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Sample records for entamoeba histolytica ptpases

  1. Cloning and partial characterization of Entamoeba histolytica PTPases

    SciTech Connect

    Herrera-Rodriguez, Sara Elisa; Baylon-Pacheco, Lidia; Talamas-Rohana, Patricia; Rosales-Encina, Jose Luis . E-mail: rosales@cinvestav.mx

    2006-04-21

    Reversible protein tyrosine phosphorylation is an essential signal transduction mechanism that regulates cell growth, differentiation, mobility, metabolism, and survival. Two genes coding for protein tyrosine phophatases, designed EhPTPA and EhPTPB, were cloned from Entamoeba histolytica. EhPTPA and EhPTPB proteins showed amino acid sequence identity of 37%, both EhPTPases showed similarity with Dictyostelium discoideum and vertebrate trasmembranal PTPases. mRNA levels of EhPTPA gene are up-regulated in trophozoites recovered after 96 h of liver abscess development in the hamster model. EhPTPA protein expressed as a glutathione S-transferase fusion protein (GST::EhPTPA) showed enzymatic activity with p-nitrophenylphosphate as a substrate and was inhibited by PTPase inhibitors vanadate and molybdate. GST::EhPTPA protein selectively dephosphorylates a 130 kDa phosphotyrosine-containing protein in trophozoite cell lysates. EhPTPA gene codifies for a 43 kDa native protein. Up-regulation of EhPTPA expression suggests that EhPTPA may play an important role in the adaptive response of trophozoites during amoebic liver abscess development.

  2. Susceptibility testing of Entamoeba histolytica

    SciTech Connect

    Cedeno, J.R.; Krogstad, D.J.

    1983-12-01

    The growth of Entamoeba histolytica in microtiter plates in vitro in a variety of environments with reduced oxygen tensions is reported. With 3% O/sub 2/, 3% CO/sub 2/, and 94% N/sub 2/, the parasite growth in microtiter plates was identical to that in screw-capped culture tubes, as measured by (/sup 3/H)thymidine incorporation and by quantitative parasite counts. There were no significant differences between the drug concentrations necessary to inhibit parasite growth by 50% based on (/sup 3/H)thymidine incorporation vs those defined by quantitative parasite counts for the 15 antimicrobial agents tested (including seven drugs used for the treatment of amebiasis). This technique provides a reproducible method to quantitate the activity of potential antiamebic agents in vitro. The isotopic method should be of particular value in defining the metabolism of the parasite and effects of antimicrobial agents on it, whereas the morphologic method may be more valuable for workers with limited resources available to them.

  3. Virulence of ’Entamoeba histolytica’ According to the Strains in Korea.

    DTIC Science & Technology

    Investigations are reported on the adaptation of Entamoeba histolytica strains to low temperature conditions. Nine strains of E. histolytica which...Electron microscopy of Entamoeba histolytica in culture and in intestinal mucosa are described. The ultrastructural differences among E. histolytica

  4. DNA characterization of simian Entamoeba histolytica-like strains to differentiate them from Entamoeba histolytica.

    PubMed

    Takano, Jun-ichiro; Tachibana, Hiroshi; Kato, Miyoko; Narita, Toyoko; Yanagi, Tetsuo; Yasutomi, Yasuhiro; Fujimoto, Koji

    2009-10-01

    Two simian Entamoeba histolytica-like strains, EHMfas1 and P19-061405, have been suggested to represent a new species based on genetic characterization. Sequence analyses of the hexokinase, glucose phosphate isomerase, and phosphoglucomutase genes supported the previous findings of isoenzyme analyses demonstrating a new zymodeme pattern. Phylogenetic studies of 18S rDNA, 5.8S rDNA, the chaperonin 60 gene, and the pyridine nucleotide transhydrogenase gene showed original clusters of simian E. histolytica-like strains below or near E. histolytica, respectively. Comparative studies of the chitinase and the serine-rich E. histolytica protein genes and locus 1-2 region revealed that most mutated units were shared among the simian E. histolytica-like strains. The similarities of each of the repeating units within the simian E. histolytica-like strains or E. histolytica and the differences of those between the both might be generated by concerted evolution. Our results indicate that EHMfas1 and P19-061405 should be considered to be the same species, despite that they were isolated from different monkey species and different habitats. Simian E. histolytica-like amebas may be endemic to macaque monkeys, as a counterpart to E. histolytica in humans, and should be differentiated from E. histolytica by the revival name Entamoeba nuttalli, as proposed for P19-061405.

  5. Seroprevalence of Entamoeba histolytica infection in China.

    PubMed

    Yang, Bin; Chen, Yingdan; Wu, Liang; Xu, Longqi; Tachibana, Hiroshi; Cheng, Xunjia

    2012-07-01

    The seroprevalence of Entamoeba histolytica infection in the residents of seven provinces in China was examined by using an enzyme-linked immunosorbent assay with a crude antigen and a recombinant surface antigen, C-Igl, of the parasites. A total of 1,312 serum samples were investigated. The positivity rates for these two antigens were 11.05% and 6.25%, respectively. There was no significant difference in the seropositivity to E. histolytica between men and women. We used a logistic regression model and maximal-likelihood methods to estimate the prevalence of E. histolytica infection from sequential serologic data. Seropositivity in Sichuan, Guizhou, and Sinkiang Provinces was higher than that in Beijing, Shanghai, and Qinghai Provinces. The present study provides an overview of seropositivity to E. histolytica infection in seven provinces in China and use the logistic regression model estimation method to achieve a more accurate measure of amebiasis prevalence.

  6. Regulation of virulence of Entamoeba histolytica.

    PubMed

    Marie, Chelsea; Petri, William A

    2014-01-01

    Entamoeba histolytica is the third-leading cause of parasitic mortality globally. E. histolytica infection generally does not cause symptoms, but the parasite has potent pathogenic potential. The origins, benefits, and triggers of amoebic virulence are complex. Amoebic pathogenesis entails depletion of the host mucosal barrier, adherence to the colonic lumen, cytotoxicity, and invasion of the colonic epithelium. Parasite damage results in colitis and, in some cases, disseminated disease. Both host and parasite genotypes influence the development of disease, as do the regulatory responses they govern at the host-pathogen interface. Host environmental factors determine parasite transmission and shape the colonic microenvironment E. histolytica infects. Here we highlight research that illuminates novel links between host, parasite, and environmental factors in the regulation of E. histolytica virulence.

  7. Erythrophagocytosis in Entamoeba histolytica and Entamoeba dispar: a comparative study.

    PubMed

    Talamás-Lara, Daniel; Chávez-Munguía, Bibiana; González-Robles, Arturo; Talamás-Rohana, Patricia; Salazar-Villatoro, Lizbeth; Durán-Díaz, Ángel; Martínez-Palomo, Adolfo

    2014-01-01

    Entamoeba histolytica is the causative agent of human intestinal and liver amebiasis. The extraordinary phagocytic activity of E. histolytica trophozoites has been accepted as one of the virulence mechanisms responsible for their invasive capacity. The recognition of the noninvasive Entamoeba dispar as a different species has raised the question as to whether the lack of pathogenic potential of this ameba correlates with a limited phagocytic capacity. We have therefore compared the process of erythrophagocytosis in both species by means of light and video microscopy, hemoglobin measurement, and the estimation of reactive oxygen species (ROS). In the present study, we confirmed that E. dispar has lower erythrophagocytic capacity. We also observed by video microscopy a new event of erythrocyte opsonization-like in both species, being more characteristic in E. histolytica. Moreover, E. dispar showed a lower capacity to produce ROS compared with the invasive species and also showed a large population of amoebae that did not engulf any erythrocyte over time. Our results demonstrate that E. histolytica has a higher phagocytic capacity than E. dispar, including a higher rate of production of ROS in the course of ingesting red blood cells.

  8. Erythrophagocytosis in Entamoeba histolytica and Entamoeba dispar: A Comparative Study

    PubMed Central

    Talamás-Lara, Daniel; Chávez-Munguía, Bibiana; González-Robles, Arturo; Talamás-Rohana, Patricia; Salazar-Villatoro, Lizbeth; Durán-Díaz, Ángel; Martínez-Palomo, Adolfo

    2014-01-01

    Entamoeba histolytica is the causative agent of human intestinal and liver amebiasis. The extraordinary phagocytic activity of E. histolytica trophozoites has been accepted as one of the virulence mechanisms responsible for their invasive capacity. The recognition of the noninvasive Entamoeba dispar as a different species has raised the question as to whether the lack of pathogenic potential of this ameba correlates with a limited phagocytic capacity. We have therefore compared the process of erythrophagocytosis in both species by means of light and video microscopy, hemoglobin measurement, and the estimation of reactive oxygen species (ROS). In the present study, we confirmed that E. dispar has lower erythrophagocytic capacity. We also observed by video microscopy a new event of erythrocyte opsonization-like in both species, being more characteristic in E. histolytica. Moreover, E. dispar showed a lower capacity to produce ROS compared with the invasive species and also showed a large population of amoebae that did not engulf any erythrocyte over time. Our results demonstrate that E. histolytica has a higher phagocytic capacity than E. dispar, including a higher rate of production of ROS in the course of ingesting red blood cells. PMID:25003123

  9. 21 CFR 866.3220 - Entamoeba histolytica serological reagents.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... Entamoeba histolytica in serum. Additionally, some of these reagents consist of antisera conjugated with a... parasite Entamoeba histolytica and provides epidemiological information on diseases caused by this parasite. The parasite may invade the skin, liver, intestines, lungs, and diaphragm, causing disease...

  10. Insights into Entamoeba histolytica virulence modulation.

    PubMed

    Padilla-Vaca, F; Anaya-Velázquez, F

    2010-08-01

    Entamoeba histolytica is able to invade human tissues by means of several molecules and biological properties related to the virulence. Pathogenic amebas use three major virulence factors, Gal/GalNAc lectin, amebapore and proteases, for lyse, phagocytose, kill and destroy a variety of cells and tissues in the host. Responses of the parasite to host components such as mucins and bacterial flora influence the behavior of pathogenic amebas altering their expression of virulence factors. The relative virulence of different strains of E. histolytica has been shown to vary as a consequence of changes in conditions of in vitro cultivation which implies substantial changes in basic metabolic aspects and factors directly and indirectly related to amebic virulence. Comparison of E. histolytica strains with different virulence phenotypes and under different conditions of growth will help to identify new virulence factor candidates and define the interplay between virulence factors and invasive phenotype. Virulence attenuate mutants of E. histolytica are useful also to uncover novel virulence determinants. The comparison of biological properties and virulence factors between E. histolytica and E. dispar, a non-pathogenic species, has been a useful approach to investigate the key factors involved in the experimental presentation of amebiasis and its complex regulation. The molecular mechanisms that regulate these variations in virulence are not yet known. Their elucidation will help us to better understand the gene expression plasticity that enables the effective adaptation of the ameba to changes in growth culture conditions and host factors.

  11. DNA hybridization probe for clinical diagnosis of Entamoeba histolytica.

    PubMed Central

    Samuelson, J; Acuna-Soto, R; Reed, S; Biagi, F; Wirth, D

    1989-01-01

    As an alternative to microscopic identification of Entamoeba histolytica parasites isolated from stool, a sensitive and species-specific DNA hybridization probe was made for rapid diagnosis of E. histolytica parasites in clinical samples directly applied to nylon membranes. The DNA hybridization probe was made by screening a genomic library of a virulent HM-1:IMSS strain of E. histolytica to detect recombinant plasmids containing highly repeated parasite DNA sequences. Four plasmid clones that reacted across Entamoeba species coded for highly repeated rRNA genes of E. histolytica. Four other plasmid clones were E. histolytica specific in that they bound to four axenized and nine xenic strains of E. histolytica but did not recognize closely related E. histolytica-like Laredo, Entamoeba moshkovskii, or Entamoeba invadens parasites. The diagnostic clones detected as few as eight cultured amoebae and did not distinguish between pathogenic and nonpathogenic zymodemes of E. histolytica. The diagnostic clones were sequenced and contained 145-base-pair sequences which appear to be tandemly repeated in the genome. No stable transcript which is homologous to the diagnostic DNA was detected. In a study of stool samples from Mexico City shown by microscopy to contain E. histolytica, Entamoeba coli, Giardia lamblia, Endolimax nana, Trichuris trichiuria, and Chilomastix mesnili parasites, the DNA hybridization probe demonstrated a sensitivity of 1.0 and a specificity of 0.93. We conclude that the DNA hybridization probe can be used for rapid and accurate diagnosis of E. histolytica parasites. Images PMID:2542361

  12. Entamoeba histolytica meningoencephalitis diagnosed by trophozoites in cerebrospinal fluid

    PubMed Central

    Goh, L M L; Marrone, J R

    2013-01-01

    Entamoeba histolytica meningoencephalitis has not been described in the modern literature, which is distinct from that caused by free-living amoebae. We report the first case of E. histolytica meningoencephalitis without liver or brain abscesses. Cerebrospinal fluid revealed 2 + very motile trophozoites. Our patient was successfully treated with intravenous metronidazole. PMID:25356319

  13. Entamoeba histolytica meningoencephalitis diagnosed by trophozoites in cerebrospinal fluid.

    PubMed

    Goh, L M L; Marrone, J R

    2013-10-01

    Entamoeba histolytica meningoencephalitis has not been described in the modern literature, which is distinct from that caused by free-living amoebae. We report the first case of E. histolytica meningoencephalitis without liver or brain abscesses. Cerebrospinal fluid revealed 2 + very motile trophozoites. Our patient was successfully treated with intravenous metronidazole.

  14. Entamoeba histolytica. Phagocytosis as a virulence factor

    PubMed Central

    1983-01-01

    In this paper, we attempted to define the role of phagocytosis in the virulence of Entamoeba histolytica. We have isolated, from a highly phagocytic and virulent strain, a clone deficient in phagocytosis. Trophozoites of wild-type strain HM1:IMSS were fed with Escherichia coli strain CR34-Thy- grown on 5-bromo,2'-deoxyuridine. The trophozoites that had incorporated the base analog through phagocytosis of the bacteria were killed by irradiation with 310 nm light. The survivors, presumably trophozoites defective in phagocytosis, were grown until log phase and submitted two more times to the selection procedure. Clone L-6, isolated from a subpopulation resulting from this selection procedure, showed 75-85% less erythrophagocytic activity than the wild-type strain. The virulence of clone L-6 and strain HM1:IMSS was measured. The inoculum required to induce liver abscesses in 50% of the newborn hamsters inoculated (AD50) of HM1:IMSS was 1.5 X 10(4) trophozoites. Clone L-6 trophozoites failed to induce liver abscesses in newborn hamsters even with inocula of 5 X 10(5) trophozoites. Virulence revertants were obtained by successive passage of L-6 trophozoites through the liver of young hamsters. The trophozoites that recovered the ability to produce liver abscesses simultaneously recuperate high erythrophagocytic rates. These results show that phagocytosis is involved in the aggressive mechanisms of E. histolytica. PMID:6313842

  15. Virulence of ’Entamoeba histolytica’ According to the Strains in Korea.

    DTIC Science & Technology

    During the years of 1969 to 1973, an investigation on the virulence of Entamoeba histolytica strains in Korea was carried out in series. Samples were...pathogenic strain of Entamoeba histolytica was recognized in Korea. (Modified author abstract)

  16. Entamoeba histolytica: observations on metabolism based on thegenome sequence

    SciTech Connect

    Anderson, Iain J.; Loftus, Brendan J.

    2005-07-01

    The sequencing of the genome of Entamoeba histolytica has allowed a reconstruction of its metabolic pathways, many of which are unusual for a eukaryote. Based on the genome sequence, it appears that amino acids may play a larger role than previously thought in energy metabolism, with roles in both ATP synthesis and NAD regeneration. Arginine decarboxylase may be involved in survival of E. histolytica during its passage through the stomach. The usual pyrimidine synthesis pathway is absent, but a partial pyrimidine degradation pathway could be part of a novel pyrimidine synthesis pathway. Ribonucleotide reductase was not found in the E. histolytica genome, but it was found in the close relatives Entamoeba invadens and Entamoeba moshkovskii, suggesting a recent loss from E. histolytica. The usual eukaryotic glucose transporters are not present, but members of a prokaryotic monosaccharide transporter family are present.

  17. Purification and cellular localization of the Entamoeba histolytica transcarboxylase.

    PubMed

    Barbosa-Cabrera, E; Salas-Casas, A; Rojas-Hernández, S; Jarillo-Luna, A; Abarca-Rojano, E; Rodríguez, M A; Campos-Rodríguez, R

    2012-09-01

    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.

  18. Molecular detection of Entamoeba histolytica and Entamoeba dispar infection among wild rats in Kuala Lumpur, Malaysia.

    PubMed

    Lau, Y L; Jamaiah, I; Rohela, M; Fong, M Y; Siti, C O S; Siti, F A

    2014-12-01

    Entamoeba histolytica infection is the third-greatest parasitic disease responsible for death in the world. Wild rats harbouring E. histolytica can be the possible reservoir hosts for human amoebiasis. There were numerous studies on prevalence of intestinal parasites among wild rats in Malaysia but none has reported E. histolytica. Rats were captured from Sentul and Chow Kit areas, Kuala Lumpur, Malaysia. The preserved stool samples were used for microscopy examination and molecular analysis. Out of 137 samples collected, 12 were positive for E. histolytica / E. dispar / E. moshkovskii microscopically. Two E. histolytica (1.4%), 1 E. dispar (0.7%) and 6 mixed infections of E. histolytica and E. dispar (4.3%) were detected using PCR. This is the first report of molecular detection of E. histolytica/dispar infection among wild rats in Malaysia. This study provides useful information about the potential risks of zoonotic agents and the importance of developing control measures to prevent zoonotic transmission.

  19. 21 CFR 866.3220 - Entamoeba histolytica serological reagents.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Entamoeba histolytica serological reagents. 866.3220 Section 866.3220 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents §...

  20. 21 CFR 866.3220 - Entamoeba histolytica serological reagents.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Entamoeba histolytica serological reagents. 866.3220 Section 866.3220 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents §...

  1. 21 CFR 866.3220 - Entamoeba histolytica serological reagents.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Entamoeba histolytica serological reagents. 866.3220 Section 866.3220 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents §...

  2. 21 CFR 866.3220 - Entamoeba histolytica serological reagents.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Entamoeba histolytica serological reagents. 866.3220 Section 866.3220 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents §...

  3. Prevalence of Entamoeba histolytica/Entamoeba dispar in the city of Campina Grande, in northeastern Brazil.

    PubMed

    Silva, Maria Teresa Nascimento; Santana, José Valfrido; Bragagnoli, Gérson; Marinho, Alexandre Magno da Nóbrega; Malagueño, Elizabeth

    2014-01-01

    There is a clear need to perform epidemiological studies to find the true prevalence of Entamoeba histolytica around the world. The evaluation of this prevalence has been hindered by the existence of two different species which are morphologically identical, but genetically different, namely E. histolytica, which causes amebiasis, and E. dispar, which is non-pathogenic. In Brazil, the E. dispar has been detected in communities in the Southeastern (SE) and Northeastern (NE) regions with poor sanitation. However, individuals infected with E. histolytica have been identified in other regions. There is an absence of reports on the prevalence of these parasites in the state of Paraíba, which also has areas with poor sanitary conditions where a high prevalence of the E. histolytica/E. dispar complex has been detected in children from urban slums. The present study evaluated the prevalence of E. histolytica and E. dispar in 1,195 asymptomatic children between two and 10 years of age, living in a sprawling urban slum in Campina Grande, in the state of Paraíba, in Northeastern Brazil. These children were examined and their feces samples were analyzed microscopically. A total of 553 children tested positive for the E. histolytica/E. dispar complex, and 456 of the positive samples were tested with the E. histolytica II® ELISA kit. All 456 samples were negative for the presence of the adhesin E. histolytica specific antigen. The evidence suggests that in this community E. histolytica is absent and E. dispar is the dominant species.

  4. A detoxifying oxygen reductase in the anaerobic protozoan Entamoeba histolytica.

    PubMed

    Vicente, João B; Tran, Vy; Pinto, Liliana; Teixeira, Miguel; Singh, Upinder

    2012-09-01

    We report the characterization of a bacterial-type oxygen reductase abundant in the cytoplasm of the anaerobic protozoan parasite Entamoeba histolytica. Upon host infection, E. histolytica is confronted with various oxygen tensions in the host intestine, as well as increased reactive oxygen and nitrogen species at the site of local tissue inflammation. Resistance to oxygen-derived stress thus plays an important role in the pathogenic potential of E. histolytica. The genome of E. histolytica has four genes that encode flavodiiron proteins, which are bacterial-type oxygen or nitric oxide reductases and were likely acquired by lateral gene transfer from prokaryotes. The EhFdp1 gene has higher expression in virulent than in nonvirulent Entamoeba strains and species, hinting that the response to oxidative stress may be one correlate of virulence potential. We demonstrate that EhFdp1 is abundantly expressed in the cytoplasm of E. histolytica and that the protein levels are markedly increased (up to ~5-fold) upon oxygen exposure. Additionally, we produced fully functional recombinant EhFdp1 and demonstrated that this enzyme is a specific and robust oxygen reductase but has poor nitric oxide reductase activity. This observation represents a new mechanism of oxygen resistance in the anaerobic protozoan pathogen E. histolytica.

  5. Entamoeba histolytica: cyst-like structures in vitro induction.

    PubMed

    Barrón-González, M P; Villarreal-Treviño, L; Reséndez-Pérez, D; Mata-Cárdenas, B D; Morales-Vallarta, M R

    2008-04-01

    The cyst of Entamoeba histolytica is responsible for amebiasis infection. However, no axenic in vitro system exists that promotes mass encystation for studying this process of this human-infecting parasite. Cyst-like structures of E. histolytica obtained in this work were induced using TYI-S-33 media in combination with enterobacterias Escherichia coli and Enterococcus faecalis conditioned media, high CO2 tension and histamine. Cyst-like structures showed the same characteristics of a typical E. histolytica cyst: aggregation, resistance to 0.15% sarcosyl for 10 min, high signal of fluorescence under UV light when stained with 10% calcofluor M2r and the surface topology showed a wrinkled wall. In addition these structures are multinucleated with condensed chromatin attached to nuclear membrane, contain big vacuoles and ribonucleoproteic helices in the cytoplasm and also present a thin cell wall. Last all characteristics are all the same as a typical of E. histolytica cyst.

  6. Differential expression of surface glycoconjugates on Entamoeba histolytica and Entamoeba dispar.

    PubMed

    Pacheco-Yépez, Judith; Campos-Rodríguez, Rafael; Rojas-Hernández, Saúl; Serrano-Luna, José J; Rivera-Aguilar, Víctor; Villa-Treviño, Saúl; Martínez-Palomo, Adolfo; Tsutsumi, Víctor; Shibayama, Mineko

    2009-06-01

    The human large intestine can harbor two morphologically similar amoebae; the invasive Entamoeba histolytica and the non-invasive Entamoeba dispar. Whereas E. histolytica can produce intestinal and extra-intestinal lesions, E. dispar is present in non-symptomatic carriers. Although biochemical, genetic and proteomic studies have identified clear differences between these Entamoebae, it has become clear that several molecules, once assumed to be involved in tissue destruction, exist in both the virulent and the avirulent species. As surface molecules may play a role in invasion and could therefore determine which amoebae are invasive, we analyzed the glycoconjugate composition of E. histolytica and E. dispar using lectins. There was a significant difference between E. histolytica and E. dispar in the expression of glycoconjugates containing d-mannose and N-acetyl-alpha-D-galactosamine residues, but not between virulent and avirulent strains of E. histolytica. N-glycoconjugates with terminal alpha (1-3)-linked mannose residues participate in the adhesion and subsequent cytotoxicity of E. histolytica to cultured hamster hepatocytes. One of them probably is the Gal/GalNAc lectin.

  7. Photoacoustic spectroscopy of Entamoeba histolytica strains

    NASA Astrophysics Data System (ADS)

    Acosta-Avalos, D.; Alvarado-Gil, J. J.; Silva, E. F.; Orozco, E.; de Menezes, L. F.; Vargas, H.

    2005-06-01

    Pathogenic and non-pathogenic strains of E. histolytica are studied using photoacoustic spectroscopy. It is shown that the pathogenic strain presents a spectrum similar to that of iron sulfur proteins. The non-pathogenic strain does not show any relevant absorption at the studied wavelength range. The differences observed between the optical absorption spectra of both strains opens the possibility of using photoacoustic spectroscopy as a reliable and simple technique to identify different types of E. histolytica strains.

  8. The nucleolus in Entamoeba histolytica and Entamoeba invadens is located at the nuclear periphery.

    PubMed

    Jhingan, Gagan Deep; Panigrahi, Sunil K; Bhattacharya, Alok; Bhattacharya, Sudha

    2009-09-01

    The ribosomal RNA genes in the human parasite Entamoeba histolytica and its reptilian counterpart Entamoeba invadens are located on extrachromosomal circles. The expression of rRNA genes generally takes place in a specialized nuclear compartment-the nucleolus. In Entamoeba species the nuclear space that may be called the nucleolus has yet to be defined. Previous studies showed that the rDNA circles are located at the nuclear periphery. Here we have raised antibodies against the E. histolytica homologue of fibrillarin, a highly conserved protein known to be a marker for nucleolus. These antibodies cross-reacted preferentially with the nuclear periphery, forming a peripheral ring. There was complete colocalization of fibrillarin with the signal obtained by antibodies against E. histolytica RNA polymerase I (but not polymerase II and III), strongly suggesting that the nucleolus in E. histolytica is indeed located at the nuclear periphery. The dynamic nature of the nucleolus was evident when cells were subjected to a variety of growth stresses. Although the peripheral nucleolar structure was retained, stress was accompanied by significant cytoplasmic localization of RNA polymerase I, and to some extent fibrillarin. The nucleolus in E. invadens was also located at the nuclear periphery. When these cells were induced to encyst the nucleolar ring structure was lost, giving way to small, fragmented foci. This study gives the first clear insight into nucleolar structure in Entamoeba.

  9. Differentiation of Entamoeba histolytica and Entamoeba dispar in cyst-passers by immunoblot.

    PubMed

    Lee, M; Hong, S T

    1996-12-01

    Differentiation of invasive strains of Entamoeba histolytica according to their pathogenicity has been a topic of long debate, but now the pathogenic species only is regarded as E. histolytica while the non pathogenic species is E. dispar. The present study applied immunoblot to differentiate infections of the two species among microscopically-detected cyst-passers in Korea. The crude extract of E. histolytica separated in 5.20% gradient gels, revealed many fractions of 94, 81, 71, 50, 44, 38.5, 37.5, 29, 19, and 18 kDa when the cysteine proteinase inhibitor, E64, was supplemented. The scrum IgG antibody of 3 proven E, histolytica cases reacted with the antigenic fractions of 117, 110, 99, 68, 66, 60, 54, 52, 46, and 45 kDa. Sera of PCR confirmed 3 cases of E. dispar reacted only to the 117 kDa fraction of the E. histolytica crude extract which was regarded as non-specific. To the antigen of monoxenic E. dispar, sera of E. dispar and E. histolytica cases showed the same immunoblot reactions. The serum IgA antibody reacted with several antigenic fractions of both E. histolytica and E. dispar, but IgM and IgE antibodies showed no reaction to either antigen. Sera of 24 symptomless amebic cyst passers were screened with the E. histolytica antigen; two were found to be infected by E. histolytica and 22 were by E. dispar. The present findings suggest that in Korea most of asymptomatic cyst passers of E. histolytica are carriers of E. dispar. Immunoblot using E. histolytica antigen is a good technique for the differentiation of E. histolytica and E. dispar infections.

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

    PubMed

    Calegar, Deiviane Aparecida; Nunes, Beatriz Coronato; Monteiro, Kerla Joeline Lima; Santos, Jéssica Pereira Dos; Toma, Helena Keiko; Gomes, Tais Ferreira; Lima, Marli Maria; Bóia, Márcio Neves; Carvalho-Costa, Filipe Anibal

    2016-02-01

    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.

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

    PubMed Central

    Calegar, Deiviane Aparecida; Nunes, Beatriz Coronato; Monteiro, Kerla Joeline Lima; dos Santos, Jéssica Pereira; Toma, Helena Keiko; Gomes, Tais Ferreira; Lima, Marli Maria; Bóia, Márcio Neves; Carvalho-Costa, Filipe Anibal

    2016-01-01

    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. PMID:26841049

  12. Taking a bite: Amoebic trogocytosis in Entamoeba histolytica and beyond.

    PubMed

    Ralston, Katherine S

    2015-12-01

    Entamoeba histolytica is a diarrheal pathogen with the ability to cause profound host tissue damage. This organism possesses contact-dependent cell killing activity, which is likely to be a major contributor to tissue damage. E. histolytica trophozoites were recently shown to ingest fragments of living human cells. It was demonstrated that this process, termed amoebic trogocytosis, contributes to cell killing. Recent advances in ex vivo and 3-D cell culture approaches have shed light on mechanisms for tissue destruction by E. histolytica, allowing amoebic trogocytosis to be placed in the context of additional host and pathogen mediators of tissue damage. In addition to its relevance to pathogenesis of amoebiasis, an appreciation is emerging that intercellular nibbling occurs in many organisms, from protozoa to mammals.

  13. Innate immunity prevents tissue invasion by Entamoeba histolytica.

    PubMed

    Shibayama, Mineko; Rivera-Aguilar, Víctor; Barbosa-Cabrera, Elizabeth; Rojas-Hernández, Saúl; Jarillo-Luna, Adriana; Tsutsumi, Víctor; Pacheco-Yepez, Judith; Campos-Rodríguez, Rafael

    2008-12-01

    Although innate and adaptive immunity both play a role in amoebiasis, the mechanisms involved in the elimination of Entamoeba histolytica are poorly understood. To provide more information about the innate immune mechanisms that may confer protection against invasive amoebiasis, we administered inflammatory substances (bacillus Calmette-Guérin, lipopolysaccharide, complete Freund's adjuvant, or mineral oil) into the peritoneum of hamsters. The animals were then challenged with pathogenic trophozoites of E. histolytica and, after 7 days, the protective host response was analysed. We found that the nonspecific inflammatory response induced in the peritoneum was sufficient to prevent liver invasion by E. histolytica. In vitro experiments showed that the killing of trophozoites was mediated by peritoneal macrophages and a protein of 68 kDa with peroxidase activity.

  14. An ecto-protein tyrosine phosphatase of Entamoeba histolytica induces cellular detachment by disruption of actin filaments in HeLa cells.

    PubMed

    Anaya-Ruiz, M; Pérez-Santos, J L M; Talamás-Rohana, P

    2003-07-01

    Actin cytoskeleton disruption in host cells has been demonstrated for PTPases from pathogenic microorganisms. In this work, we analysed whether the secreted acid phosphatase from Entamoeba histolytica has phosphotyrosine phosphatase activity and the possibility that this activity may participate in damaging host cells. The secreted acid phosphatase of E. histolytica, which catalyses p-nitrophenyl phosphate hydrolysis at acid pH values, was found to have phosphotyrosine phosphatase activity. The enzymatic properties of phosphotyrosine phosphatase and acid phosphatase were virtually identical and included: Km values of 10 x 10(-4) M, no requirement for divalent cations, and sensitivity to molybdate, vanadate, and tungstate. The phosphotyrosyl phosphatase activity caused significant levels of cell rounding and detachment correlating with disruption of the actin stress fibres in HeLa cells. Thus, our data suggest that secreted phosphotyrosine phosphatase could play a cytotoxic role during amoebic infection.

  15. PREVALENCE OF Entamoeba histolytica/Entamoeba dispar IN THE CITY OF CAMPINA GRANDE, IN NORTHEASTERN BRAZIL

    PubMed Central

    Silva, Maria Teresa Nascimento; Santana, José Valfrido; Bragagnoli, Gérson; Marinho, Alexandre Magno da Nóbrega; Malagueño, Elizabeth

    2014-01-01

    There is a clear need to perform epidemiological studies to find the true prevalence of Entamoeba histolytica around the world. The evaluation of this prevalence has been hindered by the existence of two different species which are morphologically identical, but genetically different, namely E. histolytica, which causes amebiasis, and E. dispar, which is non-pathogenic. In Brazil, the E. dispar has been detected in communities in the Southeastern (SE) and Northeastern (NE) regions with poor sanitation. However, individuals infected with E. histolytica have been identified in other regions. There is an absence of reports on the prevalence of these parasites in the state of Paraíba, which also has areas with poor sanitary conditions where a high prevalence of the E. histolytica/E. dispar complex has been detected in children from urban slums. The present study evaluated the prevalence of E. histolytica and E. dispar in 1,195 asymptomatic children between two and 10 years of age, living in a sprawling urban slum in Campina Grande, in the state of Paraíba, in Northeastern Brazil. These children were examined and their feces samples were analyzed microscopically. A total of 553 children tested positive for the E. histolytica/E. dispar complex, and 456 of the positive samples were tested with the E. histolytica II® ELISA kit. All 456 samples were negative for the presence of the adhesin E. histolytica specific antigen. The evidence suggests that in this community E. histolytica is absent and E. dispar is the dominant species. PMID:25229229

  16. Development of a thermostabilized, one-step, nested, tetraplex PCR assay for simultaneous identification and differentiation of Entamoeba species, Entamoeba histolytica and Entamoeba dispar from stool samples.

    PubMed

    Foo, Phiaw Chong; Chan, Yean Yean; See Too, Wei Cun; Tan, Zi Ning; Wong, Weng Kin; Lalitha, Pattabhiraman; Lim, Boon Huat

    2012-09-01

    Entamoeba histolytica is the only Entamoeba species that causes amoebiasis in humans. Approximately 50 million people are infected, with 100, 000 deaths annually in endemic countries. Molecular diagnosis of Entamoeba histolytica is important to differentiate it from the morphologically identical Entamoeba dispar to avoid unnecessary medication. Conventional molecular diagnostic tests require trained personnel, cold-chain transportation and/or are storage-dependent, which make them user-unfriendly. The aim of this study was to develop a thermostabilized, one-step, nested, tetraplex PCR assay for the detection of Entamoeba histolytica, Entamoeba dispar and Entamoeba species in cold-chain-free and ready-to-use form. The PCR test was designed based on the Entamoeba small subunit rRNA (SSU-rRNA) gene, which detects the presence of any Entamoeba species, and simultaneously can be used to differentiate Entamoeba histolytica from Entamoeba dispar. In addition, a pair of primers was designed to serve as an internal amplification control to help identify inhibitors in the samples. All PCR reagents together with the designed primers were thermostabilized by lyophilization and were stable at 24 °C for at least 6 months. The limit of detection of the tetraplex PCR was found to be 39 pg DNA or 1000 cells for Entamoeba histolytica and 78 pg DNA or 1000 cells for Entamoeba dispar, and the specificity was 100 %. In conclusion, this cold-chain-free, thermostabilized, one-step, nested, multiplex PCR assay was found to be efficacious in differentiating Entamoeba histolytica from other non-pathogenic Entamoeba species.

