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PubMed2003

Entamoeba histolytica: an update.

PURPOSE OF REVIEW: Over the past decade, since it was formally recognized that Entamoeba histolytica and Entamoeba dispar were two distinct species, studies in this field have made dramatic in-roads into the understanding of E. histolytica and the pathogenesis of invasive amoebiasis. Over the same period it has also become clear that the true incidence of E. histolytica infection, particularly in vulnerable populations such as low socioeconomic children, is exceedingly high. Understanding the epidemiology, pathophysiology, and the molecular and genetic biology of the organism will not only lead to improved diagnostic and treatment options but, ultimately, to the development of a safe and efficacious vaccine. RECENT FINDINGS: The recent advances in the genetic and molecular sciences have increased our understanding of the mechanisms that make E. histolytica unique among enteric protozoa in causing invasive disease. In addition, host factors, which predispose individuals or populations to infection or disease, are beginning to be elucidated. New diagnostic tools specific to E. histolytica are being exploited by clinicians and researchers to identify and treat patients as well as to add to the knowledge of the epidemiology and natural history of this infection. The ultimate goal - eradication of disease - is theoretically feasible since humans and primates are the only reservoirs of E. histolytica. Many talented and dedicated individuals are pursuing the development of an effective and safe amoebiasis vaccine. SUMMARY: E. histolytica remains an important pathogen in many populations of the world and although there has been substantial progress into understanding the disease major challenges still exist.

Animals↗
PubMed1991

Detection of HIV-1 in Entamoeba histolytica without evidence of transmission to human cells.

Intestinal protozoa like Entamoeba histolytica and Giardia lamblia have been proposed as vectors or cofactors in the development of AIDS. To determine whether these protozoa could transmit HIV, laboratory strains of protozoa were cocultured with cells chronically infected by a highly replicative strain of HIV-1. Entamoeba histolytica, but not Giardia lamblia, took up virus. Immunologically detectable HIV-1 was present in the amebae up to 48 h after exposure to infected cells, but this virus could not be transferred to uninfected human cells. Amebae isolated directly from two HIV-infected individuals were also found to be positive for HIV-1. After lysis of these protozoa and coculture with uninfected peripheral blood mononuclear cells, no transfer of virus to the human cells was observed.

Animals↗
PubMedDate not supplied

Consumption of L-arginine mediated by Entamoeba histolytica L-arginase (EhArg) inhibits amoebicidal activity and nitric oxide production by activated macrophages.

In this study we discuss the cloning and expression of Entamoeba histolytica arginase (EhArg), an enzyme that catalyses the hydrolysis of L-arginine to L-ornithine and urea. L-norvaline, a competitive inhibitor of E. histolytica L-arginase, inhibits the growth of the parasite, which suggests that the catabolism of L-arginine mediated by EhArg is essential. Nitric oxide (NO) is an antimicrobial agent that inhibits some key enzymes in the metabolism of Entamoeba histolytica. NO is synthesized by activated macrophages from L-arginine, the substrate of NO synthase (NOS-II). We show that E. histolytica inhibits NO mediated amoebicidal activity of activated macrophages by consuming L-arginine present in the medium.

Amino Acid Sequence↗
PubMed1995

Nucleotide sequence of the SrRNA gene of Entamoeba gingivalis: applications for construction of a species-specific DNA probe and phylogenetic analysis.

The small subunit ribosomal RNA (SrRNA) gene of Entamoeba gingivalis was amplified by PCR and the product of 1.9-kbp sequence was cloned into a plasmid vector pUC18. Four clones were isolated and sequenced. The insert DNAs were 1918- to 1921-bp long and A+T rich (65.5%). The four SrRNA sequences of E. gingivalis were found to be aligned with those of nine related protozoans while searching for E. gingivalis-specific sequences. A sequence of 28 oligonucleotides was chosen, chemically synthesized, and labeled with digoxigenin for use as a DNA probe. The probe thus constructed was shown to hybridize only with either the SrRNA-coding DNAs or the cells of the two E. gingivalis strains and not with those of other protozons or oral fungi tested. A representative SrRNA-sequence was analyzed and a phylogenetic tree was constructed by the neighbor-joining (NJ) method. Among the protists examined, E. gingivalis was placed next to Entamoeba histolytica as expected from the traditional taxonomy.

