Entamoeba histolytica and E. invadens: sulfhydryl-dependent proteolytic activity

Sodium dodecyl sulfate (SDS) and 2-mercaptoethanol (2-ME) activated proteolytic enzymes present in extracts of Entamoeba histolytica and E. invadens; SDS (0.5%) and 2-ME (1.4 and 715 mM) doubled the enzymatic activity when assayed on a stained insoluble substrate. Urea (4 M) did not reduce this activity, suggesting that amebic proteases are stable in the above denaturant conditions. Specific reagents for sulfhydryl (-SH) groups completely inhibited proteolytic activity regardless of pH. Inhibition with alkylating agents, such as N-ethylmaleimide and iodoacetamide, was reversed with 715 mM 2-ME as was also observed with papain. We conclude from these results that the main proteolytic enzymes contained in extracts of E. histolytica and E. invadens are dependent on free thiol groups.     

Pathogenic and Non-pathogenic Entamoeba: Pore Formation and Hemolytic Activity1

Pore-forming activity in planar lipid bilayers and liposomes of extracts from differentially pathogenic Entamoeba and the-capacity of trophozoites and subcellular fractions to lyse human red blood cells (hrbc) were investigated. In all amebas studied, the two activities paralleled each other. They were high in E. histolytica irrespective of the virulence of the particular strain, but low in non-pathogenic E. histolytica-like amebas of human origin as well as in E. invadens, which is pathogenic for reptiles, and in E. moshkovskii isolated from sewage. We conclude that the capacities to insert pores and to lyse are not sufficient for virulence although they may be necessary. The subcellular distribution of the hemolytic activity of E. histolytica and its sensitivity to a variety of inhibitors and activators differ from those of other known amebic cytotoxic activities including pore formation. Therefore, there may be an additional constituent of E. histolytica involved in the cytotoxicity of the parasite.     

Differentiation of Entamoeba histolytica: a possible role for enolase

The study of the encystation process of Entamoeba histolytica has been hampered by the lack of experimental means of inducing mature cysts in vitro. Previously we have found that cytoplasmic vesicles similar to the encystation vesicles of Entamoeba invadens are present in E. histolytica trophozoites only in amebas recovered from experimental amebic liver abscesses. Here we report that a monoclonal antibody (B4F2) that recognizes the cyst wall of E. invadens also identifies a 48 kDa protein in vesicles of E. histolytica trophozoites recovered from hepatic lesions. This protein is less expressed in trophozoites continuously cultured in axenical conditions. As previously reported for E. invadens, the B4F2 specific antigen was identified as enolase in liver-recovered E. histolytica, by two-dimensional electrophoresis, Western blot and mass spectrometry. In addition, the E. histolytica enolase mRNA was detected by RT PCR. The antigen was localized by immunoelectron microscopy in cytoplasmic vesicles of liver-recovered amebas. The B4F2 antibody also recognized the wall of mature E. histolytica cysts obtained from human samples. These results suggest that the enolase-containing vesicles are produced by E. histolytica amebas, when placed in the unfavorable liver environment that could be interpreted as an attempt to initiate the encystation process.     

Serological Comparison of Selected Parasitic and Free-Living Amoebae in vitro,Using Diffusion-Precipitation and Fluorescent-Antibody Technics*

SYNOPSIS. The present study has been directed to a serological comparison of three closely similar species of Entamoeba: E. histolytica, E. invadens and E. moshkovskii, and two free-living soil amoebae: Hartmannella rhysodes and Mayorella palestinensis. Except for E. histolytica and E. moshkovskii, the other amoebae used here were grown axenically; this is the first report of the use of antigenic extracts from axenic cultures of parasitic amoebae. The method described here provides a potent antigen that elicits a good antibody titer and is generally applicable to both parasitic and free-living amoebae. Amoebae pooled from well-grown cultures were thoroughly washed, sonicated and mixed with Freund's Adjuvant; this antigenic preparation was injected into rabbits. Two subcutaneous injections were given at three-week intervals, and 2-3 weeks thereafter blood was withdrawn to obtain antiserum. Agar-gel diffusion, cellulose acetate paper and fluorescent antibody technics were used to test the antigen-antibody (Ag-Ab) reactions. Results of the Ag-Ab reactions can be summarized as follows: 1) The homologous Ag-Ab reaction was obtained in all cases tested. 2) There was no serological reaction between the parasitic and free-living amoebae tested. 3) There was a definite serological reaction between H. rhysodes and M. palestinensis. 4) Multiple antigens were found in E. invadens (PZ strain) and E. histolytica (DKB strain) when they were tested against anti-PZ serum and anti-DKB serum, respectively, and no reaction was found when the other test antigens were exposed to these two antisera in gel-diffusion tests. 5) With the fluorescent antibody technic, E. histolytica (Laredo strain), E. moshkovskii (DSR strain) and E. histolytica (DKB strain) showed some degree of serological reaction in descending order when they were stained with conjugated anti-E. invadens serum.     