  17. Species-specific immunity induced by infection with Entamoeba histolytica and Entamoeba moshkovskii in mice.

    PubMed

    Shimokawa, Chikako; Culleton, Richard; Imai, Takashi; Suzue, Kazutomo; Hirai, Makoto; Taniguchi, Tomoyo; Kobayashi, Seiki; Hisaeda, Hajime; Hamano, Shinjiro

    2013-01-01

    Entamoeba histolytica, the parasitic amoeba responsible for amoebiasis, causes approximately 100,000 deaths every year. There is currently no vaccine against this parasite. We have previously shown that intracecal inoculation of E. histolytica trophozoites leads to chronic and non-healing cecitis in mice. Entamoeba moshkovskii, a closely related amoeba, also causes diarrhea and other intestinal disorders in this model. Here, we investigated the effect of infection followed by drug-cure of these species on the induction of immunity against homologous or heterologous species challenge. Mice were infected with E. histolytica or E. moshkovskii and treated with metronidazole 14 days later. Re-challenge with E. histolytica or E. moshkovskii was conducted seven or 28 days following confirmation of the clearance of amoebae, and the degree of protection compared to non-exposed control mice was evaluated. We show that primary infection with these amoebae induces a species-specific immune response which protects against challenge with the homologous, but not a heterologous species. These findings pave the way, therefore, for the identification of novel amoebae antigens that may become the targets of vaccines and provide a useful platform to investigate host protective immunity to Entamoeba infections.

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

    PubMed

    Chávez-Munguía, Bibiana; Talamás-Rohana, Patricia; Castañón, Guadalupe; Salazar-Villatoro, Lizbeth; Hernández-Ramírez, Verónica; Martínez-Palomo, Adolfo

    2012-07-01

    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.

  19. Feed-forward regulation of phagocytosis by Entamoeba histolytica.

    PubMed

    Sateriale, Adam; Vaithilingam, Archana; Donnelly, Liam; Miller, Peter; Huston, Christopher D

    2012-12-01

    The parasitic protozoan Entamoeba histolytica is aptly named for its capacity to destroy host tissue. When E. histolytica trophozoites invade the lamina propria of a host colon, extracellular matrices are degraded while host cells are killed and phagocytosed. The ability of E. histolytica to phagocytose host cells correlates with virulence in vivo. In order to better understand the mechanism of phagocytosis, we used an E. histolytica Affymetrix microarray chip to measure the total gene expression of phagocytic and nonphagocytic subpopulations. Using paramagnetic beads coated with a known host ligand that stimulates phagocytosis, phagocytic and nonphagocytic amoebae from a single culture were purified. Microarray analysis of the subpopulations identified 121 genes with >2-fold higher expression in phagocytic than in nonphagocytic amoebae. Functional annotation identified genes encoding proteins involved in actin binding and cytoskeletal organization as highly enriched gene clusters. Post hoc analyses of selected genes showed that the gene expression profile identified in the microarray experiment did not exist prior to cell sorting but rather was stimulated through phagocytosis. Further, these expression profiles correlated with an increase in phagocytic ability, as E. histolytica cultures exposed to an initial stimulus of phagocytosis showed increased phagocytic ability upon a second stimulus. To our knowledge, this is the first description of such feed-forward regulation of gene expression and phagocytic ability in a phagocyte.

  20. MALDI-TOF mass spectrometry for the detection and differentiation of Entamoeba histolytica and Entamoeba dispar.

    PubMed

    Calderaro, Adriana; Piergianni, Maddalena; Buttrini, Mirko; Montecchini, Sara; Piccolo, Giovanna; Gorrini, Chiara; Rossi, Sabina; Chezzi, Carlo; Arcangeletti, Maria Cristina; Medici, Maria Cristina; De Conto, Flora

    2015-01-01

    Detection of Entamoeba histolytica and its differentiation from Entamoeba dispar is an important goal of the clinical parasitology laboratory. The aim of this study was the identification and differentiation of E. histolytica and E. dispar by MALDI-TOF MS, in order to evaluate the application of this technique in routine diagnostic practice. MALDI-TOF MS was applied to 3 amebic reference strains and to 14 strains isolated from feces that had been differentiated by molecular methods in our laboratory. Protein extracts from cultures of these strains (axenic cultures for the 3 reference strains and monoxenic cultures for the 14 field isolates) were analyzed by MALDI-TOF MS and the spectra obtained were analyzed by statistical software. Five peaks discriminating between E. histolytica and E. dispar reference strains were found by protein profile analysis: 2 peaks (8,246 and 8,303 Da) specific for E. histolytica and 3 (4,714; 5,541; 8,207 Da) for E. dispar. All clinical isolates except one showed the discriminating peaks expected for the appropriate species. For 2 fecal samples from which 2 strains (1 E. histolytica and 1 E. dispar) out of the 14 included in this study were isolated, the same discriminating peaks found in the corresponding isolated amebic strains were detected after only 12h (E. histolytica) and 24h (E. dispar) of incubation of the fecal samples in Robinson's medium without serum. Our study shows that MALDI-TOF MS can be used to discriminate between E. histolytica and E. dispar using in vitro xenic cultures and it also could have potential for the detection of these species in clinical samples.

  1. Detection of Entamoeba histolytica by Recombinase Polymerase Amplification.

    PubMed

    Nair, Gayatri; Rebolledo, Mauricio; White, A Clinton; Crannell, Zachary; Richards-Kortum, R Rebecca; Pinilla, A Elizabeth; Ramírez, Juan David; López, M Consuelo; Castellanos-Gonzalez, Alejandro

    2015-09-01

    Amebiasis is an important cause of diarrheal disease worldwide and has been associated with childhood malnutrition. Traditional microscopy approaches are neither sensitive nor specific for Entamoeba histolytica. Antigen assays are more specific, but many cases are missed unless tested by molecular methods. Although polymerase chain reaction (PCR) is effective, the need for sophisticated, expensive equipment, infrastructure, and trained personnel limits its usefulness, especially in the resource-limited, endemic areas. Here, we report development of a recombinase polymerase amplification (RPA) method to detect E. histolytica specifically. Using visual detection by lateral flow (LF), the test was highly sensitive and specific and could be performed without additional equipment. The availability of this inexpensive, sensitive, and field-applicable diagnostic test could facilitate rapid diagnosis and treatment of amebiasis in endemic regions.

  2. Detection of Entamoeba histolytica by Recombinase Polymerase Amplification

    PubMed Central

    Nair, Gayatri; Rebolledo, Mauricio; White, A. Clinton; Crannell, Zachary; Richards-Kortum, R. Rebecca; Pinilla, A. Elizabeth; Ramírez, Juan David; López, M. Consuelo; Castellanos-Gonzalez, Alejandro

    2015-01-01

    Amebiasis is an important cause of diarrheal disease worldwide and has been associated with childhood malnutrition. Traditional microscopy approaches are neither sensitive nor specific for Entamoeba histolytica. Antigen assays are more specific, but many cases are missed unless tested by molecular methods. Although polymerase chain reaction (PCR) is effective, the need for sophisticated, expensive equipment, infrastructure, and trained personnel limits its usefulness, especially in the resource-limited, endemic areas. Here, we report development of a recombinase polymerase amplification (RPA) method to detect E. histolytica specifically. Using visual detection by lateral flow (LF), the test was highly sensitive and specific and could be performed without additional equipment. The availability of this inexpensive, sensitive, and field-applicable diagnostic test could facilitate rapid diagnosis and treatment of amebiasis in endemic regions. PMID:26123960

  3. Entamoeba histolytica Trophozoites and Lipopeptidophosphoglycan Trigger Human Neutrophil Extracellular Traps

    PubMed Central

    Ávila, Eva E.; Rodríguez, Mayra C.; Díaz-Godínez, César; Laclette, Juan P.; Becker, Ingeborg; Carrero, Julio C.

    2016-01-01

    Neutrophil defense mechanisms include phagocytosis, degranulation and the formation of extracellular traps (NET). These networks of DNA are triggered by several immune and microbial factors, representing a defense strategy to prevent microbial spread by trapping/killing pathogens. This may be important against Entamoeba histolytica, since its large size hinders its phagocytosis. The aim of this study was to determine whether E. histolytica and their lipopeptidophosphoglycan (EhLPPG) induce the formation of NETs and the outcome of their interaction with the parasite. Our data show that live amoebae and EhLPPG, but not fixed trophozoites, induced NET formation in a time and dose dependent manner, starting at 5 min of co-incubation. Although immunofluorescence studies showed that the NETs contain cathelicidin LL-37 in close proximity to amoebae, the trophozoite growth was only affected when ethylene glycol tetra-acetic acid (EGTA) was present during contact with NETs, suggesting that the activity of enzymes requiring calcium, such as DNases, may be important for amoeba survival. In conclusion, E. histolytica trophozoites and EhLPPG induce in vitro formation of human NETs, which did not affect the parasite growth unless a chelating agent was present. These results suggest that NETs may be an important factor of the innate immune response during infection with E. histolytica. PMID:27415627

  4. Copro prevalence and estimated risk of Entamoeba histolytica in Diarrheic patients at Beni-Suef, Egypt.

    PubMed

    Ibrahim, Shimaa S; El-Matarawy, Olfat M; Ghieth, Marwa A; Abu Sarea, Enas Y; El-Badry, Ayman A

    2015-02-01

    Amoebiasis diagnosis is usually based on microscopy that cannot differentiate pathogenic E. histolytica from morphologically identical non-pathogenic species. 194 fecal samples were collected from diarrheic &/or dysenteric patients and examined for Entamoeba complex microscopically, E. histolytica/E. dispar coproantigen using ICT and E. histolytica coproantigen using Tech lab E. histolytica II ELISA test. Entamoeba complex trophozoites/cysts, E. histolytica/E. dispar coproantigen and E. histolytica coproantigen were detected in 22.2, 14.4 and 3.6 % of samples, respectively. Microscopy and ICT method had limited sensitivity with poor PPV (9.3 and 7.1 %, respectively) and both slightly agree with ELISA test. The prevalence of E. histolytica was low (3.6 %) in studied individuals and was 14 times lower than non-pathogenic amoebae. E. histolytica detection studied individuals was positively associated with mucoid and bloody stool, which makes them disease predictors. E. histolytica fecal ELISA assay for E. histolytica detection surpassed microscopy and E. histolytica/E. dispar ICT assay. This has highlighted the need for practical non-microscopic detection methods that can differentiate between amoeba infections to avoid unnecessary and possibly harmful therapies and to determine the true prevalence and epidemiology of E. histolytica.

  5. Entamoeba histolytica cell movement: a central role for self-generated chemokines and chemorepellents.

    PubMed

    Zaki, Mehreen; Andrew, Natalie; Insall, Robert H

    2006-12-05

    Entamoeba histolytica cells, the cause of amoebic dysentery, are highly motile, and this motility is an essential feature of the pathogenesis and morbidity of amoebiasis. However, the control of E. histolytica motility within the gut and during invasion is poorly understood. We have used an improved chemotaxis assay to identify the key extracellular signals mediating Entamoeba chemotaxis. The dominant responses we observe are caused by factors generated by E. histolytica cells themselves. Medium that has been conditioned by E. histolytica growth causes both chemokinesis and negative chemotaxis. The speed of random movement is more than doubled in conditioned compared with fresh medium, and cells move efficiently away from conditioned medium by negative chemotaxis. Ethanol, the product of Entamoeba glucose metabolism, is the principal component of the chemokinetic response. The closely related but nonpathogenic Entamoeba dispar shows no change in motility in response to conditioned medium implying that these responses are central to E. histolytica pathogenesis.

  6. Development of multiplex real-time polymerase chain reaction for detection of Entamoeba histolytica, Entamoeba dispar, and Entamoeba moshkovskii in clinical specimens.

    PubMed

    Hamzah, Zulhainan; Petmitr, Songsak; Mungthin, Mathirut; Leelayoova, Saovanee; Chavalitshewinkoon-Petmitr, Porntip

    2010-10-01

    Multiplex real-time polymerase chain reaction (PCR) was developed for differential detection of Entamoeba histolytica, Entamoeba dispar, and Entamoeba moshkovskii. Specific primers were designed for all three species, and then differentiation of E. histolytica and E. dispar was achieved simultaneously using a hybridization probe and melting curve analysis, whereas E. moshkovskii was detected with a separate probe under the same condition. This assay detected as little as 0.2 pg of E. histolytica DNA and 2 pg each for E. dispar and E. moshkovskii DNA. Thirty-five clinical samples suspected to be E. histolytica infection by microscopy were tested. The results showed 32 positive samples; four samples were E. histolytica and 28 samples were E. dispar. Interestingly, one E. dispar positive sample showed a mixed infection with E. moshkovskii. This is the first report of E. moshkovskii infection from Thailand and this assay is currently the most rapid and sensitive method to differentiate these human amoebas.

  7. Entamoeba histolytica: gene expression analysis of cells invading tissues.

    PubMed

    Fernandes, Helen C; Costa, Ana F; Freitas, Michelle A R; Martins, Almir S; Pesquero, Jorge L; Rabelo, Élida M; Gomes, Maria A

    2014-01-01

    Entamoeba histolytica is a protozoan parasite that presents a risk to the health of millions of people worldwide. Due to the existence of different clinical forms caused by the parasite and also different virulence levels presented by one strain, one would expect differences in the profile of gene transcripts between virulent and nonvirulent cultures. In this study we used the differential display to select gene segments related to invasiveness of amoeba. One Brazilian strain of E. histolytica in two conditions, able or not to cause lesions in experimental animals, was used. RNA from this strain, was used to study the differential expression of genes. 29 specific gene fragments differentially expressed in the virulent strain were selected. By real-time PCR, six of these genes had confirmed their differential expression in the virulent culture. These genes may have important roles in triggering invasive amoebiasis and may be related to adaptation of trophozoites to difficulties encountered during colonization of the intestinal epithelium and liver tissue. Future studies with these genes may elucidate its actual role in tissue invasion by E. histolytica generating new pathways for diagnosis and treatment of amoebiasis.

  8. Analysis of the epithelial damage produced by Entamoeba histolytica infection.

    PubMed

    Betanzos, Abigail; Schnoor, Michael; Javier-Reyna, Rosario; García-Rivera, Guillermina; Bañuelos, Cecilia; Pais-Morales, Jonnatan; Orozco, Esther

    2014-06-12

    Entamoeba histolytica is the causative agent of human amoebiasis, a major cause of diarrhea and hepatic abscess in tropical countries. Infection is initiated by interaction of the pathogen with intestinal epithelial cells. This interaction leads to disruption of intercellular structures such as tight junctions (TJ). TJ ensure sealing of the epithelial layer to separate host tissue from gut lumen. Recent studies provide evidence that disruption of TJ by the parasitic protein EhCPADH112 is a prerequisite for E. histolytica invasion that is accompanied by epithelial barrier dysfunction. Thus, the analysis of molecular mechanisms involved in TJ disassembly during E. histolytica invasion is of paramount importance to improve our understanding of amoebiasis pathogenesis. This article presents an easy model that allows the assessment of initial host-pathogen interactions and the parasite invasion potential. Parameters to be analyzed include transepithelial electrical resistance, interaction of EhCPADH112 with epithelial surface receptors, changes in expression and localization of epithelial junctional markers and localization of parasite molecules within epithelial cells.

  9. Kaempferol inhibits Entamoeba histolytica growth by altering cytoskeletal functions.

    PubMed

    Bolaños, Verónica; Díaz-Martínez, Alfredo; Soto, Jacqueline; Marchat, Laurence A; Sanchez-Monroy, Virginia; Ramírez-Moreno, Esther

    2015-11-01

    The flavonoid kaempferol obtained from Helianthemum glomeratum, an endemic Mexican medicinal herb used to treat gastrointestinal disorders, has been shown to inhibit growth of Entamoeba histolytica trophozoites in vitro; however, the mechanisms associated with this activity have not been documented. Several works reported that kaempferol affects cytoskeleton in mammalian cells. In order to gain insights into the action mechanisms involved in the anti-amoebic effect of kaempferol, here we evaluated the effect of this compound on the pathogenic events driven by the cytoskeleton during E. histolytica infection. We also carried out a two dimensional gel-based proteomic analysis to evidence modulated proteins that could explain the phenotypical changes observed in trophozoites. Our results showed that kaempferol produces a dose-dependent effect on trophozoites growth and viability with optimal concentration being 27.7 μM. Kaempferol also decreased adhesion, it increased migration and phagocytic activity, but it did not affect erythrocyte binding nor cytolytic capacity of E. histolytica. Congruently, proteomic analysis revealed that the cytoskeleton proteins actin, myosin II heavy chain and cortexillin II were up-regulated in response to kaempferol treatment. In conclusion, kaempferol anti-amoebic effects were associated with deregulation of proteins related with cytoskeleton, which altered invasion mechanisms.

  10. Entamoeba histolytica and E. dispar infections in captive macaques (Macaca fascicularis) in the Philippines.

    PubMed

    Rivera, Windell L; Yason, John Anthony D L; Adao, Davin Edric V

    2010-01-01

    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.

  11. Literature Reference for Entamoeba histolytica (Journal of Parasitology. 1972. 58(2): 306–310)

    EPA Pesticide Factsheets

    Procedures are described for analysis of clinical samples and may be adapted for assessment of solid, particulate, liquid and water samples contaminated with Entamoeba histolytica using a culture method.

  12. Entamoeba histolytica infection and secreted proteins proteolytically damage enteric neurons.

    PubMed

    Lourenssen, Sandra; Houpt, Eric R; Chadee, Kris; Blennerhassett, Michael G

    2010-12-01

    The enteric protozoan parasite Entamoeba histolytica causes amebic colitis through disruption of the mucus layer, followed by binding to and destruction of epithelial cells. However, it is not known whether ameba infections or ameba components can directly affect the enteric nervous system. Analysis of mucosal innervations in the mouse model of cecal amebiasis showed that axon density was diminished to less than 25% of control. To determine whether amebas directly contributed to axon loss, we tested the effect of either E. histolytica secreted products (Eh-SEC) or soluble components (Eh-SOL) to an established coculture model of myenteric neurons, glia, and smooth muscle cells. Neuronal survival and axonal degeneration were measured after 48 h of exposure to graded doses of Eh-SEC or Eh-SOL (10 to 80 μg/ml). The addition of 80 μg of either component/ml decreased the neuron number by 30%, whereas the axon number was decreased by 50%. Cytotoxicity was specific to the neuronal population, since the glial and smooth muscle cell number remained similar to that of the control, and was completely abrogated by prior heat denaturation. Neuronal damage was partially prevented by the cysteine protease inhibitor E-64, showing that a heat-labile protease was involved. E. histolytica lysates derived from amebas deficient in the major secreted protease EhCP5 caused a neurotoxicity similar to that of wild-type amebas. We conclude that E. histolytica infection and ameba protease activity can cause selective damage to enteric neurons.

  13. Iron Responsive-like Elements in the parasite Entamoeba histolytica.

    PubMed

    Soto-Castro, Liliana; Plata-Guzman, Laura Yuliana; Figueroa-Angulo, Elisa Elvira; Calla-Choque, Jaeson; Reyes-Lopez, Magda; DE LA Garza, Mireya; Leon-Sicairos, Nidia; Garzon-Tiznado, Jose Antonio; Arroyo, Rossana; Leon-Sicairos, Claudia

    2017-01-18

    In Entamoeba histolytica, iron modulates virulence and gene expression via unknown regulatory mechanisms. The existence of a posttranscriptional iron regulatory system parallel to the IRE/IRP (Iron-Responsive Element/Iron Regulatory Protein) system in the protozoan Trichomonas vaginalis has recently been reported. Due to their evolutionary closeness and the importance of iron for growth and virulence in these protozoa, we hypothesized the existence of an IRE/IRP-like mechanism in E. histolytica. To determine the presence of IRE-like elements in some mRNAs from this parasite, we performed in silico analyses of the 5'- and 3'-UTRs of mRNAs encoding virulence factors and cytoskeleton, ribosomal, and metabolism proteins. The Zuker mfold software predicted IRE-like secondary structures in 52 of the 135 mRNAs analysed. However, only nine structures shared sequence similarity with the apical loop sequence (CAGUGN) of the previously reported human IRE-ferritin, whereas the GUU/UUG protozoan-specific motif was detected in 23 stem-loop structures. A new motif, AUU/AUUU, was also observed in 23 structures, suggesting the possible existence of an amoeba-specific motif. Additionally, cross-linking and RNA electrophoretic mobility shift assays showed specific RNA-protein interactions, using as a model two amoebic IRE-like elements from iron-regulated mRNAs and HeLa, T. vaginalis and E. histolytica cytoplasmic proteins. Our data suggest the presence of a posttranscriptional iron regulatory IRE/IRP-like mechanism in E. histolytica.

  14. Pathogenic and Nonpathogenic Strains of Entamoeba histolytica can be Differentiated by Monoclonal Antibodies to the Galactose-Specific Adherence Lectin

    DTIC Science & Technology

    1991-04-01

    AD- A235 913 DEVELOPMENT Ei ENGINEERING CENTER CRDEC-TR-268 PATHOGENIC AND NONPATHOGENIC STRAINS OF ENTAMOEBA HISTOLYTICA CAN BE DIFFERENTIATED BY...Pathogenic and Nonpathogenic Strains of Entamoeba Histolytica can be Differentiated by Monoclonal PR-IFJlX2XXRPEW Antibodies to the Galactose-Specific...galactose lectin produced by Entamoeba histolytica provide the basis for development of a model system for the environmental detection of adherence and

  15. Myeloperoxidase binds to and kills Entamoeba histolytica trophozoites.

    PubMed

    Pacheco-Yépez, J; Rivera-Aguilar, V; Barbosa-Cabrera, E; Rojas Hernández, S; Jarillo-Luna, R A; Campos-Rodríguez, R

    2011-05-01

    During amebic invasion, neutrophils are a key component in either protecting against invading trophozoites or contributing to tissue damage. Upon degranulating or being lysed, neutrophils release toxic substances that can kill amebas as well as damage host tissue. In a previous study we identified a protein from nonspecifically stimulated peritoneal exudates of hamster that has peroxidase and marked amebicidal activity. In the current study we analyzed the in vitro amebicidal effect of purified hamster myeloperoxidase (MPO). The results demonstrate that MPO must bind directly to the surface of Entamoeba histolytica trophozoites in order to carry out amebicidal activity by using the H(2) O(2) produced by the amebas themselves. Myeloperoxidase-incubated amebas showed important morphological and ultrastructural alterations that increased with incubation time. Changes included an increase of vacuoles in the cytoplasm, a decrease of glycogen, alterations of nuclear morphology and disturbances in the plasma membrane culminating in complete ameba destruction.

  16. Host-Parasite interactions in Entamoeba histolytica and Entamoeba dispar: what have we learned from their genomes?

    PubMed

    Wilson, I W; Weedall, G D; Hall, N

    2012-01-01

    Invasive amoebiasis caused by Entamoeba histolytica is a major global health problem. Virulence is a rare outcome of infection, occurring in fewer than 1 in 10 infections. Not all strains of the parasite are equally virulent, and understanding the mechanisms and causes of virulence is an important goal of Entamoeba research. The sequencing of the genome of E. histolytica and the related avirulent species Entamoeba dispar has allowed whole-genome-scale analyses of genetic divergence and differential gene expression to be undertaken. These studies have helped elucidate mechanisms of virulence and identified genes differentially expressed in virulent and avirulent parasites. Here, we review the current status of the E. histolytica and E. dispar genomes and the findings of a number of genome-scale studies comparing parasites of different virulence.

  17. Biochemical characterization of the DNA ligase I from Entamoeba histolytica.

    PubMed

    Cardona-Felix, Cesar S; Pastor-Palacios, Guillermo; Cardenas, Helios; Azuara-Liceaga, Elisa; Brieba, Luis G

    2010-11-01

    DNA ligases play an essential role in DNA replication and repair. Herein, we report the cloning and biochemical characterization of DNA ligase I from the protozoan parasite Entamoeba histolytica (EhDNAligI). EhDNAligI is an ATP-dependent DNA ligase of 685 amino acids with 35% identity to human DNA ligase I. This report shows that heterologous expressed EhDNAligI is able to perform the three conserved steps of a DNA ligation reaction: adenylation, binding to a 5'-phosphorylated nicked DNA substrate and sealing of the nick. EhDNAligI is strongly inhibited by NaCl and displays optimal activity at pH 7.5. EhDNAligI uses Mn2+ or Mg2+ as metal cofactors and ATP as nucleotide cofactor. EhDNAligI has a nicked DNA binding constant of 6.6microM and follows Michaelis-Menten steady-state kinetics with a K(m) ATP of 64nM and a k(cat) of 2.4min(-1). Accordingly to its properties as a family I DNA ligase, EhDNAligI is able to ligate a RNA strand upstream of a nucleic acid nick, but not in the downstream or the template position. We propose that EhDNAligI is involved in sealing DNA nicks during lagging strand synthesis and may have a role in base excision repair in E. histolytica.

  18. Immune Response of Amebiasis and Immune Evasion by Entamoeba histolytica

    PubMed Central

    Nakada-Tsukui, Kumiko; Nozaki, Tomoyoshi

    2016-01-01

    Entamoeba histolytica is a protozoan parasite and the causative agent of amebiasis. It is estimated approximately 1% of humans are infected with E. histolytica, resulting in an estimate of 100,000 deaths annually. Clinical manifestations of amebic infection range widely from asymptomatic to severe symptoms, including dysentery and extra-intestinal abscesses. Like other infectious diseases, it is assumed that only ~20% of infected individuals develop symptoms, and genetic factors of both the parasite and humans as well as the environmental factors, e.g., microbiota, determine outcome of infection. There are multiple essential steps in amebic infection: degradation of and invasion into the mucosal layer, adherence to the intestinal epithelium, invasion into the tissues, and dissemination to other organs. While the mechanisms of invasion and destruction of the host tissues by the amebae during infection have been elucidated at the molecular levels, it remains largely uncharacterized how the parasite survive in the host by evading and attacking host immune system. Recently, the strategies for immune evasion by the parasite have been unraveled, including immunomodulation to suppress IFN-γ production, elimination of immune cells and soluble immune mediators, and metabolic alterations against reactive oxygen and nitrogen species to fend off the attack from immune system. In this review, we summarized the latest knowledge on immune reaction and immune evasion during amebiasis. PMID:27242782

  19. Bioconjugated fluorescent silica nanoparticles for the rapid detection of Entamoeba histolytica.

    PubMed

    Hemadi, Ahmad; Ekrami, Alireza; Oormazdi, Hormozd; Meamar, Ahmad Reza; Akhlaghi, Lame; Samarbaf-Zadeh, Ali Reza; Razmjou, Elham

    2015-05-01

    Rapid detection of Entamoeba histolytica based on fluorescent silica nanoparticle (FSNP) indirect immunofluorescence microscopy was evaluated. Silica nanoparticles were synthesized using Stöber's method, with their surface activated to covalently bind to, and immobilize, protein A. For biolabeling, FSNP was added to conjugated E. histolytica trophozoites with monoclonal anti-E. histolytica IgG1 for microscopic observation of fluorescence. Fluorescent silica nanoparticle sensitivity was determined with axenically cultured E. histolytica serially diluted to seven concentrations. Specificity was evaluated using other intestinal protozoa. Fluorescent silica nanoparticles detected E. histolytica at the lowest tested concentration with no cross-reaction with Entamoeba dispar, Entamoeba moshkovskii, Blastocystis sp., or Giardia lamblia. Visualization of E. histolytica trophozoites with anti-E. histolytica antibody labeled with fluorescein isothiocyanate (FITC) was compared with that using anti-E. histolytica antibody bioconjugated FSNP. Although FITC and FSNP produced similar results, the amount of specific antibody required for FITC to induce fluorescence of similar intensity was fivefold that for FSNP. Fluorescent silica nanoparticles delivered a rapid, simple, cost-effective, and highly sensitive and specific method of detecting E. histolytica. Further study is needed before introducing FSNP for laboratory diagnosis of amoebiasis.

  20. Different clinical outcomes of Entamoeba histolytica in Malaysia: does genetic diversity exist?

    PubMed

    Anuar, Tengku Shahrul; Al-Mekhlafi, Hesham M; Abdul Ghani, Mohamed Kamel; Azreen, Siti Nor; Salleh, Fatmah Md; Ghazali, Nuraffini; Bernadus, Mekadina; Moktar, Norhayati

    2013-04-01

    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.

  1. Entamoeba histolytica induces cell death of HT29 colonic epithelial cells via NOX1-derived ROS.

    PubMed

    Kim, Kyeong Ah; Kim, Ju Young; Lee, Young Ah; Min, Arim; Bahk, Young Yil; Shin, Myeong Heon

    2013-02-01

    Entamoeba histolytica, which causes amoebic colitis and occasionally liver abscess in humans, is able to induce host cell death. However, signaling mechanisms of colon cell death induced by E. histolytica are not fully elucidated. In this study, we investigated the signaling role of NOX in cell death of HT29 colonic epithelial cells induced by E. histolytica. Incubation of HT29 cells with amoebic trophozoites resulted in DNA fragmentation that is a hallmark of apoptotic cell death. In addition, E. histolytica generate intracellular reactive oxygen species (ROS) in a contact-dependent manner. Inhibition of intracellular ROS level with treatment with DPI, an inhibitor of NADPH oxidases (NOXs), decreased Entamoeba-induced ROS generation and cell death in HT29 cells. However, pan-caspase inhibitor did not affect E. histolytica-induced HT29 cell death. In HT29 cells, catalytic subunit NOX1 and regulatory subunit Rac1 for NOX1 activation were highly expressed. We next investigated whether NADPH oxidase 1 (NOX1)-derived ROS is closely associated with HT29 cell death induced by E. histolytica. Suppression of Rac1 by siRNA significantly inhibited Entamoeba-induced cell death. Moreover, knockdown of NOX1 by siRNA, effectively inhibited E. histolytica-triggered DNA fragmentation in HT29 cells. These results suggest that NOX1-derived ROS is required for apoptotic cell death in HT29 colon epithelial cells induced by E. histolytica.

  2. Real-time PCR assay for rapid qualitative and quantitative detection of Entamoeba histolytica.

    PubMed

    Orosz, Erika; Perkátai, Katalin; Kapusinszky, Beatrix; Farkas, Agnes; Kucsera, István

    2012-12-01

    Simple real-time PCR assay with one set of primer and probe for rapid, sensitive qualitative and quantitative detection of Entamoeba histolytica has been used. Consensus sequences were used to amplify a species-specific region of the 16S rRNA gene, and fluorescence resonance energy transfer hybridization probes were used for detection in a LightCycler platform (Roche). The anchor probe sequence was designed to be a perfect match for the 16S rRNA gene of Entamoeba species, while the acceptor probe sequence was designed for Entamoeba histolytica, which allowed differentiation. The performed characteristics of the real-time PCR assay were compared with ELISA antigen and microscopical detection from 77 samples of individuals with suspected clinical diagnosis of imported E. histolytica infection. Stool and liver abscess pus samples were examined with analytical sensitivity of 5 parasites per PCR reaction. The melting curve means Tms (standard deviation) in clinical isolates were 54°C. The real-time assay was 100% sensitive and specific for differentiation of Entamoeba histolytica, compared with conventional ELISA or microscopy. This real-time PCR assay with melting curve analysis is rapid, and specific for the detection and differentiation of Entamoeba histolytica. The suitability for routine use of this assay in clinical diagnostic laboratories is discussed.

  3. Differential detection of Entamoeba histolytica, Entamoeba dispar and Entamoeba moshkovskii in fecal samples by nested PCR in the United Arab Emirates (UAE).

    PubMed

    ElBakri, Ali; Samie, Amidou; Ezzedine, Sinda; Odeh, Raed Abu

    2013-06-01

    Amoebiasis is one of the most important infectious diseases afflicting mainly tropical and subtropical countries. This study was carried out in the Sharjah Emirate, UAE in order to accurately detect and differentiate Entamoeba histolytica, Entamoeba dispar and E. moshkovskii in fecal samples collected from the Sharjah municipality public health clinic by ELISA and nested polymerase chain reaction (PCR). One hundred and twenty specimens were examined and the PCR was positive for E. histolytica, E. dispar and E. moshkovskii (collectively referred to as Entamoeba complex) in 19.2% (23 out of 120). Of those, 10% (12/120) were mono - infection with E. histolytica; 2.5% (3/120) with E. dispar; and 2.5% (3/120) E. moshkovskii. The nested PCR also detected mixed infections by both E. histolytica and E. dispar in 3.3% (4/120) and E. dispar and E. moshkovskii in 0.8% (1/120). The TechLab ELISA kit failed to detect E. histolytica in any of the E. histolytica PCR positive samples. Overall, the percentage of E. histolytica including those found in mixed infections was 13.3% (16/120). Compared to nested PCR, microscopy was found to have an overall sensitivity of 52.2% and a specificity of 75.2% for detection of Entamoeba complex. The present study indicates that E. histolytica is present in the UAE with an average incidence rate of 13.3%. However, larger studies need to be conducted in order to confirm these findings. We propose the use of PCR in both the routine diagnosis of amoebiasis and epidemiological survey in the UAE.

  4. Antigens in electron-dense granules from Entamoeba histolytica as possible markers for pathogenicity.

    PubMed Central

    Muñoz, M L; Lamoyi, E; León, G; Tovar, R; Pérez-García, J; De La Torre, M; Murueta, E; Bernal, R M

    1990-01-01

    In vitro interaction of Entamoeba histolytica with collagen induces intracellular formation and release of electron-dense granules (EDG) and stimulation of collagenolytic activity. Purified EDG contain 1.66 U of collagenase per mg of protein. Thus, EDG may participate in tissue destruction during invasive amebiasis. Monoclonal antibodies (MAbs) L1.1 and L7.1 reacted specifically with EDG in enzyme-linked immunosorbent assay (ELISA) and immunofluorescence and immunoelectron microscopy. MAb L7.1 immunoprecipitated three polypeptides with molecular weights of 95,000, 68,000, and 28,000 from lysates of biosynthetically labeled E. histolytica. Both MAbs recognized the pathogenic E. histolytica axenic strains HM1:IMSS, HM38:IMSS, and HK-9 but failed to react in ELISA with Entamoeba moshkovskii, Entamoeba invadens, and E. histolytica-like Laredo. In addition, MAb L7.1 reacted with one E. histolytica isolate from a symptomatic patient but did not react with four of five isolates from asymptomatic patients. EDG antigens were detected by a MAb L7.1-based ELISA in E. histolytica-containing fecal samples from symptomatic, but not asymptomatic, individuals. These results suggest that the EDG antigen detected with MAb L7.1 may be differentially expressed in pathogenic and nonpathogenic E. histolytica. Images PMID:2174899

  5. [Detection and differentiation of Entamoeba histolytica and Entamoeba dispar by polymerase chain reaction in a community in Zulia State, Venezuela].

