Introns of Entamoeba histolytica and Entamoeba dispar.

The genome of Entamoeba histolytica is considered to possess very few intervening sequences (introns), as only 5 intron-containing genes from this protozoan parasite have been reported so far. However, while sequencing a number of genomic contigs as well as three independent genes coding for ribosomal protein L27a, we have identified 9 additional intron-containing genes of E. histolytica and the closely related species Entamoeba dispar, indicating that introns are more common in these organisms than previously suggested. The various amoeba introns are relatively short comprising between 46 and 115 nucleotides only and have a higher AT-content compared to the corresponding exon sequences. In contrast to higher eukaryotes, amoeba introns do not contain a well-conserved branch point consensus, and have extended donor and acceptor splice sites of the sequences G     

Comparison of adherence, cytotoxicity, and Gal/GalNAc lectin gene structure in Entamoeba histolytica and Entamoeba dispar

The recent development of axenic culture for E. dispar allowed us to examine this ameba's ability to bind and lyse target cells and compare it to E. histolytica which has been axenically cultured for years. We found that under axenic conditions, E. dispar's adherence to target cells, ligand binding, and cytotoxicity were less than that of E. histolytica. These events were Gal/GalNAc-inhibitable supporting the idea that E. dispar expresses a lectin similar to E. histolytica. Genetic analysis showed that E. dispar had at least two members of the lectin heavy subunit family and four members of the lectin light subunit family that hybridized to ehhgl and ehlgl gene probes. A library screen produced clones which were isolated and sequenced. Derived amino acid sequences showed that the E. dispar heavy and light subunit clones were 86% and 79% identical, respectively, to their E. histolytica counterparts. In particular, the region which contains the epitopes for two adherence-enhancing monoclonal antibodies and a complement-sensitizing monoclonal antibody (amino acids 882–959 of the lectin heavy subunit) were conserved between the species.     

Close sequence identity between ribosomal DNA episomes of the non-pathogenicEntamoeba dispar and pathogenicEntamoeba histolytica

Entamoeba dispar andEntamoeba histolytica are now recognized as two distinct species-the former being nonpathogenic to humans. We had earlier studied the organization of ribosomal RNA genes inE. histolytica. Here we report the analysis of ribosomal RNA genes inE. dispar. The rRNA genes ofE. dispar, like their counterpart inE. histolytica are located on a circular rDNA molecule. From restriction map analysis, the size ofE. dispar rDNA circle was estimated to be 24·4 kb. The size was also confirmed by linearizing the circle withBsaHI, and by limited DNAseI digestion. The restriction map of theE. dispar rDNA circle showed close similarity to EhR1, the rDNA circle ofE. histolytica strain HM-1:IMSS which has two rDNA units per circle. The various families of short tandem repeats found in the upstream and downstream intergenic spacers (IGS) of EhR1 were also present inE. dispar. Partial sequencing of the cloned fragments ofE. dispar rDNA and comparison with EhR1 revealed only 2·6% to 3·8% sequence divergence in the IGS. The region Tr and the adjoiningPvuI repeats in the IGS of EhR1, which are missing in thoseE. histolytica strains that have one rDNA unit per circle, were present in theE. dispar rDNA circle. Such close similarity in the overall organization and sequence of the IGS of rDNAs of two different species is uncommon. In fact the spacer sequences were only slightly more divergent than the 18S rRNA gene sequence which differs by 1·6% in the two species. The most divergent sequence betweenE. histolytica andE. dispar was the internal transcribed spacer, ITS2. Therefore, it was concluded that probes derived from the ITS1 and ITS 2 sequences would be more reliable and reproducible than probes from the IGS regions used earlier for identifying these species.     

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.     

