Studies on the substrate specificity of alpha- and beta-amylase of Entamoeba histolytica

From the supernatant fraction of cell homogenates of Entamoeba histolytica alpha-amylase (1,4-alpha-D-glucan glucanohydrolase, EC 3.2.1.1) and beta-amylase (1,4-alpha-D-glucan maltohydrolase, EC 3.2.1.2) were separated and purified by gel filtration and isoelectric focusing, followed by DEAE- and CM-chromatography, respectively. Both enzymes catalyzed the degradation of amylose, amylopectin and glycogen. Hydrolysis of polysaccharides by alpha-amylase yielded as reaction products maltose, maltotriose, maltopentaose and maltohexaose, but no free glucose. beta-Amylase produced as main degradation product of glucopolysaccharides maltose and to a minor extend maltotriose, but no glucose. alpha- and beta-amylase were able to hydrolyze 4-nitrophenyl-alpha-D-glucooligosaccharides (Gn-pNP) containing more than two glucose units per molecule. Under identical conditions G6-pNP was cleaved at highest velocity by alpha-amylase, while beta-amylase exhibited the highest activity with G4-pNP as substrate. alpha-Amylase hydrolyzed G4-pNP, G5-pNP and G6-pNP yielding as main reaction product G2-pNP, but also the formation of G1-pNP and G3-pNP from G4-pNP, of G1-pNP, G3-pNP and G4-pNP from G5-pNP, of G1-pNP, G3-pNP, G4-pNP and G5-pNP from G6-pNP was observed. alpha-Amylase as endo-glucohydrolase attacked all glycosidic bonds in G6-pNP, G5-pNP and G4-pNP, while beta-amylase successively removed maltose units from the non-reducing ends of the glycosides.     

Differences in cap formation between invasive Entamoeba histolytica and non-invasive Entamoeba dispar

The rapid redistribution of surface antigen-antibody complexes in trophozoites of the human protozoan parasite Entamoeba histolytica, in a process known as capping, has been considered as a means of the parasite to evade the host immune response. So far, capping has been documented in the invasive E. histolytica, whereas the mobility of surface components in the non-invasive Entamoeba dispar is not known. E. dispar does not induce liver lesions in rodent experimental models, in contrast to the liver abscesses produced by E. histolytica in the same animal model. We have therefore analyzed the mobility of surface receptors to the lectin concanavalin A and of Rab11, a membrane-associated protein, in both species of Entamoebae by confocal fluorescence microscopy and transmission and scanning electron microscopy. The great majority of E. histolytica trophozoites became morphologically polarized through the formation of well-defined caps at the posterior pole of the parasite. Actin colocalized with the lectin caps. Antibodies against the membrane protein Rab 11 also produced capping. In striking contrast, in E. dispar, the mobility of concanavalin A surface receptors was restricted to the formation of irregular surface patches that did no progress to constitute well-defined caps. Also, anti-Rab 11 antibodies did not result in capping in E. dispar. Whether the failure of E. dispar to efficiently mobilize surface molecules in response to lectin or antibodies as shown in the present results is related to its non-invasive character represents an interesting hypothesis requiring further analysis.     

Purification and properties of NADPH:flavin oxidoreductase from Entamoeba histolytica

Amebal NADPH:flavin oxidoreductase was purified to apparent homogeneity. Molecular weights of 40 000 and 38 000 were estimated by gel filtration and by sodium dodecyl sulfate polyacrylamide gel electrophoresis, respectively, indicating that the enzyme is composed of a single polypeptide chain. The enzyme does not contain firmly bound flavin. It exhibited 20-fold selectivity for NADPH over NADH. With the former donor it reduced riboflavin, galactoflavin, FMN, or FAD. Aerobically the reducing equivalents were passed from reduced flavin to oxygen to form hydrogen peroxide. Intact amebae do not produce peroxide when they respire. If the title enzyme functions to reduce flavin in the intact cells some electron carrier must intervene between reduced flavin and oxygen so that the final step produces water instead of peroxide.     

Comparison of repeated DNA from strains of Entamoeba histolytica and other Entamoeba

Restriction enzyme digestion patterns of total genomic DNA revealed the presence of highly repeated DNA in Entamoeba histolytica and E. histolytica-like (Laredo type) amoebae of humans, E. invadens and E. invadens and E. terrapinae of reptiles, and E. moshkovskii isolated from sewage polluted waters. Homology with Plasmodium berghei ribosomal DNA was striking. Northern blot analysis of E. histolytica RNA provided further evidence that the highly repeated DNA codes for ribosomal RNA. The distribution of restriction enzyme sites on repeated DNA of the Entamoeba was highly polymorphic. Thus it was possible, based on EcoRI digestion patterns, to distinguish between strains of E. histolytica and the other species of Entamoeba. Additionally, these investigations at the molecular level corroborate previously reported physiological and biochemical evidence indicating the E. histolytica-like amoebae and E. histolytica are not conspecific. EcoRI fragments from repeated DNA of E. histolytica (strain HM-1: IMSS) were cloned in the plasmid vector pTZ18R. A subfragment from one of these clones proved to be a useful hybridization probe in distinguishing E. histolytica from the other Entamoeba.     