Species-specific and cross-reactive antigens of Entamoeba

Specific and cross-reactive antigens were defined in four species of Entamoeba: invadens, moshkovskii, Laredo and histolytica strains HM1, HM3, HM38 and HK9. Among these species extensive common reactivities were observed by immunoblot. Eight E. histolytica antigenic markers were revealed after blocking common specificities with antigens of other Entamoeba species. A monoclonal antibody (mAb) defined two protein markers of E. histolytica, M, 29 and 25 kDa. The four strains of E. histolytica, which varied in virulence as determined by the development of liver abscesses in hamsters, showed the same antigenic patterns with the mAb and with polyclonal antibodies.     

Localisation of thiamine pyrophosphatase within the cytoplasmic fine structure of trophozoites of Entamoeba histolytica and E. invadens

McCaul T.F. and Bird R.G. 1978. Localisation of thiamine pyrophosphatase within the cytoplasmic fine structure of trophozoites of Entamoeba histolytica and E. invadens. International Journal for Parasitology8: 501–506. The distribution of thiamine pyrophosphatase (TPPase) activity was studied in both formaldehyde and glutaraldehyde fixed trophozoites of Entamoeba histolytica and E. invadens. The activity was localised within certain vacuoles. No dense deposits for TPPase activity were seen in the small vesicles, elongated smooth-walled lacunae equated with endoplasmic reticulum, or the nucleus. The demonstration of small vesicles surrounding the larger vacuoles indicated that the Golgi-like vacuoles might be involved in the production of cell coat materials and primary lysosomes.     

Influence of Heterologous Transplant of DNA‐lacking Mitochondria from Entamoeba histolytica on Proliferation of Entamoeba invadens

In mitochondria, compatibility of proteins encoded in mitochondrial DNA and nuclear DNA is essential for the normal functioning of the organelle. Incompatibility between mitochondrial and nuclear DNA can lead to dysfunctional respiration, mitochondrial diseases, and lethal problems, which suggests that the presence of heterologous mitochondria is unfavorable. In a previous study, we established a transplant method for DNA‐lacking mitochondria (mitosomes) in the anaerobic protozoan Entamoeba histolytica. In this study, interspecies transplant of mitosomes from E. histolytica into Entamoeba invadens, which is a parasitic protozoon of reptiles, was performed using the microinjection method at various temperatures and injection volumes. When E. invadens was used as recipient, it showed higher tolerance to a lower temperature and larger injection volume, in comparison with E. histolytica. After microinjection, donor mitosomes expressing HA‐tag conjugated protein were observed in recipient cells by immunofluorescent staining. The heterologous mitosomes‐injected cells proliferated and growth rate of the microinjected‐cells was similar to that of intact cells. Therefore, we conclude that interspecies transplant of DNA‐lacking mitochondria does not result in incompatibility.     

Entamoeba histolytica and Entamoeba invadens: Effects of temperature and oxygen tension on growth and survival

The temperature ranges for axenic growth of Entamoeba histolytica strain HM-1 and Entamoeba invadens strain 165 clone II in TYI-S-33 medium were: 32 to 40 C for E. histolytica with an optimum of 35.5 to 37 C; whereas the range for E. invadens 165 II was 20 to 30 C, optimum 24 to 27 C. Growth of strain HM-1 at 40 C was dependent upon the cell density of the culture used as a source of the inoculum. Clonal growth in agar was used to assay survival of Entamoeba spp. trophozoites after exposure to deleterious physical conditions. The lowest temperature for thermal killing of E. histolytica HM-1 was 41.5 C and for E. invadens 165, 35.5 C. Both were killed rapidly at 42 C, but slowly at 0 C. Killing of E. histolytica HM-1 trophozoites by exposure to increased oxygen tensions was a function of temperature and time. At 24 C and 35.5 C, there was little loss of viability during the first 7 hr exposure, then killing was quite rapid. The cells were killed sooner if the medium was preexposed to air. In contrast, at 0 C there was less killing by oxygen. E. invadens 165 II appeared to be more oxygen sensitive at 24 than at 0 C. Other E. histolytica strains tested were similar to HM-1 in their responses to temperature and air.     

