Effect of jasplakinolide on the growth, encystation, and actin cytoskeleton of Entamoeba histolytica and Entamoeba invadens

The effect of jasplakinolide. an actin-polymerizing and filament-stabilizing drug, on the growth, encystation, and actin cytoskeleton of Entamoeba histolytica and Entamoeba invadens was examined. Jasplakinolide inhibited the growth of E. histolytica strain HM-1:IMSS and E. invadens strain IP-1 in a concentration-dependent manner, the latter being more resistant to the drug. The inhibitory effect of jasplakinolide on the growth of E. histolytica trophozoites was reversed by removal of the drug after exposure to 1 microM for 1 day. Encystation of E. invadens as induced in vitro was also inhibited by jasplakinolide. Trophozoites exposed to jasplakinolide in encystation medium for 1 day did not encyst after removal of the drug, whereas those exposed to the drug in growth medium for 7 days did encyst without the drug. The process of cyst maturation was unaffected by jasplakinolide. Large round structures were formed in trophozoites of both amoebae grown with jasplakinolide; these were identified as F-actin aggregates by staining with fluorescent phalloidin. Accumulation in trophozoites of both amoebae of actin aggregates was observed after culture in jasplakinolide. Also, E. invadens cysts formed from trophozoites treated with jasplakinolide contained the actin aggregate. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis revealed that the jasplakinolide treatment led to an increase in the proportion of F-actin associated with formation of the aggregate. The results suggest that aggregates are formed from the cortical flow of F-actin filaments, and that these filaments would normally be depolymerized but are artificially stabilized by jasplakinolide binding.     

Colonic short-chain fatty acids inhibit encystation of Entamoeba invadens

Entamoeba parasites multiply as trophozoites in the layer of mucus that overlies the colonic epithelium. In response to stimuli that are not understood, trophozoites stop multiplying and differentiate into cysts that are released to infect another host. In the colon, Entamoeba trophozoites are exposed to the large variety of biochemicals that are carried into or are produced within this organ. The normal bacterial population of the colon releases large amounts of short-chain fatty acids (SCFAs). These compounds have effects on the growth, differentiation and repair of the colonic epithelium that correlate with de-creased activity of a Class I/II histone deacetylase (HDAC). We found that the formation of cysts, but not the growth of trophozoite-stage Entamoeba invadens parasites, was inhibited by physiologic concentrations of SCFAs. Variable levels of cyst formation did occur if SCFA concentrations were lowered. Specific inhibitors of Class I/II-type HDACs also prevented encystation, and trophozoites exposed to these compounds had increased levels of acetylation of histone H4 and other nuclear proteins. These results suggest that production of the infectious cyst stage of Entamoeba parasites is regulated in part by the levels of SCFAs made by the bacterial population of the colon.     

Differentiation of Entamoeba: a new medium and optimal conditions for axenic encystation of E. invadens.

Nutritional and culturing requirements for efficient axenic encystation of Entamoeba invadens have been studied. A simple and reliable axenic encystation medium has been developed. It contains 0.5% tryptic digest of casein, 0.5% yeast extract, and 5% dialyzed serum in 5 mM potassium phosphate, pH 7.0. Mass encystation (avg 70%) occurred within 30 hr when axenically growing trophozoites of E. invadens IP-l were transferred to this medium before they entered stationary growth phase. Mass encystation of E. invadens PZ occurred similarly, but less reproducibly. Two E. histolytica strains did not encyst. Experiments established that differentiation did not depend upon changes in the external environment after amebase were transferred to encystation medium and, therefore, was initiated by the shift from growth to encystation medium.     

Encystation in Entamoeba invadens

Knowledge of the biology and pathogenesis of the human parasite Entamoeba histolytica has been limited by the lack of efficient procedures to induce axenic encystation in the laboratory. However, such methods have been developed for E. invadens, a reptilian parasite, for which encystation can be induced by mechanisms that are as yet poorly defined. This has allowed the analysis of some morphological, physiological and biochemical events that accompany differentiation into cysts. Elucidation of these changes will lead to a better understanding of the process and therefore to the possibility of controlling it. Here, Everardo López-Romero and Julio Cézar Villagómez-Castro emphasize the metabolism of cyst-wall polymers as a potential target to inhibit cyst formation with specific drugs that would be in principle, harmless to the host.     

Entamoeba invadens: in vitro axenic encystation with a serum substitute

The current media for axenic cultivation of Entamoeba histolytica and Entamoeba invadens are supplemented with bovine or equine serum, which provides several essential nutrients to amoebas. Serum has also been considered an essential component in encystation media for E. invadens. A substitute of serum, PACSR has been described as an alternative for growth of E. histolytica and also maintains growth of E. invadens. When PACSR was used instead of serum for encystation of E. invadens the efficiency was the same as for serum. Our present data show that PACSR can support the growth and induction of encystation of E. invadens strain IP-1.     