<Emphasis Type="Italic">Entamoeba histolytica</Emphasis> and <Emphasis Type="Italic">E. dispar</Emphasis> infections in captive macaques (<Emphasis Type="Italic">Macaca fascicularis</Emphasis>) in the Philippines

Entamoeba histolytica is a protozoan parasite that infects man and animals. This parasite has a global distribution and the disease it causes is usually characterized by diarrhea. In order to detect the parasite, it is necessary to differentiate it from Entamoeba dispar. E. dispar appears morphologically similar to E. histolytica but does not cause disease and tissue invasion. This study reports on the prevalence of E. histolytica and E. dispar among captive macaques in a primate facility in the Philippines. PCR was used to correctly identify both Entamoeba species. Indirect fluorescent antibody test (IFAT) was also performed to determine the seroprevalence of amebiasis in the captive macaques. Based on PCR targeting of the peroxiredoxin gene, of the 96 stool samples collected, 23 (24%) contained E. histolytica while 32 (33%) contained E. dispar. IFAT revealed 26 (27%) serum samples positive for antibodies against E. histolytica. Sequence analysis of the 18S rRNA gene showed that the 23 E. histolytica isolates were identical to human E. histolytica isolates deposited in the GenBank and not Entamoeba nuttalli as found in macaques in other recent reports. The Philippines is a major exporter of monkeys for biomedical research purposes, so screening animals before transporting them to other locations lessens the risk of spreading zoonoses to a wider area. This is the first report of the molecular detection of E. histolytica and E. dispar among macaques in the Philippines. This study complements the limited information available on the animal hosts of E. histolytica in the Philippines.     

Frequency and molecular characterisation of Entamoeba histolytica,Entamoeba dispar,Entamoeba moshkovskii,and Entamoeba hartmanni in the context of water scarcity in northeastern Brazil

This study aimed to estimate the frequency, associated factors, and molecular characterisation of Entamoeba histolytica, Entamoeba dispar, Entamoeba moshkovskii, andEntamoeba hartmanni infections. We performed a survey (n = 213 subjects) to obtain parasitological, sanitation, and sociodemographic data. Faecal samples were processed through flotation and centrifugation methods.E. histolytica, E. dispar, E. moshkovskii, and E. hartmanni were identified by nested-polymerase chain reaction (PCR). The overall prevalence of infection was 22/213 (10.3%). The infection rate among subjects who drink rainwater collected from roofs in tanks was higher than the rate in subjects who drink desalinated water pumped from wells; similarly, the infection rate among subjects who practice open defecation was significantly higher than that of subjects with latrines. Out of the 22 samples positive for morphologically indistinguishableEntamoeba species, the differentiation by PCR was successful for 21. The species distribution was as follows: 57.1% to E. dispar, 23.8% to E. histolytica, 14.3% toE. histolytica and E. dispar, and 4.8% E. dispar and E. hartmanni. These data suggest a high prevalence of asymptomatic infection by the group of morphologically indistinguishable Entamoeba histolytica/dispar/moshkovskiicomplex and E. hartmanni species. In this context of water scarcity, the sanitary and socioenvironmental characteristics of the region appear to favour transmission.     

Entamoeba histolytica and Entamoeba dispar: Morphological and Behavioral Differences Induced by Fibronectin through GTPases Activation and Actin‐Binding Proteins

Early steps of tissue invasion by Entamoeba histolytica are mediated by adhesion and migration through matrix components such as fibronectin with the participation of the actin cytoskeleton. Striking differences in their produced structures, movement, and migration were found. These observations suggest differential changes in their ability to organize the actin cytoskeleton and, therefore, to modify its morphology after adhesion to fibronectin. To understand these observations, we explore deeper the cytoskeleton pathway of E. histolytica compared to Entamoeba dispar, analyzing the activation and involvement of actin cytoskeleton regulatory proteins such as small GTPases (Rho, Rac1 and Cdc42), myosin IB, paxillin, alpha‐actinin, and ARP2/3 during interaction with fibronectin. Results showed a higher activation of Rac1 in E. histolytica compared to E. dispar, while Cdc42 and RhoA were equally activated in both amebae; besides, variations in the amount of myosin IB, paxillin, and ARP2/3 were detected among these species, coinciding and reflected in formation of lamellipodia in E. histolytica and filopodia in E. dispar. These could partially explain the higher invasive capacity of E. histolytica compared to E. dispar, due to its pleomorphic ability, high motility, migration, activation, and abundance of proteins involved in the cytoskeleton arrangement.     