Conservation and function of Rab small GTPases in Entamoeba: Annotation of E. invadens Rab and its use for the understanding of Entamoeba biology

Entamoeba invadens is a reptilian enteric protozoan parasite closely related to the human pathogen Entamoeba histolytica and a good model organism of encystation. To understand the molecular mechanism of vesicular trafficking involved in the encystation of Entamoeba, we examined the conservation of Rab small GTPases between the two species. E. invadens has over 100 Rab genes, similar to E. histolytica. Most of the Rab subfamilies are conserved between the two species, while a number of species-specific Rabs are also present. We annotated all E. invadens Rabs according to the previous nomenclature [Saito-Nakano, Y., Loftus, B.J., Hall, N., Nozaki, T., 2005. The diversity of Rab GTPases in Entamoeba histolytica. Experimental Parasitology 110, 244-252]. Comparative genomic analysis suggested that the fundamental vesicular traffic machinery is well conserved, while there are species-specific protein transport mechanisms. We also reviewed the function of Rabs in Entamoeba, and proposed the use of the annotation of E. invadens Rab genes to understand the ubiquitous importance of Rab-mediated membrane trafficking during important biological processes including differentiation in Entamoeba.     

Entamoeba invadens: Identification of ADF/cofilin and their expression analysis in relation to encystation and excystation

The differentiation processes of excystation and encystation of Entamoeba are essential for infection and completion of their life-cycle, and the processes need cell motility and its control by actin cytoskeletal reorganization. This study investigated actin depolymerizing factor (ADF)/cofilin (Cfl) family proteins, which are important molecules in actin cytoskeletal reorganization, in Entamoeba invadens in relation to the encystation and excystation. Axenic culture systems were used to induce encystation and excystation. A homology search of the E. invadens genome database and molecular cloning identified three ADF/Cfl family proteins of the parasite (named for short as EiCfl-1, EiCfl-2, and EiCfl-3). This is different from other Entamoeba species, i.e. Entamoeba histolytica and Entamoeba dispar, each of which has only one ADF/Cfl family protein. These ADF/Cfl of E. invadens do not have Ser3 (serine locates third from first methionine), similar to E. histolytica, E. dispar, Saccharomyces cerevisiae and Schizosaccharomyces pombe, although the activity of ADF/Cfl is negatively regulated by phosphorylation of the Ser3 in metazoans. Phylogenetic analysis revealed that Entamoeba Cfl formed a distinctive clade that is separate from other organisms, and the branches of the tree were separated in two consistent with the presence and absence of Ser3. Rabbit anti-EiCfl-2 serum reacted with all recombinant EiCfls and EiCfl in lysates of cysts, trophozoites and metacystic amoebae. Immunofluorescence staining with this antiserum showed co-localization of EiCfl with actin beneath the cell membrane through the life stages. Both proteins proved to be rich in pseudopodia of trophozoites and metacystic amoebae. Real-time RT-PCR showed that mRNAs of EiCfl-2 and actins were highly expressed, but there were few mRNA of EiCfl-1 and EiCfl-3. Remarkably decreased mRNA levels were observed in EiCfl-2 and actins during encystation. All three EiCfls and actins became transcribed after the induction of excystation. The mRNAs of only EiCfl-1 and EiCfl-3 increased remarkably when the excystation was induced in the presence of cytochalasin D. These findings demonstrate that EiCfl-2 and actins co-localize beneath the cell membrane in trophozoites and cysts as well as metacystic amoebae being rich in pseudopodia, that EiCfl-1 and EiCfl-3 are expressed only after the induction of excystation, and that enhanced excystation by cytochalasin D is associated with high expression of EiCfl-1 and EiCfl-3.     

Incorporation and Toxicity of 32P-Orthophosphate and Occurrence of Poly phosphate in Entamoeba Trophozoites1

We explored the requirements of inorganic phosphate (Pi), the incorporation of ³²P-orthophosphate (³²Pi), and the occurrence of inorganic polyphosphate (polyP) in axenic Entamoeba cultures. Maximal population densities and growth rates of Entamoeba histolytica trophozoites were attained in complete TP-S-1 medium. As ³²Pi concentration was increased in the medium, its own incorporation and the culture growth rate were progressively inhibited, especially in Pi-deficient medium. PolyP grains were found in the cytoplasm and occasionally in the nuclear membrane of E. histolytica, E. histolytica-like, E. invadens, and E. moshkovskii trophozoites.     

