA andmerB genes according to the published sequences of the corresponding enzymes. These DNA probes were used for the detection of specific mercury-resistant microorganisms isolated from the Rhine River which had been polluted 3 years previously in 1986. Mercuric reductase and organomercurial lyase genes persist in the bacterial genome even after the disappearance of the pollutant but are absent in axenic amoebae. A total of 49 bacterial isolates showed DNA homologies with the(32)P-labelled DNA probes and 15 free-living amoebae were selected due to their harboured symbiotic mercury-resistant bacteria.

Quantification of transformation efficiency using a new method for clonal growth and selection of axenic Dictyostelium cells

A new method for clonal growth of Dictyostelium axenic amoebae has been developed. Cells are plated in growth medium containing 1% ultra-low gelling temperature agarose. Cells grow normally in the agarose and form colonies up to several millimeters in diameter. When the colonies have grown to a sufficient size, they begin multicellular development. Pseudoplasmodia are formed, migrate to the surface of the agar, and then undergo fruiting body formation. Cells can be removed from the soft agarose colonies with a toothpick or by picking spores from the fruiting bodies. This method should be useful for drug, auxotrophic, and temperature selections where clonal maintenance of axenic colonies is important. This method has been used in combination with a selection for resistance to G418 to isolate independent colonies following DNA-mediated transformation. Several parameters in the calcium phosphate and electroporation transformation protocols have been optimized and the transformation frequency quantified. Independent transformed colonies are obtained at a frequency of 1 in 10(4) to 1 in 10(5) cells when integrating plasmids are introduced using calcium phosphate coprecipitation. The frequency is about tenfold higher when extrachromosomal shuttle vectors are introduced into cells.

The interaction of human neutrophils and Entamoeba histolytica increases cytopathogenicity for liver cell monolayers.

The in vitro interaction of neutrophils and virulent axenic Entamoeba histolytica trophozoites on Chang liver cells was studied to determine if the presence of neutrophils enhanced destruction of liver cell monolayers. Axenic amoebae (strain HM1:IMSS) destroy liver cell monolayers in a dose-dependent manner (P less than .001). Human neutrophils had no effect on the liver cell monolayers; however, when neutrophils were added to amoebae, destruction of monolayers was enhanced (P less than .05 compared with amoebae alone). Similar results were obtained when Chinese hamster ovary (CHO) cells were substituted for Chang cells (P less than .01). In this in vitro model, neutrophils were lysed by amoebae, as determined by release of 111Indium oxine from labeled neutrophils (P less than .001). The augmentation of cytopathogenicity for Chang cells was not inhibited by catalase (3,000 U/ml) and was observed with neutrophils from a patient with chronic granulomatous disease. N-Acetyl-D-galactosamine (45.0 mM) decreased monolayer destruction due to amoebae, with or without the presence of neutrophils (P less than .01). These studies establish that in vitro lysis of human neutrophils by E. histolytica enhances destruction of liver or CHO cell monolayers and that this effect is not due to release of neutrophil oxidative products.

Interaction of human leukocytes and Entamoeba histolytica. Killing of virulent amebae by the activated macrophage.

Capable effector mechanisms in the human immune response against the cytolytic, protozoan parasite Entamoeba histolytica have not been described. To identify a competent human effector cell, we studied the in vitro interactions of normal human polymorphonuclear neutrophils, peripheral blood mononuclear cells (PBMC), monocytes (MC), and MC-derived macrophages with virulent axenic amebae (strain HMI-IMSS). Amebae killed neutrophils, PBMC, MC, and MC-derived macrophages (P less than 0.001), without loss of parasite viability. The addition of heat-inactivated immune serum did not enable leukocytes to kill amebae, nor did it protect these host cells from amebae. MC-derived macrophages, activated with lymphokine elicited by the mitogens conconavalin A, phytohemagglutinin, or an amebic soluble protein preparation (strain HK9), killed 55% of amebae by 3 h in a trypan blue exclusion assay (P less than 0.001); during this time, 40% of the activated macrophages died. Lysis of amebae was confirmed using 111Indium oxine radiolabeled parasites and was antibody independent. Macrophage death appeared to be due to the deleterious effect of lysed amebae rather than the contact-dependent effector mechanisms of E. histolytica. Adherence between activated macrophages and amebae was greater than that between other leukocytes and amebae (P less than 0.001). Microscopic observations, kinetic analysis of the killing of amebae by activated macrophages, and suspension of amebae with adherent activated macrophages in a 10% dextran solution indicated that contact by activated macrophages was necessary to initiate the killing of amebae. Catalase but not superoxide dismutase inhibited the amebicidal capacity of activated macrophages (P less than 0.001). However, activated macrophages from an individual with chronic granulomatous disease were able to kill amebae, but not as effectively as normal cells (P less than 0.01). In summary, activated MC-derived macrophages killed virulent E. histolytica trophozoites through a contact-dependent, antibody-independent mechanism involving oxidative-dependent and -independent processes.