EhGEF2, a Dbl-RhoGEF from Entamoeba histolytica has atypical biochemical properties and participates in essential cellular processes

Dbl proteins are a family of factors that exchange the guanine nucleotide which promote the activation of Rho small GTPases. This paper reports the molecular, structural, biochemical and functional characterization of EhGEF2, a new member of the Dbl family. EhGEF2 is the second GEF studied in parasites and in the protozoan Entamoeba histolytica, and it is also the first member of the Dbl family that was found to have Arm repeats. The catalytic domain (DH) of EhGEF2 has the conserved residues T421, N590 and E591, which are important for the activation of the GTPases. Biochemical studies on EhGEF2 showed that it could activate in vitro the amoebic GTPases EhRacA, EhRacB, EhRacC, EhRacD, EhRacG, EhRacH and EhCdc42, being EhRacG its main target. It was found that the DH domain binds specifically phosphatidic acid (PA); docking and lipid dot blot studies indicated that this binding does not interfere with the contact surface of EhRacG. Functional studies showed that both the Arm repeats and the catalytic domain of EhGEF2 participate in its localization at the amoebic membrane. Expression of a negative dominant version of EhGEF2 protein in E. histolytica provoked a 30% decrease in its ability to phagocyte human erythrocytes as well as severe effects on both the proliferation and the cellular chemotaxis which suggest that EhGEF2 participates in these cellular processes.     

Isolation and characterization of polymorphic DNA from Entamoeba histolytica

An important gap in our understanding of the epidemiology of amebiasis is what determines the outcome of Entamoeba histolytica infections. To investigate the possible existence of invasive and noninvasive strains as one factor, the ability to differentiate individual isolates of E. histolytica is necessary. Two new loci containing internal repeats, locus 1-2 and locus 5-6, have been isolated. Each contains a single repeat block with two types of related direct repeats arranged in tandem. Southern blot analysis suggests that both loci are multicopy and may themselves be arranged in tandem arrays. Three other previously reported, internally repetitive loci containing at least two repeat blocks each with one or more related repeat units were also investigated. PCR was used to study polymorphism at each of these loci, which was detected to various degrees in each case. Variation was seen in the total number of bands obtained per isolate and their sizes. Nucleotide sequence comparison of loci 1-2 and 5-6 in five axenic isolates revealed differences in the number of repeat units, which correlated with the observed PCR product size variation, and in repeat sequence. Use of multiple loci collectively allowed differentiation of a majority of the 13 isolates studied, and we believe that these loci have the potential to be used as polymorphic molecular markers for investigating the epidemiology of E. histolytica and the potential existence of genetically distinct invasive and noninvasive strains.     

Protection of gerbils from amebic liver abscess by immunization with a recombinant Entamoeba histolytica antigen.

Amebiasis, infection by the intestinal protozoan parasite Entamoeba histolytica, is a leading parasitic cause of death. As a step in the development of a recombinant antigen vaccine to prevent E. histolytica infection, we looked at the ability of a recombinant version of the serine-rich E. histolytica protein (SREHP) to elicit a protective immune response against invasive amebic disease. Gerbils, a standard model for amebic liver abscess, were immunized with either a recombinant SREHP/maltose-binding protein (MBP) fusion, recombinant MBP alone, or phosphate-buffered saline (PBS), all combined with complete Freund's adjuvant. In the first trial (group 1), gerbils received a primary and two booster immunizations intraperitoneally; in the second trial (group 2), gerbils were immunized by a single intradermal injection. SREHP/MBP-immunized gerbils in both groups produced antibody to native SHEHP and developed delayed-type hypersensitivity responses to recombinant SREHP. All gerbils were challenged by an intrahepatic injection with 5 x 10(4) virulent E. histolytica HM1-IMSS trophozoites. Complete protection from amebic liver abscess was seen in 64% of the SHEHP/MBP-immunized gerbils in group 1 and in 100% of the SREHP/MBP-immunized gerbils in group 2. There was no protection observed in MBP- or PBS-immunized gerbils in either group. Our results indicate that the SREHP molecule has potential as a vaccine to prevent amebic infection and demonstrate that successful vaccination of animals with recombinant E. histolytica antigen vaccines is possible.     