    PubMed

    Rivero, Zulbey; Bracho, Angela; Calchi, Marinella; Díaz, Iris; Acurero, Ellen; Maldonado, Adriana; Chourio, Glenis; Arráiz, Nailet; Corzo, Gilbert

    2009-01-01

    Differential identification of Entamoeba histolytica and Entamoeba dispar is essential for both appropriate patient treatment and epidemiological purposes. To determine the prevalence of these amoeba infections in Santa Rosa de Agua (Maracaibo, Zulia State, Venezuela), a PCR assay using specific primers for each species was standardized and applied. 204 stool samples were analyzed through direct microscopic examination with SSF (0.85%) and lugol, formol-ether concentration, and PCR. Under direct microscopy, 42 individuals (20.58%) presented the E. histolytica/E. dispar complex. Meanwhile PCR showed 47 positive cases for these amoebas: 22 E. histolytica (10.78%), 16 E. dispar (7.84%), and 9 (4.41%) mixed infections. There was no significant difference in the presence of E. histolytica and/or E. dispar according to either gender or age. There were no cases of these amoebas in children under 2 years of age. Observed frequency of E. histolytica (31/204) shows the endemic nature of amoeba infection in this community.

  6. Epithelial cell apoptosis facilitates Entamoeba histolytica infection in the gut.

    PubMed

    Becker, Stephen M; Cho, Kyou-Nam; Guo, Xiaoti; Fendig, Kirsten; Oosman, Mohammed N; Whitehead, Robert; Cohn, Steven M; Houpt, Eric R

    2010-03-01

    Entamoeba histolytica is the protozoan parasite that causes amebic colitis. The parasite triggers apoptosis on contact with host cells; however, the biological significance of this event during intestinal infection is unclear. We examined the role of apoptosis in a mouse model of intestinal amebiasis. Histopathology revealed that abundant epithelial cell apoptosis occurred in the vicinity of amoeba in histological specimens. Epithelial cell apoptosis occurred rapidly on co-culture with amoeba in vitro as measured by annexin positivity, DNA degradation, and mitochondrial dysfunction. Administration of the pan caspase inhibitor ZVAD decreased the rate and severity of amebic infection in CBA mice by all measures (cecal culture positivity, parasite enzyme-linked immunosorbent assay, and histological scores). Similarly, caspase 3 knockout mice on the resistant C57BL/6 background exhibited even lower cecal parasite antigen burden and culture positive rates than wild type mice. The permissive effect of apoptosis on infection could be tracked to the epithelium, in that transgenic mice that overexpressed Bcl-2 in epithelial cells were more resistant to infection as measured by cecal parasite enzyme-linked immunosorbent assay and histological scores. We concluded that epithelial cell apoptosis in the intestine facilitates amebic infection in this mouse model. The parasite's strategy for inducing apoptosis may point to key virulence factors, and therapeutic maneuvers to diminish epithelial apoptosis may be useful in amebic colitis.

  7. Immunology of Entamoeba histolytica in human and animal hosts.

    PubMed

    Trissl, D

    1982-01-01

    Although Entamoeba histolytica induces humoral and cellular immune responses in both human and animal hosts, there is no indication of postinfection immunity in humans; in contrast, several other mammals are protected by prior infection or immunization. The exacerbation of the disease by immunosuppression suggests a protective function of still-unknown defense mechanisms. Specific local and circulating antibodies are produced regularly during invasive amebiasis. Although serum antibodies, together with complement, are lytic to the trophozoites in vitro, the poor correlation of these antibodies with resistance contradicts a protective capacity in vivo. The parasite may evade harm by shedding antigen-antibody complexes from its surface. Demonstration of immediate-type skin reactions, elevated IgE titers, and specific antiamebic IgE suggests that anaphylaxis occurs. The function of the anaphylactic reaction in pathology and resistance remains to be studied. Delayed hypersensitivity parallels healing or resistance and is retarded in human hepatic amebiasis. This observation is consistent with a protective role of cell-mediated immunity.

  8. Trophozoites of Entamoeba histolytica express a CD59-like molecule in human colon.

    PubMed

    Ventura-Juárez, J; Campos-Rodríguez, R; Jarillo-Luna, R A; Muñoz-Fernández, L; Escario-G-Trevijano, J A; Pérez-Serrano, J; Quintanar, J L; Salinas, E; Villalobos-Gómez, F R

    2009-03-01

    In vitro studies have proved the presence of epitopes of CD59 in the surface of trophozoites of Entamoeba histolytica (E. histolytica). However, it has not been proved if CD59 molecules are expressed in the surface during the trophozoites' tissue invasion. The aim of the present study was to determine whether the complement-regulatory protein CD59 is present on trophozoites of E. histolytica in human colon. Eleven specimens of amoebic colitis were studied by immunohistochemistry and electron microscopy techniques with a monoclonal antibody against human CD59 molecule. Our results show that a CD59-like molecule is expressed in trophozoites of E. histolytica found in colonic amebic lesions. Also, a CD59-like molecule was detected by western blot analysis in whole lysate of E. histolytica as well as on the plasma membrane by immunocytochemistry. These results suggest that E. histolytica can use CD59-like protein against the lytic action of membrane attack complex.

  9. Characterization of a monoclonal antibody that selectively recognizes a subset of Entamoeba histolytica isolates.

    PubMed Central

    Bhattacharya, A; Ghildyal, R; Bhattacharya, S; Diamond, L S

    1990-01-01

    Monoclonal antibody 2D7.10 recognized an antigen present in seven of nine isolates of axenically cultured Entamoeba histolytica and absent in all other Entamoeba isolates studied. The antigen was absent in two isolates: 200:NIH and Rahman. All nine isolates belonged to pathogenic zymodeme II. Western blot (immunoblot) analysis and treatment with periodate and the proteolytic enzyme trypsin suggest that the antigen recognized by 2D7.10 is a carbohydrate moiety. Images PMID:1698182

  10. Serosurvey of Entamoeba Histolytica Exposure among Tepehuanos Population in Durango, Mexico

    PubMed Central

    Alvarado-Esquivel, Cosme; Hernández-Tinoco, Jesús; Francisco Sánchez-Anguiano, Luis; Ramos-Nevárez, Agar; Margarita Cerrillo-Soto, Sandra; Alberto Guido-Arreola, Carlos

    2015-01-01

    The seroepidemiology of Entamoeba histolytica infection in Tepehuanos population in Mexico is largely unknown. This study aimed to study the seroprevalence and correlates of E. histolytica antibodies in Tepehuanos in Durango, Mexico. Through a cross-sectional study, we determined the frequency of E. histolytica IgG antibodies in 156 Tepehuanos people in Durango, Mexico using an enzyme-linked immunoassay. Furthermore, we studied the association of E. histolytica seroprevalence with the socio-demographic, clinical, and behavioral characteristics of the Tepehuanos studied. Forty-four (28.2%) Tepehuanos with mean age of 31.03 ± 16.71 years old had anti- E. histolytica IgG antibodies. Multivariate analysis showed that E. histolytica exposure was positively associated with laborer occupation (Odds ratio=2.77; 95% CI: 1.15, 6.66; p=0.02), and history of lymphadenopathy (Odds ratio=4.97; 95% CI: 1.74, 14.13; p=0.002), and negatively associated with soil contact (Odds ratio=0.13; 95% CI: 0.03, 0.53; p=0.004). Other behavioral characteristics including drinking untreated water or unpasteurized milk, and consumption of unwashed raw vegetables or fruits were not associated with E. histolytica exposure. The seroprevalence of E. histolytica infection in Tepehuanos in Durango is higher thanseroprevalences reported in national surveys. The factors associated with E. histolytica seropositivity reported in the present study might aid for the planning and implementation of effective measures against E. histolytica infection. PMID:26199578

  11. Knockdown of Five Genes Encoding Uncharacterized Proteins Inhibits Entamoeba histolytica Phagocytosis of Dead Host Cells.

    PubMed

    Sateriale, Adam; Miller, Peter; Huston, Christopher D

    2016-04-01

    Entamoeba histolytica is the protozoan parasite that causes invasive amebiasis, which is endemic to many developing countries and characterized by dysentery and liver abscesses. The virulence of E. histolytica correlates with the degree of host cell engulfment, or phagocytosis, and E. histolytica phagocytosis alters amebic gene expression in a feed-forward manner that results in an increased phagocytic ability. Here, we used a streamlined RNA interference screen to silence the expression of 15 genes whose expression was upregulated in phagocytic E. histolytica trophozoites to determine whether these genes actually function in the phagocytic process. When five of these genes were silenced, amebic strains with significant decreases in the ability to phagocytose apoptotic host cells were produced. Phagocytosis of live host cells, however, was largely unchanged, and the defects were surprisingly specific for phagocytosis. Two of the five encoded proteins, which we named E. histolytica ILWEQ (EhILWEQ) and E. histolytica BAR (EhBAR), were chosen for localization via SNAP tag labeling and localized to the site of partially formed phagosomes. Therefore, both EhILWEQ and EhBAR appear to contribute to E. histolytica virulence through their function in phagocytosis, and the large proportion (5/15 [33%]) of gene-silenced strains with a reduced ability to phagocytose host cells validates the previously published microarray data set demonstrating feed-forward control of E. histolytica phagocytosis. Finally, although only limited conclusions can be drawn from studies using the virulence-deficient G3 Entamoeba strain, the relative specificity of the defects induced for phagocytosis of apoptotic cells but not healthy cells suggests that cell killing may play a rate-limiting role in the process of Entamoeba histolytica host cell engulfment.

  12. Current and future perspectives on the chemotherapy of the parasitic protozoa Trichomonas vaginalis and Entamoeba histolytica.

    PubMed

    Moya, Ignace A; Su, Zhengding; Honek, John F

    2009-07-01

    Trichomonas vaginalis and Entamoeba histolytica are clinically important protozoa that affect humans. T. vaginalis produces sexually transmitted infections and E. histolytica is the causative agent of amebic dysentery. Metronidazole, a compound first used to treat T. vaginalis in 1959, is still the main drug used worldwide to treat these pathogens. It is essential to find new biochemical differences in these organisms that could be exploited to develop new antiprotozoal chemotherapeutics. Recent findings associated with T. vaginalis and E. histolytica biochemistry and host-pathogen interactions are surveyed. Knowledge concerning the biochemistry of these parasites is serving to form the foundation for the development of new approaches to control these important human pathogens.

  13. Characterization of the protein tyrosine phosphatase PRL from Entamoeba histolytica.

    PubMed

    Ramírez-Tapia, Ana Lilia; Baylón-Pacheco, Lidia; Espíritu-Gordillo, Patricia; Rosales-Encina, José Luis

    2015-12-01

    Protein tyrosine phosphatase of regenerating liver (PRL) is a group of phosphatases that has not been broadly studied in protozoan parasites. In humans, PRLs are involved in metastatic cancer, the promotion of cell migration and invasion. PTPs have been increasingly recognized as important effectors of host-pathogen interactions. We characterized the only putative protein tyrosine phosphatase PRL (PTP EhPRL) in the eukaryotic human intestinal parasite Entamoeba histolytica. Here, we reported that the EhPRL protein possessed the classical HCX5R catalytic motif of PTPs and the CAAX box characteristic of the PRL family and exhibited 31-32% homology with the three human PRL isoforms. In amebae, the protein was expressed at low but detectable levels. The recombinant protein (rEhPRL) had enzymatic activity with the 3-o-methyl fluorescein phosphate (OMFP) substrate; this enzymatic activity was inhibited by the PTP inhibitor o-vanadate. Using immunofluorescence we showed that native EhPRL was localized to the cytoplasm and plasma membrane. When the trophozoites interacted with collagen, EhPRL relocalized over time to vesicle-like structures. Interaction with fibronectin increased the presence of the enzyme in the cytoplasm. Using RT-PCR, we demonstrated that EhPRL mRNA expression was upregulated when the trophozoites interacted with collagen but not with fibronectin. Trophozoites recovered from amoebic liver abscesses showed higher EhPRL mRNA expression levels than normal trophozoites. These results strongly suggest that EhPRL may play an important role in the biology and adaptive response of the parasite to the host environment during amoebic liver abscess development, thereby participating in the pathogenic mechanism.

  14. Multisite clinical evaluation of a rapid test for Entamoeba histolytica in stool.

    PubMed

    Verkerke, Hans P; Hanbury, Blake; Siddique, Abdullah; Samie, Amidou; Haque, Rashidul; Herbein, Joel; Petri, William A

    2015-02-01

    Rapid point-of-care detection of enteric protozoa in diarrheal stool is desirable in clinical and research settings to efficiently determine the etiology of diarrhea. We analyzed the ability of the third-generation E. histolytica Quik Chek assay developed by Techlab to detect amebic antigens in fecal samples collected from independent study populations in South Africa and Bangladesh. We compared the performance of this recently released rapid test to that of the commercially available ProSpecT Entamoeba histolytica microplate assay from Remel and the E. histolytica II enzyme-linked immunosorbent assay (ELISA) from Techlab, using real-time and nested-PCR for Entamoeba species to resolve any discrepant results. After discrepant resolution, The E. histolytica Quik Chek assay exhibited sensitivity and specificity compared to the E. histolytica II ELISA of 98.0% (95% confidence interval [CI], 92.9% to 99.8%) and 100% (95% CI, 99.0% to 100%), respectively. Compared to the ProSpecT microplate assay, the E. histolytica Quik Chek (Quik Chek) assay exhibited 97.0% sensitivity (95% CI, 91.5% to 99.4%) and 100% specificity (95% CI, 99.0% to 100%). Our results indicate that the Quik Chek is a robust assay for the specific detection of E. histolytica trophozoites in unfixed frozen clinical stool samples.

  15. A monoclonal antibody for distinction of invasive and noninvasive clinical isolates of Entamoeba histolytica.

    PubMed Central

    Gonzalez-Ruiz, A; Haque, R; Rehman, T; Aguirre, A; Jaramillo, C; Castañon, G; Hall, A; Guhl, F; Ruiz-Palacios, G; Warhurst, D C

    1992-01-01

    Approximately 10% of the world population is infected with Entamoeba histolytica, but only 10% of the carriers develop symptomatic amebiasis. This discrepancy could be explained by the genotypic differences between the morphologically indistinguishable invasive and noninvasive strains of E. histolytica currently identified by zymodeme analysis, a technique that is unsuitable for routine diagnostic laboratories. Here we report the production of a monoclonal antibody against E. histolytica and its use in an immunofluorescence assay to identify invasive isolates cultured from stool samples of infected patients in several regions where amebiasis is endemic: Bangladesh, Colombia, and Mexico. After testing a total of 88 E. histolytica isolates, the correlation between zymodeme characterization and the immunofluorescence assay with the invasive isolate-specific monoclonal antibody was 100%. The epitope detected by the invasive isolate-specific monoclonal antibody resides in a previously undescribed internal protein with molecular masses of 84 and 81 kDa in axenic and polyxenic E. histolytica strains, respectively. Images PMID:1452651

  16. Outcome of untreated infection with Entamoeba histolytica in homosexual men with and without HIV antibody.

    PubMed Central

    Allason-Jones, E.; Mindel, A.; Sargeaunt, P.; Katz, D.

    1988-01-01

    Among homosexual men the prevalence of infection with Entamoeba histolytica is high. To determine the clinical importance of this infection 55 homosexual men carrying the parasite were investigated in detail. No clinical, serological, or histological evidence of invasive amoebiasis was found in any of them. The patients were not treated and were followed up for 12 to 29 months (mean 21.6 months), during which period none developed symptoms that could be attributed to E histolytica. Spontaneous loss of the parasite occurred in 17 patients, some of whom later became reinfected. Sixteen patients had antibody to human immunodeficiency virus, and infection with E histolytica showed the same benign course in them as in the patients who did not have antibody. Throughout the study classification of the isolates of E histolytica consistently showed that they belonged only to non-pathogenic zymodemes. The findings provide further evidence that E histolytica in homosexual men is a commensal organism. PMID:2902889

  17. Value of microscopy in the diagnosis of dysentery associated with invasive Entamoeba histolytica.

    PubMed Central

    González-Ruiz, A; Haque, R; Aguirre, A; Castañón, G; Hall, A; Guhl, F; Ruiz-Palacios, G; Miles, M A; Warhurst, D C

    1994-01-01

    AIMS--To assess the reliability of the detection of erythrophagocytic amoebic trophozoites in stool samples in the diagnosis of dysentery associated with invasive Entamoeba histolytica. METHODS--Amoebic culture was carried out on single stool samples collected from patients from Mexico, Colombia, and Bangladesh. The stools had been examined by light microscopy. Amoebic dysentery was diagnosed when erythrophagocytic E histolytica trophozoites were observed in a case of bloody diarrhoea. E histolytica isolates were characterised by isoenzyme electrophoresis and results correlated with microscopical findings in stools. Statistical analysis was performed using the chi 2 test. RESULTS--Where erythrophagocytic amoebae had been observed in dysenteric stool specimens the E histolytica phenotype was invariably invasive (p < 0.0001). Observation of erythrophagocytic amoebae in dysentery is 100% specific and predictive of infection with invasive E histolytica. When amoebic culture-positive cases only are considered it is 96% sensitive. In this study E histolytica of zymodeme XIV was more commonly associated with amoebic dysentery than zymodeme II. There was no significant difference between the carriage rate of invasive and non-invasive E histolytica in non-dysenteric diarrhoea. Asymptomatic subjects carried non-invasive E histolytica more frequently than invasive E histolytica. Patients with non-amoebic dysentery, when shown to be infected with E histolytica, carried non-invasive strains (12%). CONCLUSIONS--Sensitivity and specificity of microscopical examination of a single stool specimen for diagnosing amoebic dysentery is very high; intestinal carriage of invasive E histolytica detected by culture is not necessarily an indication of active disease as patients with diarrhoea and asymptomatic subjects shed invasive and non-invasive E histolytica. There are possibly two subpopulations of invasive E histolytica with different pathogenic potential which can be differentiated

  18. Literature Reference for Entamoeba histolytica (Journal of Clinical Microbiology. 2005. 43(11): 5491–5497)

    EPA Pesticide Factsheets

    Procedures are described for analysis of clinical samples and may be adapted for assessment of solid, particulate, liquid and water samples. The method is a real-time PCR assay that targets the 18S rRNA gene sequence of Entamoeba histolytica.

  19. Risk of underdiagnosing amebic dysentery due to false-negative Entamoeba histolytica antigen detection.

    PubMed

    Prim, Núria; Escamilla, Pilar; Solé, Roser; Llovet, Teresa; Soriano, Germán; Muñoz, Carmen

    2012-08-01

    Entamoeba histolytica antigen assays on stool are widely used to diagnose amebiasis. We report a case of confirmed amebic colitis with a false-negative antigen detection that became positive after treatment. Our results indicate that these assays may underdiagnose acute amebic infection when used alone and should be used cautiously.

  20. Discovery of Entamoeba histolytica hexokinase 1 inhibitors through homology modeling and virtual screening.

    PubMed

    Saucedo-Mendiola, María Leticia; Salas-Pacheco, José Manuel; Nájera, Hugo; Rojo-Domínguez, Arturo; Yépez-Mulia, Lilián; Avitia-Domínguez, Claudia; Téllez-Valencia, Alfredo

    2014-06-01

    Entamoeba histolytica, the parasite which causes amebiasis is responsible for 110,000 deaths a year. Entamoeba histolytica depends on glycolysis to obtain ATP for cellular work. According to metabolic flux studies, hexokinase exerts the highest flux control of this metabolic pathway; therefore, it is an excellent target in the search of new antiamebic drugs. To this end, a tridimensional model of E. histolytica hexokinase 1 (EhHK1) was constructed and validated by homology modeling. After virtual screening of 14,400 small molecules, the 100 with the best docking scores were selected, purchased and assessed in their inhibitory capacity. The results showed that three molecules (compounds 2921, 11275 and 2755) inhibited EhHK1 with an I50 of 48, 91 and 96 µM, respectively. Thus, we found the first inhibitors of EhHK1 that can be used in the search of new chemotherapeutic agents against amebiasis.

  1. Seroprevalence of Entamoeba histolytica Infection among Chinese Men Who Have Sex with Men

    PubMed Central

    Li, Xiangwei; Yang, Yu; Gao, Cong; Jin, Qi; Gao, Lei

    2013-01-01

    Background Men who have sex with men (MSM) were found to be one of the high-risk populations for Entamoeba histolytica (E. histolytica) infection. Accompanied by the prevalence of human immunodeficiency virus (HIV) among MSM, invasive amebiasis caused by E. histolytica has been paid attention to as an opportunistic parasitic infection. However, the status of E. histolytica infection among MSM has been barely studied in mainland China. Methods Seroprevalance of E. histolytica was determined using an enzyme-linked immunosorbent assay based on a cross-sectional study conducted in Beijing and Tianjin, China. Factors potentially associated with E. histolytica infection were identified by logistic regression analysis. Results A total of 602 MSM were included in the study and the laboratory data on serostatus of E. histolytica were available for 599 of them (99.5%). 246 (41.1%) and 51 (8.5%) of the study participants were E. histolytica seropositive and HIV seropositive, respectively. Univariate analyses suggested preferred anal sex behaviors were associated with E. histolytica seropositivity. In multivariate logistic regression analysis, “only has receptive anal sex” (OR: 2.03; 95% CI: 1.22 3.37), “majority receptive anal sex” (OR: 1.83; 95% CI: 1.13, 2.95), and “sadomasochistic behavior (SM)” (OR: 2.30; 95% CI: 1.04, 5.13) were found to be significantly associated with E. histolytica infection. Conclusions High seroprevalence of E. histolytica infection was observed among MSM from Beijing and Tianjin, China. Receptive anal sex behavior and SM were identified as potential predictors. Therefore, E. histolytica and HIV co-infection needs to be concerned among MSM due to their sharing the common risk behaviors. PMID:23717699

  2. Evaluation of a new single-tube multiprobe real-time PCR for diagnosis of Entamoeba histolytica and Entamoeba dispar.

    PubMed

    Liang, Shih-Yu; Hsia, Kan-Tai; Chan, Yun-Hsien; Fan, Chia-Kwung; Jiang, Donald Dah-Shyong; Landt, Olfert; Ji, Dar-Der

    2010-08-01

    A single-tube multiprobe real-time PCR assay for simultaneous detection of Entamoeba histolytica and Entamoeba dispar was developed. One primer pair with 2 species-specific probes was designed based on new SSU RNA regions of the ribosomal DNA-containing episome. The sensitivity is 1 parasite per milliliter of feces and thus superior to the conventional nested PCR and comparable to other published real-time PCR protocols. The applicability for clinical diagnosis was validated with 218 stool specimens from patients. A total of 51 E. histolytica and 39 E. dispar positive samples was detected by the multiprobe real-time PCR compared to 39 and 22 by routine nested PCR diagnosis. The detection rate of Entamoeba species for the multiprobe real-time PCR assays was significantly higher than the nested PCR (40.8% vs. 28.0%, P < 0.01). The test did not show cross reactivity with DNA from Entamoeba moshkovskii, Giardia lamblia , Cryptosporidium sp., Escherichia coli , or other nonpathogenic enteric parasites. The multiprobe real-time PCR assay is simple and rapid and has high specificity and sensitivity. The assay could streamline the laboratory diagnosis procedure and facilitate epidemiological investigation.

  3. Identification of Entamoeba histolytica by Molecular Method in Surface Water of Rasht City, Iran

    PubMed Central

    HEMMATI, Arash; HOOSHMAND, Elham; HOSSEINI, Mohammad Javad

    2015-01-01

    Background: This study was conducted with the aim of determining surface water contamination with cysts of Entamoeba histolytica using PCR in Rasht City, Northern Iran. Methods: In this cross-sectional study, 49 water samples including 18 rivers and 6 wetlands were collected from different regions near the city of Rasht in autumn of 2012. After filtration using 0.22 μm nitrate cellulose membrane filters, the samples were examined using microscope and PCR method. Results: In microscopic examination, four samples of the 49 samples were positive for cysts of E. (histolytica / dispar / muschkovskii). By using PCR method and molecular analysis, one sample was positive for E. histolytica. Conclusion: In the molecular analysis, contamination by E. histolytica was proved in the waters of Rasht City. Further investigations including more samples and necessary preparations must be applied to prevent contamination. PMID:25905058

  4. A severe combined immunodeficient (SCID) mouse model for infection with Entamoeba histolytica

    PubMed Central

    1992-01-01

    We used severe combined immunodeficient (SCID) mice to study resistance to invasive infection with Entamoeba histolytica. Seven of seven SCID mice developed liver abscesses when challenged intrahepatically with virulent HM1:IMSS strain E. histolytica trophozoites. Only one of seven similarly challenged immunocompetent congenic C.B-17 mice developed an abscess. Adoptive transfer of polyclonal rabbit anti-E. histolytica antiserum, but not preimmune rabbit serum, completely protected 7 of 12 SCID mice from intrahepatic challenge with ameba. These results demonstrate that lymphocyte-based immunity is important in protection against amebic liver abscess, and that anti-E. histolytica antibody can protect against amebic infection in this system. The SCID mouse may provide a powerful model for studying the components of protective immunity to invasive amebiasis. PMID:1460420

  5. Role of the Gut Microbiota of Children in Diarrhea Due to the Protozoan Parasite Entamoeba histolytica

    PubMed Central

    Gilchrist, Carol A.; Petri, Sarah E.; Schneider, Brittany N.; Reichman, Daniel J.; Jiang, Nona; Begum, Sharmin; Watanabe, Koji; Jansen, Caroline S.; Elliott, K. Pamela; Burgess, Stacey L.; Ma, Jennie Z.; Alam, Masud; Kabir, Mamun; Haque, Rashidul; Petri, William A.

    2016-01-01

    Background. An estimated 1 million children die each year before their fifth birthday from diarrhea. Previous population-based surveys of pediatric diarrheal diseases have identified the protozoan parasite Entamoeba histolytica, the etiological agent of amebiasis, as one of the causes of moderate-to-severe diarrhea in sub-Saharan Africa and South Asia. Methods. We prospectively studied the natural history of E. histolytica colonization and diarrhea among infants in an urban slum of Dhaka, Bangladesh. Results. Approximately 80% of children were infected with E. histolytica by the age of 2 years. Fecal anti-galactose/N-acetylgalactosamine lectin immunoglobulin A was associated with protection from reinfection, while a high parasite burden and expansion of the Prevotella copri level was associated with diarrhea. Conclusions. E. histolytica infection was prevalent in this population, with most infections asymptomatic and diarrhea associated with both the amount of parasite and the composition of the microbiota. PMID:26712950

  6. Prevalence of amoebiasis in a model research community and its confirmation using stool antigen elisa for Entamoeba histolytica.

    PubMed

    Akhtar, Tasleem; Khan, Aamir Ghafoor; Ahmed, Israr; Nazli, Rubina; Haider, Jamila

    2016-09-01

    Entamoeba histolytica (E. histolytica) produces an invasive disease called amoebiasis, which commonly produces diarrhea with or without blood in both children and adults, leading to high morbidity and mortality. Entamoeba dispar (E. Dispar) is a non invasive, non pathogenic organism. Both Entamoeba histolytica and Entamoeba Dispar look alike on microscopy and therefore cannot be differentiated unless checked on ELISA, PCR or other specific method. To calculate the actual prevalence of pathogenic amoebiasis in children by comparing the stool microscopy with ELISA stool antigen i.e. gold standard. Across sectional, comparative study. Children under five years in a community village Budhni, District Peshawar. A sample of 288 children aged <5 years were randomly selected. Information's were collected on the age and gender of the children. Fresh stool specimens were examined microscopically and with stool antigen kit of ELISA for detection of Entamoeba histolytica. The specificity and sensitivity of microscopic method was calculated against ELISA. Data was analyzed using statistical computer software package SPSS version 10.0. A total of 288 stool specimens were collected and examined for Entamoeba histolytica. Out of these 36(12.5%) stools were positive for E. histolyticaon microscopy while 14(4.9%) were positive on ELISA. Out of 14 ELISA positive samples, 10 samples were also positive on microscopy while 4 were ELISA positive but microscopy negative. About 22 samples, which were positive on microscopy were negative on ELISA indicating that these samples might have been of E. Dispar which is non pathogenic protozoa. The sensitivity and specificity of microscopic method was 71.4% and 90.5% respectively, as against stool antigen test. Actual prevalence of Entamoeba histolytica is low in the area. Stool ELISA was able to differentiate between pathogenic Entamoeba histolytica and the non-pathogenic Entamoeba dispar and thus can minimize unnecessary antiamoebic treatment in

  7. The Entamoeba histolytica genome: primary structure and expression of proteolytic enzymes

    PubMed Central

    Tillack, Manuela; Biller, Laura; Irmer, Henriette; Freitas, Michelle; Gomes, Maria A; Tannich, Egbert; Bruchhaus, Iris

    2007-01-01

    Background A number of studies have shown that peptidases and in particular cysteine peptidases constitute major pathogenicity factors in Entamoeba histolytica. Recent studies have suggested that a considerable number of genes coding for proteolytic enzymes are present within the E. histolytica genome and questions remain about the mode of expression of the various molecules. Results By homology search within the recently published amoeba genome, we identified a total of 86 E. histolytica genes coding for putative peptidases, including 46 recently described peptidase genes. In total these comprise (i) 50 cysteine peptidases of different families but most of which belong to the C1 papain superfamily, (ii) 22 different metallo peptidases from at least 11 different families, (iii) 10 serine peptidases belonging to 3 different families, and (iv) 4 aspartic peptidases of only one family. Using an oligonucleotide microarray, peptidase gene expression patterns of 7 different E. histolytica isolates as well as of heat stressed cells were analysed. A total of 21 out of 79 amoeba peptidase genes analysed were found to be significantly expressed under standard axenic culture conditions whereas the remaining are not expressed or at very low levels only. In heat-stressed cells the expression of 2 and 3 peptidase genes, respectively, were either decreased or increased. Only minor differences were observed between the various isolates investigated, despite the fact that these isolates were originated from asymptomatic individuals or from patients with various forms of amoebic diseases. Conclusion Entamoeba histolytica possesses a large number of genes coding for proteolytic enzymes. Under standard culture conditions or upon heat-stress only a relatively small number of these genes is significantly expressed and only very few variations become apparent between various clinical E. histolytica isolates, calling into question the importance of these enzymes in E. histolytica

  8. Identification and Characterization of microRNAS from Entamoeba histolytica HM1-IMSS

    PubMed Central

    Salinas-Hernández, Jannet E.; Taméz-Guerrero, Marcela M.; Barrón-González, María P.; Morales-Rubio, Eufemia; Treviño-Neávez, Jaime; Verduzco-Martínez, Jorge A.; Morales-Vallarta, Mario R.; Reséndez-Pérez, Diana

    2013-01-01

    Background Entamoeba histolytica is the causative agent of amebiasis, a disease that is a major source of morbidity and mortality in the developing world. MicroRNAs (miRNAs) are a large group of non-coding RNAs that play important roles in regulating gene expression and protein translation in animals. Genome-wide identification of miRNAs is a critical step to facilitating our understanding of genome organization, genome biology, evolution, and post-transcriptional regulation. Methodology/Principal Findings We sequenced a small RNA library prepared from a culture of trophozoites of Entamoeba histolytica Strain HM1-IMSS using a deep DNA sequencing approach. Deep sequencing yielded 16 million high-quality short sequence reads containing a total of 5 million non-redundant sequence reads. Based on a bioinformatics pipeline, we found that only 0.5% of these non-redundant small RNA reads were a perfect match with the drafted E. histolytica genome. We did not find miRNA homologs in plant or animal miRNAs. We discovered 199 new potential Entamoeba histolytica miRNAs. The expression and sequence of these Ehi-miRNAs were further validated through microarray by µParaflo Microfluidic Biochip Technology. Ten potential miRNAs were additionally confirmed by real time RT-PCR analysis. Prediction of target genes matched 32 known genes and 34 hypothetical genes. Conclusions/Significance These results show that there is a number of regulatory miRNAs in Entamoeba histolytica. The collection of miRNAs in this parasite could be used as a new platform to study genomic structure, gene regulation and networks, development, and host-parasite interactions. PMID:23874540

  9. In vitro amoebicidal activity of borage (Borago officinalis) extract on Entamoeba histolytica.

    PubMed

    Leos-Rivas, Catalina; Verde-Star, M Julia; Torres, Lidia Osuna; Oranday-Cardenas, Azucena; Rivas-Morales, Catalina; Barron-Gonzalez, M Porfiria; Morales-Vallarta, Mario R; Cruz-Vega, Delia E

    2011-01-01

    Borage (Borago officinalis) is a plant with nutritional value that is also used in traditional medicine to treat gastrointestinal disease. This study investigated the amoebicidal activity of a methanol extract of borage. The 50% inhibitory concentration (IC₅₀) of the extract for Entamoeba histolytica was 33 μg/mL. The 50% lethal dose of the extract for brine shrimp was greater than 1,000 μg/mL. The IC₅₀ of the extract for Vero cells was 203.9 μg/mL. These results support the use of borage to prevent diseases associated with E. histolytica infection.

  10. Identification of exocytotic membrane proteins, syntaxin-1 and SNAP-25, in Entamoeba histolytica from hamster liver.

    PubMed

    Ventura-Juárez, Javier; Salinas, Eva; Campos-Rodríguez, Rafael; Kouri, Juan B; Quintanar, Luis

    2007-06-01

    Entamoeba histolytica is a protozoan parasite causing dysentery and in some cases liver abscesses. These effects have been attributed to cytolytic substances released by exocytosis. In this study, the presence of the proteins syntaxin-1 and SNAP-25, which are assumed to be involved in exocytosis, were examined by immunohistochemistry, immunoelectron microscopy and western blot analysis. Syntaxin-1 and SNAP-25 were expressed in the vesicular, vacuolar and plasma membranes of E. histolytica trophozoites. It can be concluded that these proteins might be involved in exocytosis processes.