Entamoeba histolytica: Impedance measurements and cytotoxicity in the presence of bepridil, verapamil, and cytochalasin D

Entamoeba histolytica, and invasive enteric protozoa, kills mammalian target cells by sequential adherence and cytolytic events. Using platinum plate electrodes with an alternating current source placed in a Wheatstone bridge circuit, the impedance (resistance to ion flow) of a cell suspension of axenic amebae (strain HM1-IMSS) was measured. The impedance of the amebic cell suspension, expressed as resistivity (in ohm-cm), was significantly greater than the test solution and increased with decreasing temperature or greater cell packing (P less than 0.01), indicating that the resistivity measurements reflected the impedance of the amebic surface membrane. Cytochalasin D (10 micrograms/ml), a microfilament inhibitor which inhibited amebic in vitro adherence and cytolysis of target Chinese hamster ovary (CHO) cells (P less than 0.001), also increased resistivity of the amebic suspension (P less than 0.01). Exposure of amebae to bepridil (10(5) M), a slow-channel blocker, inhibited amebic killing of target cells (P less than 0.01) and also increased the resistivity of the amebic suspension (P less than 0.01), but both to a lesser degree than cytochalasin D (P less than 0.001). In contrast, exposure of amebae to verapamil followed by washing had no effect on amebic killing of target cells or resistivity of the amebic suspension. The increased resistivity measured in cytochalasin D or following exposure to bepridil was not due to a change in cell density of the amebic suspension. These studies indicate that changes in impedance of the amebic surface membrane are produced by bepridil and cytochalasin D. The effect of these agents on membrane impedance may contribute directly to the concurrent observed alteration in amebic cytopathogenic capacity or may serve as a parallel marker for the cell membrane alterations induced by such pharmacologic agents which inhibit amebic microfilament function or calcium flux.

Intestinal invasive amebiasis: an experimental model in rodents using axenic or monoxenic strains of Entamoeba histolytica.

A new experimental model for the production of early stages of invasive intestinal amebiasis in hamsters or guinea pigs with axenic or monoxenic cultures of Entamoeba histolytica of the HM1:IMSS strain is reported. The model is called the washed-closed cecal loop, because it involves the washing out of the cecum contents of conventionally raised animals and the formation of a closed loop, into which amebas are inoculated. Colonization occurred in all inoculated animals. After 48 hr, macroscopic ulcerations of the cecal mucosa were found in approximately one-half (5/12) of the animals inoculated with axenic amebas and in more than two-thirds (10/12) of those inoculated with monoxenic parasites. The amebic nature of the mucosal ulcerations was confirmed by light and transmission electron microscopy, as well as by scanning electron microscopy (SEM) of fractured samples of cecal mucosa. SEM provided a rapid and reliable technique to assess both the extent and the nature of the lesions. This model is particularly useful for the study of the pathogenesis of early intestinal lesions produced by virulent amebas, and may also be applied to experimental studies on the immunology of invasive intestinal amebiasis.

N-Acetyl-D-galactosamine-inhibitable adherence lectin of Entamoeba histolytica. II. Mitogenic activity for human lymphocytes.