Entamoeba histolytica infection in children and protection from subsequent amebiasis

The contribution of amebiasis to the burden of diarrheal disease in children and the degree to which immunity is acquired from natural infection were assessed in a 4-year prospective observational study of 289 preschool children in an urban slum in Dhaka, Bangladesh. Entamoeba histolytica infection was detected at least once in 80%, and repeat infection in 53%, of the children who completed 4 years of observation. Annually there were 0.09 episodes/child of E. histolytica-associated diarrhea and 0.03 episodes/child of E. histolytica-associated dysentery. Fecal immunoglobulin A (IgA) anti-parasite Gal/GalNAc lectin carbohydrate recognition domain (anti-CRD) was detected in 91% (183/202) of the children at least once and was associated with a lower incidence of infection and disease. We concluded that amebiasis was a substantial burden on the overall health of the cohort children. Protection from amebiasis was associated with a stool anti-CRD IgA response. The challenge of producing an effective vaccine will be to improve upon naturally acquired immunity, which does not provide absolute protection from reinfection.     

PCR detection of Entamoeba histolytica, Entamoeba dispar, and Entamoeba moshkovskii in stool samples from Sydney, Australia

This study investigated the presence of Entamoeba histolytica, Entamoeba dispar, and Entamoeba moshkovskii in stool samples from a patient population in Sydney, Australia. Stool samples were tested by microscopy and PCR. Five patients were found with E. histolytica infections, while E. dispar and E. moshkovskii were observed in 63 (70.8%) and 55 (61.8%) patients, respectively, by PCR. This is the first study in Australia using molecular techniques to determine the presence of E. histolytica, E. dispar, and E. moshkovskii.     

Cloning and characterization of an unusual elongation factor-1 alpha cDNA from Entamoeba histolytica

The coding sequence deduced from two overlapping cDNA inserts obtained from a pathogenic strain of Entamoeba histolytica revealed a striking homology (greater than 85%) with elongation factor EF-1 alpha from Saccharomyces cerevisiae and Artemia salina. The deduced amino acid sequence predicted a size of 49 kDa, and antibodies raised against the S. cerevisiae EF-1 alpha cross-reacted with an amoebic protein of similar size (45-47 kDa). Sequence analysis of the cDNA revealed that the 5' untranslated region contained a stretch of 190 nucleotides which was perfectly complementary to a segment of the 3' terminal coding region situated 1015 bases downstream of the methionine initiation codon. Electron microscopy of self-renatured cDNA confirmed the potential of such molecules to form a stem-loop secondary structure. The presence of the complementary sequences was confirmed at the genomic level by sequence analysis of polymerase chain reaction-amplified segments which span both the 3' and 5' terminal complementary regions. Comparison of the deduced amino acid sequence of E. histolytica EF-1 alpha with Ef-Tu from Escherichia coli and EF-1 alpha from different sources, suggested that the major functional domains of the protein are located within the loop structure.     

Protection of gerbils from amebic liver abscess by immunization with recombinant Entamoeba histolytica 29-kilodalton antigen.

The goal of our study was to obtain a highly conserved Entamoeba histolytica recombinant antigen for study as a subunit amebiasis vaccine. We screened a Uni-Zap cDNA library of E. histolytica (strain HM1:IMSS) with human immune sera and isolated a dominant 804-bp cDNA clone. A 33-kDa fusion protein expressed from the cDNA clone was determined by monoclonal antibody binding, DNA hybridization, and nucleotide sequence to be the complete E. histolytica 29-kDa antigen. Serum antibodies to the recombinant protein were detected by enzyme-linked immunosorbent assay in 80% of subjects from Egypt and South Africa with amebic liver abscess. Similar results were found with the native 29-kDa protein. Native and recombinant 29-kDa antigens induced proliferation of lymphocytes harvested from patients with amebic liver abscess (P < 0.01 compared with controls). Intraperitoneal immunization of gerbils with the recombinant fusion protein (10 micrograms) with Titermax adjuvant elicited an antigen-specific serum immunoglobulin G antibody response and was partially protective (54%) against intrahepatic challenge with 5 x 10(5) virulent axenic trophozoites (strain HM1:IMSS). In summary, the recombinant form of the E. histolytica 29-kDa antigen demonstrated serologic specificity for amebic liver abscess, exhibited conserved T-cell epitopes, and was effective as a subunit vaccine in an experimental animal model of amebic liver abscess.     