Animals↗
PubMedDate not supplied

The pathogenicity of Entamoeba histolytica is related to the capacity of evading innate immunity.

The host and parasite factors that influence susceptibility to Entamoeba histolytica infection and disease are not well understood. Entamoeba histolytica pathogenicity has been considered by focusing principally on parasite rather than host factors. Thus, research has concentrated on explaining the molecular differences between pathogenic E. histolytica and non-pathogenic E. dispar. However, the amoeba molecules considered most important for host tissue destruction (amoebapore, galactose/N-acetyl galactosamine inhibitable lectin, and cysteine proteinases) are present in both pathogenic E. histolytica and non-pathogenic E. dispar. In addition, the genetic differences in pathogenicity among E. histolytica isolates are unlikely to completely explain the different outcomes of infection. Considering that the principal difference between pathogenic and non-pathogenic amoebas lies in their surface coats, we propose that pathogenicity of the amoebas is related to the composition and properties of the surface coat components (or pathogen-associated molecular patterns, PAMPs), and the ability of innate immune response to recognize these components and eliminate the parasite. According to this hypothesis, a key feature that may distinguish pathogenic (E. histolytica) from non-pathogenic (E. dispar) strains is whether or not they can overcome innate immune defences. A corollary of this hypothesis is that in susceptible individuals the PAMPs are either not recognized or they are recognized by a set of Toll-like receptors (TLRs) that leads to an inflammatory response. In both cases, the result is tissue damage. On the contrary, in resistant individuals the innate/inflammatory response, induced through the activation of a different set of TLRs, eliminates the parasite.

Animals↗
PubMed2005

A lysine- and glutamic acid-rich protein, KERP1, from Entamoeba histolytica binds to human enterocytes.

Contact-dependent cytolysis of host cells by Entamoeba histolytica is an important hallmark of amoebiasis that points out the importance of molecules involved in the interaction between the parasite and the human cells. To decipher the molecular and cellular mechanisms supporting the invasion of the intestinal epithelium by E. histolytica, we analysed proteins involved in the interaction of the parasite with enterocytes. Affinity chromatography revealed several amoebic proteins interacting with purified brush border of differentiated Caco2 cells. Among them were found the intermediate subunit of the Gal/GalNAc lectin, an alpha-actinin-like protein and two new proteins KERP1 and KERP2 rich in lysine and glutamic acid. In silico analysis revealed the presence of KERP2 in the closely related non-pathogenic amoeba species Entamoeba dispar but not of KERP1. In additon, polymerase chain reaction analysis allowed to suggest the absence of kerp1 homologous gene in E. dispar. Therefore, we concentrated on the cellular analysis of KERP1. Cloning of the KERP1-encoding gene, production of a recombinant protein in Escherichia coli and production of a specific antibody allowed us to show the following properties: (i) purified KERP1 binds to epithelial cell surface, (ii) KERP1 is located on the plasma membrane and in vesicles of trophozoites and (iii) KERP1 is delivered in the interstitial area between the trophozoites and the intestinal cells.

Amino Acid Sequence↗
PubMed1979

Studies on a morphologically distinct colchicine-resistance variant of Entamoeba sp.

Colchicine has a temperature-dependent cytotoxic effect on Entamoeba sp. (Laredo isolate) that is most apparent when the drug is applied during the initiation of cultures at a concentration of 7.5 mM or higher. Continued transfer of cultures in medium containing progressively increasing concentrations of colchicine has resulted in a variant that grows prolifically in the presence of colchicine (7.5 mM) with a generation time comparable to that of the parent stock, Comparison of a number of parameters of the 2 variants revealed that colchicine resistance was accompanied by a change in cell shape, a reduced membrane permeability, which could partially be overcome by the addition of dimethyl sulfoxide (DMSO), and a reduced tolerance to osmotic stress. However, the parent strain and resistant variant were equally susceptible to cycloheximide and puromycin suggesting that the acquired colchicine resistance may not be explained on the basis of an entirely unspecific generalized reduced ability for drug uptake. Colchicine resistance and altered structure were found to be stable over a long period of time. The possible interdependence of these 2 parameters and their relation to cell motility in Entamoeba sp. are discussed.