A soluble protein preparation from virulent axenic amoebae (strain HM1-IMSS) has been demonstrated to be mitogenic for normal human lymphocytes; mitogenesis was optimal at 100 micrograms/ml when incubation was maintained for five to seven days with monocytes (P less than .001). After gel filtration chromatography mitogenic activity of fractions was associated with N-acetyl-D-galactosamine-inhibitable lectin activity (agglutination of Chinese hamster ovary cells; P less than .001). Lymphocyte proliferation induced by these active fractions was specifically inhibited by asialofetuin (P less than .001), which contains three terminal beta 1-4 linked galactose residues. In four strains of axenic amoebae mitogenic activity of soluble protein preparations correlated with in vitro virulence and lectin activity for Chinese hamster ovary cells. The amoebic N-acetyl-D-galactosamine-inhibitable lectin appears to be responsible for the mitogenic activity of soluble amoebic protein preparations; alteration of cell-mediated immunity by this amoebic moiety could potentiate in vivo virulence of the parasite.

N-Acetyl-D-galactosamine-inhibitable adherence lectin of Entamoeba histolytica. I. Partial purification and relation to amoebic virulence in vitro.

Adherence of axenic Entamoeba histolytica, strain HM1-IMSS, to Chinese hamster ovary (CHO) cells is mediated by an amoebic adhesin that is inhibited by N-acetyl-D-galactosamine (GalNAc). The in vitro virulence for CHO cells and human neutrophils (PMNs) of four strains of axenic amoebae was strain HM1-IMSS greater than H303-NIH = 200-NIH greater than nonvirulent Laredo (P less than .001). The HM1 strain had the greatest sensitivity to GalNAc-mediated inhibition of adherence (P less than .001). GalNAc (1.0 g/100 ml) inhibited the killing of CHO cells and PMNs by HM1 amoebae (P less than .001) and allowed PMNs to kill the amoebae (P less than .0047). Gel filtration chromatography of a soluble fraction of amoebic sonicate demonstrated a GalNAc-inhibitable amoebic lectin of 43,000-67,000 daltons that agglutinated CHO cells, erythrocytes, and PMNs. Strain HM1 contained greater specific lectin activity than did strains 303, 200, and Laredo amoebae (P less than .0016). The in vitro virulence of E. histolytica is associated with a GalNAc-inhibitable amoebic adhesin; this report is the first of a soluble GalNAc-inhibitable amoebic lectin.

Induced division synchrony in Entamoeba histolytica. Effects of hydroxyurea and serum deprivation.

Synchronous growth of axenic Entamoeba histolytica was induced by nutritional deprivation and/or hydroxyurea. Axenic amebae maintained in 2% serum culture medium for 17-25 hours and returned to complete TP-S-1 (10% serum) medium showed a single synchronous cycle with a mean division time of 8.8 hours and a synchrony index of 0.47. Exposure to 10 mM hydroxyurea for 20-26 hours induced a single synchronous cycle with a division time of 3.0 hours and a synchrony index of 0.85. Hydroxyurea was toxic to 56% of the cells in the culture. The control generation time in TP-S-1 medium was 14.8 hours. Cells maintained for 44 hours in a nucleotide-deficient medium followed by a 10 mM hydroxyurea treatment exhibited two synchronous cycles. The first cycle had a generation time of 5.0 hours, a division time of 2.2 hours, and a synchrony index of 0.84.

Pathogenicity of Axenically Cultivated Entamoeba histolytica, Strain 200: NIH, in the Hamster

Entamoeba histolytica strain 200:NIH cultured axenically for about 15 years produced lesions and abscesses in inbred adult hamsters following intrahepatic, intraperitoneal, and intradermal inoculation. These amebae, even in much higher doses, inoculated into the ceca of adult and young hamsters failed to produce lesions or to become established there. On the other hand, 200:NIH axenic amebae passaged through hamster liver (substrain 200:NIH-L) were able to become established and to produce lesions in the ceca of very young hamsters (1- and 3-week-old), the degree of pathogenicity appearing to be inversely proportional to age of the hamster.