  11. Prevalence of antibodies against Entamoeba histolytica in Mexico measured by ELISA

    PubMed Central

    González, C. R.; Isibasi, A.; Ortiz-Navarrete, V.; Paniagua, J.; García, J. A.; Ramirez, A.; Salvatierra, B.; Tapia, R.; Sepúlveda, J.; Gutiérrez, G.; Kumate, J.

    1995-01-01

    The prevalence of antibodies against Entamoeba histolytica was studied in the Mexican population using an immunoenzyme assay in solid phase (ELISA) and semiautomatic equipment. The antigen was a mixture of membrane proteins obtained by Triton X-100 extraction from an axenic culture of Entamoeba histolytica HM1-IMSS. The method was standardized by comparing serum samples from amoebic liver abscess patients with healthy volunteers. From the 60538 samples supplied by the National Seroepidemiology Survey, antibodies were found in 4·49% (4·32—4·65% at 95% confidence limit). More significant titres occurred in the central region of the country. The ratio female to male was 1·25:1. The population living in metropolitan areas had probably been infected at a younger age than those living in the country. Important differences were found in the seroprevalence obtained by ELISA compared with a study which used indirect haemagglutination (IHA) in the same sample frame. PMID:8557086

  12. Novel hemagglutinating, hemolytic and cytotoxic activities of the intermediate subunit of Entamoeba histolytica lectin.

    PubMed

    Kato, Kentaro; Yahata, Kazuhide; Gopal Dhoubhadel, Bhim; Fujii, Yoshito; Tachibana, Hiroshi

    2015-09-10

    Galactose and N-acetyl-D-galactosamine (Gal/GalNAc) inhibitable lectin of Entamoeba histolytica, a common protozoan parasite, has roles in pathogenicity and induction of protective immunity in mouse models of amoebiasis. The lectin consists of heavy (Hgl), light (Lgl), and intermediate (Igl) subunits. Hgl has lectin activity and Lgl does not, but little is known about the activity of Igl. In this study, we assessed various regions of Igl for hemagglutinating activity using recombinant proteins expressed in Escherichia coli. We identified a weak hemagglutinating activity of the protein. Furthermore, we found novel hemolytic and cytotoxic activities of the lectin, which resided in the carboxy-terminal region of the protein. Antibodies against Igl inhibited the hemolytic activity of Entamoeba histolytica trophozoites. This is the first report showing hemagglutinating, hemolytic and cytotoxic activities of an amoebic molecule, Igl.

  13. Structure of uridine diphosphate N-acetylglucosamine pyrophosphorylase from Entamoeba histolytica.

    PubMed

    Edwards, Thomas E; Gardberg, Anna S; Phan, Isabelle Q H; Zhang, Yang; Staker, Bart L; Myler, Peter J; Lorimer, Donald D

    2015-05-01

    Uridine diphosphate N-acetylglucosamine pyrophosphorylase (UAP) catalyzes the final step in the synthesis of UDP-GlcNAc, which is involved in cell-wall biogenesis in plants and fungi and in protein glycosylation. Small-molecule inhibitors have been developed against UAP from Trypanosoma brucei that target an allosteric pocket to provide selectivity over the human enzyme. A 1.8 Å resolution crystal structure was determined of UAP from Entamoeba histolytica, an anaerobic parasitic protozoan that causes amoebic dysentery. Although E. histolytica UAP exhibits the same three-domain global architecture as other UAPs, it appears to lack three α-helices at the N-terminus and contains two amino acids in the allosteric pocket that make it appear more like the enzyme from the human host than that from the other parasite T. brucei. Thus, allosteric inhibitors of T. brucei UAP are unlikely to target Entamoeba UAPs.

  14. Thermophilic alcohol dehydrogenase from the mesophile Entamoeba histolytica: crystallization and preliminary X-ray characterization.

    PubMed

    Shimon, Linda J W; Peretz, Moshe; Goihberg, Edi; Burstein, Yigal; Frolow, Felix

    2002-03-01

    The tetrameric NADP(+)-dependent secondary alcohol dehydrogenase from Entamoeba histolytica has been crystallized in its apo form. The crystals belong to space group C222(1), with unit-cell parameters a = 76.89, b = 234.24, c = 96.24 A, and diffract to 1.9 A at liquid-nitrogen temperature. Analysis of the Patterson self-rotation function shows that the crystals contain one dimer per asymmetric unit.

  15. Detection of beta-tubulin in the cytoplasm of the interphasic Entamoeba histolytica trophozoites.

    PubMed

    Gómez-Conde, Eduardo; Vargas-Mejía, Miguel Ángel; Díaz-Orea, María Alicia; Hernández-Rivas, Rosaura; Cárdenas-Perea, María Elena; Guerrero-González, Tayde; González-Barrios, Juan Antonio; Montiel-Jarquín, Álvaro José

    2016-08-01

    It is known that the microtubules (MT) of Entamoeba histolytica trophozoites form an intranuclear mitotic spindle. However, electron microscopy studies and the employment of anti-beta-tubulin (β-tubulin) antibodies have not exhibited these cytoskeletal structures in the cytoplasm of these parasites. The purpose of this work was to detect β-tubulin in the cytoplasm of interphasic E. histolytica trophozoites. Activated or non-activated HMI-IMSS-strain E. histolytica trophozoites were used and cultured for 72 h at 37 °C in TYI-S-33 medium, and then these were incubated with the anti-β-tubulin antibody of E. histolytica. The anti-β-tubulin antibody reacted with the intranuclear mitotic spindle of E. histolytica-activated trophozoites as control. In contrast, in non-activated interphasic parasites, anti-β-tubulin antibody reacted with diverse puntiform structures in the cytoplasm and with ring-shaped structures localized in the cytoplasm, cellular membrane and endocytic stomas. In this work, for the first time, the presence of β-tubulin is shown in the cytoplasm of E. histolytica trophozoites.

  16. Entamoeba histolytica adaptation to glucose starvation: a matter of life and death.

    PubMed

    Baumel-Alterzon, Sharon; Ankri, Serge

    2014-08-01

    Parasites are often challenged by constant changes of the glucose concentration in their different hosts and/or within the different biotopes in the same host. During its life cycle, Entamoeba histolytica, the causative protozoan parasite of human amoebiasis, is exposed to both a glucose-poor environment in the colon and a glucose-rich environment in the liver. High-throughput 'omics' technologies are now widely used to characterize the cell's global response to various stresses and these technologies can survey E. histolytica's global response to fluctuations in glucose concentration in its environment. In this review, we discuss the phenotypic and metabolic responses of E. histolytica to glucose challenges, and compare these responses to those of other protozoan parasites.

  17. Differential expression and immunolocalization of antioxidant enzymes in Entamoeba histolytica isolates during metronidazole stress.

    PubMed

    Iyer, Lakshmi Rani; Singh, Nishant; Verma, Anil Kumar; Paul, Jaishree

    2014-01-01

    Entamoeba histolytica infections are endemic in the Indian subcontinent. Five to eight percent of urban population residing under poor sanitary conditions suffers from Entamoeba infections. Metronidazole is the most widely prescribed drug used for amoebiasis. In order to understand the impact of metronidazole stress on the parasite, we evaluated the expression of two antioxidant enzymes, peroxiredoxin and FeSOD, in Entamoeba histolytica isolates during metronidazole stress. The results reveal that, under metronidazole stress, the mRNA expression levels of these enzymes did not undergo any significant change. Interestingly, immunolocalization studies with antibodies targeting peroxiredoxin indicate differential localization of the protein in the cell during metronidazole stress. In normal conditions, all the Entamoeba isolates exhibit presence of peroxiredoxin in the nucleus as well as in the membrane; however with metronidazole stress the protein localized mostly to the membrane. The change in the localization pattern was more pronounced when the cells were subjected to short term metronidazole stress compared to cells adapted to metronidazole. The protein localization to the cell membrane could be the stress response mechanism in these isolates. Colocalization pattern of peroxiredoxin with CaBp1, a cytosolic protein, revealed that the membrane and nuclear localization was specific to peroxiredoxin during metronidazole stress.

  18. Entamoeba histolytica: a snapshot of current research and methods for genetic analysis.

    PubMed

    Morf, Laura; Singh, Upinder

    2012-08-01

    Entamoeba histolytica represents one of the leading causes of parasitic death worldwide. Although identified as the causative agent of amebiasis since 1875, the molecular mechanisms by which the parasite causes disease are still not fully understood. Studying Entamoeba reveals insights into a eukaryotic cell that differs in many ways from better-studied model organisms. Thus, much can be learned from this protozoan parasite on evolution, cell biology, and RNA biology. In this review we discuss selected research highlights in Entamoeba research and focus on the development of molecular biological techniques to study this pathogen. We end by highlighting some of the many questions that remain to be answered in order to fully understand this important human pathogen.

  19. Risk factors for Entamoeba histolytica infection in an agricultural community in Hanam province, Vietnam

    PubMed Central

    2011-01-01

    Background Entamoeba histolytica is an important protozoan intestinal infection in resource-poor settings, including Vietnam. The study objective was to assess risk factors of E. histolytica infection in a community in Vietnam, where wastewater and human excreta are used in agriculture. A case-control study was conducted among residents of Hanam province, Northern Vietnam. Cases (n = 46) infected with E. histolytica and non-infected controls (n = 138) were identified in a cross-sectional survey among 794 randomly selected individuals and matched for age, sex and place of residence. Potential risk factors including exposure to human and animal excreta and household wastewater were assessed with a questionnaire. Results People from households with an average socio-economic status had a much higher risk of E. histolytica infection (odds ratio [OR]=4.3, 95% confidence interval [CI]: 1.3-14.0) compared with those from households with a good socioeconomic status. Those individuals who never or rarely used soap for hand washing had a 3.4 times higher risk for infection (OR=3.4, 95% CI: 1.1-10.0), compared to those who used always soap. In contrast, none of the factors related to use of human or animal excreta was statistically significant associated with E. histolytica infection. People having close contact with domestic animals presented a greater risk of E. histolytica infection (OR = 5.9, 95% CI: 1.8-19.0) than those without animal contact. E. histolytica infection was not associated with direct contact with Nhue river water, pond water and household's sanitary conditions, type of latrine or water source used. Conclusions Our study suggests that in settings where human and animal excreta and Nhue River water are intensively used in agriculture, socio-economic and personal hygiene factors determine infection with E. histolytica, rather than exposure to human and animal excreta in agricultural activities. PMID:21663665

  20. Genomic and cDNA actin sequences from a virulent strain of Entamoeba histolytica.

    PubMed Central

    Edman, U; Meza, I; Agabian, N

    1987-01-01

    Invasiveness of Entamoeba histolytica strains that cause acute amoebiasis is characterized by aggressive behavior associated with cell motility and actin function. Analysis of actin genes from E. histolytica was initiated by devising methods for the isolation of biologically active nucleic acids, which allowed the preparation of cDNA and genomic DNA libraries. E. histolytica actin-encoding cDNAs and genomic clones have been isolated from libraries prepared from the virulent HM1:IMSS strain using a heterologous actin probe. Nucleotide sequence analysis of three independent cDNA clones and one genomic clone reveals a highly unusual codon bias and the absence of intervening sequences in E. histolytica actin. The coding sequence of the genomic clone is identical to that of two of the three cDNA clones. These represent at least two distinct mRNAs differing only by five silent changes in the protein coding sequence. Multiple genomic copies of the actin gene can be detected by Southern hybridization. E. histolytica actin exhibits a higher degree of homology to cytoplasmic than to muscle actin. Although the protein has been shown not to bind DNase I, the inferred amino acid sequence indicates conservation of all residues implied to participate in this binding. Images PMID:2883657

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

    PubMed Central

    Zhang, T; Cieslak, P R; Stanley, S L

    1994-01-01

    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. Images PMID:8132322

  2. Murine T-cell clones against Entamoeba histolytica: in vivo and in vitro characterization.

    PubMed Central

    Denis, M; Chadee, K

    1989-01-01

    Eleven T-cell clones were raised from the spleens of BALB/c mice hyperimmunized against a crude soluble extract of Entamoeba histolytica trophozoites. Seven clones were of the Lyt-1+, and four of the Lyt-23+ phenotype. All clones proliferated in the presence of E. histolytica antigens but not to a purified protein derivative; five clones proliferated to a crude extract of the E. histolytica-like Laredo amoebae. Ten clones secreted T-cell growth factors in response to E. histolytica antigens. Two clones (Lyt-23+) mediated direct lymphocytotoxicity (73% and 86%) against amoebic trophozoites that was inhibited with rabbit anti-mouse TNF-alpha. Supernatants of five of the clones (all Lyt-1+) activated mouse peritoneal macrophages (Mphi) to kill E. histolytica trophozoites in vitro, seemingly independent of secreted reactive oxygen intermediates (O2- and H2O2) in the case of three clones supernatants. All of the clones that were activating Mphi to kill amoeba in vitro also mediated a local DTH reaction in mouse footpad. Our results demonstrate direct lymphocyte cytotoxicity via a cytolytic molecule antigenically related to TNF-alpha and lymphokines activating Mphi for amoebic killing by oxidative and non-oxidative mechanisms, the latter process mediated by a macrophage-activating factor (MAF) distinct from interferon-gamma (IFN-gamma). PMID:15493266

  3. Effect of the sesquiterpene lactone incomptine A in the energy metabolism of Entamoeba histolytica.

    PubMed

    Velázquez-Domínguez, José; Marchat, Laurence A; López-Camarillo, Cesar; Mendoza-Hernández, Guillermo; Sánchez-Espíndola, Esther; Calzada, Fernando; Ortega-Hernández, Alfredo; Sánchez-Monroy, Virginia; Ramírez-Moreno, Esther

    2013-11-01

    Entamoeba histolytica is the causative agent of human amoebiasis, which mainly affects developing countries. Although several drugs are effective against E. histolytica trophozoites, the control of amoebiasis requires the development of new and better alternative therapies. Medicinal plants have been the source of new molecules with remarkable antiprotozoal activity. Incomptine A isolated from Decachaeta incompta leaves, is a sesquiterpene lactone of the heliangolide type which has the major in vitro activity against E. histolytica trophozoites. However the molecular mechanisms involved in its antiprotozoal activity are still unknown. Using a proteomic approach based on two-dimensional gel electrophoresis and mass spectrometry (ESI-MS/MS) analysis, we evidenced that 21 E. histolytica proteins were differentially expressed in response to incomptine A treatment. Notably, three glycolytic enzymes, namely enolase, pyruvate:ferredoxin oxidoreductase and fructose-1,6-biphosphate aldolase, were down-regulated. Moreover, ultrastructural analysis of trophozoites through electronic microscopy showed an increased number of glycogen granules. Taken together, our data suggested that incomptine A could affect E. histolytica growth through alteration of its energy metabolism.

  4. Entamoeba histolytica contains an occludin-like protein that can alter colonic epithelial barrier function.

    PubMed

    Goplen, Michael; Lejeune, Manigandan; Cornick, Steve; Moreau, France; Chadee, Kris

    2013-01-01

    The exact mechanism by which Entamoeba histolytica disrupts the human colonic epithelium and invades the mucosa has yet to be clearly elucidated. E. histolytica produces a diverse array of putative virulent factors such as glycosidase, cysteine proteinases and amebapore that can modulate and/or disrupt epithelial barrier functions. However, it is currently thought that E. histolytica produces numerous other molecules and strategies to disrupt colonic mucosal defenses. In this study, we document a putative mechanism whereby the parasite alters the integrity of human epithelium by expressing a cognate tight junction protein of the host. We detected this protein as "occludin-like" as revealed by immunoblotting and immunoprecipitation studies and visualization by confocal microscopy using antibodies highly specific for human occludin. We propose that E. histolytica occludin-like protein might displace mucosal epithelial occludin-occludin tight junction interactions resulting in epithelial disruption analogous to sub mucosal human dendritic cells sampling luminal contents. These results indicate that E. histolytica occludin is a putative virulent component that can play a role in the pathogenesis of intestinal amebiasis.

  5. The MAK16 gene of Entamoeba histolytica and its identification in isolates from patients.

    PubMed

    Crisóstomo-Vázquez, María del Pilar; Marevelez-Acosta, Víctor Alberto; Flores-Luna, Andrés; Jiménez-Cardoso, Enedina

    2014-08-01

    To identify sequences of Entamoeba histolytica associated with the development of amebic liver abscess (ALA) in hamsters, subtractive hybridization of cDNA from E. histolytica HM-1:IMSS under 2 growth conditions was performed: 1) cultured in axenic medium and 2) isolated from experimental ALA in hamsters. For this procedure, 6 sequences were obtained. Of these sequences, the mak16 gene was selected for amplification in 29 cultures of E. histolytica isolated from the feces of 10 patients with intestinal symptoms and 19 asymptomatic patients. Only 5 of the 10 isolates obtained from symptomatic patients developed ALA and amplified the mak16 gene, whereas the 19 isolates from asymptomatic patients did not amplify the mak16 gene nor did they develop ALA. Based on the results of Fisher's exact test (P<0.001), an association was inferred between the presence of the mak16 gene of E. histolytica and the ability to develop ALA in hamsters and with the patient's symptoms (P=0.02). The amplification of the mak16 gene suggests that it is an important gene in E. histolytica because it was present in the isolates from hamsters that developed liver damage.

  6. Use of Liposomes for Directed Drug Delivery against Entamoeba Histolytica

    DTIC Science & Technology

    1992-01-31

    suspended at 2 X 104 cells/ml in TYI ( culture medium minus serum). Two ml of amoeba cell suspension were placed in 2.5 ml screw capped vials to which...histolytica trophozoites in culture and protect cultured mammalian cells from destruction by the parasite; develop an animal model and use this to...THIS PAGE Item 19. Abstract (continued) N-acetylgalactosamine termini stimulated rapid (90 sec) polymerization of amoeba actin. Glycans with terminal N

  7. Preparation of Recombinant Human Monoclonal Antibody Fab Fragments Specific for Entamoeba histolytica

    PubMed Central

    Tachibana, Hiroshi; Cheng, Xun-Jia; Watanabe, Katsuomi; Takekoshi, Masataka; Maeda, Fumiko; Aotsuka, Satoshi; Kaneda, Yoshimasa; Takeuchi, Tsutomu; Ihara, Seiji

    1999-01-01

    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. PMID:10225840

  8. Comparison of a Stool Antigen Detection Kit and PCR for Diagnosis of Entamoeba histolytica and Entamoeba dispar Infections in Asymptomatic Cyst Passers in Iran

    PubMed Central

    Solaymani-Mohammadi, Shahram; Rezaian, Mostafa; Babaei, Zahra; Rajabpour, Azam; Meamar, Ahmad R.; Pourbabai, Ahmad A.; Petri, William A.

    2006-01-01

    The present study was conducted to compare stool antigen detection with PCR for the diagnosis of Entamoeba sp. infection in asymptomatic cyst passers from Iran. Entamoeba dispar and, in one case, E. moshkovskii were the Entamoeba spp. found in the amebic cyst passers. There was a 100% correlation between the results from the TechLab E. histolytica II stool antigen kit and those from nested PCR. We concluded that E. dispar is much more common in asymptomatic cyst passers in Iran and that antigen detection and PCR are comparable diagnostic modalities. PMID:16757634

  9. Mitosis and polykaryon formation of Entamoeba histolytica in axenic cultures.

    PubMed

    Márquez-Monter, H; Becker, I; Carrillo-Farga, J

    1990-01-01

    Axenic cultures of E. histolytica were established in Coplin jars containing glass slides. The cultures were harvested after 72 hours, fixed and stained with Giemsa and acridine orange. Sedimented parasites were used for inclusion in methacrylate for thin sections that were stained with acridine orange, and epon-araldite for ultrathin sections for electron microscope study. The Giemsa stain showed trophozoites of different sizes and variation in the shape, size and number of the nuclei. Division occurred by budding, sometimes by fission, with occasionally apparent extrusion of nuclear material into the cytoplasm. Acridine orange demonstrated DNA in the center of the nuclei and within intranuclear small vesicles. The electron microscope showed mono and multinucleated amoebae with irregular nuclear membranes, coarse material attached to the nuclear membrane, and sometimes intranuclear parallel microtubules without nuclear attachment. There were structures around the karyosome in some nuclei resembling chromosomic material of superior eukaryotes, without kinetochore, as well as intranuclear small vesicles containing sausage-shaped structures. The findings suggest that E. histolytica undergoes polyploidy evidenced by giant nuclei, followed by segregation of chromatin in smaller nuclei, by budding and production of polykaryons of different nuclear size. Multiple small intranuclear vesicles containing structures that may represent forms of rRNA thus contributing to ribosomes. Absence of cytokinesis is the cause of the production of polykaryons with an asynchronous rate of nuclear and cytoplasmic division. Polynucleation also could be related in the origin of cyst formation in amoeba, as unknown selective forces may induce amoebae the synthesis of chitin capsule.

  10. Identification of calcium-transporting ATPases of Entamoeba histolytica and cellular localization of the putative SERCA.

    PubMed

    Martinez-Higuera, Aarón; Salas-Casas, Andrés; Calixto-Gálvez, Mercedes; Chávez-Munguía, Bibiana; Pérez-Ishiwara, D Guillermo; Ximénez, Cecilia; Rodríguez, Mario A

    2013-09-01

    Calcium has an important role on signaling of different cellular processes in the protozoa parasite Entamoeba histolytica, including development and pathogenesis. However, the systems that control calcium responses in this parasite are incompletely understood. Calcium-ATPases (Ca(2+)-ATPases) are proteins that play an important role in calcium homeostasis by catalyzing the active efflux of this ion from cytoplasm and are essential to the correct functioning of the cell machinery. Here, we reported the identification of five E. histolytica genes encoding putative Ca(2+)-ATPases, three related to PMCA, and two related to organellar ATPases. RT-PCR assays showed that all those genes are expressed in trophozoites and specific antibodies against the SERCA-like member located this protein in a continuous cytoplasmic network, supporting the hypothesis that it corresponds to the Ca(2+)-ATPase responsible to sequester calcium in the endoplasmic reticulum of this parasite.

  11. Chew on this: Amoebic trogocytosis and host cell killing by Entamoeba histolytica

    PubMed Central

    Ralston, Katherine S.

    2015-01-01

    Entamoeba histolytica was named “histolytica” (histo-: tissue; lytic-: dissolving) for its ability to destroy host tissues. Direct killing of host cells by the amoebae is likely to be the driving factor that underlies tissue destruction, but the mechanism was unclear. We recently showed that after attaching to host cells, amoebae bite off and ingest distinct host cell fragments, and that this contributes to cell killing. Here we review this process, termed “amoebic trogocytosis” (trogo-: nibble), and how this process interplays with phagocytosis, or whole cell ingestion, in this organism. “Nibbling” processes have been described in other microbes and in multicellular organisms. The discovery of amoebic trogocytosis in E. histolytica may also shed light on an evolutionarily conserved process for intercellular exchange. PMID:26070402

  12. Structural and functional studies of a noncanonical Dicer from Entamoeba histolytica

    PubMed Central

    Yu, Xiang; Li, Xuhang; Zheng, Lina; Ma, Jinbiao; Gan, Jianhua

    2017-01-01

    RNaseIII proteins are dsRNA-specific endonucleases involved in many important biological processes, such as small RNA processing and maturation in eukaryotes. Various small RNAs have been identified in a protozoan parasite Entamoeba histolytica. EhRNaseIII is the only RNaseIII endonuclease domain (RIIID)-containing protein in E. histolytica. Here, we present three crystal structures that reveal several unique structural features of EhRNaseIII, especially the interactions between the two helixes (α1 and α7) flanking the RIIID core domain. Structure and sequence analysis indicate that EhRNaseIII is a noncanonical Dicer and it lacks a dsRBD in the C-terminal region (CTR). In vitro studies suggest that EhRNaseIII prefers to bind and cleave longer dsRNAs, generating products around 25 nucleotides in length. Truncation of the CTR or attaching the dsRBD of Aquifex aeolicus RNaseIII can enhance the binding and cleavage activities of EhRNaseIII. In combination with in vitro crosslinking assay, our results suggested that EhRNaseIII functions in a cooperative mode. We speculate that some partner proteins may exist in E. histolytica and regulates the activity of EhRNaseIII through interaction with its CTR. Our studies support that EhRNaseIII plays an important role in producing small RNAs in E. histolytica. PMID:28317870

  13. ANTIPARASITIC ACTIVITY OF SILVER AND COPPER OXIDE NANOPARTICLES AGAINST ENTAMOEBA HISTOLYTICA AND CRYPTOSPORIDIUM PARVUM CYSTS.

    PubMed

    Saad, Halim A; Soliman, Mohamed I; Azzam, Ahmed M; Mostafa, B

    2015-12-01

    Nanoparticles (NPs) have received more attention as antiparasitic agents. In the present study, silver and copper nanoparticles were synthesized and characterized using scanning electron microscopy (SEM), transmission electron microscope (TEM) and X-ray fluorescence (XRF). The antiparasitic activity of Ag and CuO nanoparticles were tested against two of the most environmentally spread parasites in Egypt (Entamoeba histolytica and Cryptosporidium parvum). The average sizes of synthesized Ag NPs and CuO NPs were 9 & 29 nm respectively and a significant reduction for cysts viability (p > 0.05) was observed for CuO NPs against E. histolytica cysts and Ag NPs against C. parvum oocysts. Moreover, LC50-3h of CuO NPs for E. histolytica and C. parvum were 0.13 and 0.72 mg/l, while Ag NPs recorded 0.34 and 0.54 mg/l respectively. Accordingly, these NPs could be suggested as a new nanoform agent for safe and effective treatment of E. histolytica and C. parvum parasites.

  14. Biochemical characterization of rat colonic mucins secreted in response to Entamoeba histolytica.

    PubMed

    Tse, S K; Chadee, K

    1992-04-01

    Invasion of the colonic mucosa by Entamoeba histolytica trophozoites is preceded by colonic mucus depletion. The aim of our studies was to determine whether E. histolytica caused a differential secretion of mucin species in a rat colonic loop model. Mucus secretion in response to amoebae was followed by release of acid-precipitable 3H-glucosamine metabolically labelled glycoproteins and in vitro labelling of glycoprotein secretion with NaB3H4. The secretory response consisted of high-Mr goblet cell mucins and an increase in the secretion of low-Mr nonmucin glycoproteins as determined by Sepharose 4B column chromatography. High-Mr mucins subfractionated by Cellex-E (ECTEOLA) ion-exchange chromatography demonstrated a minor neutral and a major acidic mucin (greater than 98%) species. Marked differences between the neutral and acidic mucin species were indicated by immunogenicity and amino acid compositions. Thin-section histochemistry of rat colons confirmed secretion of neutral and acidic mucins in response to E. histolytica and demonstrated secretory activity from goblet cells from both the crypts and interglandular epithelium. E. histolytica mucus secretagogue activity was generalized and may function to deplete the host's protective mucus layer, facilitating invasion by the parasites.

  15. Gene expression profiling in Entamoeba histolytica identifies key components in iron uptake and metabolism.

    PubMed

    Hernández-Cuevas, Nora Adriana; Weber, Christian; Hon, Chung-Chau; Guillen, Nancy

    2014-01-01

    Entamoeba histolytica is an ameboid parasite that causes colonic dysentery and liver abscesses in humans. The parasite encounters dramatic changes in iron concentration during its invasion of the host, with relatively low levels in the intestinal lumen and then relatively high levels in the blood and liver. The liver notably contains sources of iron; therefore, the parasite's ability to use these sources might be relevant to its survival in the liver and thus the pathogenesis of liver abscesses. The objective of the present study was to identify factors involved in iron uptake, use and storage in E. histolytica. We compared the respective transcriptomes of E. histolytica trophozoites grown in normal medium (containing around 169 µM iron), low-iron medium (around 123 µM iron), iron-deficient medium (around 91 µM iron), and iron-deficient medium replenished with hemoglobin. The differentially expressed genes included those coding for the ATP-binding cassette transporters and major facilitator transporters (which share homology with bacterial siderophores and heme transporters) and genes involved in heme biosynthesis and degradation. Iron deficiency was associated with increased transcription of genes encoding a subset of cell signaling molecules, some of which have previously been linked to adaptation to the intestinal environment and virulence. The present study is the first to have assessed the transcriptome of E. histolytica grown under various iron concentrations. Our results provide insights into the pathways involved in iron uptake and metabolism in this parasite.

  16. Heterotrimeric G-protein Signaling Is Critical to Pathogenic Processes in Entamoeba histolytica

    PubMed Central

    Muller, Robin E.; Giguère, Patrick M.; Machius, Mischa; Willard, Francis S.; Temple, Brenda R. S.; Siderovski, David P.

    2012-01-01

    Heterotrimeric G-protein signaling pathways are vital components of physiology, and many are amenable to pharmacologic manipulation. Here, we identify functional heterotrimeric G-protein subunits in Entamoeba histolytica, the causative agent of amoebic colitis. The E. histolytica Gα subunit EhGα1 exhibits conventional nucleotide cycling properties and is seen to interact with EhGβγ dimers and a candidate effector, EhRGS-RhoGEF, in typical, nucleotide-state-selective fashions. In contrast, a crystal structure of EhGα1 highlights unique features and classification outside of conventional mammalian Gα subfamilies. E. histolytica trophozoites overexpressing wildtype EhGα1 in an inducible manner exhibit an enhanced ability to kill host cells that may be wholly or partially due to enhanced host cell attachment. EhGα1-overexpressing trophozoites also display enhanced transmigration across a Matrigel barrier, an effect that may result from altered baseline migration. Inducible expression of a dominant negative EhGα1 variant engenders the converse phenotypes. Transcriptomic studies reveal that modulation of pathogenesis-related trophozoite behaviors by perturbed heterotrimeric G-protein expression includes transcriptional regulation of virulence factors and altered trafficking of cysteine proteases. Collectively, our studies suggest that E. histolytica possesses a divergent heterotrimeric G-protein signaling axis that modulates key aspects of cellular processes related to the pathogenesis of this infectious organism. PMID:23166501

  17. Suppression of T-lymphocyte responses to Entamoeba histolytica antigen by immune sera.

    PubMed Central

    Salata, R A; Martinez-Palomo, A; Canales, L; Murray, H W; Trevino, N; Ravdin, J I

    1990-01-01

    Lymphocytes from patients cured of amebic liver abscesses proliferate and produce gamma interferon upon incubation with soluble Entamoeba histolytica antigen: however, amebic liver abscesses exhibit a relentless progression without treatment. To determine whether suppressive factors are present in sera, we studied T-lymphocyte responses to total soluble E. histolytica antigen by using cells from five patients treated for amebic liver abscesses in the presence of 15 different immune sera and 10 control sera. In the presence of immune sera, E. histolytica antigen-induced lymphocyte proliferation decreased by 63% and production of gamma interferon was reduced by 93.2% (P less than 0.01). Immune sera had no effect on the mitogenic responses of patient lymphocytes to phytohemagglutinin or on the proliferative responses of control lymphocytes to phytohemagglutinin or tetanus toxoid. The suppressive activity of immune sera diminished as the time between therapy for amebic liver abscesses and serum collection increased (P less than 0.05). Suppressive activity did not correlate with the titers of serum anti-amebic antibody and was not affected when serum was absorbed with viable amebic trophozoites. In conclusion, soluble factors present in the sera of amebic liver abscess patients suppressed in vitro lymphocyte responses to E. histolytica antigen and may have contributed to the lack of development of effective in vivo cell-mediated immune responses following the onset of amebic liver abscesses. Images PMID:2123828

  18. Entamoeba histolytica antigenic protein detected in pus aspirates from patients with amoebic liver abscess.

    PubMed

    Othman, Nurulhasanah; Mohamed, Zeehaida; Yahya, Maya Mazuwin; Leow, Voon Meng; Lim, Boon Huat; Noordin, Rahmah

    2013-08-01

    Entamoeba histolytica is a causative agent of amoebic liver abscess (ALA) and is endemic in many underdeveloped countries. We investigated antigenic E. histolytica proteins in liver abscess aspirates using proteomics approach. Pus samples were first tested by real-time PCR to confirm the presence of E. histolytica DNA and the corresponding serum samples tested for E. histolytica-specific IgG by a commercial ELISA. Proteins were extracted from three and one pool(s) of pus samples from ALA and PLA (pyogenic liver abscess) patients respectively, followed by analysis using isoelectric focussing, SDS-PAGE and Western blot. Unpurified pooled serum samples from infected hamsters and pooled human amoebic-specific IgG were used as primary antibodies. The antigenic protein band was excised from the gel, digested and analysed by MALDI-TOF/TOF and LC-MS/MS. The results using both primary antibodies showed an antigenic protein band of ∼14kDa. Based on the mass spectrum analysis, putative tyrosine kinase is the most probable identification of the antigenic band.

  19. Differentially expressed genes of virulent and nonvirulent Entamoeba histolytica strains identified by suppression subtractive hybridization.

    PubMed

    Freitas, Michelle A R; Alvarenga, Ângela C; Fernandes, Helen C; Gil, Frederico F; Melo, Maria N; Pesquero, Jorge L; Gomes, Maria A

    2014-01-01

    Entamoeba histolytica is a parasite which presents capacity to degrade tissues and therefore has a pathogenic behavior. As this behavior is not shown by all strains, there have been several studies investigating molecular basis of the cytotoxicity process. Using the suppression subtractive hybridization (SSH) technique, differential gene expressions of two E. histolytica strains, one virulent (EGG) and one nonvirulent (452), have been analyzed with the purpose of isolating genes which may be involved with amoebic virulence. Nine cDNA fragments presenting high homology with E. histolytica previously sequenced genes were subtracted. Of these, four genes were confirmed by RT-PCR. Two coding for hypothetical proteins, one for a cysteine-rich protein, expressed only in the virulent strain, EGG and another one, coding for grainin 2 protein, exclusive from 452 strain. This study provided new insight into the proteins differences in the virulent and nonvirulent E. histolytica strains. We believe that further studies with these proteins may prove association of them with tissue damage, providing new perceptions to improve treatment or diagnosis of the invasive disease.

  20. Data set for the proteomics analysis of the endomembrane system from the unicellular Entamoeba histolytica.

    PubMed

    Perdomo, Doranda; Aït-Ammar, Nawel; Syan, Sylvie; Sachse, Martin; Jhingan, Gagan Deep; Guillén, Nancy

    2014-12-01

    Entamoeba histolytica is the protozoan parasite agent of amebiasis, an infectious disease of the human intestine and liver. This parasite contact and kills human cells by an active process involving pathogenic factors. Cellular traffic and secretion activities are poorly characterized in E. histolytica. In this work, we took advantage of a wide proteomic analysis to search for principal components of the endomembrane system in E. histolytica. A total of 5683 peptides matching with 1531 proteins (FDR of 1%) were identified which corresponds to roughly 20% of the total amebic proteome. Bioinformatics investigations searching for domain homologies (Smart and InterProScan programs) and functional descriptions (KEGG and GO terms) allowed this data to be organized into distinct categories. This data represents the first in-depth proteomics analysis of subcellular compartments in E. histolytica and allows a detailed map of vesicle traffic components in an ancient single-cell organism that lacks a stereotypical ER and Golgi apparatus to be established. The data are related to [1].