The intestinal protozoan parasite Entamoeba histolytica contains 20 cysteine protease genes,of which only a small subset is expressed during in vitro cultivation

Cysteine proteases are known to be important pathogenicity factors of the protozoan parasite Entamoeba histolytica. So far, a total of eight genes coding for cysteine proteases have been identified in E. histolytica, two of which are absent in the closely related nonpathogenic species E. dispar. However, present knowledge is restricted to enzymes expressed during in vitro cultivation of the parasite, which might represent only a subset of the entire repertoire. Taking advantage of the current E. histolytica genome-sequencing efforts, we analyzed databases containing more than 99% of all ameba gene sequences for the presence of cysteine protease genes. A total of 20 full-length genes was identified (including all eight genes previously reported), which show 10 to 86% sequence identity. The various genes obviously originated from two separate ancestors since they form two distinct clades. Despite cathepsin B-like substrate specificities, all of the ameba polypeptides are structurally related to cathepsin L-like enzymes. None of the previously described enzymes but 7 of the 12 newly identified proteins are unique compared to cathepsins of higher eukaryotes in that they are predicted to have transmembrane or glycosylphosphatidylinositol anchor attachment domains. Southern blot analysis revealed that orthologous sequences for all of the newly identified proteases are present in E. dispar. Interestingly, the majority of the various cysteine protease genes are not expressed in E. histolytica or E. dispar trophozoites during in vitro cultivation. Therefore, it is likely that at least some of these enzymes are required for infection of the human host and/or for completion of the parasite life cycle.     

Molecular Identification and Prevalence of Entamoeba histolytica,Entamoeba dispar and Entamoeba moshkovskii in Erbil City,Northern Iraq

The present study was conducted to evaluate the infection rates of Entamoeba histolytica, Entamoeba dispar, and Entamoeba moshkovskii among asymptomatic individuals in Erbil City, northern Iraq. The research intent was to discover whether pathogenic or nonpathogenic species cause a high rate of symptomless Entamoeba infections. Stool samples were microscopically examined, and the 18S-rRNA gene was targeted utilizing the nested PCR technique in the positive specimens. Initial results based on morphological features showed that the Entamoeba prevalence rate was 7.4%. Significantly higher rates of infections were seen in females than in males and in low-income people than in moderate-income people. The incidence rates among the asymptomatic individuals, as determined by molecular analysis, were as follows: E. histolytica – 6%, E. dispar – 4.3%, and E. moshkovskii – 0.3%. Of all the Entamoeba positive samples, a single infection with E. histolytica was identified in 41.4% samples; the single infection with E. dispar in 18.6% samples, 35.7% samples had mixed infections with two Entamoeba species, and 4.3% had mixed infections with three species. The current study concluded that 7.4% of healthy people, who live in the endemic area under investigation, carry Entamoeba species asymptomatically. Additionally, the majority of asymptomatic Entamoeba infections were caused by the pathogenic E. histolytica (81.4%) compared to E. dispar (58.6%), and E. moshkovskii with the lowest rate of infection. Single and co-infections with E. histolytica and E. dispar were noted. E. moshkovskii, which was identified for the first time in the region, was only seen in mixed infections.Key words: Entamoeba histolytica, Entamoeba dispar, Entamoeba moshkovskii, epidemiology, asymptomatic infections     

Short hairpin RNA-mediated knockdown of protein expression in Entamoeba histolytica

Background  

Entamoeba histolytica is an intestinal protozoan parasite of humans. The genome has been sequenced, but the study of individual gene products has been hampered by the lack of the ability to generate gene knockouts. We chose to test the use of RNA interference to knock down gene expression in Entamoeba histolytica.     

A novel nested multiplex polymerase chain reaction (PCR) assay for differential detection of <Emphasis Type="Italic">Entamoeba histolytica</Emphasis>, <Emphasis Type="Italic">E. moshkovskii</Emphasis> and <Emphasis Type="Italic">E. dispar</Emphasis> DNA in stool samples

Background  

E. histolytica, a pathogenic amoeba, is indistinguishable in its cyst and trophozoite stages from those of non-pathogenic E. moshkovskii and E. dispar by light microscopy. We have developed a nested multiplex PCR targeting a 16S-like rRNA gene for differential detection of all the three morphologically similar forms of E. histolytica, E. moshkovskii and E. dispar simultaneously in stool samples.     

Different Clinical Outcomes of Entamoeba histolytica in Malaysia: Does Genetic Diversity Exist?