High-resolution electron microscopical study of cyst walls of Entamoeba spp

Knowledge of the fine structural organization, molecular composition and permeability properties of the cell surface of intestinal protozoan cysts is important to understand the biologic basis of their resistance. Recent studies on the biology of the cyst walls of Entamoeba histolytica and Entamoeba invadens have considerably advanced knowledge on the cellular processes involved in the transport and surface deposition of the main cyst wall components. Using transmission electron microscopy, cytochemistry, scanning electron microscopy and freeze-fracture techniques, we have obtained new information. In mature cysts the permeability of Entamoeba cysts is limited to small molecules not by the cyst wall, but by the plasma membrane, as demonstrated with the use of ruthenium red as an electron-dense tracer. Cell walls of E. histolytica cysts are made up of five to seven layers of unordered fibrils 7-8 nm thick. Alcian blue stains a regular mesh of fibrils approximately 4 nm thick, running perpendicularly to the cyst wall. In addition, abundant ionogenic groups are seen in cyst walls treated with cationized ferritin. In the mature cysts of E. histolytica and E. invadens small cytoplasmic vesicles with granular material were in close contact with the plasma membrane, suggesting a process of fusion and deposition of granular material to the cell wall. The plasma membrane of mature cysts is devoid of intramembrane particles when analyzed with the freeze-fracture technique. When viewed with scanning electron microscopy the surface of E. histolytica cysts clearly differs from that of Entamoeba coli and E. invadens.     

The enteric parasite Entamoeba uses an autocrine catecholamine system during differentiation into the infectious cyst stage.

Enteric amoebae of the genus Entamoeba travel from host to host in an encysted form. We previously showed that in vitro cyst development of Entamoeba invadens requires the addition of defined amounts of multivalent galactose-terminated molecules, such as mucin, to the cultures. The amoeba surface lectin that binds mucin is presumed to convey transmembrane signals when clustered by the ligand, but the signaling molecules that function downstream of the lectin are not known. We report here that Entamoeba encystation was induced in the absence of galactose ligand when catecholamines were added to the encystation medium. Micromolar amounts of both epinephrine and norepinephrine induced encystation. Of a variety of synthetic catecholamine agonists tested, only beta(1)-adrenergic receptor agonists supported encystation, whereas alpha- and beta(2)-adrenergic receptor agonists did not. Only beta(1)-adrenergic receptor antagonists inhibited encystation, and did so even when exogenous catecholamines were not added, indicating that catecholamine binding is required for encystation and suggesting an endogenous source of the ligand. High performance liquid chromatography analysis of Entamoeba extracts showed that the amoebae themselves contain catecholamines and at least one of these is released when the cells are stimulated to encyst with galactose-terminated ligands. The presence of catecholamine binding sites on the surface of amoeba trophozoites was confirmed using radiolabeled catecholamine antagonist. Amoeba encystment was inhibited by addition of beta(1)-adrenergic receptor antagonist to cells that were stimulated to differentiate with either galactose ligand or catecholamines, but not with dibutyryl cAMP. This suggests that the amoeba catecholamine receptor functions downstream of the galactose lectin and upstream of adenylyl cyclase. This enteric protozoan parasite, therefore, contains the components of an autocrine catecholamine ligand-receptor system that may act in conjunction with a galactose lectin to regulate differentiation into the infectious cyst stage.     

Roles for the galactose-/N-acetylgalactosamine-binding lectin of Entamoeba in parasite virulence and differentiation

Entamoeba histolytica, an intestinal protozoan parasite, is a major cause of morbidity and mortality in developing countries. The pathology of the disease is caused by the colonization of the large intestine by the amoebic trophozoites and the invasion of the intestinal epithelium. Some of the trophozoites will eventually differentiate into the infectious cyst form, allowing them to be transmitted out of the bowel and into water supplies to be passed from person to person. Both the virulence of the organism and the differentiation process relies on a galactose-/N-acetylgalactosamine (GalNAc)-binding lectin that is expressed on the surface of trophozoites. The functional activity of this lectin has been shown to be involved in host cell binding, cytotoxicity, complement resistance, induction of encystation, and generation of the cyst wall. The role of the lectin in both differentiation and virulence suggests that it may be a pivotal molecule that determines the severity of the infection from a commensal state resulting from increased encystation to an invasive state. The lectin-glycan interactions that initiate these diverse processes are discussed with emphasis on comparing the binding of host ligands and the interactions involved in encystation.     