  1. Structures of a histidine triad family protein from Entamoeba histolytica bound to sulfate, AMP and GMP.

    PubMed

    Lorimer, Donald D; Choi, Ryan; Abramov, Ariel; Nakazawa Hewitt, Stephen; Gardberg, Anna S; Van Voorhis, Wesley C; Staker, Bart L; Myler, Peter J; Edwards, Thomas E

    2015-05-01

    Three structures of the histidine triad family protein from Entamoeba histolytica, the causative agent of amoebic dysentery, were solved at high resolution within the Seattle Structural Genomics Center for Infectious Disease (SSGCID). The structures have sulfate (PDB entry 3oj7), AMP (PDB entry 3omf) or GMP (PDB entry 3oxk) bound in the active site, with sulfate occupying the same space as the α-phosphate of the two nucleotides. The C(α) backbones of the three structures are nearly superimposable, with pairwise r.m.s.d.s ranging from 0.06 to 0.13 Å.

  2. Atg8 is involved in endosomal and phagosomal acidification in the parasitic protist Entamoeba histolytica.

    PubMed

    Picazarri, Karina; Nakada-Tsukui, Kumiko; Tsuboi, Kumiko; Miyamoto, Eri; Watanabe, Naoko; Kawakami, Eiryo; Nozaki, Tomoyoshi

    2015-10-01

    Autophagy is one of two major bulk protein degradation systems and is conserved throughout eukaryotes. The protozoan Entamoeba histolytica, which is a human intestinal parasite, possesses a restricted set of autophagy-related (Atg) proteins compared with other eukaryotes and thus represents a suitable model organism for studying the minimal essential components and ancestral functions of autophagy. E. histolytica possesses two conjugation systems: Atg8 and Atg5/12, although a gene encoding Atg12 is missing in the genome. Atg8 is considered to be the central and authentic marker of autophagosomes, but recent studies have demonstrated that Atg8 is not exclusively involved in autophagy per se, but other fundamental mechanisms of vesicular traffic. To investigate this question in E. histolytica, we studied on Atg8 during the proliferative stage. Atg8 was constitutively expressed in both laboratory-maintained and recently established clinical isolates and appeared to be lipid-modified in logarithmic growth phase, suggesting a role of Atg8 in non-stress and proliferative conditions. These findings are in contrast to those for Entamoeba invadens, in which autophagy is markedly induced during an early phase of differentiation from the trophozoite into the cyst. The repression of Atg8 gene expression in En. histolytica by antisense small RNA-mediated transcriptional gene silencing resulted in growth retardation, delayed endocytosis and reduced acidification of endosomes and phagosomes. Taken together, these results suggest that Atg8 and the Atg8 conjugation pathway have some roles in the biogenesis of endosomes and phagosomes in this primitive eukaryote.

  3. Protection of gerbils from amebic liver abscess by immunization with a recombinant protein derived from the 170-kilodalton surface adhesin of Entamoeba histolytica.

    PubMed Central

    Zhang, T; Stanley, S L

    1994-01-01

    The protozoan parasite Entamoeba histolytica causes extensive morbidity and mortality worldwide through intestinal infection and amebic liver abscess. Here we show that vaccination of gerbils, a standard model for amebic liver abscess, with recombinant proteins derived from the 170-kDa galactose-binding adhesin of E. histolytica and the serine-rich E. histolytica protein or a combination of the two recombinant antigens provides excellent protection against subsequent hepatic challenge with virulent E. histolytica trophozoites. PMID:8188384

  4. Molecular epidemiology of amoebiasis in Malaysia: highlighting the different risk factors of Entamoeba histolytica and Entamoeba dispar infections among Orang Asli communities.

    PubMed

    Anuar, Tengku Shahrul; Al-Mekhlafi, Hesham M; Abdul Ghani, Mohamed Kamel; Abu Bakar, Edariah; Azreen, Siti Nor; Salleh, Fatmah Md; Ghazali, Nuraffini; Bernadus, Mekadina; Moktar, Norhayati

    2012-12-01

    Currently, species-specific information on Entamoeba infections is unavailable in Malaysia and is restricted worldwide due to the re-description of pathogenic Entamoeba histolytica and non-pathogenic Entamoeba dispar and Entamoeba moshkovskii. Therefore, this cross-sectional study was conducted to provide the first known documented data on the true prevalence of these three species in western Malaysia using a molecular method. Another aim of this study was to determine the association of potential risk factors associated with each Entamoeba sp. A total of 500 stool samples from three Orang Asli tribes were randomly collected. The overall prevalence of E. histolytica, E. dispar and E. moshkovskii determined by microscopy was 18.6% (93/500). Molecular analysis revealed that while most Entamoeba-positive individuals were infected with E. dispar (13.4%), followed by E. histolytica (3.2%) and E. moshkovskii (1.0%), the present findings show low prevalence rates of mixed infections with E. histolytica and E. dispar (2%), E. dispar and E. moshkovskii (1.2%) and association infections of E. histolytica, E. dispar and E. moshkovskii (0.4%). Logistical regression analysis indicates that the dynamics of the transmission of the three Entamoeba spp. was different. Of six statistically significant variables observed in the univariate analysis, three were retained as significant risk factors for E. histolytica infection in the logistical regression model. These factors were (i) not washing hands after playing with soil or gardening (Odds ratio (OR)=4.7; 95% confidence level (CI)=1.38, 16.14; P=0.013), (ii) indiscriminate defecation in the river or bush (OR=5.7; 95% CI=1.46, 21.95; P=0.012) and (iii) close contact with domestic animals (OR=5.4; 95% CI=1.36, 2.51; P=0.017). However, subjects with family members who were infected with E. histolytica/E. dispar/E. moshkovskii (OR=3.8; 95 CI=2.11, 6.86; P<0.001) and those who consumed raw vegetables (OR=1.8; 95% CI=1.01, 3.23; P=0

  5. Amoebic PI3K and PKC is required for Jurkat T cell death induced by Entamoeba histolytica.

    PubMed

    Lee, Young Ah; Kim, Kyeong Ah; Min, Arim; Shin, Myeong Heon

    2014-08-01

    The enteric protozoan parasite Entamoeba histolytica is the causative agent of human amebiasis. During infection, adherence of E. histolytica through Gal/GalNAc lectin on the surface of the amoeba can induce caspase-3-dependent or -independent host cell death. Phosphorylinositol 3-kinase (PI3K) and protein kinase C (PKC) in E. histolytica play an important function in the adhesion, killing, or phagocytosis of target cells. In this study, we examined the role of amoebic PI3K and PKC in amoeba-induced apoptotic cell death in Jurkat T cells. When Jurkat T cells were incubated with E. histolytica trophozoites, phosphatidylserine (PS) externalization and DNA fragmentation in Jurkat cells were markedly increased compared to those of cells incubated with medium alone. However, when amoebae were pretreated with a PI3K inhibitor, wortmannin before being incubated with E. histolytica, E. histolytica-induced PS externalization and DNA fragmentation in Jurkat cells were significantly reduced compared to results for amoebae pretreated with DMSO. In addition, pretreatment of amoebae with a PKC inhibitor, staurosporine strongly inhibited Jurkat T cell death. However, E. histolytica-induced cleavage of caspase-3, -6, and -7 were not inhibited by pretreatment of amoebae with wortmannin or staurosporin. In addition, we found that amoebic PI3K and PKC have an important role on amoeba adhesion to host compartment. These results suggest that amebic PI3K and PKC activation may play an important role in caspase-independent cell death in Entamoeba-induced apoptosis.

  6. Entamoeba histolytica: a unicellular organism containing two active genes encoding for members of the TBP family.

    PubMed

    Castañon-Sanchez, Carlos Alberto; Luna-Arias, Juan Pedro; de Dios-Bravo, Ma Guadalupe; Herrera-Aguirre, Maria Esther; Olivares-Trejo, Jose J; Orozco, Esther; Hernandez, Jose Manuel

    2010-03-01

    Entamoeba histolytica is the protozoan parasite which causes human amoebiasis. In this parasite, few encoding genes for transcription factors have been cloned and characterized. The E. histolytica TATA-box binding protein (EhTBP) is the first basal transcription factor that has been studied. To continue with the identification of other members of the basal transcription machinery, we performed an in silico analysis of the E. histolytica genome and found three loci encoding for polypeptides with similarity to EhTBP. One locus has a 100% identity to the previously Ehtbp gene reported by our group. The second locus encodes for a 212 aa polypeptide that is 100% identical to residues 23-234 from EhTBP. The third one encodes for a 216 aa polypeptide of 24kDa that showed 42.6% identity and 73.7% similarity to EhTBP. This protein was named E. histolytica TBP-related factor 1 (EhTRF1). Ehtrf1 gene was expressed in bacteria and the purified 28kDa recombinant polypeptide showed the capacity to bind to TATTTAAA-box by electrophoretic mobility shift assays. K(D) values for rEhTBP and rEhTRF1 were (1.71+/-2.90)x10(-12)M and (1.12+/-0.160)x10(-11)M, respectively. Homology modeling of EhTRF1 and EhTBP revealed that, although they were very similar, they showed some differences on their surfaces. Thus, E. histolytica is a unicellular organism having two members of the TBP family.

  7. 1H, 13C and 15N NMR assignments of a calcium-binding protein from Entamoeba histolytica.

    PubMed

    Verma, Deepshikha; Bhattacharya, Alok; Chary, Kandala V R

    2016-04-01

    We report almost complete sequence specific (1)H, (13)C and (15)N NMR assignments of a 150-residue long calmodulin-like calcium-binding protein from Entamoeba histolytica (EhCaBP6), as a prelude to its structural and functional characterization.

  8. Crystal structure and putative substrate identification for the Entamoeba histolytica low molecular weight tyrosine phosphatase.

    PubMed

    Linford, Alicia S; Jiang, Nona M; Edwards, Thomas E; Sherman, Nicholas E; Van Voorhis, Wesley C; Stewart, Lance J; Myler, Peter J; Staker, Bart L; Petri, William A

    2014-01-01

    Entamoeba histolytica is a eukaryotic intestinal parasite of humans, and is endemic in developing countries. We have characterized the E. histolytica putative low molecular weight protein tyrosine phosphatase (LMW-PTP). The structure for this amebic tyrosine phosphatase was solved, showing the ligand-induced conformational changes necessary for binding of substrate. In amebae, it was expressed at low but detectable levels as detected by immunoprecipitation followed by immunoblotting. A mutant LMW-PTP protein in which the catalytic cysteine in the active site was replaced with a serine lacked phosphatase activity, and was used to identify a number of trapped putative substrate proteins via mass spectrometry analysis. Seven of these putative substrate protein genes were cloned with an epitope tag and overexpressed in amebae. Five of these seven putative substrate proteins were demonstrated to interact specifically with the mutant LMW-PTP. This is the first biochemical study of a small tyrosine phosphatase in Entamoeba, and sets the stage for understanding its role in amebic biology and pathogenesis.

  9. Relationship between free radicals produced by entamoeba histolytica and its proteases complex activity.

    PubMed

    Crisóstomo-Vázquez, M P; Cervantes-Cervantes, M P; Jiménez-Cardoso, E; Muñoz-Sánchez, J L

    2002-01-01

    Entamoeba histolytica is a parasite which causes health problems and there has been many approaches to know what is the factor causing its pathogenicity. In the present work, we assayed if the production of free radicals by the amoeba, has a relationship with the proteases activity. When we test the DMSO action (free radicals quenching activity) the specific activity of the proteases complex of the parasite were affected also. At 33.3% (V/V) concentration of DMSO it was present a maximal decrease of the initial activity (about 46% decrease), for to a higher concentrations existing a trend to recuperate the original activity, suggesting that the free radicals are an important factor for the hydrolysis grade of the protein substrate. All the differences except those between 46.7 and 66.6%, were significantly different compared with the control without any addition. The effects of Probucol and Probucol plus DMSO, compared to those caused by Metronidazol (MZ). We can observe that the quenchers caused a decrease on proteases activity similar to that of MZ (which is an antiparasite drug) and it was of c.a. 58% of activity decrease. These data suggest that the action of both, free radicals and proteases complex of Entamoeba histolytica, can account for the pathogenicity of the parasite.

  10. Evidence for a bacterial lipopolysaccharide-recognizing G-protein-coupled receptor in the bacterial engulfment by Entamoeba histolytica.

    PubMed

    Brewer, Matthew T; Agbedanu, Prince N; Zamanian, Mostafa; Day, Tim A; Carlson, Steve A

    2013-11-01

    Entamoeba histolytica is the causative agent of amoebic dysentery, a worldwide protozoal disease that results in approximately 100,000 deaths annually. The virulence of E. histolytica may be due to interactions with the host bacterial flora, whereby trophozoites engulf colonic bacteria as a nutrient source. The engulfment process depends on trophozoite recognition of bacterial epitopes that activate phagocytosis pathways. E. histolytica GPCR-1 (EhGPCR-1) was previously recognized as a putative G-protein-coupled receptor (GPCR) used by Entamoeba histolytica during phagocytosis. In the present study, we attempted to characterize EhGPCR-1 by using heterologous GPCR expression in Saccharomyces cerevisiae. We discovered that bacterial lipopolysaccharide (LPS) is an activator of EhGPCR-1 and that LPS stimulates EhGPCR-1 in a concentration-dependent manner. Additionally, we demonstrated that Entamoeba histolytica prefers to engulf bacteria with intact LPS and that this engulfment process is sensitive to suramin, which prevents the interactions of GPCRs and G-proteins. Thus, EhGPCR-1 is an LPS-recognizing GPCR that is a potential drug target for treatment of amoebiasis, especially considering the well-established drug targeting to GPCRs.

  11. Prevalence of Entamoeba histolytica, Giardia lamblia, and Cryptosporidium spp. in Libya: 2000–2015

    PubMed Central

    Ghenghesh, Khalifa Sifaw; Ghanghish, Khaled; BenDarif, Elloulu T.; Shembesh, Khaled; Franka, Ezzadin

    2016-01-01

    Introduction The intestinal protozoa Entamoeba histolytica, Giardia lamblia, and Cryptosporidium spp. are the causative agents of giardiasis, amebiasis, and cryptosporidiosis, respectively. Adequate knowledge of the geographical distribution of parasites and the demographic variables that influence their prevalence is important for effective control of infection in at-risk populations. Methods The data were obtained by an English language literature search of Medline and PubMed for papers using the search terms ‘intestinal parasites and Libya, G. lamblia and Libya, E. histolytica and Libya and Cryptosporidium and Libya’ for the period 2000–2015. Results The data obtained for the period 2000–2015 showed prevalence rates of 0.8–36.6% (mean 19.9%) for E. histolytica/dispar, 1.2–18.2% (mean 4.6%) for G. lamblia and 0.9–13% (mean 3.4%) for Cryptosporidium spp. among individuals in Libya with gastroenteritis (GE). On the other hand, prevalence rates of 0.8–16.3% (mean 8.3%), 1.8–28.8% (mean 4.8%), and 1.0–2.5% (mean=2.4), respectively, were observed for individuals without GE. The mean prevalence rate of E. histolytica/dispar was significantly higher among individuals with GE compared with those without GE (p<0.0000001, OR=2.74). No significant difference in prevalence rate of the three organisms was found according to gender, but most of infections were observed in children aged 10 years or younger. Conclusion The reviewed data suggest that E. histolytica, G. lamblia, and Cryptosporidium spp. may play a minor role in GE in Libya. The observed high prevalence rates of E. histolytica/dispar reported from Libya could be due mainly to the non-pathogenic E. dispar and E. moshkovskii. However, more studies are needed in the future using E. histolytica-specific enzyme immunoassays and/or molecular methods to confirm this observation. PMID:27363524

  12. Inhibitory Effects of Iranian Thymus vulgaris Extracts on in Vitro Growth of Entamoeba histolytica

    PubMed Central

    Behnia, Maryam; Komeylizadeh, Hossein; Tabaei, Seyyed-Javad Seyyed; Abadi, Alireza

    2008-01-01

    One of the most common drugs used against a wide variety of anaerobic protozoan parasites is metronidazole. However, this drug is mutagenic for bacteria and is a potent carcinogen for rodents. Thymus vulgaris is used for cough suppression and relief of dyspepsia. Also it has antibacterial and antifungal properties. The aim of this study was to investigate antiamebic effect of Thymus vulgaris against Entamoeba histolytica in comparison with metronidazole. One hundred gram air-dried T. vulgaris plant was obtained and macerated at 25℃ for 14 days using n-hexane and a mixture of ethanol and water. For essential oil isolation T. vulgaris was subjected to hydrodistillation using a clevenger-type apparatus for 3 hr. E. histolytica, HM-1: IMSS strain was used in all experiments. It was found that the minimal inhibitory concentration (MIC) for T. vulgaris hydroalcoholic, hexanic extracts, and the essential oil after 24 hr was 4 mg/mL, 4 mg/mL, and 0.7 mg/mL, respectively. After 48 hr the MIC for T. vulgaris hydroalcoholic and hexanic extracts was 3 and 3 mg/mL, respectively. Therefore, it can be concluded that the Iranian T. vulgaris is effective against the trophozoites of E. histolytica. PMID:18830054

  13. Inhibitory effects of Iranian Thymus vulgaris extracts on in vitro growth of Entamoeba histolytica.

    PubMed

    Behnia, Maryam; Haghighi, Ali; Komeylizadeh, Hossein; Tabaei, Seyyed-Javadi Seyyed; Abadi, Alireza

    2008-09-01

    One of the most common drugs used against a wide variety of anaerobic protozoan parasites is metronidazole. However, this drug is mutagenic for bacteria and is a potent carcinogen for rodents. Thymus vulgaris is used for cough suppression and relief of dyspepsia. Also it has antibacterial and antifungal properties. The aim of this study was to investigate antiamebic effect of Thymus vulgaris against Entamoeba histolytica in comparison with metronidazole. One hundred gram air-dried T. vulgaris plant was obtained and macerated at 25 degrees C for 14 days using n-hexane and a mixture of ethanol and water. For essential oil isolation T. vulgaris was subjected to hydrodistillation using a clevenger-type apparatus for 3 hr. E. histolytica, HM-1: IMSS strain was used in all experiments. It was found that the minimal inhibitory concentration (MIC) for T. vulgaris hydroalcoholic, hexanic extracts, and the essential oil after 24 hr was 4 mg/mL, 4 mg/mL, and 0.7 mg/mL, respectively. After 48 hr the MIC for T. vulgaris hydroalcoholic and hexanic extracts was 3 and 3 mg/mL, respectively. Therefore, it can be concluded that the Iranian T. vulgaris is effective against the trophozoites of E. histolytica.

  14. Nanoimaging and ultra structure of Entamoeba histolytica and its pseudopods by using atomic force microscope

    NASA Astrophysics Data System (ADS)

    Joshi, Narahari V.; Medina, Honorio; Urdaneta, H.; Barboza, J.

    2000-04-01

    Nan-imaging of Entamoeba histolytica was carried out by using Atomic Force Microscope (AFM). The structure of the nucleus, endoplasm and ectoplasm were studied separately. The diameter of the nucleus in living E. histolytica was found to be of the order of 10 micrometers which is slightly higher than the earlier reported value. The presence of karysome was detected in the nucleus. Well-organized patterns of chromatoid bodies located within the endoplasm, were detected and their repetitive patterns were examined. The organized structure was also extended within the ectoplasm. The dimensions and form of the organization suggest that chromatic bodies are constituted with ribosomes ordered in the form of folded sheet. Such structures were found to be absent in non-living E. histolytica. AFM images were also captured just in the act when ameba was extending its pseudopods. Alteration in the ultrastructure caused during the process of extension was viewed. Well marked canals of width 694.05 nm. And height 211.05 nm are clearly perceptible towards the direction of the pseudopods. 3D images are presented to appreciate the height variation, which can not be achieved by conventional well-established techniques such as electron microscopy.

  15. Entamoeba histolytica: an ecto-phosphatase activity regulated by oxidation-reduction reactions.

    PubMed

    de Sá Pinheiro, Ana Acacia; Amazonas, Juliana Natal; de Souza Barros, Fernanda; De Menezes, Lúcia Feitosa; Batista, Evander J O; Silva, Edward Felix; De Souza, Wanderley; Meyer-Fernandes, José Roberto

    2007-04-01

    In this work, an ecto-phosphatase activity of Entamoeba histolytica was characterized using intact cells. This activity presented the following biochemical characteristics: (i) it hydrolyzes p-NPP with V(max) of 8.00+/-0.22 nmol p-NP x h(-1) x 10(-5) cells and K(m) of 2.68+/-0.25 mM; (ii) it is inhibited by acid phosphatase inhibitors, such as sodium molybdate (K(i)=1.70+/-0.24 microM) and sodium fluoride (K(i)=0.25+/-0.02 mM); (iii) it also showed high sensitivity to phosphotyrosine phosphatase inhibitors, such as sodium orthovanadate (K(i)=1.07+/-0.14 microM), bpV-PHEN (K(i)=0.38+/-0.02 microM) and mpV-PIC (K(i)=0.39+/-0.04 microM). Zn(2+), an oxidizing agent, decreased the enzymatic activity in 50%. DTT and GSH, two reducing agents, enhanced the activity twofold. The non-invasive E. histolytica and free-living E. moshkovskii were less efficient in hydrolyzing p-NPP than the pathogenic E. histolytica suggesting that this enzyme could represent a virulence marker for this cell.

  16. Prospective evaluation of lactose malabsorption by lactose hydrogen breath test in individuals infected with Entamoeba histolytica and passing cysts.

    PubMed

    Rana, Satya Vati; Bhasin, Deepak Kumar; Vinayak, Virender Kumar

    2004-08-01

    The aim of the present prospective study was to detect lactose malabsorption in subjects in northern India infected with Entamoeba histolytica and passing cysts. The study group included forty-one patients with E. histolytica cysts in at least one of three consecutive faecal samples. Lactose malabsorption was detected by a lactose H2 breath test. The results were compared with those of forty controls subjects. Thirty-two of forty-one (78.0 %) subjects passing E. histolytica cysts had lactose malabsorption compared with seventeen of forty (42.5 %) control subjects (P<0.01). In conclusion, the present study shows that lactose malabsorption is significantly more common in individuals infected with E. histolytica and passing cysts compared with control subjects.

  17. Cell-matrix interactions of Entamoeba histolytica and E. dispar. A comparative study by electron-, atomic force- and confocal microscopy

    SciTech Connect

    Talamás-Lara, Daniel; Talamás-Rohana, Patricia; Fragoso-Soriano, Rogelio Jaime; Espinosa-Cantellano, Martha; Chávez-Munguía, Bibiana; González-Robles, Arturo; Martínez-Palomo, Adolfo

    2015-10-01

    Invasion of tissues by Entamoeba histolytica is a multistep process that initiates with the adhesion of the parasite to target tissues. The recognition of the non-invasive Entamoeba dispar as a distinct, but closely related protozoan species raised the question as to whether the lack of its pathogenic potential could be related to a weaker adhesion due to limited cytoskeleton restructuring capacity. We here compared the adhesion process of both amebas to fibronectin through scanning, transmission, atomic force, and confocal microscopy. In addition, electrophoretic and western blot assays of actin were also compared. Adhesion of E. histolytica to fibronectin involves a dramatic reorganization of the actin network that results in a tighter contact to and the subsequent focal degradation of the fibronectin matrix. In contrast, E. dispar showed no regions of focal adhesion, the cytoskeleton was poorly reorganized and there was little fibronectin degradation. In addition, atomic force microscopy using topographic, error signal and phase modes revealed clear-cut differences at the site of contact of both amebas with the substrate. In spite of the morphological and genetic similarities between E. histolytica and E. dispar the present results demonstrate striking differences in their respective cell-to-matrix adhesion processes, which may be of relevance for understanding the invasive character of E. histolytica. - Highlights: • Striking differences in adhesion to FN between E. histolytica and E. dispar. • A greater degree of cell stiffness in E. histolytica with respect to E. dispar. • E. histolytica but not E. dispar forms regions of close contact with FN. • The actin cytoskeleton is involved in the pathogenicity of E. histolytica.

  18. Development and initial evaluation of a lateral flow dipstick test for antigen detection of Entamoeba histolytica in stool sample.

    PubMed

    Saidin, Syazwan; Yunus, Muhammad Hafiznur; Othman, Nurulhasanah; Lim, Yvonne Ai-Lian; Mohamed, Zeehaida; Zakaria, Nik Zairi; Noordin, Rahmah

    2017-03-24

    Entamoeba histolytica infection remains a public health concern in developing countries. Early diagnosis of amoebiasis can avoid disease complications, thus this study was aimed at developing a test that can rapidly detect the parasite antigens in stool samples. Rabbits were individually immunized with recombinant pyruvate phosphate dikinase (rPPDK) and E. histolytica excretory-secretory antigens to produce polyclonal antibodies. A rapid dipstick test was produced using anti-rPPDK PAb lined on the dipstick as capture reagent and anti-EhESA PAb conjugated to colloidal gold as the detector reagent. Using E. histolytica-spiked in stool sample of a healthy individual, the detection limit of the dipstick test was found to be 1000 cells ml(-1). Meanwhile when rPPDK was spiked in the stool sample, the minimum concentration detected by the dipstick test was 0.1 μg ml(-1). The performances of the dipstick, commercial Techlab E. histolytica II enzyme-linked immunosorbent assays (ELISA) and real-time PCR were compared using 70 stool samples from patients infected with Entamoeba species (n = 45) and other intestinal pathogens (n = 25). When compared to real-time PCR, the diagnostic sensitivity of the dipstick for detection of E. histolytica was 65.4% (n = 17/26); while the diagnostic specificity when tested with stool samples containing other intestinal pathogens was 92% (23/25). In contrast, Techlab E. histolytica II ELISA detected 19.2% (5/26) of the E. histolytica-positive samples as compared to real-time PCR. The lateral flow dipstick test produced in this study enabled rapid detection of E. histolytica, thus it showed good potential to be further developed into a diagnostic tool for intestinal amoebiasis.

  19. Behavior of DNA-lacking mitochondria in Entamoeba histolytica revealed by organelle transplant

    PubMed Central

    Kazama, Makoto; Ogiwara, Sanae; Makiuchi, Takashi; Yoshida, Kazuhiro; Nakada-Tsukui, Kumiko; Nozaki, Tomoyoshi; Tachibana, Hiroshi

    2017-01-01

    The anaerobic protozoan parasite Entamoeba histolytica has mitosomes that are mitochondria lacking some canonical functions and organelle DNA. Mitosomes play an important role in the life cycle of the parasite. The distribution of proteins in mitosomes is not uniform, and how mitosomes are maintained and retained is unknown. To answer these questions, we developed a transplant method for mitosomes with hemagglutinin-tagged protein into recipient cells containing mitosomes with Myc-tagged protein. Immunofluorescence staining showed that the two protein tags colocalized in single mitosomes in some recipient cells. These results suggest that our transplant method can be used in anaerobic protozoa and that donor mitosomes may obtain recipient proteins through fusion with other mitosomes or through de novo synthesis of proteins in recipient cells. PMID:28287148

  20. Loss of susceptibility to complement lysis in Entamoeba histolytica HM1 by treatment with human serum.

    PubMed Central

    Calderon, J; Tovar, R

    1986-01-01

    Entamoeba histolytica HM1, exposed to a series of treatment with normal human serum (NHS), progressively lost susceptibility to complement lysis. Trophozoites were incubated daily with unheated or heat-inactivated NHS for 2 hr at 36 degrees, starting with 10% v/v serum and increasing by 5% every 3 days up to 40% NHS. Resistance to complement lysis was also obtained with two different HM1 clones but not with the low virulent strain HK9. Induction of resistance dependent on the number of NHS treatments, with a maximal 50% reduction occurring after 11 treatments. Susceptibility to complement-dependent lysis was regained 6 weeks after serum treatments were terminated, suggesting that resistance to lysis was an acquired rather than a genetic property. PMID:2874111

  1. Isolation of the galactose-binding lectin that mediates the in vitro adherence of Entamoeba histolytica.

    PubMed Central

    Petri, W A; Smith, R D; Schlesinger, P H; Murphy, C F; Ravdin, J I

    1987-01-01

    Entamoeba histolytica adheres to human colonic mucus, colonic epithelial cells, and other target cells via a galactose (Gal) or N-acetyl-D-galactosamine (GalNAc) inhibitable surface lectin. Blockade of this adherence lectin with Gal or GalNAc in vitro prevents amebic killing of target cells. We have identified and purified the adherence lectin by two methods: affinity columns derivatized with galactose monomers or galactose terminal glycoproteins, and affinity columns and immunoblots prepared with monoclonal antibodies that inhibit amebic adherence. By both methods the adherence lectin was identified as a 170-kD secreted and membrane-bound amebic protein. The surface location of the lectin was confirmed by indirect immunofluorescence. Purified lectin competitively inhibited amebic adherence to target cells by binding to receptors on the target Chinese hamster ovary cells in a Gal-inhibitable manner. Images PMID:2890654

  2. Entamoeba histolytica and Trichomonas vaginalis: trophozoite growth inhibition by metronidazole electro-transferred water.

    PubMed

    Heredia-Rojas, J Antonio; Torres-Flores, Antonio Cayetano; Rodríguez-De la Fuente, Abraham O; Mata-Cárdenas, Benito David; Rodríguez-Flores, Laura E; Barrón-González, María Porfiria; Torres-Pantoja, Antonio Cayetano; Alcocer-González, Juan M

    2011-01-01

    The influence of low-frequency electromagnetic (LF-EM) waves on microorganisms has been a subject of experimental investigations for more than two decades and the results are promising. In parallel, an interesting procedure known as biophysical-information-therapy or bioresonance therapy (BRT) which in principle is based on LF-EM stimulation, has emerged. BRT was discovered in the late 1980's but it is still poorly studied. This paper demonstrates that by transferring metronidazole information to water samples by an electronic amplifier (BRT device), the growth of axenically cultured trophozoites of Entamoeba histolytica and Trichomonasvaginalis is significantly inhibited, compared with those cultures treated with non and sham electro-transferred water samples. A positive control of metronidazole, a well-known cytotoxic drug against parasites, was used as a reference.

  3. Behavior of DNA-lacking mitochondria in Entamoeba histolytica revealed by organelle transplant.

    PubMed

    Kazama, Makoto; Ogiwara, Sanae; Makiuchi, Takashi; Yoshida, Kazuhiro; Nakada-Tsukui, Kumiko; Nozaki, Tomoyoshi; Tachibana, Hiroshi

    2017-03-13

    The anaerobic protozoan parasite Entamoeba histolytica has mitosomes that are mitochondria lacking some canonical functions and organelle DNA. Mitosomes play an important role in the life cycle of the parasite. The distribution of proteins in mitosomes is not uniform, and how mitosomes are maintained and retained is unknown. To answer these questions, we developed a transplant method for mitosomes with hemagglutinin-tagged protein into recipient cells containing mitosomes with Myc-tagged protein. Immunofluorescence staining showed that the two protein tags colocalized in single mitosomes in some recipient cells. These results suggest that our transplant method can be used in anaerobic protozoa and that donor mitosomes may obtain recipient proteins through fusion with other mitosomes or through de novo synthesis of proteins in recipient cells.

  4. Phenolic Acid Composition and Anti-Parasitic Effects of Four Peucedanum Species on Entamoeba histolytica Trophozoites

    PubMed Central

    DEGERLI, Serpil; TEPE, Bektas

    2015-01-01

    Background: The aim of this study was to determine phenolic acid composition and anti-parasitic effects of Peucedanum caucasicum, P. palimbioides, P. longibracteolatum and P. chryseum on Entamoeba histolytica. Methods: Methanol extracts of the plant species were prepared by soxhlet extraction. Phenolic acid compositions were determined by HPLC. Anti-proliferative effect of extracts on trophozoites was determined by using trypan blue dye exclusion test. For counting the cells, approximately a hundred of E. histolytica trophozoites were examined in each time. The data were presented as mean values with standard deviations and analyzed by repeated measures of ANOVA followed by Tukey test for post-hoc pairwise comparisons. The P-value was set at 0.05 for significance level. Results: All of the extracts showed a time and dose dependent amoebicidal action on trophozoites. Among the extracts tested, P. longibracteolatum showed the strongest amoebicidal effect on the trophozoites. As expected, this plant species also exhibited time and dose dependent activity on the trophozoites. At 4.0 mg/ml extract concentration, all of the trophozoites were killed by the extract in 72nd hour. Gallic (11.144 mg/g), P-hydroxybenzoic (17.646 mg/g), and o-coumaric acids (14.442 mg/g) were determined as the major phenolic acids of P. longibracteolatum. Gallic and P-hydroxybenzoic acids found in P. longibracteolatum could not be determined in other extracts. Therefore, high activity potential of this plant could probably be attributed to the presence of these phytochemicals. Conclusion: P. longibracteolatum can be further evaluated as potential therapeutic drugs for the treatment of Entamoeba infections. PMID:26622297

  5. Entamoeba invadens and E. histolytica: separation and purification of precysts and cysts by centrifugation on discontinuous density gradients of Percoll.

    PubMed

    Avron, B; Bracha, R; Deutsch, M R; Mirelman, D

    1983-06-01

    The different cell forms in the life cycle of Entamoeba invadens (trophozoites, precysts, and cysts) were rapidly and quantitatively separated on density step gradients of polyvinylpyrolidone-coated colloidal silica particles (Percoll). With this method, the gradual process of encystation by E. invadens trophozoites could be monitored. Percoll gradients were also efficient in separating trophozoites of Entamoeba histolytica and bacteria. After purification on Percoll, trophozoites display no evidence of damage when examined by light microscopy and no loss in viability as judged by their ability to multiply.