The present study was conducted to investigate the clinical outcomes of Entamoeba histolytica infection in symptomatic and asymptomatic Orang Asli (aborigine) communities in Malaysia. Examination was performed on 500 stool samples obtained from Orang Asli communities in 3 different states using formalin-ether concentration, trichrome staining, and single-round PCR techniques. Out of 500 stool samples, single infection of E. histolytica, Entamoeba dispar, and Entamoeba moshkovskii was identified in 3.2%, 13.4%, and 1%, respectively. In addition, 10 samples had mixed infections with E. histolytica and E. dispar. Six samples containing E. dispar were also positive for E. moshkovskii, and only 2 samples had E. histolytica in association with E. dispar and E. moshkovskii. Seventeen E. histolytica-positive samples were from symptomatic subjects, whereas the remaining 11 samples came from asymptomatic subjects. These findings suggest a predominant distribution of pathogenic potential of E. histolytica strains in this community. Therefore, further studies on genotyping of E. histolytica is required, to find out association between E. histolytica genotype and the outcome of the infection.     

Prevalence of Entamoeba nuttalli infection in wild rhesus macaques in Nepal and characterization of the parasite isolates

We have recently resurrected the name Entamoeba nuttalli Castellani, 1908 for a potentially virulent ameba isolate, P19-061405, obtained from a rhesus macaque in Kathmandu, Nepal. The ameba was morphologically indistinguishable from Entamoeba histolytica/Entamoeba dispar/Entamoeba moshkovskii, but located phylogenetically between E. histolytica and E. dispar. To evaluate the prevalence of E. nuttalli infection in wild rhesus macaques, 112 fecal samples were collected in four locations of the Kathmandu Valley. PCR analysis of DNA extracted from the feces showed positive rates of E. nuttalli, E. dispar, E. histolytica and E. moshkovskii of 51%, 12%, 0% and 0%, respectively. A total of 14 E. nuttalli isolates were obtained from four locations, of which 6 were established as axenic cultures. The sequences of the serine-rich protein gene of E. nuttalli isolates differed among four locations although no differences were found in the composition of sequence motifs. Isoenzyme pattern was analyzed in 8 isolates obtained from three locations. In hexokinase, the mobility of the slower migrating band was located between E. histolytica and E. dispar regardless of the culture conditions. These results demonstrate that E. nuttalli is highly prevalent in wild rhesus macaques in Nepal. Rhesus macaques appear to be one of the natural hosts and heterogeneity of the serine-rich protein gene might be useful for geographical typing of isolates.     

Growth studies on xenic cultures of Entamoeba gingivalis using established media

Wantland's egg medium, modified Shaffer-Frye (MSF) medium and Tryptose-Trypticase-Yeast Extract-Serum-Blood (TTY-SB) medium were compared with variations of the latter two media for their ability to support xenic growth of Entamoeba gingivalis. Wantland's egg medium was unsuitable for growth of E. gingivalis. Accompanying bacteria became resistant to penicillin and streptomycin, overwhelming the amoeba culture. MSF medium was also unsuitable for the cultivation of E. gingivalis. Bacterial growth was heavy and protozoan growth sparse. MSF medium without mercaptosuccinic acid, but with rice starch, dextran or levan substituted for glucose and with Yersinia enterocolitica added, supported limited growth of the amoeba. Unmodified TTY-SB medium did not sustain growth of E. gingivalis. However, when rice starch suspension was substituted for glucose, l-cysteine HCl was deleted, and a Crithidia sp. was added to the E. gingivalis culture grown xenically, enhanced growth of the oral amoeba resulted in this modified TTY-SB medium. E. gingivalis is very sensitive to changes in incubation temperature. Optimum growth was found to be in the narrow range from 34.5 to 35°C for all media tested.     