Animals↗
PubMed1986

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.

Animals↗
PubMedDate not supplied

Circular DNA of Entamoeba histolytica encodes ribosomal RNA.

The presence of repeated DNA sequences encoding RNA in Entamoeba histolytica has been reported. In the present study we demonstrate by agarose gel electrophoresis. DNase digestion and electron microscopic analysis that these genes are located on extrachromosomal circular DNA molecules with an approximate size of 26 kb. Detection of replication intermediates suggests the episomal nature of these molecules. Amplified, extrachromosomal rRNA genes appear to be a common feature among the lower eukaryotes, occurring more commonly as linear molecules and less commonly as circles. Entamoeba histolytica is 1 of the few organisms studied in which rRNA genes are located predominantly on extrachromosomal circles.

Animals↗
PubMedDate not supplied

Entamoeba dispar: cultivation with sterilized Crithidia fasciculata.

Four isolates of Entamoeba dispar identified by their hexokinase and phosphoglucomutase isoenzyme profile and by their failure to react with Entamoeba histolytica-specific monoclonal antibody (4G6) could be grown in either Diamond's BI-S-33 medium, newly developed BCSI-S (Biosate cysteine starch iron-serum) medium, or casein-free YI-S medium in the presence of Crithidia fasciculata (ReF-1:PRR) sterilized by heating 56 degrees C for 30 min and subsequent incubation with 1% hydrogen peroxide for 24 hours at 4 degrees C. After the cultures were maintained for over 50 passages, the amebae were identified as E. dispar by isoenzyme analysis, polymerase chain reaction with E. histolytica- and E. dispar-specific primers, i.e. p11 plus p12 and p13 plus p14, respectively, and by negative reactivity with monoclonal antibody 4G6. The flagellates added to the culture were judged to be metabolically inactive based on the results of nuclear magnetic resonance spectroscopy, electron microscopy, and polarographic analysis. All of these findings suggest that E. dispar can grow in vitro with metabolically inactive C. fasciculata as a culture associate.

Animals↗
PubMedDate not supplied

Analysis of genes of mitochondrial origin in the genus Entamoeba.

The amitochondriate protistan parasite Entamoeba histolytica has lost most mitochondrial functions secondarily but has retained a reduced organelle of mitochondrial origin, the mitosome. We here investigate the presence, origins, and expression in other species of Entamoeba of three genes of mitochondrial origin--pyridine nucleotide transhydrogenase and the mitochondrial-type chaperonins cpn60 and hsp70. The genes appear to be present in all species and specifically related, confirming that the E. histolytica mitosomal genes were not acquired recently by lateral transfer from another organism. Detection of expression was not possible in all cases under the culture conditions used, but several genes were induced during recovery from exposure to a heat shock. This includes the transhydrogenase, which to our knowledge has not been shown previously to be a heat-shock protein.

Animals↗
PubMed1983

Entamoeba histolytica causes intestinal secretion: role of serotonin.

Lysates of the protozoan parasite Entamoeba histolytica altered active electrolyte transport when present on the serosal surface of rabbit ileum and rat colon. The lysate-induced effects on electrolyte transport were similar to those caused by serotonin, and were blocked by bufotenine, an analog known to inhibit the action of serotonin. The transport effects were partially inhibited by antibody to serotonin. The amebic lysates were shown to contain serotonin by radioimmunoassay, high-performance liquid chromatography, and thin-layer chromatography. These results suggest that the serotonin present in Entamoeba histolytica may be important in the diarrhea seen in amebiasis.

Amebiasis↗
PubMed1984

Entamoeba histolytica: a eukaryote without glutathione metabolism.