Entamoeba invadens: the requirement for galactose ligands during encystment

During periods of stress, trophozoites of Entamoeba invadens (strain IP-1) undergo a process of differentiation (encystment) that results in a dormant cyst with a chitin-containing cyst wall. Encystment can be induced by resuspension of trophozoites from growth medium into a diluted glucose-free medium (47% LG) containing 5% adult bovine serum (ABS). ABS is thought to be a source of gal-terminated ligands that are required for high levels of encystment. After resuspension of trophozoites in 47% LG, encystment cultures were examined every 2h for responses to the (i) addition of 10mM free-galactose, (ii) resuspension of cells to serum-free medium, (iii) and dilution of encysting cultures to cell densities below that known to support full encystment (from 5 x 10(5) to 1 x 10(4)cells/ml). The role of serum components (and the gal-terminated ligand asialofetuin; ASF) adsorbed onto the surface upon which encystment proceeds, and their effect on the multi-cellular aggregation patterns formed during encystment, were also investigated. The addition of free-galactose reduced the levels of encystment (compared with the control) even when added at 10h after resuspension of trophozoites in 47% LG. The requirement for the presence of ABS during encystment was lost within 6h, with levels of encystment of cells washed free of serum reaching 80% of the control. The ability of cells to encyst when diluted to a cell density below that normally thought to support encystment reached over 50% by 8h. Efficient encystment could be obtained in 47% LG in the absence of ABS or ASF using pre-treated glass culture tubes. Encystment (47% LG; 5% ABS) using ultra low attachment plates was poor, suggesting attachment of cells to a surface via gal-terminated ligands was important for efficient encystment. The results suggest that ABS is probably not the only source of gal-terminated ligands necessary for high levels of encystment in 47% LG. While serum may provide a source of ligands which enhance the levels of encystment initially, other gal-terminated ligands possibly released by the encysting cells are still required for the completion of the encystment process and the formation of mature cysts. In addition, the gal-terminated ligands necessary for encystment efficiency may be adsorbed onto the glass surface of culture tubes and aid the initial aggregation process, as well as be involved in cell signaling during the encystment process.     

Mitosomes in trophozoites and cysts of the reptilian parasite Entamoeba invadens

Heat shock protein genes led to the discovery of mitosomes in Entamoeba histolytica, but mitosomes have not been described for any other Entamoeba species, nor have they been identified in the cyst stage. Here, we show that the distantly related reptilian pathogen Entamoeba invadens contains mitosomes, in both trophozoites and cysts, suggesting all Entamoeba species contain these organelles.     

Responses of Entamoeba invadens to heat shock and encystation are related

An Entamoeba invadens gene encoding a homologue of BiP/GRP78, a 70-kDa heat shock protein or chaperonin was cloned. The predicted E. invadens BiP contained an ATP-binding site, a substrate-recognition domain, and a carboxy-terminal KDEL-peptide. Messenger RNAs of E. invadens for BiP, for a 70-kDa heat shock cognate, for a cyst wall glycoprotein (Jacob), and for chitinase were all induced by heat shock and by encystation medium. The presence of Jacob in heat-shocked amebae was confirmed by confocal microscopy and suggests that heat shock and encystation responses in E. invadens are related.     

Actin mRNA Levels and Actin Synthesis During the Encystation of Entamoeba invadens

ABSTRACT. Parasitic amebas propagate among hosts through cysts, the resistant forms in their life cycle. In spite of their key role in infection, little is known about the encystation process and the mechanisms involved in reaching this stage. Two features drastically affected by encystation are motility and cell shape, both of which are determined by the cytoskeleton, composed mainly of actin in these organisms. Therefore, we studied the occurrence and relative levels of actin and actin synthesis during encystation of Entamoeba invadens. Using a cDNA actin probe obtained from a library of E. histolytica and a monoclonal antibody against actin, we found that, while the total actin levels sharply decrease as encystation proceeds, the levels of actin mRNA are reduced only in mature cysts. Moreover, actin synthesis does not take place in precysts and the later stages of cyst formation. In contrast, the levels of other proteins remain stable in trophozoites, precysts and cysts, and stage specific peptides are actively synthesized in precysts. The results indicate that encystation is accompanied by a preferential down-regulation of actin synthesis and a decrease in actin levels. The reorganization of the cytoskeleton occurring as trophozoites transform into round, quiescent cells, could be a regulatory factor in the observed changes.     