Histopathological and immunohistochemical study of the hepatic lesions experimentally induced by Entamoeba dispar

The sequence of hepatic necrotic-inflammatory events produced by Entamoeba dispar are originally described in this work. For the first time the experimental lesions produced by E. dispar were described in details, as well as the distribution of the trophozoites detected by the immunohistochemistry. Animals experimentally infected with E. dispar presented necrosis, thrombosis and chronic granulomatous inflammation. Immunoreactive products derived from trofozoites were observed close or associated with trophozoites, epithelioid cells, leucocytes and hepatocytes. Few are the articles on the literature about virulence of E. dispar, which is approximately 9 times more frequent than to Entamoeba histolytica (E. histolytica). Variation in the virulence is therefore expected and signalizing the need of the continuity of studies with E. dispar strains from different places in the world. Taking into account that E. dispar is a closely related species to E. histolytica, these studies could determine new elements involved with E. histolytica pathogenesis, helping us to better understand the disease.Key words: Entamoeba dispar, hepatic necrosis, granuloma, pathogenicity.     

Strategies of the protozoan parasite<Emphasis Type="Italic">Entamoeba histolytica</Emphasis> to evade the innate immune responses of intestinal epithelial cells

Molecules expressed by the pathogenic ameobaEntamoeba histolytica but weakly expressed or absent from the non-pathogenic ameobaEntamoeba dispar could be used by intestinal epithelial cells to discriminate between the two species and to initiate an appropriate inflammatory response. Among the possible molecules involved in this identification are the Gal/GalNac lectin and the lipophosphoglycan. Once the inflammatory response is initiated,E. histolytica trophozoites have to protect themselves against reactive nitrogen intermediates produced by intestinal epithelial cells, oxygen intermediates, and cytotoxic molecules released by activated neutrophils. By screening theE. histolytica genome, we have identified proteins that may play a role in the defence strategy of the parasite. One of these proteins, a serine proteinase inhibitor, inhibits human neutrophil cathepsin G, a key component of the host defence.     

A Redescription of Entamoeba Histolytica Schaudinn, 1903 (Emended Walker, 1911) Separating It From Entamoeba Dispar Brumpt, 19251

ABSTRACT. Explaining the low incidence of invasive disease (10%) in humans infected with Entamoeba histolytica has occupied the attention of generations of both clinical and nonclinical investigators. One possible explanation would be the existence of two morphologically identical species—one an invasive pathogen, the other noninvasive. This was first proposed by Brumpt in 1925, but his explanation was virtually ignored until 1978 when the first of several publications appeared suggesting that E. histolytica did indeed consist of two species. We have reexamined Brumpt's claim in light of recent biochemical, immunological and genetic studies and conclude that the data derived from these investigations provide unequivocal evidence supporting his hypothesis. With this in mind, we redescribe the invasive parasite retaining the name Entamoeba histolytica Schaudinn, 1903 (Emended Walker, 1911), and set it apart from the noninvasive parasite described by Brumpt, Entamoeba dispar Brumpt, 1925.     

An ex-vivo Human Intestinal Model to Study Entamoeba histolytica Pathogenesis

Amoebiasis (a human intestinal infection affecting 50 million people every year) is caused by the protozoan parasite Entamoeba histolytica. To study the molecular mechanisms underlying human colon invasion by E. histolytica, we have set up an ex vivo human colon model to study the early steps in amoebiasis. Using scanning electron microscopy and histological analyses, we have established that E. histolytica caused the removal of the protective mucus coat during the first two hours of incubation, detached the enterocytes, and then penetrated into the lamina propria by following the crypts of Lieberkühn. Significant cell lysis (determined by the release of lactodehydrogenase) and inflammation (marked by the secretion of pro-inflammatory molecules such as interleukin 1 beta, interferon gamma, interleukin 6, interleukin 8 and tumour necrosis factor) were detected after four hours of incubation. Entamoeba dispar (a closely related non-pathogenic amoeba that also colonizes the human colon) was unable to invade colonic mucosa, lyse cells or induce an inflammatory response. We also examined the behaviour of trophozoites in which genes coding for known virulent factors (such as amoebapores, the Gal/GalNAc lectin and the cysteine protease 5 (CP-A5), which have major roles in cell death, adhesion (to target cells or mucus) and mucus degradation, respectively) were silenced, together with the corresponding tissue responses. Our data revealed that the signalling via the heavy chain Hgl2 or via the light chain Lgl1 of the Gal/GalNAc lectin is not essential to penetrate the human colonic mucosa. In addition, our study demonstrates that E. histolytica silenced for CP-A5 does not penetrate the colonic lamina propria and does not induce the host''s pro-inflammatory cytokine secretion.     

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.