Entamoeba histolytica was found to grow normally without producing glutathione and the main enzymes of glutathione metabolism, indicating that glutathione is not essential for many eukaryotic processes. This parasitic amoeba is an unusual eukaryote whose special features may help define the crucial functions of glutathione in those eukaryotes that do use it. Since Entamoeba histolytica lacks mitochondria and the usual aerobic respiratory pathways, the finding that it grows without glutathione and other evidence support the hypothesis that a primary function of glutathione in eukaryotes involves protection against oxygen toxicity associated with mitochondria and suggest that eukaryotes may have acquired glutathione metabolism at the time that they acquired mitochondria.

Animals↗
PubMed1999

Efficacy of new 5-nitroimidazoles against metronidazole-susceptible and -resistant Giardia, Trichomonas, and Entamoeba spp.

The efficacies of 12 5-nitroimidazole compounds and 1 previously described lactam-substituted nitroimidazole with antiparasitic activity, synthesized via SRN1 and subsequent reactions, were assayed against the protozoan parasites Giardia duodenalis, Trichomonas vaginalis, and Entamoeba histolytica. Two metronidazole-sensitive lines and two metronidazole-resistant lines of Giardia and one line each of metronidazole-sensitive and -resistant Trichomonas were tested. All except one of the compounds were as effective or more effective than metronidazole against Giardia and Trichomonas, but none was as effective overall as the previously described 2-lactam-substituted 5-nitroimidazole. None of the compounds was markedly more effective than metronidazole against Entamoeba. Significant cross-resistance between most of the drugs tested and metronidazole was evident among metronidazole-resistant lines of Giardia and Trichomonas. However, some drugs were lethal to metronidazole-resistant Giardia and had minimum lethal concentrations similar to that of metronidazole for drug-susceptible parasites. This study emphasizes the potential in developing new nitroimidazole drugs which are more effective than metronidazole and which may prove to be useful clinical alternatives to metronidazole.

Animals↗
PubMed1999

Preparation of recombinant human monoclonal antibody Fab fragments specific for Entamoeba histolytica.

Genes coding for human antibody Fab fragments specific for Entamoeba histolytica were cloned and expressed in Escherichia coli. Lymphocytes were separated from the peripheral blood of a patient with an amebic liver abscess. Poly(A)+ RNA was isolated from the lymphocytes, and then genes coding for the light chain and Fd region of the heavy chain were amplified by a reverse transcriptase PCR. The amplified DNA fragments were ligated with a plasmid vector and were introduced into Escherichia coli. Three thousand colonies were screened for the production of antibodies to E. histolytica HM-1:IMSS by an indirect fluorescence-antibody (IFA) test. Lysates from five Escherichia coli clones were positive. Analysis of the DNA sequences of the five clones showed that three of the five heavy-chain sequences and four of the five light-chain sequences differed from each other. When the reactivities of the Escherichia coli lysates to nine reference strains of E. histolytica were examined by the IFA test, three Fab fragments with different DNA sequences were found to react with all nine strains and another Fab fragment was found to react with seven strains. None of the four human monoclonal antibody Fab fragments reacted with Entamoeba dispar reference strains or with other enteric protozoan parasites. These results indicate that the bacterial expression system reported here is effective for the production of human monoclonal antibodies specific for E. histolytica. The recombinant human monoclonal antibody Fab fragments may be applicable for distinguishing E. histolytica from E. dispar and for use in the serodiagnosis of amebiasis.

Amino Acid Sequence↗
PubMed2006

Unique posttranslational modifications of chitin-binding lectins of Entamoeba invadens cyst walls.