Behavior of Axenic IP-106 Strain of Entamoeba Histolytica in the Golden Hamster

Strain IP-106 of Entamoeba histolytica was isolated 12 yr ago from a case of amebic dysentery and has been maintained in axenic culture for the past 11 yr. Hamsters were inoculated intrahepatically, intraperitoneally, and intracecally with 6.5, 5.0, and 8.0 X 10(4) axenic trophozoites, respectively. With the first two routes of inoculation all the animals developed liver abscesses, and most developed amebic metastases to other sites. After intracecal inoculation only 3 out of 6 animals developed abscesses at the primary site and metastases to other sites. Most numerous metastases were obtained following intrahepatic inoculation. Intrahepatic inoculation with 1 and 2 X 10(4) axenic amebae resulted in an increase in the size of the liver abscess for a period of 2 wk and its apparent total resorption by day 17 post-inoculation, accompanied by a corresponding gain of body weight. With 4 and 6 X 10(4) amebae there was a steady increase of the size of the liver abscess and corresponding loss of body weight. High antiamebic indirect hemagglutination titers were observed only on day 17 post infection in animals inoculated with 2, 4, and 6 X 10(4) amebae. The extent of amebic metastases was roughly proportional to the size of the inoculum.

An energy-conserving pyruvate-to-acetate pathway in Entamoeba histolytica. Pyruvate synthase and a new acetate thiokinase.

Under anaerobic conditions, cells of Entamoeba histolytica grown with bacteria produce H2 and acetate while cells grown axenically produce neither. Aerobically, acetate is produced and O2 is consumed by amebae from either type of cells. Centrifuged extracts, 2.4 x 106 x g x min, from both types of cells contain pyruvate synthase (EC 1.2.7.1) and an acetate thiokinase which, together, form a system capable of converting pyruvate to acetate. Pyruvate synthase catalyzes the reaction: pyruvate + CoA leads to CO2 + acetyl-CoA + 2E. Electron acceptors which function with this enzyme are FAD, FMN, riboflavin, ferredoxin, and methyl viologen, but not NAD or NADP. The amebal acetate thiokinase catalyzes the reaction acetyl-CoA + ADP + Pi leads to acetate + ATP + CoA. For this apparently new enzyme we suggest the trivial name acetyl-CoA-synthetase (ADP-forming). Extracts from axenic amebae do not contain hydrogenase, but extracts from cells grown with bacteria do. It is postulated that in bacteria-grown amebae electrons generated at the pyruvate synthase step are utilized anaerobically to produce H2 via the hydrogenase and that the acetyl-CoA is converted to acetate in an energy-conserving step catalyzed by amebal acetyl-CoA synthetase. Aerobically, cells grown under either regimen may utilize the energy-conserving pyruvate-to-acetate pathway since O2 then serves as the ultimate electron acceptor.

Virulence of bacteria-associated, Crithidia-associated, and axenic Entamoeba histolytica: Experimental hamster liver infections with strains from patients and carriers

Experimental infections of the hamster liver were carried out with five strains from patients with clinical amoebiasis and ten strains from asymptomatic carriers. Inocula of comparatively small size (12000-36000 amoebae) were injected under the liver capsule. 1. The virulence of the patient strains varied from 21-96% (see article) and declined sharply within 7-15 weeks after elimination of the associated bacterial flora. The virulence of the carrier strains varied from 0-100%, probably fluctuating with changes in the concomitant bacterial flora (Table 1). 2. The interrelation between size of inoculum, period of bacteria-free growth, and virulence was demonstrated with a Crithidia-associated patient strains (Table 2). 3. A patient strain showed a faster decrease of virulence during axenic than in Crithidia-associated cultivation (Table 3). 4. Two successive passages through hamster liver resulted in a marked increase of virulence of two bacteria-free strains, lasting for several months (Table 4). 5. A significant enhancement of virulence of Crithidia-associated and axenic amoebae by reassociation with a mixed bacterial flora during two weeks, followed by elimination of the bacteria, was demonstrated with two strains. The restored virulence was lost again within a few weeks (Table 5). 6. The virulence of an attenuated patient strain did not become manifest by adding large numbers of dead amoebae to the inoculum (Table 6). 7. The pathology of the different lesions caused in the hamster liver by the amoebae is described, including one of a granulomatous type, frequently found after inoculation with bacteria-free amoebae. 8. In an attempt to explain the occurrence of strains differing in pathogenicity an hypothesis is put forward based on the idea of selection of virulence and avirulent amoebae.