Characterization of protein geranylgeranyltransferase I from the enteric protist Entamoeba histolytica

Entamoeba histolytica is a unique protozoan parasite possessing both protein farnesyltransferase and geranylgeranyltrasferase I (GGT-I) for isoprenylation of small GTPases. In this study, we demonstrated unique enzymological properties of the amebic GGT-I (EhGGT-I), including substrate specificity and insensitivity to known mammalian inhibitors. Some of important residues of the catalytic beta subunit implicated in the specificity for GTPase acceptors and prenyl donors are substituted in EhGGT-I. Recombinant alpha and beta subunits of EhGGT-I, co-expressed in Escherichia coli, showed activity to transfer geranylgeranyl to both human wild-type (CVLS) and mutant (CVLL) H-Ras, while the mammalian GGT-I geranylgeranylated, but not farnesylated, only mutant H-Ras. All the representative amebic Ras and Rho/Rac small GTPases with phenylalanine, leucine, methionine, or alanine terminus were preferentially geranylgeranylated by EhGGT-I. This indicates that the acceptor specificity of the amebic GGT-I is remarkably broader than that of its mammalian counterpart. In contrast to EhFT, which farnesylates but not geranylgeranlylates solely EhRas4-CVVA, EhGGT-I also showed significant farnesyltransferase activity against Ras GTPase acceptors. EhGGT-I showed remarkable resistance to peptidomimetics known to inhibit mammalian GGT-I. Together with our previous observation that this parasite does not appear to depend on farnesylation for a majority of Ras and Rho/Rac, these data indicate that biological and biochemical advantages leading to the evolutional selection of this isoprenyl modification must exist uniquely in this parasitic protist. Finally, remarkable biochemical differences in binding to substrates and inhibitors between amebic and mammalian GGT-I highlight this enzyme as an attractive target for the development of new chemotherapeutics against amebiasis.     

Entamoeba histolytica stimulates the alarmin molecule HMGB1 from macrophages to amplify innate host defenses

Even though Entamoeba histolytica (Eh)-induced host pro-inflammatory responses play a critical role in disease, we know very little about the host factors that regulate this response. Direct contact between host cell and Eh signify the highest level of danger, and to eliminate this threat, the host immune system elicits an augmented immune response. To understand the mechanisms of this response, we investigated the induction and release of the endogenous alarmin molecule high-mobility group box 1 (HMGB1) that act as a pro-inflammatory cytokine and chemoattractant during Eh infection. Eh in contact with macrophage induced a dose- and time-dependent secretion of HMGB1 in the absence of cell death. Secretion of HMGB1 was facilitated by Eh surface Gal-lectin-activated phosphoinositide 3-kinase and nuclear factor-κB signaling and up-regulation of histone acetyltransferase activity to trigger acetylated HMGB1 translocation from the nucleus. Unlike lipopolysaccharide, Eh-induced HMGB1 release was independent of caspase-1-mediated inflammasome and gasdermin D pores. In vivo, Eh inoculation in specific pathogen-free but not germ-free mice was associated with high levels of pro-inflammatory cytokines such as tumor necrosis factor-α, interleukin-1β, and keratinocyte-derived chemokine, which was suppressed with HMGB1 neutralization. This study reveals that Eh-induced active secretion of the HMGB1 plays a key role in shaping the pro-inflammatory landscape critical in innate host defense against amebiasis.     

Molecular biology of the hexokinase isoenzyme pattern that distinguishes pathogenic Entamoeba histolytica from nonpathogenic Entamoeba dispar

The electrophoretic patterns of hexokinase and phosphoglucomutase have been widely used to distinguish Entamoeba histolytica from Entamoeba dispar isolates. Although E. histolytica and E. dispar, previously called pathogenic and nonpathogenic Entamoeba histolytica, differ clearly in sequences of many homologous genes, a conversion between the two has been reported by several laboratories, in each case showing the conversion of hexokinase (ATP, d-hexose 6-phosphotransferase, EC 2.7.1.1) isoenzyme patterns. An apparent mobility shift of this enzyme may either be due to posttranslational modification or processing, or to the appearance of a new isoform encoded by a second gene. In this study we observed that the four observed bands in the isoenzyme patterns of pathogenic and nonpathogenic forms of Entamoeba were correlated with four different cDNAs, and that the four recombinant hexokinases produced in Escherichia coli comigrated with their natural counterparts. Polymerase chain reaction (PCR) experiments did not reveal hidden genes which might be responsible for conversion phenomena. These results strongly support the redefinition of pathogenic and nonpathogenic Entamoeba histolytica as two closely related species Entamoeba histolytica and Entamoeba dispar.     