Electrophoretic Isoenzyme Patterns of Entamoeba histolytica and Entamoeba chattoni in a Primate Survey1

Stocks of Entamoeba histolytica grown in a monoxenic culture system from the feces of nonhuman primates are compared with the eleven zymodemes of E. histolytica so far demonstrated from man. In a similar fashion, Entamoeba chattoni has also been grown and identified. Both E. histolytica and E. chattoni have been demonstrated in keepers of the primate collections. Comparisons have been made using the electrophoretic patterns of three enzymes: glucosephosphate isomerase [(GPI) E.C.5.3.1.9], phosphoglucomutase [(PGM) E.C.2.7.5.1], and L-malate—NADP⁺ oxidoreductase (oxaloacetate-decarboxylating) [(ME) E.C. 1.1.1.40]. Enzyme patterns of E. histolytica from the apes were found to be identical with three of those already demonstrated from man. The enzyme pattern of E. chattoni was distinctly different from that of any of the E. histolytica zymodemes. Other protozoa found in the single fecal sample examined from each subject are also listed.     

Characterization of alkaline proteases from a novel alkali-tolerant bacterium Bacillus patagoniensis

Summary The extracellular proteolytic activity produced by a moderately alkaliphilic bacterium, Bacillus patagoniensis PAT 05^T, was characterized. This strain, grown in a highly alkaline and saline medium, produced important levels of proteolytic activity. SDS-PAGE and zymogram analyses revealed two proteolytic active bands. Through isoelectricfocusing (IEF)-zymogram, an active band with alkaline pI and two slighter active bands with acid pI values were detected. The alkaline active enzyme in the IEF was purified and characterized. It showed a molecular mass of 29.4 kDa and its pI value was >‰10.3. Proteolytic activity of the culture supernatant showed an optimal temperature of approximately 60 °C and a plateau of maximum activity between pH 9.0 and 12.0. Such activity was not affected by H₂O₂ (10% v/v), 1,10-phenanthroline (10 mM), Triton X-100 (1% v/v) and Tween 20 (1% v/v), under the assay conditions. More than 80% of the activity was retained in 10 mM EDTA, 73% in 1 % (w/v) SDS and 63% in 2 M NaCl. The enzyme was inhibited by PMSF, indicating serine-protease activity. The proteolytic activity of the crude supernatant was thermosensitive with a half-life of 2.3 min at 70 °C, while high activity was detected at moderate temperatures. Considering PAT 05^T proteolytic activity characteristics, such as high optimum pH, high stability and residual activity in presence of oxidant, surfactant and chelating agents, this strain could be a potential source of enzymes for use as additives in detergent formulations or in the leather industry.     

Entamoeba histolytica: Effect on virulence, growth and gene expression in response to monoxenic culture with Escherichia coli 055

Monoxenic cultivation of pathogenic Entamoeba histolytica trophozoites with Escherichia coli serotype 055 which binds strongly to the Gal/GalNAc amoebic lectin, markedly improved the growth of E. histolytica and produced a significant decrease in cysteine proteinase activity and a lower cytopathic activity on monolayer cells after 3 months of monoxenic culture. However, after long term monoxenic culture (12 months) the proteolytic and cytopathic activities were recovered and the amoebic growth reached the maximum yield. Employing the GeneFishing^R technology and DNA macroarrays we detected differentially gene expression related to the amoebic interaction with bacteria. A number of differentially expressed genes encoding metabolic enzymes, ribosomal proteins, virulence factors and proteins related with cytoskeletal and vesicle trafficking were found. These results suggest that E. coli 055 has a nutritional role that strongly supports the amoebic growth, and is also able to modulate some biological activities related with amoebic virulence.     

Characterization of rat lymphocyte primary culture for the development of an in-vitro mutagenesis assay: Effect of interleukin-2 and 2-mercaptoethanol on the activities of intermediary metabolism enzymes and cell proliferation