Entamoeba histolytica, which causes amebic dysentery and liver abscesses, is spread via chitin-walled cysts. The most abundant protein in the cyst wall of Entamoeba invadens, a model for amebic encystation, is a lectin called EiJacob1. EiJacob1 has five tandemly arrayed, six-Cys chitin-binding domains separated by low-complexity Ser- and Thr-rich spacers. E. histolytica also has numerous predicted Jessie lectins and chitinases, which contain a single, N-terminal eight-Cys chitin-binding domain. We hypothesized that E. invadens cyst walls are composed entirely of proteins with six-Cys or eight-Cys chitin-binding domains and that some of these proteins contain sugars. E. invadens genomic sequences predicted seven Jacob lectins, five Jessie lectins, and three chitinases. Reverse transcription-PCR analysis showed that mRNAs encoding Jacobs, Jessies, and chitinases are increased during E. invadens encystation, while mass spectrometry showed that the cyst wall is composed of an approximately 30:70 mix of Jacob lectins (cross-linking proteins) and Jessie and chitinase lectins (possible enzymes). Three Jacob lectins were cleaved prior to Lys at conserved sites (e.g., TPSVDK) in the Ser- and Thr-rich spacers between chitin-binding domains. A model peptide was cleaved at the same site by papain and E. invadens Cys proteases, suggesting that the latter cleave Jacob lectins in vivo. Some Jacob lectins had O-phosphodiester-linked carbohydrates, which were one to seven hexoses long and had deoxysugars at reducing ends. We concluded that the major protein components of the E. invadens cyst wall all contain chitin-binding domains (chitinases, Jessie lectins, and Jacob lectins) and that the Jacob lectins are differentially modified by site-specific Cys proteases and O-phosphodiester-linked glycans.

Amino Acid Sequence↗
PubMed2006

Expression and function of a family of transmembrane kinases from the protozoan parasite Entamoeba histolytica.

The signaling proteome of Entamoeba histolytica is made of transmembrane kinases (TMKs) that are rarely found in unicellular eukaryotes. There are 90 TMK genes reported for E. histolytica, and these have been grouped into nine distinct families based on motifs present on both extracellular and kinase domains. Of these, the B1 family was chosen for further analysis. Genomic sequencing revealed the presence of 28 members belonging to this family. Genes corresponding to the majority of these were truncated and not considered for further analysis. Only five members were full length and contained both extracellular and cytosolic kinase domains. BLAST analysis revealed the presence of homologs of these B1 TMKs in the nonpathogenic Entamoeba dispar. However, the ligand binding domains of the orthologous B1 TMKs of the two species showed considerable divergence, indicating the possibility of a correlation with the pathogenic potential of the organism. Only two of the five full-length copies (B1.I.1 and B1.I.2) were expressed in E. histolytica under the culture conditions used. Antisera generated against the extracellular domain of B1.I.1 stained the cell surface, particularly the areas of contact between the trophozoites. Staining was also seen in the frontal and posterior regions of the motile amoeba. An amoebic cell line expressing a truncated version of the B1.I.1 that lacked the kinase domain was generated. Inducible expression of the truncated TMK resulted in a decrease in cellular proliferation and an increase in sensitivity to serum starvation. Our data indicate that the B1.I class of TMKs is involved in parasite proliferation.

Animals↗
PubMed1985

Entamoeba histolytica trophozoites in the lumen and mucus blanket of rat colons studied in vivo.

Trophozoites of Entamoeba histolytica HM-1 were cultivated axenically in TYI-S medium. The amoebae were then transferred into this medium lacking serum (TYI) and inoculated into in vivo colon loops of adult Sprague-Dawley rats. The trophozoites were rapidly absorbed by the mucus, and few were found free in the luminal fluid by 1 h. By 4 h, the amoebae began to reappear in the lumen, aggregated in sloughed mucus blanket fragments. The colon was examined histologically and by scanning electron microscopy. There was no evidence of invasion or even brush-border attachment by the trophozoites within 4 h. In TYI, trophozoite motility was low. Exposure to the colonic lumen environment for 5 min in this medium significantly increased motility. However, as the trophozoites became absorbed to mucus fragments, their observed motility virtually ceased despite some morphological evidence of pseudopod extension. Erythrophagocytosis was not significantly affected by either exposing trophozoites to TYI washings of the colonic lumen, or by the more complete medium, TYI-S, in which the amoebae were significantly more motile. Two major mucus glycoprotein oligosaccharide end-group sugars, L-fucose and N-acetyl-neuraminic acid, were tested for their effects on trophozoite motility in both TYI and TYI-S. L-Fucose reduced motility; the sialic acid increased motility. It is concluded that the intestinal lumen contains several compartments, including the luminal fluid and the mucus blanket, and that Entamoeba trophozoites exist in a highly motile state in the former and a low motility state in the latter. The mucus blanket provided a significant barrier to trophozoite access to intestinal epithelium target tissue.

Amebiasis↗