  6. Cyclosporin A inhibits calcineurin (phosphatase 2B) and P-glycoprotein activity in Entamoeba histolytica.

    PubMed

    Carrero, Julio C; Lugo, Haydee; Pérez, D Guillermo; Ortiz-Martínez, César; Laclette, Juan P

    2004-08-01

    Cyclosporin A (CsA) inhibits the proliferation of several protozoan parasites through blocking the activity of calcineurin (Cn) or P-glycoproteins (Pgp). We report here, that inhibition of the proliferation of Entamoeba histolytica trophozoites, the causal agent of human amebiasis, is due to interference of the phosphatase activity of Cn, in a similar fashion to the effect of this immunosuppressive drug on T lymphocytes. The non-immunosuppressive CsA analog PSC-833, which binds Pgp without interfering the function of Cn, did not inhibit the proliferation of HM1:IMSS trophozoites. Moreover, phosphatase activity of amebic Cn, detected using the phosphopeptide RII, was drastically affected by incubation with CsA, but not with PSC-833. On the other hand, both drugs were also tested on clone C2 trophozoites, which grow in the presence of emetine due to over-expression of Pgp. The effect of CsA was similar to that observed on HM1:IMSS trophozoites, whereas PSC-833 only affected the proliferation and viability of clone C2 when the trophozoites were grown in the presence of 40 microM of emetine, suggesting an interference of the Pgp activity. This suggestion was confirmed by results from experiments of Pgp-dependent effux of rhodamine from pre-loaded trophozoites, in the presence of either of these drugs. Therefore, CsA inhibition of E. histolytica trophozoite proliferation is more likely due to Cn than Pgp activity inhibition.

  7. Rat and human colonic mucins bind to and inhibit adherence lectin of Entamoeba histolytica.

    PubMed Central

    Chadee, K; Petri, W A; Innes, D J; Ravdin, J I

    1987-01-01

    Establishment of adherence by Entamoeba histolytica is mediated by a 170-kD Gal/GalNAc inhibitable lectin and is required for cytolysis and phagocytosis of mammalian target cells. We studied the biochemical mechanisms of the in vitro interaction between rat and human colonic mucins and axenic E. histolytica trophozoites. Crude mucus prevented amebic adherence to Chinese hamster ovary (CHO) cells by up to 70%. Purification of the colonic mucins by Sepharose 4B chromatography, nuclease digestion, and cesium chloride gradient centrifugation resulted in a 1,000-fold enrichment of the inhibitory mucins. Purified rat mucin inhibited amebic adherence to and cytolysis of homologous rat colonic epithelial cells. Oxidation and enzymatic cleavage of rat mucin Gal and GalNAc residues completely abrogated mucin inhibition of amebic adherence. The binding of rat 125I-mucin to amebae was galactose specific, saturable, reversible, and pH dependent. A monoclonal antibody specific for the 170-kD amebic Gal/GalNAc lectin completely inhibited the binding of rat 125I-mucin. Rat mucin bound to Affigel affinity purified the amebic lectin from conditioned medium. Colonic mucin glycoproteins act as an important host defense by binding to the parasite's adherence lectin, thus preventing amebic attachment to and cytolysis of host epithelial cells. Images PMID:2890655

  8. Trogocytosis by Entamoeba histolytica contributes to cell killing and tissue invasion.

    PubMed

    Ralston, Katherine S; Solga, Michael D; Mackey-Lawrence, Nicole M; Somlata; Bhattacharya, Alok; Petri, William A

    2014-04-24

    Entamoeba histolytica is the causative agent of amoebiasis, a potentially fatal diarrhoeal disease in the developing world. The parasite was named "histolytica" for its ability to destroy host tissues, which is probably driven by direct killing of human cells. The mechanism of human cell killing has been unclear, although the accepted model was that the parasites use secreted toxic effectors to kill cells before ingestion. Here we report the discovery that amoebae kill by ingesting distinct pieces of living human cells, resulting in intracellular calcium elevation and eventual cell death. After cell killing, amoebae detach and cease ingestion. Ingestion of human cell fragments is required for cell killing, and also contributes to invasion of intestinal tissue. The internalization of fragments of living human cells is reminiscent of trogocytosis (from Greek trogo, nibble) observed between immune cells, but amoebic trogocytosis differs because it results in death. The ingestion of live cell material and the rejection of corpses illuminate a stark contrast to the established model of dead cell clearance in multicellular organisms. These findings change the model for tissue destruction in amoebiasis and suggest an ancient origin of trogocytosis as a form of intercellular exchange.

  9. Repurposing the Open Access Malaria Box To Discover Potent Inhibitors of Toxoplasma gondii and Entamoeba histolytica

    PubMed Central

    Fokou, Patrick V. T.; Tchokouaha, Lauve R. Y.; Spangenberg, Thomas; Mfopa, Alvine N.; Kouipou, Ruffin M. T.; Mbouna, Cedric J.; Donfack, Valerie F. Donkeng; Zollo, Paul H. A.

    2014-01-01

    Toxoplasmosis and amebiasis are important public health concerns worldwide. The drugs currently available to control these diseases have proven limitations. Therefore, innovative approaches should be adopted to identify and develop new leads from novel scaffolds exhibiting novel modes of action. In this paper, we describe results from the screening of compounds in the Medicines for Malaria Venture (MMV) open access Malaria Box in a search for new anti-Toxoplasma and anti-Entamoeba agents. Standard in vitro phenotypic screening procedures were adopted to assess their biological activities. Seven anti-Toxoplasma compounds with a 50% inhibitory concentration (IC50) of <5 μM and selectivity indexes (SI) of >6 were identified. The most interesting compound was MMV007791, a piperazine acetamide, which has an IC50 of 0.19 μM and a selectivity index of >157. Also, we identified two compounds, MMV666600 and MMV006861, with modest activities against Entamoeba histolytica, with IC50s of 10.66 μM and 15.58 μM, respectively. The anti-Toxoplasma compounds identified in this study belong to scaffold types different from those of currently used drugs, underscoring their novelty and potential as starting points for the development of new antitoxoplasmosis drugs with novel modes of action. PMID:25049259

  10. Entamoeba histolytica calreticulin: an endoplasmic reticulum protein expressed by trophozoites into experimentally induced amoebic liver abscesses.

    PubMed

    González, Enrique; de Leon, Maria del Carmen García; Meza, Isaura; Ocadiz-Delgado, Rodolfo; Gariglio, Patricio; Silva-Olivares, Angelica; Galindo-Gómez, Silvia; Shibayama, Mineko; Morán, Patricia; Valadez, Alicia; Limón, Angelica; Rojas, Liliana; Hernández, Eric G; Cerritos, René; Ximenez, Cecilia

    2011-02-01

    Entamoeba histolytica calreticulin (EhCRT) is remarkably immunogenic in humans (90-100% of invasive amoebiasis patients). Nevertheless, the study of calreticulin in this protozoan is still in its early stages. The exact location, biological functions, and its role in pathogenesis are yet to be fully understood. The aim of the present work is to determine the location of EhCRT in virulent trophozoites in vivo and the expression of the Ehcrt gene during the development of experimentally induced amoebic liver abscesses (ALA) in hamsters. Antibodies against recombinant EhCRT were used for the immunolocalization of EhCRT in trophozoites through confocal microscopy; immunohistochemical assays were also performed on tissue sections of ALAs at different times after intrahepatic inoculation. The expression of the Ehcrt gene during the development of ALA was estimated through both in situ RT-PCR and real-time RT-PCR. Confocal assays of virulent trophozoites showed a distribution of EhCRT in the cytoplasmic vesicles of different sizes. Apparently, EhCRT is not exported into the hepatic tissue. Real-time RT-PCR demonstrated an over-expression of the Ehcrt gene at 30 min after trophozoite inoculation, reaching a peak at 1-2 h; thereafter, the expression fell sharply to its original levels. These results demonstrate for the first time in an in vivo model of ALA, the expression of Ehcrt gene in E. histolytica trophozoites and add evidence that support CRT as a resident protein of the ER in E. histolytica species. The in vivo experiments suggest that CRT may play an important role during the early stages of the host-parasite relationship, when the parasite is adapting to a new environment, although the protein seems to be constitutively synthesized. Moreover, trophozoites apparently do not export EhCRT into the hepatic tissue in ALA.

  11. Nitroimidazole carboxamides as antiparasitic agents targeting Giardia lamblia, Entamoeba histolytica and Trichomonas vaginalis.

    PubMed

    Jarrad, A M; Debnath, A; Miyamoto, Y; Hansford, K A; Pelingon, R; Butler, M S; Bains, T; Karoli, T; Blaskovich, M A T; Eckmann, L; Cooper, M A

    2016-09-14

    Diarrhoeal diseases caused by the intestinal parasites Giardia lamblia and Entamoeba histolytica constitute a major global health burden. Nitroimidazoles are first-line drugs for the treatment of giardiasis and amebiasis, with metronidazole 1 being the most commonly used drug worldwide. However, treatment failures in giardiasis occur in up to 20% of cases and development of resistance to metronidazole is of concern. We have re-examined 'old' nitroimidazoles as a foundation for the systematic development of next-generation derivatives. Using this approach, derivatisation of the nitroimidazole carboxamide scaffold provided improved antiparasitic agents. Thirty-three novel nitroimidazole carboxamides were synthesised and evaluated for activity against G. lamblia and E. histolytica. Several of the new compounds exhibited potent activity against G. lamblia strains, including metronidazole-resistant strains of G. lamblia (EC50 = 0.1-2.5 μM cf. metronidazole EC50 = 6.1-18 μM). Other compounds showed improved activity against E. histolytica (EC50 = 1.7-5.1 μM cf. metronidazole EC50 = 5.0 μM), potent activity against Trichomonas vaginalis (EC50 = 0.6-1.4 μM cf. metronidazole EC50 = 0.8 μM) and moderate activity against the intestinal bacterial pathogen Clostridium difficile (0.5-2 μg/mL, cf. metronidazole = 0.5 μg/mL). The new compounds had low toxicity against mammalian kidney and liver cells (CC50 > 100 μM), and selected antiparasitic hits were assessed for human plasma protein binding and metabolic stability in liver microsomes to demonstrate their therapeutic potential.

  12. A Novel Alpha Kinase EhAK1 Phosphorylates Actin and Regulates Phagocytosis in Entamoeba histolytica

    PubMed Central

    Mansuri, M. Shahid; Bhattacharya, Sudha; Bhattacharya, Alok

    2014-01-01

    Phagocytosis plays a key role in nutrient uptake and virulence of the protist parasite Entamoeba histolytica. Phagosomes have been characterized by proteomics, and their maturation in the cells has been studied. However, there is so far not much understanding about initiation of phagocytosis and formation of phagosomes at the molecular level. Our group has been studying initiation of phagocytosis and formation of phagosomes in E. histolytica, and have described some of the molecules that play key roles in the process. Here we show the involvement of EhAK1, an alpha kinase and a SH3 domain containing protein in the pathway that leads to formation of phagosomes using red blood cell as ligand particle. A number of approaches, such as proteomics, biochemical, confocal imaging using specific antibodies or GFP tagged molecules, expression down regulation by antisense RNA, over expression of wild type and mutant proteins, were used to understand the role of EhAK1 in phagocytosis. EhAK1 was found in the phagocytic cups during the progression of cups, until closure of phagosomes, but not in the phagosomes themselves. It is recruited to the phagosomes through interaction with the calcium binding protein EhCaBP1. A reduction in phagocytosis was observed when EhAK1 was down regulated by antisense RNA, or by over expression of the kinase dead mutant. G-actin was identified as one of the major substrates of EhAK1. Phosphorylated actin preferentially accumulated at the phagocytic cups and over expression of a phosphorylation defective actin led to defects in phagocytosis. In conclusion, we describe an important component of the pathway that is initiated on attachment of red blood cells to E. histolytica cells. The main function of EhAK1 is to couple signalling events initiated after accumulation of EhC2PK to actin dynamics. PMID:25299184

  13. Interaction of human leukocytes and Entamoeba histolytica. Killing of virulent amebae by the activated macrophage.

    PubMed Central

    Salata, R A; Pearson, R D; Ravdin, J I

    1985-01-01

    Capable effector mechanisms in the human immune response against the cytolytic, protozoan parasite Entamoeba histolytica have not been described. To identify a competent human effector cell, we studied the in vitro interactions of normal human polymorphonuclear neutrophils, peripheral blood mononuclear cells (PBMC), monocytes (MC), and MC-derived macrophages with virulent axenic amebae (strain HMI-IMSS). Amebae killed neutrophils, PBMC, MC, and MC-derived macrophages (P less than 0.001), without loss of parasite viability. The addition of heat-inactivated immune serum did not enable leukocytes to kill amebae, nor did it protect these host cells from amebae. MC-derived macrophages, activated with lymphokine elicited by the mitogens conconavalin A, phytohemagglutinin, or an amebic soluble protein preparation (strain HK9), killed 55% of amebae by 3 h in a trypan blue exclusion assay (P less than 0.001); during this time, 40% of the activated macrophages died. Lysis of amebae was confirmed using 111Indium oxine radiolabeled parasites and was antibody independent. Macrophage death appeared to be due to the deleterious effect of lysed amebae rather than the contact-dependent effector mechanisms of E. histolytica. Adherence between activated macrophages and amebae was greater than that between other leukocytes and amebae (P less than 0.001). Microscopic observations, kinetic analysis of the killing of amebae by activated macrophages, and suspension of amebae with adherent activated macrophages in a 10% dextran solution indicated that contact by activated macrophages was necessary to initiate the killing of amebae. Catalase but not superoxide dismutase inhibited the amebicidal capacity of activated macrophages (P less than 0.001). However, activated macrophages from an individual with chronic granulomatous disease were able to kill amebae, but not as effectively as normal cells (P less than 0.01). In summary, activated MC-derived macrophages killed virulent E. histolytica

  14. Adherence of Entamoeba histolytica trophozoites to rat and human colonic mucosa.

    PubMed Central

    Ravdin, J I; John, J E; Johnston, L I; Innes, D J; Guerrant, R L

    1985-01-01

    We studied the adherence of [3H]thymidine-labeled axenic Entamoeba histolytica (strain HM1-IMSS) to in vitro preparations of rat and human colonic mucosa. Studies were performed with fixed or unfixed rat colonic mucosa, unfixed rat mucosa exposed to trypsin, unfixed rat submucosa, and fixed human colonic mucosa. Twenty percent of the amebae adhered to fixed rat colonic mucosa; adherence was specifically inhibited by N-acetyl-D-galactosamine (GalNAc), galactose, and asialofetuin. The adherence of amebae to fixed human colonic mucosa was also GalNAc inhibitable. Greater adherence was found with unfixed rat colonic mucosa (40.9%) and was not GalNAc inhibitable unless the tissue was first exposed to trypsin. However, GalNAc did inhibit the adherence of amebae to unfixed rat submucosa. Glutaraldehyde fixation of amebae inactivates known amebic adhesion proteins; there was a markedly decreased adherence of fixed amebae to trypsin-exposed mucosa or fixed rat colonic mucosa. However, fixed or viable amebae had equal levels of adherence to unfixed rat colonic mucosa, suggesting the presence of a host adhesion protein that binds to receptors on amebae. Human (10%) and rabbit (5%) immune sera reduced the adherence of viable amebae to fixed rat colonic mucosa. We concluded that the GalNAc-inhibitable adhesion protein on the surface of E. histolytica trophozoites mediated adherence to fixed rat mucosa, fixed human colonic mucosa, trypsin-exposed unfixed rat mucosa, and unfixed rat submucosa. The surface of unfixed rat colonic mucosa contained a glutaraldehyde- and trypsin-sensitive host adhesion protein, perhaps in the overlying mucus blanket, which bound viable or fixed E. histolytica trophozoites. Images PMID:2580787

  15. Production of mouse monoclonal antibodies which inhibit in vitro adherence of Entamoeba histolytica trophozoites.

    PubMed Central

    Ravdin, J I; Petri, W A; Murphy, C F; Smith, R D

    1986-01-01

    Adherence by axenic Entamoeba histolytica trophozoites to mammalian cells is mediated by an N-acetylgalactosamine (GalNAc)-inhibitable adhesin on the surface of the parasite. We isolated 35 hybridoma cell lines producing antibodies to E. histolytica as indicated by ELISA with sonicated amebic protein or by immunofluorescence assay with fixed whole trophozoites. Tissue culture supernatants were further screened for subcloning by the ability to bind to Chinese hamster ovary (CHO) cells which were first exposed to a partially purified soluble preparation of the amebic GalNAc-inhibitable lectin. Eight tissue culture supernatants were positive in this assay. Antibodies from four subcloned cell lines (D3-14, H8-5, I12-2, and I1-21) inhibited amebic adherence to CHO cells (P less than 0.01). Of the original 35 tissue culture supernatants, 3 also inhibited amebic adherence (P less than 0.01; F1, F14, and J10); monoclonal antibodies in these supernatants did not bind to lectin-exposed CHO cells. Three purified monoclonal antibodies (H8-5, I12-2, and I1-21) inhibited amebic adherence at greater than or equal to 2 micrograms/10(4) amebae (P less than 0.05). None of these inhibitory monoclonal antibodies immunoprecipitated with a soluble amebic protein preparation following sodium dodecyl sulfate-polyacrylamide gel electrophoresis under denaturing conditions. Monoclonal antibodies which inhibit in vitro adherence by E. histolytica will be useful in purification of the GalNAc-inhibitable lectin. PMID:2873102

  16. Worldwide genealogy of Entamoeba histolytica: an overview to understand haplotype distribution and infection outcome.

    PubMed

    Zermeño, Valeria; Ximénez, Cecilia; Morán, Patricia; Valadez, Alicia; Valenzuela, Olivia; Rascón, Edgar; Diaz, Daniel; Cerritos, René

    2013-07-01

    Although Entamoeba histolytica is one of the most prevalent intestinal parasites, how the different strains of this species are distributed all over the world and how different genotypes are associated with the infection outcome are yet to be fully understood. Recently, the use of a number of molecular markers has made the characterization of several genotypes in those regions with high incidence of amoebiasis possible. This work proposes the first genealogy of E. histolytica, with an haplotype network based on two tRNA gene-linked array of Short Tandem Repeats (STRs) reported until today, and 47 sequences from 39 new isolates of Mexican Amoebic Liver Abscesses (ALA) samples. One hundred and three sequences were obtained from D-A locus, their information about the geographic region of isolation as well as clinical diagnosis were also collected. One hundred and five sequences from N-K2 locus were also obtained as well as the region of isolation, but the information about clinical diagnosis was not available in all cases. The most abundant and widely distributed haplotype in the world is the one of E. histolytica HM1:IMSS strain. This was found in Mexico, Bangladesh, Japan, China and USA and is associated to symptomatic patients as well as asymptomatic cyst passers. Many other haplotypes were found only in a single country. Both genealogies suggest that there are no lineages within the networks that may be related to a particular geographic region or infection outcome. A concatenated analysis of the two molecular markers revealed 12 different combinations, which suggests the possibility of genetic recombination events. The present study is the first to propose a global genealogy of this species and suggests that there are still many genotypes to be discovered. The genotyping of new isolates will help to understand the great diversity and genetic structure of this parasite.

  17. A novel alpha kinase EhAK1 phosphorylates actin and regulates phagocytosis in Entamoeba histolytica.

    PubMed

    Mansuri, M Shahid; Bhattacharya, Sudha; Bhattacharya, Alok

    2014-10-01

    Phagocytosis plays a key role in nutrient uptake and virulence of the protist parasite Entamoeba histolytica. Phagosomes have been characterized by proteomics, and their maturation in the cells has been studied. However, there is so far not much understanding about initiation of phagocytosis and formation of phagosomes at the molecular level. Our group has been studying initiation of phagocytosis and formation of phagosomes in E. histolytica, and have described some of the molecules that play key roles in the process. Here we show the involvement of EhAK1, an alpha kinase and a SH3 domain containing protein in the pathway that leads to formation of phagosomes using red blood cell as ligand particle. A number of approaches, such as proteomics, biochemical, confocal imaging using specific antibodies or GFP tagged molecules, expression down regulation by antisense RNA, over expression of wild type and mutant proteins, were used to understand the role of EhAK1 in phagocytosis. EhAK1 was found in the phagocytic cups during the progression of cups, until closure of phagosomes, but not in the phagosomes themselves. It is recruited to the phagosomes through interaction with the calcium binding protein EhCaBP1. A reduction in phagocytosis was observed when EhAK1 was down regulated by antisense RNA, or by over expression of the kinase dead mutant. G-actin was identified as one of the major substrates of EhAK1. Phosphorylated actin preferentially accumulated at the phagocytic cups and over expression of a phosphorylation defective actin led to defects in phagocytosis. In conclusion, we describe an important component of the pathway that is initiated on attachment of red blood cells to E. histolytica cells. The main function of EhAK1 is to couple signalling events initiated after accumulation of EhC2PK to actin dynamics.

  18. Changes in isoenzyme patterns of a cloned culture of nonpathogenic Entamoeba histolytica during axenization.

    PubMed Central

    Mirelman, D; Bracha, R; Wexler, A; Chayen, A

    1986-01-01

    The axenization of an Entamoeba histolytica isolate with a nonpathogenic isoenzyme electrophoretic pattern (zymodeme) was recently achieved for the first time (15). Forty days after the cells were transferred to the medium used for axenic cultivation, the amebae developed virulence properties, and the zymodeme converted to a pathogenic pattern. To exclude the possibility that the original isolate consisted of two zymodeme populations and that conditions of growth selected for a particular population, the experiment was repeated with a cloned culture of a nonpathogenic (zymodeme III) strain, E. histolytica SAW 1734R clAR, isolated by and obtained from P. G. Sargeaunt. Axenization was accomplished, as before, by transferring trophozoites to TYI-S-33 medium containing a mixture of antibiotics to suppress the growth of the associated bacterial flora and a nutritional supplement consisting of gamma-irradiated bacteria. A change in the hexokinase and phosphoglucomutase isoenzyme pattern was observed 21 days after the amebae had been transferred to the axenic medium but before complete axenization of the amebae had occurred. The change in zymodeme was accompanied by an increase in virulence, as evidenced by the ability of fewer amebae to induce hepatic abscesses in hamsters. A reverse conversion to a nonpathogenic zymodeme was also accomplished by reassociating and subculturing the newly converted pathogenic trophozoites of strain SAW 1734R clAR with the bacterial flora that accompanied this ameba in the original xenic culture. The electromobilities of the hexokinase isoenzymes changed back to their original pattern 7 days after the amebae were returned to xenic growth conditions. Our in vitro results demonstrate that culture conditions and bacterial flora can cause changes in the zymodeme and virulence of a cloned ameba isolate and raise the concern that this could happen also in vivo. Thus, the finding of a particular zymodeme in a culture of E. histolytica isolated

  19. Endoplasmic Reticulum Stress-Sensing Mechanism Is Activated in Entamoeba histolytica upon Treatment with Nitric Oxide

    PubMed Central

    Santi-Rocca, Julien; Smith, Sherri; Weber, Christian; Pineda, Erika; Hon, Chung-Chau; Saavedra, Emma; Olivos-García, Alfonso; Rousseau, Sandrine; Dillies, Marie-Agnès; Coppée, Jean-Yves; Guillén, Nancy

    2012-01-01

    The Endoplasmic Reticulum stores calcium and is a site of protein synthesis and modification. Changes in ER homeostasis lead to stress responses with an activation of the unfolded protein response (UPR). The Entamoeba histolytica endomembrane system is simple compared to those of higher eukaryotes, as a canonical ER is not observed. During amoebiasis, an infection of the human intestine and liver by E. histolytica, nitric oxide (NO) triggers an apoptotic-like event preceded by an impairment of energy production and a loss of important parasite pathogenic features. We address the question of how this ancient eukaryote responds to stress induced by immune components (i.e. NO) and whether stress leads to ER changes and subsequently to an UPR. Gene expression analysis suggested that NO triggers stress responses marked by (i) dramatic up-regulation of hsp genes although a bona fide UPR is absent; (ii) induction of DNA repair and redox gene expression and iii) up-regulation of glycolysis-related gene expression. Enzymology approaches demonstrate that NO directly inhibits glycolysis and enhance cysteine synthase activity. Using live imaging and confocal microscopy we found that NO dramatically provokes extensive ER fragmentation. ER fission in E. histolytica appears as a protective response against stress, as it has been recently proposed for neuron self-defense during neurologic disorders. Chronic ER stress is also involved in metabolic diseases including diabetes, where NO production reduces ER calcium levels and activates cell death. Our data highlighted unique cellular responses of interest to understand the mechanisms of parasite death during amoebiasis. PMID:22384074

  20. Identification of phosphatidylcholine transfer protein-like in the parasite Entamoeba histolytica.

    PubMed

    Piña-Vázquez, Carolina; Reyes-López, Magda; Mendoza-Hernández, Guillermo; Bermúdez-Cruz, Rosa María; de la Garza, Mireya

    2014-12-01

    Caveolin is the protein marker of caveola-mediated endocytosis. Previously, we demonstrated by immunoblotting and immunofluorescence that an anti-chick embryo caveolin-1 monoclonal antibody (mAb) recognizes a protein in amoeba extracts. Nevertheless, the caveolin-1 gene is absent in the Entamoeba histolytica genome database. In this work, the goal was to isolate, identify and characterize the protein that cross-reacts with chick embryo caveolin-1. We identified the protein using a proteomic approach, and the complete gene was cloned and sequenced. The identified protein, E. histolytica phosphatidylcholine transfer protein-like (EhPCTP-L), is a member of the StAR-related lipid transfer (START) protein superfamily. The human homolog binds and transfers phosphatidylcholine (PC) and phosphatidylethanolamine (PE) between model membranes in vitro; however, the physiological role of PCTP-L remains elusive. Studies in silico showed that EhPCTP-L has a central START domain and also contains a C-terminal intrinsically disordered region. The anti-rEhPCTP-L antibody demonstrated that EhPCTP-L is found in the plasma membrane and cytosol, which is in agreement with previous reports on the human counterpart. This result points to the plasma membrane as one possible target membrane for EhPCTP-L. Furthermore, assays using filipin and nystatin showed down regulation of EhPCTP-L, in an apparently cholesterol-independent way. Interestingly, EhPCTP-L binds primarily to anionic phospholipids phosphatidylserine (PS) and phosphatidic acid (PA), while its mammalian counterpart HsPCTP-L binds neutral phospholipids PC and PE. The present study provides information that helps reveal the possible function and regulation of PCTP-L expression in the primitive eukaryotic parasite E. histolytica.

  1. The macrophage migration inhibitory factor homolog of Entamoeba histolytica binds to and immunomodulates host macrophages.

    PubMed

    Moonah, Shannon N; Abhyankar, Mayuresh M; Haque, Rashidul; Petri, William A

    2014-09-01

    The host inflammatory response contributes to the tissue damage that occurs during amebic colitis, with tumor necrosis factor alpha (TNF-α) being a key mediator of the gut inflammation observed. Mammalian macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine that plays an important role in the exacerbation of a wide range of inflammatory diseases, including colitis. We identified a MIF gene homolog in the Entamoeba histolytica genome, raising the question of whether E. histolytica MIF (EhMIF) has proinflammatory activity similar to that of mammalian MIF. In this report, we describe the first functional characterization of EhMIF. Antibodies were prepared against recombinantly expressed EhMIF and used to demonstrate that EhMIF is expressed as a 12-kDa protein localized to the cytoplasm of trophozoites. In a manner similar to that of mammalian MIF, EhMIF interacted with the MIF receptor CD74 and bound to macrophages. EhMIF induced interleukin-6 (IL-6) production. In addition, EhMIF enhanced TNF-α secretion by amplifying TNF-α production by lipopolysaccharide (LPS)-stimulated macrophages and by inhibiting the glucocorticoid-mediated suppression of TNF-α secretion. EhMIF was expressed during human infection, as evidenced by the presence of anti-EhMIF antibodies in the sera of children living in an area where E. histolytica infection is endemic. Anti-EhMIF antibodies did not cross-react with human MIF. The ability of EhMIF to modulate host macrophage function may promote an exaggerated proinflammatory immune response and contribute to the tissue damage seen in amebic colitis.

  2. Humoral immune response against proteophosphoglycan surface antigens of Entamoeba histolytica elicited by immunization with synthetic mimotope peptides.

    PubMed

    Melzer, Helen; Baier, Karin; Felici, Franco; von Specht, Bernd Ulrich; Wiedermann, Gerhard; Kollaritsch, Herwig; Wiedermann, Ursula; Duchêne, Michael

    2003-07-15

    The protozoan parasite Entamoeba histolytica, which is responsible for intestinal amebiasis and amebic liver abscess, is causing significant morbidity and mortality worldwide. Proteophosphoglycans (PPGs, also known as lipophosphoglycans, LPGs, or lipopeptidophosphoglycans, LPPGs) are major surface components of E. histolytica. Passive immunization with a monoclonal antibody (EH5) directed against the PPGs protected severe combined immune-deficient mice from amebic liver abscess. The structure of the PPGs is very complex and only known in part. To find peptide mimics of E. histolytica PPG antigens, we had screened phage-displayed random peptide libraries with the antibody EH5. We identified various peptide mimics of E. histolytica PPGs, all sharing a consensus sequence Gly-Thr-His-Pro-X-Leu. Several of the phage clones induced a significant, specific IgG response against membrane antigens of E. histolytica after immunization of mice with whole phage particles. In the present work, in order to avoid the use of phage particles for immunization, we coupled two selected chemically synthesized peptides to keyhole limpet hemocyanin (KLH). The two KLH-conjugated peptides were immunogenic in mice and induced the production of high titers of anti-peptide antibodies, and one of the two peptides was also able to induce significant titers of antibodies against E. histolytica PPGs. Our results demonstrate that the KLH-conjugated peptides are able to mimic the EH5 epitope without the M13 phage sequences flanking the peptide inserts and independent of the structural framework of the phage.

  3. Molecular prevalence of Entamoeba histolytica/dispar infection among patients attending four health centres in north-west Ethiopia.

    PubMed

    Yimer, Mulat; Zenebe, Yohannes; Mulu, Wondemagegn; Abera, Bayeh; Saugar, José M

    2017-01-01

    The prevalence of amoebiasis is often overestimated owing to its epidemiological overlap with the non-pathogenic Entamoeba dispar To provide evidence for this conjecture, a cross-sectional study was conducted from November 2013 to January 2015. A range of 180-200 µg of semi-solid and formed stools and 200 µL of diarrhoeic stool samples were used for DNA extraction from microscopically E. histolytica/dispar positive samples using the QIAamp® DNA Stool Mini Kit according to manufacturers' instructions. Nested PCR targeting 18S ribosomal RNA gene was used. In 422 microscopically positive E. histolytica/dispar stools, molecular prevalence revealed that E. histolytica infestation was present in only 1.7% (95% confidence interval [CI], 0.47-2.93) and E. dispar was found in 42.2% (95% CI, 37.49-46.91), while 56.2% (95% CI, 51.47-60.93) had neither E. histolytica nor E. dispar (P < 0.001). We conclude that infestation with E. histolytica is rarer in our study areas than was previously believed. Hence, accurate differentiation of E. histolytica and E. dispar is crucial.

  4. Molecular modeling on pyruvate phosphate dikinase of Entamoeba histolytica and in silico virtual screening for novel inhibitors

    NASA Astrophysics Data System (ADS)

    Stephen, Preyesh; Vijayan, Ramachandran; Bhat, Audesh; Subbarao, N.; Bamezai, R. N. K.

    2008-09-01

    Pyruvate phosphate dikinase (PPDK) is the key enzyme essential for the glycolytic pathway in most common and perilous parasite Entamoeba histolytica. Inhibiting the function of this enzyme could control the wide spread of intestinal infections caused by Entamoeba histolytica in humans. With this objective, we modeled the three dimensional structure of the PPDK protein. We used templates with 51% identity and 67% similarity to employ homology-modeling approach. Stereo chemical quality of protein structure was validated by protein structure validation program PROCHECK and VERIFY3D. Experimental proof available in literature along with the in silico studies indicated Lys21, Arg91, Asp323, Glu325 and Gln337 to be the probable active sites in the target protein. Virtual screening was carried out using the genetic docking algorithm GOLD and a consensus scoring function X-Score to substantiate the prediction. The small molecule libraries (ChemDivision database, Diversity dataset, Kinase inhibitor database) were used for screening process. Along with the high scoring results, the interaction studies provided promising ligands for future experimental screening to inhibit the function of PPDK in Entamoeba histolytica. Further, the phylogeny study was carried out to assess the possibility of using the proposed ligands as inhibitors in related pathogens.

  5. Identification of natural inhibitors of Entamoeba histolytica cysteine synthase from microbial secondary metabolites

    PubMed Central

    Mori, Mihoko; Jeelani, Ghulam; Masuda, Yui; Sakai, Kazunari; Tsukui, Kumiko; Waluyo, Danang; Tarwadi; Watanabe, Yoshio; Nonaka, Kenichi; Matsumoto, Atsuko; Ōmura, Satoshi; Nozaki, Tomoyoshi; Shiomi, Kazuro

    2015-01-01

    Amebiasis is a common worldwide diarrheal disease, caused by the protozoan parasite, Entamoeba histolytica. Metronidazole has been a drug of choice against amebiasis for decades despite its known side effects and low efficacy against asymptomatic cyst carriers. E. histolytica is also capable of surviving sub-therapeutic levels of metronidazole in vitro. Novel drugs with different mode of action are therefore urgently needed. The sulfur assimilatory de novo L-cysteine biosynthetic pathway is essential for various cellular activities, including the proliferation and anti-oxidative defense of E. histolytica. Since the pathway, consisting of two reactions catalyzed by serine acetyltransferase (SAT) and cysteine synthase (CS, O-acetylserine sulfhydrylase), does not exist in humans, it is a rational drug target against amebiasis. To discover inhibitors against the CS of E. histolytica (EhCS), the compounds of Kitasato Natural Products Library were screened against two recombinant CS isozymes: EhCS1 and EhCS3. Nine compounds inhibited EhCS1 and EhCS3 with IC50 values of 0.31–490 μM. Of those, seven compounds share a naphthoquinone moiety, indicating the structural importance of the moiety for binding to the active site of EhCS1 and EhCS3. We further screened >9,000 microbial broths for CS inhibition and purified two compounds, xanthofulvin and exophillic acid from fungal broths. Xanthofulvin inhibited EhCS1 and EhCS3. Exophillic acid showed high selectivity against EhCS1, but exhibited no inhibition against EhCS3. In vitro anti-amebic activity of the 11 EhCS inhibitors was also examined. Deacetylkinamycin C and nanaomycin A showed more potent amebicidal activity with IC50 values of 18 and 0.8 μM, respectively, in the cysteine deprived conditions. The differential sensitivity of trophozoites against deacetylkinamycin C in the presence or absence of L-cysteine in the medium and the IC50 values against EhCS suggest the amebicidal effect of deacetylkinamycin C is due to

  6. Technical advances in trigger-induced RNA interference gene silencing in the parasite Entamoeba histolytica.

    PubMed

    Khalil, Mohamed I; Foda, Bardees M; Suresh, Susmitha; Singh, Upinder

    2016-03-01

    Entamoeba histolytica has a robust endogenous RNA interference (RNAi) pathway. There are abundant 27 nucleotide (nt) anti-sense small RNAs (AS sRNAs) that target genes for silencing and the genome encodes many genes involved in the RNAi pathway such as Argonaute proteins. Importantly, an E. histolytica gene with numerous AS sRNAs can function as a "trigger" to induce silencing of a gene that is fused to the trigger. Thus, the amebic RNAi pathway regulates gene expression relevant to amebic biology and has additionally been harnessed as a tool for genetic manipulation. In this study we have further improved the trigger-induced gene silencing method. We demonstrate that rather than using the full-length gene, a short portion of the coding region fused to a trigger is sufficient to induce silencing; the first 537 bp of the E. histolytica rhomboid gene (EhROM1) fused in-frame to the trigger was sufficient to silence EhROM1. We also demonstrated that the trigger method could silence two amebic genes concomitantly; fusion of the coding regions of EhROM1 and transcription factor, EhMyb, in-frame to a trigger gene resulted in both genes being silenced. Alternatively, two genes can be silenced sequentially: EhROM1-silenced parasites with no drug selection plasmid were transfected with trigger-EhMyb, resulting in parasites with both EhROM1 and EhMyb silenced. With all approaches tested, the trigger-mediated silencing was substantive and silencing was maintained despite loss of the G418 selectable marker. All gene silencing was associated with generation of AS sRNAs to the silenced gene. We tested the reversibility of the trigger system using inhibitors of histone modifications but found that the silencing was highly stable. This work represents a technical advance in the trigger gene silencing method in E. histolytica. Approaches that readily silence multiple genes add significantly to the genetic toolkit available to the ameba research community.