Efficient energy utilization is essential for cell growth; in an attempt to improve the growth conditions of the rat T-lymphocyte culture model for potential use in studying the mutagenic activity of carcinogens in vitro, we have investigated the effects of phytohemagglutinin (PHA), interleukin-2 (IL-2) and 2-mercaptoethanol (2-ME) on the activities of intermediary metabolism enzymes and cell proliferation. Isolated lymphocytes were cultured in the presence and absence of PHA, IL-2, or 2-ME. The intermediary metabolism enzymes investigated were glutamate dehydrogenase, glutamate-pyruvate transaminase, malate dehydrogenase, isocitrate dehydrogenase, lactate dehydrogenase, pyruvate kinase, and fatty acid synthetase (FAS). Measurable activity of all enzymes investigated, except for FAS, was detected in PHA-stimulated cells cultured with IL-2 or 2-ME. The unstimulated lymphocytes had significantly lower enzyme activity than stimulated cells. The combination of all three agents showed increased enzyme activity. This increase in activity brought about by the combination of the three agents was not reproduced by either agent acting alone. In general, the increase in enzyme activity correlated with cell proliferation as measured by [³H]thymidine uptake in PHA-stimulated cultures containing IL-2 and/or 2-ME. The results suggest that the addition of exogenous IL-2 and 2-ME enhances metabolic function and may be beneficial in in vitro culture of rat lymphocytes.Abbreviations PHA phytohemagglutinin - IL-2 interleukin-2 - 2-ME 2-mercaptoethanol - GDH glutamate dehydrogenase - GPT glutamate-pyruvate transaminase - MDH malate dehydrogenase - ICD isocitrate dehydrogenase - LDM lactate dehydrogenase - PK pyruvate kinase - FAS fatty acid synthetase     

Chitin synthesis inhibitors prevent cyst formation by Entamoeba trophozoites

Cysts of Entamoeba invadens obtained under axenic culture conditions have been reported to be similar to cysts of the human intestinal parasite E histolytica both in morphology and chitin presence in their wails. Mature E. invadens cyst forms, isolated from cultures following discontinuous Percoll gradient sedimentation were resistant (>80%) to detergent treatment. Addition of chitin synthesis inhibitors such as Polyoxin D and Nikkomycin (50 μg/ml) to cultures in encystation media markedly inhibited (>85%) the formation of detergent resistant cysts and prevented the incorporation of radiolabeled chitin precursor N-acetyl[³H]glucosamine. These findings suggest that chitin synthesis inhibitors may serve as drugs which specifically block the life cycle of the Entamoeba parasite.     

Entamoeba histolytica sirtuin EhSir2a deacetylates tubulin and regulates the number of microtubular assemblies during the cell cycle

We have discovered four sirtuin genes in Entamoeba histolytica, two of which are similar to eukaryotic sirtuins and two to bacterial and archaeal sirtuins. The eukaryotic sirtuin homologue, EhSir2a, showed NAD⁺‐dependent deacetylase activity and was sensitive to class III HDAC inhibitors. Localization of EhSir2a at different cellular sites suggested that this deacetylase could have multiple targets. Using an E. histolytica cDNA library in the yeast two‐hybrid genetic screen, we identified several proteins that bound to EhSir2a. These proteins included Eh α‐tubulin, whose interaction with EhSir2a was validated in E. histolytica. We have shown that EhSir2a deacetylated tubulin and localized with microtubules in E. histolytica. Increased expression levels of EhSir2a in stable transformants led to reduced number of microtubular assemblies in serum synchronized cells. This effect was abrogated by mutations in the deacetylase domain of EhSir2a, showing that EhSir2a deacetylase activity affected the stability and number of microtubular assemblies during the cell cycle of E. histolytica. Our results suggest that epigenetic modification of tubulin by EhSir2a is one of the mechanisms that regulates microtubular assembly in E. histolytica.     

Entamoeba invadens: Dynamics of DNA synthesis during differentiation from trophozoite to cyst

The DNA dynamics which mediate conversion of uni-nucleate trophozoite into quadrinucleate cyst in Entamoeba histolytica is not well understood. Here, we have addressed this question in Entamoeba invadens (a model system for encystation) through a detailed time course study of the differentiation process. We combined flow cytometric analysis with the change in rate of thymidine incorporation and the number of nuclei per cell. Our data shows that during encystment the cell population passes through three phases: (1) Early phase (0–8 h); of rapid DNA synthesis which may correspond to completion of ongoing DNA replication. Bi-nucleated cells increase with concomitant drop in uni-nucleated cells. (2) Commitment phase (8–24 h); in which DNA synthesis rate slows down. Possibly new rounds of replication are initiated which proceed slowly, followed by mitosis at 20 h. After this the number of bi- and uni-nucleated cells gradually decline and the tri- and tetra-nucleated cells begin to increase. (3) Consolidation phase (24–72 h); in which the rate of DNA synthesis shows a small increase till 32 h and then begins to decline. The G2/M peak reappears at 48 h, showing that more rounds of DNA replication may be getting completed, followed by nuclear division. By 72 h the encystment is virtually complete. The bi-nucleated stage could be an intermediate both in the conversion of trophozoite to cyst and back. Our study provides a comprehensive view of DNA dynamics during encystation and excystation of E. invadens.