Monoxenic and axenic cultivation of carrier and patient strains of Entamoeba histolytica

All of five strains of Entamoeba histolytica, isolated from symptomatic cases of amoebiasis, could be adapted to axenic growth on the TP-S-1 medium of Diamond (1968). Four axenic strains were started from amoeba-Crithidia cultures; one could be axenized directly after isolation from a case of cutaneous amoebiasis. Attempts to monoxenize, resp. axenize strains, isolated from Dutch, asymptomatic carriers, were less successful. Only three out of ten strains could be submitted to bacteria-free growth. These three strains, however, originated probably from a recent case of intestinal amoebiasis. The results, suggesting that highly virulent strains can be easier cultivated bacteria-free than those with low or no virulence, are further discussed. The yield of axenic amoebae per tube fluctuates largely depending on many factors such as the strain, the number of transfers (i.e. degree of establishment), the quality of Panmede liver digest and serum in the TP-S-1 medium, and the care of manipulating the cultures. For optimal growth, a more acid medium was required in an amoeba-Crithidia culture than in an axenic culture. Multinucleated, giant amoebae were frequently observed in axenic cultures.

Results of Intracecal Inoculation of Germfree and Conventional Guinea Pigs and Germfree Rats with Axenically Cultivated Entamoeba histolytica

SYNOPSIS. Germfree and conventional guinea pigs and germfree rats were inoculated with large numbers of axenically cultivated Entamoeba histolytica. Amebic lesions were not found in any of the animals, and there was no indication that the ameba had established lumen infections in any instance. The axenic amebae appeared to have lost their pathogenicity. This loss was believed to depend, in part at least, upon the fact that they did not encyst and thus did not complete their life cycle in axenic culture.

Cytochemical comparison of distribution of proteins and lipids in axenically vs. monoxenically grown Entamoeba histolytica

Ninhydrin-Schiff (NS) method revealed a higher level of protein-bound amino acids in diffuse and particulate form in cytoplasm of 72-hr, axenically grown amebae as compared to 72-hr cells from monoxenic cultures. Younger (24-hr) monoxenically grown amebae had more such cytoplasmic NS-staining material than older cells. Nuclear NS-positive material in both axenic and monoxenic cells, which was restricted to the peripheral chromatin and endosome, was more intense than that in the cytoplasm. Tyrosine-containing proteins, as determined by Millon's reaction, were present in small amounts in the cytoplasm of both axenic and monoxenic amebae. As was true of the NS method, more intense staining was observed in the nuclei of both cell types. In contrast to the NS technic, however, the nuclear stain was evenly dispersed. Axenic 72-hr amebae had more intensely stained granular and diffuse sudanophilic material (stained with Sudan black B) in the cytoplasm than 72-hr monoxenic cells. Twenty-four-hour monoxenic cells had more sudanophilic inclusions than 48- and 72-hr amebae, primarily associated with large bodies. In some amebae (axenic and monoxenic) intense sudanophilic material was in close association with the nuclear membrane, rarely with the endosome. Cold acetone extracted all sudanophilic material from axenic amebae but left some lightly stained cytoplasmic inclusions in monoxenic cells, especially younger (24-hr) cells. Hot chloroform:methanol extracted all lipids from all cells. The phosphine 3R secondary fluorescence technic used to demonstrate neutral fats gave negative results with both cell types except for a faint reaction in young (24-hr) monoxenic cells. The amount of neutral fat detected was very low. Oil red O demonstrated only a faint diffuse and particulate reaction in 72-hr axenic and 72-hr monoxenic amebae. However, younger (24-, 48-hr) monoxenic amebae had more neutral fat (= oil red Ostaining material) than 72-hr cells. In some young cells (24-, 48-hr) oil red O-positive material was often in association with the nuclear membrane. Phospholipids could not be demonstrated in either cell type with Nile blue sulfate.