  7. Structure of an ADP-ribosylation factor, ARF1, from Entamoeba histolytica bound to Mg(2+)-GDP.

    PubMed

    Serbzhinskiy, Dmitry A; Clifton, Matthew C; Sankaran, Banumathi; Staker, Bart L; Edwards, Thomas E; Myler, Peter J

    2015-05-01

    Entamoeba histolytica is the etiological agent of amebiasis, a diarrheal disease which causes amoebic liver abscesses and amoebic colitis. Approximately 50 million people are infected worldwide with E. histolytica. With only 10% of infected people developing symptomatic amebiasis, there are still an estimated 100,000 deaths each year. Because of the emergence of resistant strains of the parasite, it is necessary to find a treatment which would be a proper response to this challenge. ADP-ribosylation factor (ARF) is a member of the ARF family of GTP-binding proteins. These proteins are ubiquitous in eukaryotic cells; they generally associate with cell membranes and regulate vesicular traffic and intracellular signalling. The crystal structure of ARF1 from E. histolytica has been determined bound to magnesium and GDP at 1.8 Å resolution. Comparison with other structures of eukaryotic ARF proteins shows a highly conserved structure and supports the interswitch toggle mechanism of communicating the conformational state to partner proteins.

  8. Cell-matrix interactions of Entamoeba histolytica and E. dispar. A comparative study by electron-, atomic force- and confocal microscopy.

    PubMed

    Talamás-Lara, Daniel; Talamás-Rohana, Patricia; Fragoso-Soriano, Rogelio Jaime; Espinosa-Cantellano, Martha; Chávez-Munguía, Bibiana; González-Robles, Arturo; Martínez-Palomo, Adolfo

    2015-10-01

    Invasion of tissues by Entamoeba histolytica is a multistep process that initiates with the adhesion of the parasite to target tissues. The recognition of the non-invasive Entamoeba dispar as a distinct, but closely related protozoan species raised the question as to whether the lack of its pathogenic potential could be related to a weaker adhesion due to limited cytoskeleton restructuring capacity. We here compared the adhesion process of both amebas to fibronectin through scanning, transmission, atomic force, and confocal microscopy. In addition, electrophoretic and western blot assays of actin were also compared. Adhesion of E. histolytica to fibronectin involves a dramatic reorganization of the actin network that results in a tighter contact to and the subsequent focal degradation of the fibronectin matrix. In contrast, E. dispar showed no regions of focal adhesion, the cytoskeleton was poorly reorganized and there was little fibronectin degradation. In addition, atomic force microscopy using topographic, error signal and phase modes revealed clear-cut differences at the site of contact of both amebas with the substrate. In spite of the morphological and genetic similarities between E. histolytica and E. dispar the present results demonstrate striking differences in their respective cell-to-matrix adhesion processes, which may be of relevance for understanding the invasive character of E. histolytica.

  9. Simple differential detection of Entamoeba histolytica and Entamoeba dispar in fresh stool specimens by sodium acetate-acetic acid-formalin concentration and PCR.

    PubMed

    Troll, H; Marti, H; Weiss, N

    1997-07-01

    Amoebiasis is caused by two distinct species, a pathogenic form (Entamoeba histolytica) and a nonpathogenic form (Entamoeba dispar), which are morphologically identical. Although the distinction between these two species is of great clinical importance, the methods developed for this purpose either are very time-consuming or involve laborious procedures for isolation of the DNA. We report here a simple PCR method starting with fresh stool specimen that allows for the sensitive and reliable distinction between E. histolytica and E. dispar. After initial concentration by the sodium acetate-acetic acid-formalin (SAF) method and digestion with proteinase K, a 0.88-kb sequence of the multicopy 16S rRNA gene served as a target for PCR amplification. The method starting with unpreserved specimens proved to be very sensitive and was not influenced by the quick exposure to SAF fixative during the initial concentration step. However, storage in SAF fixative prior to testing resulted in a decreased sensitivity within 2 days. The detection limit of the method was as low as one copy of the 16S rRNA gene. No cross-reactivity was observed with other common intestinal protozoa. Mixed infections involving both E. histolytica and E. dispar could easily be detected at a ratio of 1:10,000 by agarose gel electrophoresis or a DNA hybridization immunoassay.

  10. Immunization with the Entamoeba histolytica surface metalloprotease EhMSP-1 protects hamsters from amebic liver abscess.

    PubMed

    Roncolato, Eduardo C; Teixeira, José E; Barbosa, José E; Zambelli Ramalho, Leandra N; Huston, Christopher D

    2015-02-01

    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.

  11. [Comparison of direct microscopy, culture, ELISA and molecular methods for diagnosis of Entamoeba histolytica].

    PubMed

    Tüzemen, Nazmiye Ulkü; Doğan, Nihal

    2014-01-01

    Amebiasis, a parasitic infection caused by Entamoeba histolytica, is one of the most common parasitic infections worldwide. Since it is still an important public health problem in developing countries, rapid differential diagnosis of amebiasis is crucial in terms of treatment. The most frequently used method for laboratory diagnosis is direct microscopy, however more reliable and specific methods are needed in order to differentiate the apathogenic Entamoeba dispar under the microscope. This study was conducted to compare the results of different methods namely, direct microscopy, culture, ELISA and PCR for the detection of E.histolytica in stool samples and to evaluate the performances of those methods. A total of 1049 stool samples collected from pediatric and adult patients who were admitted to hospital with diarrhea complaint between January 2011-March 2013, and randomly selected samples from primary school children, were included in the study. Direct microscopic examination was performed by native-lugol, physiological saline, modified formol-ethyl acetate sedimentation and trichrome staining methods. The stool samples were also inoculated into TYI-S-33 media for axenic cultivation of amoeba. The presence of amebic antigens in the samples were screened by a commercial ELISA kit (TechLab, E.histolytica II, USA). For the molecular diagnosis, a multiplex tandem real-time PCR (MT-PCR) kit (AusDiagnostics Pty Ltd, Australia) was used, after the extraction of DNAs with QIAamp DNA Stool Mini Kit (Qiagen, USA). A total of 354 samples which could be evaluated by all of the methods, were included in the study. Of the 354 stool samples, 84 (23.7%) were found E.histolytica/E.dispar positive by direct microscopy, 61 (17.2%) by trichrome staining, 46 (12.9%) by culture, 31 (8.7%) by ELISA and 9 (2.5%) by MT-PCR. Of direct microscopy positive samples 54.7% (46/84) were also positive with trichrome staining, 39.3% (33/84) with culture, 15.5% (13/84) with ELISA and 7.1% (6

  12. Downregulation of an Entamoeba histolytica rhomboid protease reveals roles in regulating parasite adhesion and phagocytosis.

    PubMed

    Baxt, Leigh A; Rastew, Elena; Bracha, Rivka; Mirelman, David; Singh, Upinder

    2010-08-01

    Entamoeba histolytica is a deep-branching eukaryotic pathogen. Rhomboid proteases are intramembrane serine proteases, which cleave transmembrane proteins in, or in close proximity to, their transmembrane domain. We have previously shown that E. histolytica contains a single functional rhomboid protease (EhROM1) and has unique substrate specificity. EhROM1 is present on the trophozoite surface and relocalizes to internal vesicles during erythrophagocytosis and to the base of the cap during surface receptor capping. In order to further examine the biological function of EhROM1 we downregulated EhROM1 expression by >95% by utilizing the epigenetic silencing mechanism of the G3 parasite strain. Despite the observation that EhROM1 relocalized to the cap during surface receptor capping, EhROM1 knockdown [ROM(KD)] parasites had no gross changes in cap formation or complement resistance. However, ROM(KD) parasites demonstrated decreased host cell adhesion, a result recapitulated by treatment of wild-type parasites with DCI, a serine protease inhibitor with activity against rhomboid proteases. The reduced adhesion phenotype of ROM(KD) parasites was noted exclusively with healthy cells, and not with apoptotic cells. Additionally, ROM(KD) parasites had decreased phagocytic ability with reduced ingestion of healthy cells, apoptotic cells, and rice starch. Decreased phagocytic ability is thus independent of the reduced adhesion phenotype, since phagocytosis of apoptotic cells was reduced despite normal adhesion levels. The defect in host cell adhesion was not explained by altered expression or localization of the heavy subunit of the Gal/GalNAc surface lectin. These results suggest no significant role of EhROM1 in complement resistance but unexpected roles in parasite adhesion and phagocytosis.

  13. The Entamoeba histolytica Dnmt2 homolog (Ehmeth) confers resistance to nitrosative stress.

    PubMed

    Hertz, Rivi; Tovy, Ayala; Kirschenbaum, Michael; Geffen, Meirav; Nozaki, Tomoyoshi; Adir, Noam; Ankri, Serge

    2014-04-01

    Nitric oxide (NO) has antimicrobial properties against many pathogens due to its reactivity as an S-nitrosylating agent. It inhibits many of the key enzymes that are involved in the metabolism and virulence of the parasite Entamoeba histolytica through S-nitrosylation of essential cysteine residues. Very little information is available on the mechanism of resistance to NO by pathogens in general and by this parasite in particular. Here, we report that exposure of the parasites to S-nitrosoglutathione (GSNO), an NO donor molecule, strongly reduces their viability and protein synthesis. However, the deleterious effects of NO were significantly reduced in trophozoites overexpressing Ehmeth, the cytosine-5 methyltransferase of the Dnmt2 family. Since these trophozoites also exhibited high levels of tRNA(Asp) methylation, the high levels suggested that Ehmeth-mediated tRNA(Asp) methylation is part of the resistance mechanism to NO. We previously reported that enolase, another glycolytic enzyme, binds to Ehmeth and inhibits its activity. We observed that the amount of Ehmeth-enolase complex was significantly reduced in GSNO-treated E. histolytica, which explains the aforementioned increase of tRNA methylation. Specifically, we demonstrated via site-directed mutagenesis that cysteine residues 228 and 229 of Ehmeth are susceptible to S-nitrosylation and are crucial for Ehmeth binding to enolase and for Ehmeth-mediated resistance to NO. These results indicate that Ehmeth has a central role in the response of the parasite to NO, and they contribute to the growing evidence that NO is a regulator of epigenetic mechanisms.

  14. Structural and functional analysis of the Entamoeba histolytica EhrabB gene promoter

    PubMed Central

    Romero-Díaz, Mónica; Gómez, Consuelo; López-Reyes, Israel; Martínez, Máximo B; Orozco, Esther; Rodríguez, Mario A

    2007-01-01

    Background The Entamoeba histolytica EhrabB gene encodes for a Rab GTPase involved in phagocytosis. It is located at a virulence locus where the Ehcp112 gene is in the complementary strand at 332 bp of EhrabB start codon, suggesting a finely regulated transcription of both genes. However, the transcription regulation in this parasite is poorly understood. Results To initiate the knowledge of EhrabB gene expression regulation, here we studied the structural characteristics of its gene promoter and its control transcription elements. In silico searches of the EhrabB 5'-flanking region revealed that it contains a motif similar to the upstream regulatory element 1 (URE1) of the E. histolytica hgl5 gene. It also has sequences with homology to C/EBP and GATA1 binding sites, and heat shock elements (HSE). Primer extension experiments revealed that EhrabB has at least four transcription initiation sites. The elements at the 5'-flanking region that drive EhrabB gene expression were detected and characterized using transitory transfected trophozoites with a plasmid carrying the CAT reporter gene. EhrabB transcription is negatively regulated by a sequence located between positions -491 to -428 with respect to the first transcription initiation site. We also showed that the URE1-like motif activates EhrabB transcription. In addition, heat shock activated the EhrabB promoter in episomal constructs and lead to an increase in de novo EhrabB transcription. Conclusion The data suggest that EhrabB transcription is controlled negatively by an unidentified sequence, but it is activated by an URE1-like motif. Our analyses also revealed the presence of activator HSE that function under stress. PMID:17883848

  15. Unique Structural and Nucleotide Exchange Features of the Rho1 GTPase of Entamoeba histolytica

    SciTech Connect

    Bosch, Dustin E.; Wittchen, Erika S.; Qiu, Connie; Burridge, Keith; Siderovski, David P.

    2012-08-10

    The single-celled human parasite Entamoeba histolytica possesses a dynamic actin cytoskeleton vital for its intestinal and systemic pathogenicity. The E. histolytica genome encodes several Rho family GTPases known to regulate cytoskeletal dynamics. EhRho1, the first family member identified, was reported to be insensitive to the Rho GTPase-specific Clostridium botulinum C3 exoenzyme, raising the possibility that it may be a misclassified Ras family member. Here, we report the crystal structures of EhRho1 in both active and inactive states. EhRho1 is activated by a conserved switch mechanism, but diverges from mammalian Rho GTPases in lacking a signature Rho insert helix. EhRho1 engages a homolog of mDia, EhFormin1, suggesting a role in mediating serum-stimulated actin reorganization and microtubule formation during mitosis. EhRho1, but not a constitutively active mutant, interacts with a newly identified EhRhoGDI in a prenylation-dependent manner. Furthermore, constitutively active EhRho1 induces actin stress fiber formation in mammalian fibroblasts, thereby identifying it as a functional Rho family GTPase. EhRho1 exhibits a fast rate of nucleotide exchange relative to mammalian Rho GTPases due to a distinctive switch one isoleucine residue reminiscent of the constitutively active F28L mutation in human Cdc42, which for the latter protein, is sufficient for cellular transformation. Nonconserved, nucleotide-interacting residues within EhRho1, revealed by the crystal structure models, were observed to contribute a moderating influence on fast spontaneous nucleotide exchange. Collectively, these observations indicate that EhRho1 is a bona fide member of the Rho GTPase family, albeit with unique structural and functional aspects compared with mammalian Rho GTPases.

  16. [A Case of Peristomal Cutaneous Ulcer Following Amebic Colitis Caused by Entamoeba histolytica].

    PubMed

    Sasaki, Yu; Yoshida, Tetsuya; Suzuki, Jun; Kobayashi, Seiki; Sato, Tomotaka

    2016-01-01

    A 66-year-old Japanese male with a history of a rectal ulcer and rectovesical fistula following brachytherapy and radiotherapy for prostate cancer, who had undergone colostomy and vesicotomy presented with a painful peristomal ulcer of approximately 5 x 2.5cm adjacent to the direction of 6 o'clock of the stoma in his left lower abdomen. Although he was admitted to be treated with intravenous antibiotics and topical debridement, the ulcer was rapidly increasing. In the laboratory findings, WBC was 12,400/μL, CRP was 16.9 mg/dL, ESR was 105mm in the first hour. Contrast enhanced CT images showed a wide high density area of skin and subcutaneous tissue around the stoma and dillitation of the transverse and descending colon. Colonoscopy showed furred profound ulcers in the rectum. A biopsy from the ulcer floor submitted to histopathology showed necrotic tissue with a mixed inflammatory infiltrates mainly composed of neutrophils and lymphocytes in the dermis. We suspected pyoderma gangrenosum with an inflammatory bowel disease in the beginning. Although he was started on oral prednisolone 60 mg daily, the ulcer did not respond to treatment. Additional methylprednisolone pulse therapy, intravenous cyclosporine and granulocytapheresis were also ineffective. A biopsy specimen from the skin ulcer margin showed erythrophagocytosis by trophozoites of amebae which were identified on PAS stained slides. The PCR method and stool examination showed positive for Entamoeba histolytica (E. histolytica), but serum antibodies were negative. Within two weeks of treatment with oral metronidazole 2,250 mg/day and topical metronidazole ointment, resolution of the ulcer was observed, then the prednisolone dosage was tapered. A split-thickness skin graft was used to cover the ulcer with a successful result. Even though we originally misdiagnosed this case, we finally reached a diagnosis of amebiasis. It is important to take account of amebiasis in the differential diagnosis of intractable

  17. Control of Entamoeba histolytica adherence involves metallosurface protease 1, an M8 family surface metalloprotease with homology to leishmanolysin.

    PubMed

    Teixeira, Jose E; Sateriale, Adam; Bessoff, Kovi E; Huston, Christopher D

    2012-06-01

    Invasive amebiasis due to Entamoeba histolytica infection is an important cause of morbidity in developing countries. The E. histolytica genome contains two homologues to the metalloprotease leishmanolysin gene, Entamoeba histolytica MSP-1 (EhMSP-1) and EhMSP-2, while the commensal ameba Entamoeba dispar has lost EhMSP-1. In this study, we sought to characterize E. histolytica metallosurface protease 1 (EhMSP-1). Using immunoprecipitation and a model substrate, we found that EhMSP-1 was a functional metalloprotease. Confocal microscopy and flow cytometry revealed that EhMSP-1 localized to the cell surface and revealed the existence of distinct, nonclonal trophozoite populations with high and low EhMSP-1 surface abundance that became synchronized following serum starvation. Phenotypic assays were performed after silencing EhMSP-1. Adherence of EhMSP-1-deficient trophozoites to tissue culture cell monolayers was more than five times greater than that of control amebas, but surface staining of several antigens, including the galactose adherence lectin, was unchanged. EhMSP-1 silencing similarly increased adherence to both viable and apoptotic Jurkat lymphocytes. Tissue culture cell monolayer destruction was reduced by EhMSP-1 silencing, although it was blocked almost completely by inhibiting cysteine proteases. Consistent with a primary defect in regulation of amebic adherence, EhMSP-1 silencing also resulted in reduced mobility on tissue culture cell monolayers and in increased phagocytosis. In conclusion, EhMSP-1 was shown to be a surface metalloprotease involved in regulation of amebic adherence, with additional effects on cell motility, cell monolayer destruction, and phagocytosis.

  18. In Entamoeba histolytica, a BspA family protein is required for chemotaxis toward tumour necrosis factor

    PubMed Central

    Silvestre, Anne; Plaze, Aurélie; Berthon, Patricia; Thibeaux, Roman; Guillen, Nancy; Labruyère, Elisabeth

    2015-01-01

    Background: Entamoeba histolytica cell migration is essential for the development of human amoebiasis (an infectious disease characterized by tissue invasion and destruction). The tissue inflammation associated with tumour necrosis factor (TNF) secretion by host cells is a well-documented feature of amoebiasis. Tumour necrosis factor is a chemoattractant for E. histolytica, and the parasite may have a TNF receptor at its cell surface. Methods: confocal microscopy, RNA Sequencing, bioinformatics, RNA antisense techniques and histological analysis of human colon explants were used to characterize the interplay between TNF and E. histolytica. Results: an antibody against human TNF receptor 1 (TNFR1) stained the E. histolytica trophozoite surface and (on immunoblots) binds to a 150-kDa protein. Proteome screening with the TNFR1 sequence revealed a BspA family protein in E. histolytica that carries a TNFR signature domain and six leucine-rich repeats (named here as "cell surface protein", CSP, in view of its cellular location). Cell surface protein shares structural homologies with Toll-Like receptors, colocalizes with TNF and is internalized in TNF-containing vesicles. Reduction of cellular CSP levels abolished chemotaxis toward TNF and blocked parasite invasion of human colon. Conclusions: there is a clear link between TNF chemotaxis, CSP and pathogenesis. PMID:28357299

  19. Proteomic Study of Entamoeba histolytica Trophozoites, Cysts, and Cyst-Like Structures

    PubMed Central

    Luna-Nácar, Milka; Navarrete-Perea, José; Moguel, Bárbara; Bobes, Raúl J.; Laclette, Juan P.; Carrero, Julio C.

    2016-01-01

    The cyst stage of Entamoeba histolytica is a promising therapeutic target against human amoebiasis. Our research team previously reported the production in vitro of Cyst-Like Structures (CLS) sharing structural features with cysts, including rounded shape, size reduction, multinucleation, and the formation of a chitin wall coupled to the overexpression of glucosamine 6-phosphate isomerase, the rate-limiting enzyme of the chitin synthesis pathway. A proteomic study of E. histolytica trophozoites, cysts, and in vitro-produced CLS is reported herein to determine the nature of CLS, widen our knowledge on the cyst stage, and identify possible proteins and pathways involved in the encystment process. Total protein extracts were obtained from E. histolytica trophozoites, CLS, and partially purified cysts recovered from the feces of amoebic human patients; extracts were trypsin-digested and analyzed by LC-MS/MS. In total, 1029 proteins were identified in trophozoites, 550 in CLS, and 411 in cysts, with 539, 299, and 84 proteins unique to each sample, respectively, and only 74 proteins shared by all three stages. About 70% of CLS proteins were shared with trophozoites, even though differences were observed in the relative protein abundance. While trophozoites showed a greater abundance of proteins associated to a metabolically active cell, CLS showed higher expression of proteins related to proteolysis, redox homeostasis, and stress response. In addition, the expression of genes encoding for the cyst wall proteins Jessie and Jacob was detected by RT-PCR and the Jacob protein identified by Western blotting and immunofluorescence in CLS. However, the proteomic profile of cysts as determined by LC-MS/MS was very dissimilar to that of trophozoites and CLS, with almost 40% of hypothetical proteins. Our global results suggest that CLS are more alike to trophozoites than to cysts, and they could be generated as a rapid survival response of trophozoites to a stressful condition

  20. The Tudor Staphylococcal Nuclease Protein of Entamoeba histolytica Participates in Transcription Regulation and Stress Response

    PubMed Central

    Cázares-Apátiga, Javier; Medina-Gómez, Christian; Chávez-Munguía, Bibiana; Calixto-Gálvez, Mercedes; Orozco, Esther; Vázquez-Calzada, Carlos; Martínez-Higuera, Aarón; Rodríguez, Mario A.

    2017-01-01

    Entamoeba histolytica is the protozoa parasite responsible of human amoebiasis, disease that causes from 40,000 to 100,000 deaths annually worldwide. However, few are known about the expression regulation of molecules involved in its pathogenicity. Transcription of some virulence-related genes is positively controlled by the cis-regulatory element named URE1. Previously we identified the transcription factor that binds to URE1, which displayed a nuclear and cytoplasmic localization. This protein belongs to the Tudor Staphyococcal nuclease (TSN) family, which in other systems participates in virtually all pathways of gene expression, suggesting that this amoebic transcription factor (EhTSN; former EhURE1BP) could also play multiple functions in E. histolytica. The aim of this study was to identify the possible cellular events where EhTSN is involved. Here, we found that EhTSN in nucleus is located in euchromatin and close to, but not into, heterochromatin. We also showed the association of EhTSN with proteins involved in transcription and that the knockdown of EhTSN provokes a diminishing in the mRNA level of the EhRabB gene, which in its promoter region contains the URE1 motif, confirming that EhTSN participates in transcription regulation. In cytoplasm, this protein was found linked to the membrane of small vesicles and to plasma membrane. Through pull-down assays and mass spectrometry we identity thirty two candidate proteins to interact with EhTSN. These proteins participate in transcription, metabolism, signaling, and stress response, among other cellular processes. Interaction of EhTSN with some candidate proteins involved in metabolism, and signaling was validated by co-immunoprecipitation or co-localization. Finally we showed the co-localization of EhTSN and HSP70 in putative stress granules during heat shock and that the knockdown of EhTSN increases the cell death during heat shock treatment, reinforcing the hypothesis that EhTSN has a role during stress

  1. cDNA sequence analysis of a 29-kDa cysteine-rich surface antigen of pathogenic Entamoeba histolytica

    SciTech Connect

    Torian, B.E.; Stroeher, V.L.; Stamm, W.E. ); Flores, B.M. ); Hagen, F.S. )

    1990-08-01

    A {lambda}gt11 cDNA library was constructed from poly(U)-Spharose-selected Entamoeba histolytica trophozoite RNA in order to clone and identify surface antigens. The library was screened with rabbit polyclonal anti-E. histolytica serum. A 700-base-pair cDNA insert was isolated and the nucleotide sequence was determined. The deduced amino acid sequence of the cDNA revealed a cysteine-rich protein. DNA hybridizations showed that the gene was specific to E. histolytica since the cDNA probe reacted with DNA from four axenic strains of E. histolytica but did not react with DNA from Entamoeba invadens, Acanthamoeba castellanii, or Trichomonas vaginalis. The insert was subcloned into the expression vector pGEX-1 and the protein was expressed as a fusion with the C terminus of glutathione S-transferase. Purified fusion protein was used to generate 22 monoclonal antibodies (mAbs) and a mouse polyclonal antiserum specific for the E. histolytica portion of the fusion protein. A 29-kDa protein was identified as a surface antigen when mAbs were used to immunoprecipitate the antigen from metabolically {sup 35}S-labeled live trophozoites. The surface location of the antigen was corroborated by mAb immunoprecipitation of a 29-kDa protein from surface-{sup 125}I-labeled whole trophozoites as well as by the reaction of mAbs with live trophozoites in an indirect immunofluorescence assay performed at 4{degree}C. Immunoblotting with mAbs demonstrated that the antigen was present on four axenic isolates tested. mAbs recognized epitopes on the 29-kDa native antigen on some but not all clinical isolates tested.

  2. Binding and Endocytosis of Bovine Hololactoferrin by the Parasite Entamoeba histolytica

    PubMed Central

    Ortíz-Estrada, Guillermo; Calderón-Salinas, Víctor; Shibayama-Salas, Mineko; León-Sicairos, Nidia; de la Garza, Mireya

    2015-01-01

    Entamoeba histolytica is a human parasite that requires iron (Fe) for its metabolic function and virulence. Bovine lactoferrin (B-Lf) and its peptides can be found in the digestive tract after dairy products are ingested. The aim of this study was to compare virulent trophozoites recently isolated from hamster liver abscesses with nonvirulent trophozoites maintained for more than 30 years in cultures in vitro regarding their interaction with iron-charged B-Lf (B-holo-Lf). We performed growth kinetics analyses of trophozoites in B-holo-Lf and throughout several consecutive transfers. The virulent parasites showed higher growth and tolerance to iron than nonvirulent parasites. Both amoeba variants specifically bound B-holo-Lf with a similar Kd. However, averages of 9.45 × 105 and 6.65 × 106 binding sites/cell were found for B-holo-Lf in nonvirulent and virulent amoebae, respectively. Virulent amoebae bound more efficiently to human and bovine holo-Lf, human holo-transferrin, and human and bovine hemoglobin than nonvirulent amoebae. Virulent amoebae showed two types of B-holo-Lf binding proteins. Although both amoebae endocytosed this glycoprotein through clathrin-coated vesicles, the virulent amoebae also endocytosed B-holo-Lf through a cholesterol-dependent mechanism. Both amoeba variants secreted cysteine proteases cleaving B-holo-Lf. These data demonstrate that the B-Lf endocytosis is more efficient in virulent amoebae. PMID:26090404

  3. Calpain-like: A Ca(2+) dependent cystein protease in Entamoeba histolytica cell death.

    PubMed

    Monroy, Virginia Sánchez; Flores, Olivia Medel; García, Consuelo Gómez; Maya, Yesenia Chávez; Fernández, Tania Domínguez; Pérez Ishiwara, D Guillermo

    2015-12-01

    Entamoeba histolytica programmed cell death (PCD) induced by G418 is characterized by the release of important amounts of intracellular calcium from reservoirs. Nevertheless, no typical caspases have been detected in the parasite, the PCD phenotype is inhibited by the cysteine protease inhibitor E-64. These results strongly suggest that Ca(2+)-dependent proteases could be involved in PCD. In this study, we evaluate the expression and activity of a specific dependent Ca(2+) protease, the calpain-like protease, by real-time quantitative PCR (RTq-PCR), Western blot assays and a enzymatic method during the induction of PCD by G418. Alternatively, using cell viability and TUNEL assays, we also demonstrated that the Z-Leu-Leu-Leu-al calpain inhibitor reduced the rate of cell death. The results demonstrated 4.9-fold overexpression of calpain-like gene 1.5 h after G418 PCD induction, while calpain-like protein increased almost two-fold with respect to basal calpain-like expression after 3 h of induction, and calpain activity was found to be approximately three-fold higher 6 h after treatment compared with untreated trophozoites. Taken together, these results suggest that this Ca(2+)-dependent protease could be involved in the executory phase of PCD.

  4. Ligand heterogeneity of the cysteine protease binding protein family in the parasitic protist Entamoeba histolytica.

    PubMed

    Marumo, Konomi; Nakada-Tsukui, Kumiko; Tomii, Kentaro; Nozaki, Tomoyoshi

    2014-08-01

    Lysosomal soluble proteins are targeted to endosomes and lysosomes by specific receptors resident in the endoplasmic reticulum and/or the Golgi apparatus. The enteric protozoan parasite Entamoeba histolytica has a novel class of lysosomal targeting receptors, named the cysteine protease binding protein family (CPBF). Among 11 CPBFs (CPBF1-11), ligands for three members, CPBF1, CPBF6 and CPBF8, were previously shown to be cysteine proteases, α- and γ- amylases, and β-hexosaminidase and lysozymes, respectively. To further understand the heterogeneity of the ligands of CPBFs, we attempted to isolate and identify the ligands for other members of CPBFs, namely CPBF2, 3, 4, 5, 7, 9, 10 and 11, by immunoprecipitation and mass spectrometric analysis. We found that CPBF2 and CPBF10 bound to α-amylases while CPBF7 bound to β-hexosaminidases. It is intriguing that cysteine protease are exclusively recognised by CPBF1, whereas three α-amylases and β-hexosaminidases are redundantly recognised by three and two CPBFs, respectively. It was shown by bioinformatics analysis and phylogenetic reconstruction that each CPBF contains six prepeptidase carboxyl-terminal domains, and the domain configuration is evolutionarily conserved among CPBFs. Taken together, CPBFs with unique and conserved domain organisation have a remarkable ligand heterogeneity toward cysteine protease and carbohydrate degradation enzymes. Further structural studies are needed to elucidate the structural basis of the ligand specificity.

  5. In vivo identification of the steps that control energy metabolism and survival of Entamoeba histolytica.

    PubMed

    Pineda, Erika; Encalada, Rusely; Vázquez, Citlali; Néquiz, Mario; Olivos-García, Alfonso; Moreno-Sánchez, Rafael; Saavedra, Emma

    2015-01-01

    The steps that control the Entamoeba histolytica glycolytic flux were here identified by elasticity analysis, an experimental approach of metabolic control analysis. The concentrations of glycolytic metabolites were gradually varied in live trophozoites by (a) feeding with different glucose concentrations and (b) inhibiting the final pathway steps; in parallel, the changes in the pathway flux were determined. From the metabolite concentration-flux relationship, the elasticity coefficients of individual or groups of pathway reactions were determined and used to calculate their respective degrees of control on the glycolytic flux (flux control coefficients). The results indicated that the pathway flux was mainly controlled (72-86%) by the glucose transport/hexokinase/glycogen degradation group of reactions and by bifunctional aldehyde-alcohol dehydrogenase (ADHE; 18%). Further, inhibition of the first pathway reactions with 2-deoxyglucose (2DOG) decreased the glycolytic flux and ATP content by 75% and 50%, respectively. Cell viability was also decreased by 2DOG (25%) and more potently (50%) by 2DOG plus the ADHE inhibitor tetraethylthiuram disulfide (disulfiram). Biosate as an alternative carbon (amino acid) source was unable to replace glucose for ATP supply, which indicated that glucose was the main nutrient for amoebal ATP synthesis and survival. These results indicated that glycolysis in the parasite is mainly controlled by the initial pathway reactions and that their inhibition can decrease the parasite energy load and survival.

  6. Characterization of pH-induced transitions of Entamoeba histolytica D-phosphoglycerate dehydrogenase.

    PubMed

    Mishra, Vibhor; Kumar, Ashutosh; Ali, Vahab; Zhang, Kam Y J; Nozaki, Tomoyoshi

    2015-08-01

    Entamoeba histolytica D-phosphoglycerate dehydrogenase (EhPGDH) exists as a functionally active homodimer at pH 7. Our earlier studies have shown that ionic interactions are essentially required for the oligomeric status and activity of the protein. Present study focuses on pH associated structural modulations of EhPGDH. Far-UV CD spectra showed loss in the secondary structure of the protein as a function of low pH, however, the protein was not completely unfolded even at pH 2. Energy minimized average simulated models of EhPGDH at different pH show stable secondary structure elements in the nucleotide binding domain (NBD) however, the substrate binding domain (SBD) was more sensitive toward acidic pH and completely unfolds at pH 2. The data suggest presence of partially folded/unfolded intermediate state at pH 2. Size exclusion chromatography shows that this intermediate has larger hydrodynamic radius compared with dimer (pH 7) or monomer (pH 5). The intermediate has poor tertiary organization with significantly exposed hydrophobic patches monitored by pH-dependent fluorescence spectroscopy and molecular dynamic simulations. Collectively, the results suggest that the two domains (NBD and SBD) of EhPGDH have independent pH-dependent structural transitions with stabilization of an intermediate state at pH 2.