Immunization with the Entamoeba histolytica Surface Metalloprotease EhMSP-1 Protects Hamsters from Amebic Liver Abscess

Diarrhea and amebic liver abscesses due to invasive Entamoeba histolytica infections are an important cause of morbidity and mortality in the developing world. Entamoeba histolytica adherence and cell migration, two phenotypes linked to virulence, are both aberrant in trophozoites deficient in the metallosurface protease EhMSP-1, which is a homologue of the Leishmania vaccine candidate leishmanolysin (GP63). We examined the potential of EhMSP-1 for use as a vaccine antigen to protect against amebic liver abscesses. First, existing serum samples from South Africans naturally infected with E. histolytica were examined by enzyme-linked immunosorbent assay (ELISA) for the presence of EhMSP-1-specific IgG. Nine of 12 (75%) people with anti-E. histolytica IgG also had EhMSP-1-specific IgG antibodies. We next used a hamster model of amebic liver abscess to determine the effect of immunization with a mixture of four recombinant EhMSP-1 protein fragments. EhMSP-1 immunization stimulated a robust IgG antibody response. Furthermore, EhMSP-1 immunization of hamsters reduced development of severe amebic liver abscesses following intrahepatic injection of E. histolytica by a combined rate of 68% in two independent animal experiments. Purified IgG from immunized compared to control animals bound to the surface of E. histolytica trophozoites and accelerated amebic lysis via activation of the classical complement cascade. We concluded that EhMSP-1 is a promising antigen that warrants further study to determine its full potential as a target for therapy and/or prevention of invasive amebiasis.     

Purification and cellular localization of the Entamoeba histolytica transcarboxylase

Genome analysis of Entamoeba histolytica predicts the presence of acetyl-CoA carboxylase. Using Western blot, histochemistry, and confocal microscopy, we demonstrated the presence of a biotin-containing protein in the cytoplasm of E. histolytica, with a molecular weight of 136 kDa and biotin–carboxylase activity. This protein probably corresponds to a transcarboxylase that catalyzes the rate-limiting reaction leading to fatty acid elongation.     

Characterization of nucleoside uptake and transport in Entamoeba histolytica

The uptake and transportation of purine and pyrimidine based nucleosides by trophozoites of axenically grown Entamoeba histolytica (HMI-IMSS) were studied. The trophozoites transported adenosine and its analog tubercidin (1 μM) at a significant rate but poor transportation was observed in case of uridine (about 10% relative rate), inosine (3%?), thymidine (2%?) and formycin B (1%?). The Km for adenosine was 160?±?42 μM. Unlabeled nucleosides (100 μM) inhibited adenosine and tubercidin transport. Adenosine related compounds 5′-deoxyadenosine and nebularin inhibited adenosine and tubercidine transport by 50%? or more. However, inosine related compounds guanosine, 3′-deoxyinosine and formycin B were less inhibitory. The pyrimidine nucleosides uridine, thymidine and cytidine were poorly inhibitory. 6-[(4 nitrobenzyl)-mercapto] purine ribonucleoside, an inhibitor of mammalian nucleoside transporter, inhibited adenosine or tubercidin transport in E. histolytica variably between 0–30%?at 10?μM, but dilazep, a known inhibitor, was inactive upto 10 μM. Increase in temperature from 22°C to 33°C enhanced the rate of transport of adenosine 4.5 fold, tubercidin 7.3 fold and of inosine 4 fold. These findings along with the structure activity figures suggested that transport was mediated and not passive.     

Correlation of Virulence and Collagenolytic Activity in Entamoeba histolytica

The levels of collagenolytic activity of strains HM-1:1 MSS (HM-1), (HM-1), 200-NIH, and HK-9 of Entamoeba histolytica were compared. Collagen degradation was evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Conditioned media as well as extracts of the highly virulent strain HM-1 effectively degraded native type I collagen. Significantly lower activity was found in the analogous fractions of strains 200-NIH and HK-9, which are not as virulent. The collagenolytic activity of strain HM-1 was associated with the isolated plasma membrane fraction and could be eluted from the membranes by buffers of high ionic strength, indicating that it is not an integral membrane protein. Unlike the vertebrate and the clostridial collagenases, the collagenolytic activity of E. histolytica HM-1 was enhanced in presence of dithiothreitol and was inhibited by N-ethylmaleimide. The correlation between virulence of the individual strains and their collagenolytic activity suggests that collagenase might play a role in pathogenesis of amoebiasis. The localization of the enzyme on the plasma membrane and its presence in the extracellular medium favor this view.