Entamoeba histolytica: ultrastructure of trophozoites recovered from experimental liver lesions

Ultrastructural studies on Entamoeba histolytica have been carried out mostly with trophozoites cultured for many years. Under these conditions, the availability of nutrients and the absence of environmental stimuli may switch off some phenotypic characteristics of the parasite. As a result, virulence of E. histolytica diminishes with prolonged culture passages, and the ability to form cysts disappears in axenically maintained trophozoites. The present analysis by transmission electron microscopy of trophozoites recovered from experimental amebic liver lesions in hamsters revealed two types of cytoplasmic changes. On the one hand, the number of peripheral electron dense granules significantly increased in amebas obtained from liver lesions 15 min and 6h after inoculation. On the other hand, large cytoplasmic vesicles with a microfibrillar content appeared in trophozoites cultured from 72 or 96 h hepatic lesions. With fluorescence microscopy, a chitin-like material was identified in these vesicles by reactivity with calcofluor M2R. Ultrastructurally, these cytoplasmic components resemble the encystation vesicles of Entamoeba invadens and Giardia lamblia. The release of large amounts of electron dense granules, known to contain collagenase activity, probably contributes to degrade extracellular matrix components during tissue invasion. In addition, under the conditions mentioned above, amebas form encystation-like vesicles in an incomplete process of differentiation into cysts, which are the resistant form of the parasite.     

Naegleria fowleri: enolase is expressed during cyst differentiation

Cysts of Naegleria fowleri present an external single-layered cyst wall. To date, little information exists on the biochemical components of this cyst wall. Knowledge of the cyst wall composition is important to understand its resistance capacity under adverse environmental conditions. We have used of a monoclonal antibody (B4F2 mAb) that specifically recognizes enolase in the cyst wall of Entamoeba invadens. By Western blot assays this antibody recognized in soluble extracts of N. fowleri cysts a 48-kDa protein with similar molecular weight to the enolase reported in E. invadens cysts. Immunofluorescence with the B4F2 mAb revealed positive cytoplasmic vesicles in encysting amebas, as well as a positive reaction at the cell wall of mature cysts. Immunoelectron microscopy using the same monoclonal antibody confirmed the presence of enolase in the cell wall of N. fowleri cysts and in cytoplasmic vesicular structures. In addition, the B4F2 mAb had a clear inhibitory effect on encystation of N. fowleri.     

Inhibition of excystation and metacystic development of Entamoeba invadens by the dinitroaniline herbicide oryzalin

The effect of oryzalin on excystation and metacystic development of Entamoeba invadens strain IP-1 was examined by transfer of cysts to a growth medium containing the drug. Excystation, which was assessed by counting the number of metacystic amoebae after induction of excystation, was inhibited by oryzalin in a concentration-dependent manner. Metacystic development, which was determined by the number of nuclei in metacystic amoebae, was also inhibited by oryzalin because the percentage of 4-nucleate amoebae at day 1 remained unchanged at day 3. The addition of oryzalin after the induction of excystation decreased the number of metacystic amoebae, compared with control cultures. When cysts were incubated for 1 day in growth medium plus oryzalin, little increase in the number of metacystic amoebae was observed after removal of the drug. Excystation and metacystic development were further inhibited when the cysts were incubated for 30 min in encystation medium containing oryzalin before transfer to growth medium with the drug. When cysts were incubated for 30 min in encystation medium before transfer to growth medium without the drug, metacystic amoebae decreased in number. Pretreatment of cysts with oryzalin for 30 min in phosphate-buffered saline markedly reduced viability and prevented excystation in growth medium with or without the drug. The results indicate that oryzalin inhibits excystation and metacystic development of E. invadens, suggesting that it may be an inhibitor of Entamoeba infection.     

Encystation of Entamoeba invadens IP-1 is induced by lowering the osmotic pressure and depletion of nutrients from the medium

Trophozoites of Entamoeba invadens IP-1 can be induced to encyst in simple solutions composed of semipermeable constituents (buffer, salts, or sugars) provided that their osmotic pressure is in the range of 60-160 mosmol/kg. Optimal yield of mature cysts was obtained when the osmotic pressure of the medium was 110 mosmol/kg. Encystation could be obtained in the absence of serum although higher yields were obtained in its presence. No difference in the yield of mature cysts was found when either dialyzed or full serum was used. High yields of encystation were obtained (greater than 70%) in the presence of 5% serum in solutions of NaCl, KCl, or MgSO4, suggesting that the mechanism of encystation is not induced via sodium or potassium channels. Cysts were obtained in the presence of 72 mM glucose, indicating that depletion of a carbon source is not the only requirement for encystation. A rapid change in the density of the Entamoeba cells was observed upon transfer of trophozoites (density 1.061-1.073 g/ml) from growth medium to the low osmotic pressure encystation solutions. Within the first 2 min their density decreased (to 1.050 g/ml), but it soon increased, reaching within 30 min a density higher than 1.120 g/ml. As the encystation process continued to completion, the density of the cells gradually decreased, the mature cysts reaching a density of 1.049-1.061 g/ml.