  7. Activity, stability and folding analysis of the chitinase from Entamoeba histolytica.

    PubMed

    Muñoz, Patricia L A; Minchaca, Alexis Z; Mares, Rosa E; Ramos, Marco A

    2016-02-01

    Human amebiasis, caused by the parasitic protozoan Entamoeba histolytica, remains as a significant public health issue in developing countries. The life cycle of the parasite compromises two main stages, trophozoite and cyst, linked by two major events: encystation and excystation. Interestingly, the cyst stage has a chitin wall that helps the parasite to withstand harsh environmental conditions. Since the amebic chitinase, EhCHT1, has been recognized as a key player in both encystation and excystation, it is plausible to consider that specific inhibition could arrest the life cycle of the parasite and, thus, stop the infection. However, to selectively target EhCHT1 it is important to recognize its unique biochemical features to have the ability to control its cellular function. Hence, to gain further insights into the structure-function relationship, we conducted an experimental approach to examine the effects of pH, temperature, and denaturant concentration on the enzymatic activity and protein stability. Additionally, dependence on in vivo oxidative folding was further studied using a bacterial model. Our results attest the potential of EhCHT1 as a target for the design and development of new or improved anti-amebic therapeutics. Likewise, the potential of the oxidoreductase EhPDI, involved in oxidative folding of amebic proteins, was also confirmed.

  8. The parasite Entamoeba histolytica exploits the activities of human matrix metalloproteinases to invade colonic tissue.

    PubMed

    Thibeaux, Roman; Avé, Patrick; Bernier, Michèle; Morcelet, Marie; Frileux, Pascal; Guillén, Nancy; Labruyère, Elisabeth

    2014-10-07

    Intestinal invasion by the protozoan parasite Entamoeba histolytica is characterized by remodelling of the extracellular matrix (ECM). The parasite cysteine proteinase A5 (CP-A5) is thought to cooperate with human matrix metalloproteinases (MMPs) involved in ECM degradation. Here, we investigate the role CP-A5 plays in the regulation of MMPs upon mucosal invasion. We use human colon explants to determine whether CP-A5 activates human MMPs. Inhibition of the MMPs' proteolytic activities abolishes remodelling of the fibrillar collagen structure and prevents trophozoite invasion of the mucosa. In the presence of trophozoites, MMPs-1 and -3 are overexpressed and are associated with fibrillar collagen remodelling. In vitro, CP-A5 performs the catalytic cleavage needed to activate pro-MMP-3, which in turn activates pro-MMP-1. Ex vivo, incubation with recombinant CP-A5 was enough to rescue CP-A5-defective trophozoites. Our results suggest that MMP-3 and/or CP-A5 inhibitors may be of value in further studies aiming to treat intestinal amoebiasis.

  9. Small GTPase Rab21 mediates fibronectin induced actin reorganization in Entamoeba histolytica: implications in pathogen invasion.

    PubMed

    Emmanuel, Merlyn; Nakano, Yumiko Saito; Nozaki, Tomoyoshi; Datta, Sunando

    2015-03-01

    The protozoan parasite Entamoeba histolytica causes a wide spectrum of intestinal infections. In severe cases, the trophozoites can breach the mucosal barrier, invade the intestinal epithelium and travel via the portal circulation to the liver, where they cause hepatic abscesses, which can prove fatal if left untreated. The host Extra Cellular Matrix (ECM) plays a crucial role in amoebic invasion by triggering an array of cellular responses in the parasite, including induction of actin rich adhesion structures. Similar actin rich protrusive structures, known as 'invadosomes', promote chemotactic migration of the metastatic cancer cells and non-transformed cells by remodeling the ECM. Recent studies showed a central role for Rab GTPases, the master regulators of vesicular trafficking, in biogenesis of invadosomes. Here, we showed that fibronectin, a major host ECM component induced actin remodeling in the parasite in a Rab21 dependent manner. The focalized actin structures formed were reminiscent of the mammalian invadosomes. By using various approaches, such as immunofluorescence confocal microscopy and scanning electron microscopy, along with in vitro invasion assay and matrix degradation assay, we show that the fibronectin induced formation of amoebic actin dots depend on the nucleotide status of the GTPase. The ECM components, fibronectin and collagen type I, displayed differential control over the formation of actin dots, with fibronectin positively and collagen type I negatively modulating it. The cell surface adhesion molecule Gal/GalNAc complex was also found to impose additional regulation on this process, which might have implication in collagen type I mediated suppression of actin dots.

  10. Recombinant expression and biochemical characterization of an NADPH:flavin oxidoreductase from Entamoeba histolytica.

    PubMed Central

    Bruchhaus, I; Richter, S; Tannich, E

    1998-01-01

    The gene encoding a putative NADPH:flavin oxidoreductase of the protozoan parasite Entamoeba histolytica (Eh34) was recombinantly expressed in Escherichia coli. The purified recombinant protein (recEh34) has a molecular mass of about 35 kDa upon SDS/PAGE analysis, exhibits a flavoprotein-like absorption spectrum and contains 1 mol of non-covalently bound FMN per mol of protein. RecEh34 reveals two different enzymic activities. It catalyses the NADPH-dependent reduction of oxygen to hydrogen peroxide (H2O2), as well as of disulphides such as 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) and cystine. The disulphide reductase but not the H2O2-forming NADPH oxidase activity is inhibitable by sulphydryl-active compounds, indicating that a thiol component is part of the active site for the disulphide reductase activity, whereas for the H2O2-forming NADPH oxidase activity only the flavin is required. Compared with the recombinant protein, similar activities are present in amoebic extracts. Native Eh34 is active in a monomeric as well as in a dimeric state. In contrast to recEh34, no flavin was associated with the native protein. However, both NADPH oxidase as well as DTNB reductase activity were found to be dependent on the addition of FAD or FMN. PMID:9494088

  11. Nanoformulation of synergistic TLR ligands to enhance vaccination against Entamoeba histolytica

    PubMed Central

    Noor, Zannatun; Tomai, Mark A.; Elvecrog, James; Fox, Christopher B.

    2017-01-01

    Diarrheal infectious diseases represent a major cause of global morbidity and mortality. There is an urgent need for vaccines against diarrheal pathogens, especially parasites. Modern subunit vaccines rely on combining a highly purified antigen with an adjuvant to increase their efficacy. In the present study, we evaluated the ability of a nanoliposome adjuvant system to trigger a strong mucosal immune response to the Entamoeba histolytica Gal/GalNAc lectin LecA antigen. CBA/J mice were immunized with alum, emulsion or liposome based formulations containing synthetic TLR agonists. A liposome formulation containing TLR4 and TLR7/8 agonists was selected based on its ability to generate intestinal IgA, plasma IgG2a/IgG1, IFN-γ and IL-17A. Immunization with a mucosal prime followed by a parenteral boost generated a high mucosal IgA response that inhibited adherence of parasites to mammalian cells. Inclusion of the immune potentiator all-trans retinoic acid in the regimen further improved the mucosal IgA response. Immunization protected from infection with up to 55% efficacy. Our results show that a nanoliposome delivery system coupled to TLR agonists is a promising prospect for the development of vaccines against enteric pathogens, especially when a multifaceted immune response is desired. PMID:28089548

  12. Nanoformulation of synergistic TLR ligands to enhance vaccination against Entamoeba histolytica.

    PubMed

    Abhyankar, Mayuresh M; Noor, Zannatun; Tomai, Mark A; Elvecrog, James; Fox, Christopher B; Petri, William A

    2017-02-07

    Diarrheal infectious diseases represent a major cause of global morbidity and mortality. There is an urgent need for vaccines against diarrheal pathogens, especially parasites. Modern subunit vaccines rely on combining a highly purified antigen with an adjuvant to increase their efficacy. In the present study, we evaluated the ability of a nanoliposome adjuvant system to trigger a strong mucosal immune response to the Entamoeba histolytica Gal/GalNAc lectin LecA antigen. CBA/J mice were immunized with alum, emulsion or liposome based formulations containing synthetic TLR agonists. A liposome formulation containing TLR4 and TLR7/8 agonists was selected based on its ability to generate intestinal IgA, plasma IgG2a/IgG1, IFN-γ and IL-17A. Immunization with a mucosal prime followed by a parenteral boost generated a high mucosal IgA response that inhibited adherence of parasites to mammalian cells. Inclusion of the immune potentiator all-trans retinoic acid in the regimen further improved the mucosal IgA response. Immunization protected from infection with up to 55% efficacy. Our results show that a nanoliposome delivery system containing TLR agonists is a promising prospect for the development of vaccines against enteric pathogens, especially when a multifaceted immune response is desired.

  13. In vitro susceptibility of Entamoeba histolytica and Giardia lamblia to plants used in Mexican traditional medicine for the treatment of gastrointestinal disorders.

    PubMed

    Calzada, Fernando; Yépez-Mulia, Lilian; Aguilar, Abigaíl

    2006-12-06

    In our search for new antiprotozoal chemotherapy, we collected a selection of 26 plants used in Mexican traditional medicine for the treatment of gastrointestinal disorders. Methanolic extracts of these species were screened for their antiprotozoal activity against Entamoeba histolytica and Giardia lamblia trophozoites using in vitro tests. Among the tested extracts, the derivates of following species showed selectivity and significant antiprotozoal activity: Chiranthodendron pentadactylon, Annona cherimola and Punica granatum were the most active on Entamoeba histolytica with IC50 < 30 microg/ml. Dorstenia contrajerva, Senna villosa and Ruta chalepensis were the most active toward Giardia lamblia with IC50 < 38 microg/ml. The potency of Chiranthodendron pentadactylon (IC50 2.5 microg/ml) on Entamoeba histolytica was close that of to emetine, but far less than metronidazole, drugs used as control. The results of the antiprotozoal screening support the popular uses of the studied species for the treatment of diarrhoea and dysentery in Mexican traditional medicine.

  14. Identification of a pathogenic isolate-specific 30,000-Mr antigen of Entamoeba histolytica by using a monoclonal antibody.

    PubMed Central

    Tachibana, H; Kobayashi, S; Kato, Y; Nagakura, K; Kaneda, Y; Takeuchi, T

    1990-01-01

    A monoclonal antibody (MAb) produced against trophozoites of Entamoeba histolytica strain HM-1:IMSS, reacted with all of 42 isolates and 4 clones showing pathogenic zymodeme (Z) patterns, i.e., Z-II, Z-II alpha-, Z-II (glucose phosphate isomerase: gamma +), Z-VII, Z-VII (glucose phosphate isomerase: alpha lack, gamma +), Z-XI, Z-XIV, and Z-XIX, regardless of culture conditions, geographical origins, or host symptoms in an indirect fluorescence antibody test. In contrast, the MAb failed to react with 14 isolates possessing nonpathogenic zymodemes Z-I and Z-VIII and did not react with other enteric protozoan parasites, such as E. histolytica-like Laredo, Entamoeba hartmanni, Entamoeba coli, Endolimax nana, Dientamoeba fragilis, Trichomonas hominis, and Giardia lamblia. Western immunoblotting analysis showed that the molecular weight of the antigenic component recognized by the MAb was exclusively 30,000 in pathogenic isolates of different zymodemes. These results suggest that the 30,000-molecular-weight antigen is a marker of pathogenic isolates and that the indirect fluorescent-antibody test with the MAb is useful for the accurate discrimination of pathogenic amebae. Images PMID:2180826

  15. Evidence that the Entamoeba histolytica Mitochondrial Carrier Family Links Mitosomal and Cytosolic Pathways through Exchange of 3'-Phosphoadenosine 5'-Phosphosulfate and ATP.

    PubMed

    Mi-ichi, Fumika; Nozawa, Akira; Yoshida, Hiroki; Tozawa, Yuzuru; Nozaki, Tomoyoshi

    2015-11-01

    Entamoeba histolytica, a microaerophilic protozoan parasite, possesses mitosomes. Mitosomes are mitochondrion-related organelles that have largely lost typical mitochondrial functions, such as those involved in the tricarboxylic acid cycle and oxidative phosphorylation. The biological roles of Entamoeba mitosomes have been a long-standing enigma. We previously demonstrated that sulfate activation, which is not generally compartmentalized to mitochondria, is a major function of E. histolytica mitosomes. Sulfate activation cooperates with cytosolic enzymes, i.e., sulfotransferases (SULTs), for the synthesis of sulfolipids, one of which is cholesteryl sulfate. Notably, cholesteryl sulfate plays an important role in encystation, an essential process in the Entamoeba life cycle. These findings identified a biological role for Entamoeba mitosomes; however, they simultaneously raised a new issue concerning how the reactions of the pathway, separated by the mitosomal membranes, cooperate. Here, we demonstrated that the E. histolytica mitochondrial carrier family (EhMCF) has the capacity to exchange 3'-phosphoadenosine 5'-phosphosulfate (PAPS) with ATP. We also confirmed the cytosolic localization of all the E. histolytica SULTs, suggesting that in Entamoeba, PAPS, which is produced through mitosomal sulfate activation, is translocated to the cytosol and becomes a substrate for SULTs. In contrast, ATP, which is produced through cytosolic pathways, is translocated into the mitosomes and is a necessary substrate for sulfate activation. Taking our findings collectively, we suggest that EhMCF functions as a PAPS/ATP antiporter and plays a crucial role in linking the mitosomal sulfate activation pathway to cytosolic SULTs for the production of sulfolipids.

  16. Exploring the Possible Role of Lysine Acetylation on Entamoeba histolytica Virulence: A Focus on the Dynamics of the Actin Cytoskeleton

    PubMed Central

    López-Contreras, L.; Hernández-Ramírez, V. I.; Lagunes-Guillén, A. E.; Montaño, Sarita; Chávez-Munguía, B.; Sánchez-Ramírez, B.; Talamás-Rohana, P.

    2013-01-01

    Cytoskeleton remodeling can be regulated, among other mechanisms, by lysine acetylation. The role of acetylation on cytoskeletal and other proteins of Entamoeba histolytica has been poorly studied. Dynamic rearrangements of the actin cytoskeleton are crucial for amebic motility and capping formation, processes that may be effective means of evading the host immune response. Here we report the possible effect of acetylation on the actin cytoskeleton dynamics and in vivo virulence of E. histolytica. Using western blot, immunoprecipitation, microscopy assays, and in silico analysis, we show results that strongly suggest that the increase in Aspirin-induced cytoplasm proteins acetylation reduced cell movement and capping formation, likely as a consequence of alterations in the structuration of the actin cytoskeleton. Additionally, intrahepatic inoculation of Aspirin-treated trophozoites in hamsters resulted in severe impairment of the amebic virulence. Taken together, these results suggest an important role for lysine acetylation in amebic invasiveness and virulence. PMID:24078923

  17. Exploring the possible role of lysine acetylation on Entamoeba histolytica virulence: a focus on the dynamics of the actin cytoskeleton.

    PubMed

    López-Contreras, L; Hernández-Ramírez, V I; Lagunes-Guillén, A E; Montaño, Sarita; Chávez-Munguía, B; Sánchez-Ramírez, B; Talamás-Rohana, P

    2013-01-01

    Cytoskeleton remodeling can be regulated, among other mechanisms, by lysine acetylation. The role of acetylation on cytoskeletal and other proteins of Entamoeba histolytica has been poorly studied. Dynamic rearrangements of the actin cytoskeleton are crucial for amebic motility and capping formation, processes that may be effective means of evading the host immune response. Here we report the possible effect of acetylation on the actin cytoskeleton dynamics and in vivo virulence of E. histolytica. Using western blot, immunoprecipitation, microscopy assays, and in silico analysis, we show results that strongly suggest that the increase in Aspirin-induced cytoplasm proteins acetylation reduced cell movement and capping formation, likely as a consequence of alterations in the structuration of the actin cytoskeleton. Additionally, intrahepatic inoculation of Aspirin-treated trophozoites in hamsters resulted in severe impairment of the amebic virulence. Taken together, these results suggest an important role for lysine acetylation in amebic invasiveness and virulence.

  18. Evidence for a link between locus R-R sequence type and outcome of infection with Entamoeba histolytica.

    PubMed

    Ali, I K M; Haque, R; Alam, F; Kabir, M; Siddique, A; Petri, W A

    2012-07-01

    The results of Entamoeba histolytica infections range from asymptomatic colonization to variable disease outcomes. However, markers that may predict infection outcomes are not known. Here, we investigated sequence types of a non-coding tRNA-linked locus R-R to identify surrogate markers that may show association with infection outcomes. Among 112 clinical samples--21 asymptomatic, 20 diarrhoea/dysentery and 71 liver abscesses--we identified 11 sequence types. Sequence type 5RR was mostly associated with asymptomatic samples, and sequence type 10RR was predominantly associated with the symptomatic (diarrhoea/dysentery and liver abscess) samples. This is the first report that identifies markers that may predict disease outcomes in E. histolytica infection.

  19. [Adaptation of a sensitive DNA extraction method for detection of Entamoeba histolytica by real-time polymerase chain reaction].

    PubMed

    Pınar, Ahmet; Akyön, Yakut; Alp, Alpaslan; Ergüven, Sibel

    2010-07-01

    This study was aimed to adapt a sensitive DNA extraction protocol in stool samples for real-time polymerase chain reaction (PCR) detection of Entamoeba histolytica which causes important morbidity and mortality worldwide. Stool extraction is a problematic step and has direct effects on PCR sensitivity. In order to improve the sensitivity of E.histolytica detection by real-time PCR, "QIAamp DNA stool minikit (Qiagen, Germany)" was modified by adding an overnight incubation step with proteinase K and sodium dodecyl sulfate (SDS) in this study. Three different extraction methods [(1) original method, (2) cetyltrimethyl-ammonium bromide (CTAB) method, (3) modified method] were evaluated for effects on sensitivity in real-time quantitative PCR (Artus RealArt TM E.histolytica RG PCR Kit, Qiagen Diagnostics, Germany). For this purpose, several concentrations of standard E.histolytica DNA were spiked in parasite-free stool samples and three different extraction protocols were performed. Detection sensitivities of "QIAamp DNA stool minikit" was found 5000 copies/ml and of CTAB method was found 500 copies/ml. Detection sensitivity of the extraction was improved to 5 copies/mL by modified "QIAamp DNA stool minikit" protocol. Since detection sensitivities of nucleic acid extraction protocols from stool samples directly affect the sensitivity of PCR amplification, different extraction protocols for different microorganisms should be evaluated.

  20. Genetic variability of the serine-rich Entamoeba histolytica protein gene in clinical isolates from the United Arab Emirates.

    PubMed

    ElBakri, Ali; Samie, Amidou; Ezzedine, Sinda; Odeh, Ra'ed Abu

    2014-06-01

    The genetic diversity of 20 Entamoeba histolytica isolates from asymptomatic individuals from the UAE was investigated by analyzing polymorphism in the serine-rich E. histolytica gene (SREHP) by nested polymerase chain reaction (PCR) amplification followed by restriction fragment length polymorphism (RFLP) on DNA extracted directly from stool samples. The SREHP gene was successfully amplified in 15 out of 20 E. histolytica-positive samples. Four out of the remaining five isolates did not amplify for the SREHP gene. Despite successful amplification of the SREHP gene in the fifth isolate, AluI digestion of the amplified PCR product revealed no bands. As a result, all five samples were excluded from the study. Twelve different profiles were obtained from the 15 successfully amplified isolates. Thus, demonstrating extensive genetic variability and reinforcing the argument that E. histolytica has an extremely polymorphic genetic structure. Despite the sample size limitation, a finding in the study was the occurrence of one profile common to one Indian isolate while another profile common to one Pakistani isolate; indicating the possibility of clonal infection. Furthermore, we found one isolate from a Bangladeshi expatriate identical to 2 asymptomatic Bangladeshi isolates reported in an earlier study. No clear association between the different genotypes and the study population demographics was noted. The results also indicated the possibility of strains clustering by region.

  1. Effect of the silencing of the Ehcp112 gene on the in vitro virulence of Entamoeba histolytica

    PubMed Central

    2013-01-01

    Background Entamoeba histolytica is an intestinal protozoan parasite that causes amoebiasis in humans, affecting up to 50 million people worldwide each year and causing 40,000 to 100,000 deaths annually. EhCP112 is a cysteine proteinase of E. histolytica able to disrupt cell monolayers and digest extracellular matrix proteins, it is secreted by trophozoites and it can be active in a wide range of temperature and pH. These characteristics have encouraged the use of EhCP112 in the design and production of possible vaccines against amoebiasis, obtaining promising results. Nevertheless, we have no conclusive information about the role of EhCP112 in the E. histolytica pathogenesis. Methods A set of three specific siRNA sequences were used to silence the Ehcp112 gene via the soaking system. Silencing was evaluated by Western blot using an antibody against the EhCP112 recombinant protein. Finally, we analyzed the protease activity, the phagocytosis rate and the ability to destroy MDCK cells of the EhCP112-silenced trophozoites. Results The highest silencing effect on EhCP112 was detected at 16 h of treatment; time enough to perform the in vitro virulence assays, which showed that EhCP112 silencing produces a significant reduction in cytolysis and phagocytosis of target cells, indicating the participation of this proteinase in these events. Conclusions EhCP112 is involved in the in vitro virulence of E. histolytica. PMID:23981435

  2. Catalysis by N-Acetyl-d-glucosaminylphosphatidylinositol De-N-acetylase (PIG-L) from Entamoeba histolytica

    PubMed Central

    Ashraf, Mohammad; Sreejith, Perinthottathil; Yadav, Usha; Komath, Sneha Sudha

    2013-01-01

    We showed previously that Entamoeba histolytica PIG-L exhibits a novel metal-independent albeit metal-stimulated activity. Using mutational and biochemical analysis, here we identify Asp-46 and His-140 of the enzyme as being important for catalysis. We show that these mutations neither affect the global conformational of the enzyme nor alter its metal binding affinity. The defect in catalysis, due to the mutations, is specifically due to an effect on Vmax and not due to altered substrate affinity (or Km). We propose a general acid-base pair mechanism to explain our results. PMID:23341455

  3. [Epidemiologic and molecular study of Entamoeba histolytica and Entamoeba dispar strains in pacients with diarrhea in Cumana, Sucre state, Venezuela].

    PubMed

    Mora, Leonor; García, Ana; De Donato, Marcos; Urdaneta, Haideé

    2008-06-01

    An epidemiological and molecular study on E. histolytica and E. dispar was carried out in 428 patients with gastrointestinal symptomatology of diarrhea from different health centers in Cumana, Sucre state. The samples were processed through: direct examination with 0.85% physiological saline solution, temporal lugol staining, trichromic staining and the Ritchie method of concentration; a sucrose gradient was used for cyst isolation. The small subunit of the 16S RNA was amplified by nested, multiplex PCR for the molecular detection. The E. histolytica/E. dispar prevalences according to the direct, Ritchie and trichromic staining methods were 20.09, 13.79 and 12.15%, respectively; while prevalences according to PCR for E. histolytica and E. dispar were 6.31% and 4.44%, respectively, also detecting four cases of mixed infection. Sequencing of the amplified fragments of E. histolytica showed 100% homology with the sequences with strains from Merida (Venezuela), USA, Brazil, Mexico and GenBank. The infections by E. histolytica and E. dispar were statistically associated with age but not with sex. The presence of mucus, blood and abdominal pain were only associated to E. histolytica infection. The moderate prevalence of E. histolytica shows the endemic status of this population and warns about the potential problem as a morbidity and mortality in Sucre state. The frequency of E. dispar in this population suggests the existence of an overestimation problem in the diagnosis of amoebiasis with its clinical and epidemiological implications, and shows the poor knowledge about the true prevalences of this protozoan. The PCR allowed for the differential identification of E. histolytica and E. dispar, as well as the presence of mixed infections, making a great tool for epidemiological amoebiasis studies.

  4. Structure-based identification of inositol polyphosphate 1-phosphatase from Entamoeba histolytica.

    PubMed

    Faisal Tarique, Khaja; Arif Abdul Rehman, Syed; Betzel, Christian; Gourinath, Samudrala

    2014-11-01

    Inositol polyphosphate 1-phosphatase from Entamoeba histolytica (EhIPPase) is an Mg(2+)-dependent and Li(+)-sensitive enzyme that catalyzes the hydrolysis of inositol 1,4-bisphosphate [Ins(1,4)P2] into myo-inositol 1-monophosphate and PO4(3-). In the present work, EhIPPase has been biochemically identified and its crystal structure has been determined in the presence of Mg(2+) and PO4(3-) at 2.5 Å resolution. This enzyme was previously classified as a 3'(2'),5'-bisphosphate nucleotidase in the NCBI, but its biochemical activity and structural analysis suggest that this enzyme behaves more like an inositol polyphosphate 1-phosphatase. The ability of EhIPPase to hydrolyze the smaller Ins(1,4)P2 better than the bulkier 3'-phosphoadenosine 5'-phosphate (PAP) is explained on the basis of the orientations of amino-acid residues in the binding site. This structure is the first of its class to be determined from any protozoan parasite, and is the third to determined among all organisms, following its rat and bovine homologues. The three-dimensional fold of EhIPPase is similar to those of other members of the inositol monophosphatase superfamily, which also includes inositol monophosphatase, 3'(2'),5'-bisphosphate nucleotidase and fructose-1,6-bisphosphate 1-phosphatase. They all share conserved residues essential for metal binding and substrate hydrolysis, with the motif D-Xn-EE-Xn-DP(I/L)DG(S/T)-Xn-WD-Xn-GG. The structure is divided into two domains, namely α+β and α/β, and the substrate and metal ions bind between them. However, the ability of each enzyme class to act specifically on its cognate substrate is governed by the class-specific amino-acid residues at the active site.

  5. Functional manipulation of a calcium-binding protein from Entamoeba histolytica guided by paramagnetic NMR.

    PubMed

    Rout, Ashok K; Patel, Sunita; Somlata; Shukla, Manish; Saraswathi, Deepa; Bhattacharya, Alok; Chary, Kandala V R

    2013-08-09

    EhCaBP1, one of the calcium-binding proteins from Entamoeba histolytica, is a two-domain EF-hand protein. The two domains of EhCaBP1 are structurally and functionally different from each other. However, both domains are required for structural stability and a full range of functional diversity. Analysis of sequence and structure of EhCaBP1 and other CaBPs indicates that the C-terminal domain of EhCaBP1 possesses a unique structure compared with other family members. This had been attributed to the absence of a Phe-Phe interaction between highly conserved Phe residues at the -4 position in EF-hand III (F[-4]; Tyr(81)) and at the 13th position in EF-hand IV (F[+13]; Phe(129)) of the C-terminal domain. Against this backdrop, we mutated the Tyr residue at the -4th position of EF III to the Phe residue (Y81F), to bring in the Phe-Phe interaction and understand the nature of structural and functional changes in the protein by NMR spectroscopy, molecular dynamics (MD) simulation, isothermal titration calorimetry (ITC), and biological assays, such as imaging and actin binding. The Y81F mutation in EhCaBP1 resulted in a more compact structure for the C-terminal domain of the mutant as in the case of calmodulin and troponin C. The compact structure is favored by the presence of a π-π interaction between Phe(81) and Phe(129) along with several hydrophobic interactions of Phe(81), which are not seen in the wild-type protein. Furthermore, the biological assays reveal preferential membrane localization of the mutant, loss of its colocalization with actin in the phagocytic cups, whereas retaining its ability to bind G- and F-actin.

  6. Identification and characterisation of early reactions of asparagine-linked oligosaccharide assembly in Entamoeba histolytica.

    PubMed

    Vargas-Rodríguez, L; Villagómez-Castro, J C; Flores-Carreón, A; López-Romero, E

    1998-09-01

    Sequential incubation of a mixed membrane fraction isolated from Entamoeba histolytica trophozoites with the nonionic detergents Brij 35 and Igepal CA-630 rendered a soluble fraction with the ability to transfer N-acetylglucosamine (GlcNAc) from UDP-GlcNAc to dolichol phosphate to form a lipid saccharide that was identified as a mixture of dolichol-P-P-GlcNAc and dolichol-P-P-(GlcNAc)2 as follows. (a) The reaction occurred only in the presence of exogenously added dolichol phosphate and was strongly inhibited by tunicamycin and amphomycin; (b) Over 90% of the aminosugar moiety of the lipid saccharide was released by mild acid hydrolysis and was identified as a mixture of GlcNAc and diacetylchitobiose [(GlcNAc)2]; (c) Time course experiments revealed that dolichol-P-P-(GlcNAc)2 accumulated at the expense of a parallel decrease in dolichol-P-P-GlcNAc revealing the tandem operation of UDPGlcNAc:dolichol-P GlcNAc-1-P transferase and UDPGlcNAc:dolichol-P GlcNAc transferase. Mg2+ and to a lower extent Mn2+ were required for catalytic activity and were optimal at 2.5 mM and 1.25 mM, respectively. Common phospholipids with different head groups failed to increase catalytic activity and phosphatidylglycerol was inhibitory. At low concentration, nucleotides such as ATP, GMP and GTP brought about stimulations of 24-54% but higher concentrations were inhibitory. Others were inhibitory at all concentrations the strongest being those containing a uridine base.

  7. Isolation, purification, and partial characterization of an enterotoxin from extracts of Entamoeba histolytica trophozoites.

    PubMed Central

    Feingold, C; Bracha, R; Wexler, A; Mirelman, D

    1985-01-01

    Soluble cell-free extracts of pathogenic Entamoeba histolytica, as well as serum-free minimal media in which trophozoites are incubated, contain substances that cause the rapid rounding up and detachment of tissue-cultured monolayers of mammalian cells (cytopathic activity) and induce fluid secretion in ligated intestinal loops of indomethacin-pretreated rats (enterotoxic activity). A semiquantitative assay for the determination of the cytopathic activity based on the rate of detachment of tissue-cultured baby hamster kidney cells was developed. Two peaks containing cytopathic activity were obtained upon gel filtration of the soluble extracts: peak I, with over 60% of the activity, emerged in the 30,000 to 50,000 molecular weight region, and peak II, containing the remaining activity, was in the 15,000 to 25,000 molecular weight region. The activity of peak I was found to be heat labile and inhibited by sialoglycoproteins such as fetuin and mucin (5 mg/ml), as well as by sialic acid. Protease inhibitors such as antitrypsin, pepstatin, phenylmethylsulfonyl fluoride, metaloprotease inhibitors, and bacitracin had no effect on the cytopathic activity. Marked inhibition of cytopathic activity was observed, however, with iodoacetamide and p-chloromercuribenzoate, which affect sulfhydryl groups. The toxic material in peak II was found to have ionophoric activity and was not inhibited by sialic acid-containing compounds. The materials from both peaks had enterotoxic activity in intestinal ligated loops. The active substance from peak I was further purified (200X) on an agarose-fetuin affinity column, yielding one major protein band with an apparent molecular weight of ca. 30,000 on sodium dodecyl sulfate. Amino acid analysis revealed that the protein was very poor in sulfur amino acids. The sialic acid-sensitive toxic activity was higher in known virulent strains such as HM-1:IMSS and could be markedly augmented after preincubation of the trophozoites with certain

  8. Affinity purification and biochemical characterization of histolysin, the major cysteine proteinase of Entamoeba histolytica.

    PubMed Central

    Luaces, A L; Barrett, A J

    1988-01-01

    We report a one-step method for the purification to homogeneity of a cysteine proteinase of Entamoeba histolytica (histolysin) by affinity chromatography of the soluble extract of the parasite on immobilized phenylalanyl(2-phenyl)aminoacetaldehyde semicarbazone. The enzyme has an apparent Mr of 26,000 by SDS/polyacrylamide-gel electrophoresis and 29,000 by gel chromatography. Its pH optimum varies widely, from 5.5 with azocasein to approx. 7 with other protein substrates and benzyloxycarbonylphenylalanyl-L-citrullylaminomethylcourmarin++ + (Z-Phe-Cit-NHMec), and to 9.5 with benzyloxycarbonylphenylalanylarginylaminomethylcoumarin (Z-Phe-Arg-NHMec) and benzyloxycarbonylarginylarginylaminomethylcourmarin (Z-Arg-Arg-NHMec). Values of Km, kcat. and kcat/Km are 1.5 microM, 130 s-1 and 87 X 10(6) M-1.s-1 for Z-Arg-Arg-NHMec, and 32 microM, 0.4 s-1 and 0.012 x 10(6) M-1.s-1 for Z-Phe-Arg-NHMec, respectively, at pH 7.5 and 37 degrees C. The enzyme is inhibited by leupeptin and such inhibitors of cysteine proteinases as L-transepoxysuccinyl-L-leucylamido-4-(guanidino)butane, peptidyldiazomethanes, iodoacetic acid and chicken cystatin. The tentative N-terminal amino acid sequence of the enzyme closely resembles that of papain. Histolysin does not degrade type I collagen or elastin, but it is active against cartilage proteoglycan and kidney glomerular basement-membrane collagen. It also detaches cells from their substratum in vitro, and could well play a role in tissue invasion. Images Fig. 2. Fig. 4. PMID:2898937

  9. Gene migration for re-emerging amebiasis in Iran's northwest-Iraq borders: a microevolutionary scale for reflecting epidemiological drift of Entamoeba histolytica metapopulations.

    PubMed

    Mohammadzadeh, Asad; Spotin, Adel; Mahami-Oskouei, Mahmoud; Haghighi, Ali; Zebardast, Nozhat; Kohansal, Kobra

    2017-01-01

    In the microevolutionary scales of Entamoeba isolates, the gene migration shows how Entamoeba spp. has epidemiologically drifted among border countries. Five hundred fecal samples were taken from patients suffering gastrointestinal disorders, abdominal pain, and diarrhea at Saggez, northwest Iran located within the border Iraq country. Following parasitological techniques, DNA samples were extracted and amplified by polymerase chain reaction (PCR) of 18S rRNA region to identify Entamoeba infections. To distinguish the Entamoeba spp., a multiplex PCR was conducted. Amplicons were sequenced to reconfirm their heterogeneity traits and phylogenetic analysis. Additionally, Entamoeba histolytica sequences of Iraq were retrieved from GenBank database. The suspected isolates were diagnosed as E. histolytica (2.2 %), Entamoeba moshkovskii (1 %), and Entamoeba dispar (0.4 %). Mixed Entamoeba infections did not detect among isolates. A parsimonious network of the sequence haplotypes displayed star-like features in the overall isolates containing E.h1, E.d2, and E.m3 as the most common haplotypes. According to analysis of molecular variance (AMOVA) test, high partial value of haplotype diversity (0.700 to 0.800) of E. histolytica was shown the total genetic variability within populations while nucleotide diversity was low among Iranian and Iraqi metapopulations. Neutrality indices of the 18S rRNA were shown negative values in E. histolytica populations which indicating significant deviations from neutrality. A pairwise fixation index (F-statistics [Fst]) as a degree of gene flow had a low value for all populations (0.001) while the number of migrants was 2.48. The statistically Fst value indicates that E. histolytica isolates are not genetically differentiated among shared isolates of Iran and Iraq. Occurrence of E.h1 between two regional populations indicates that there is dawn of Entamoeba flow due to transfer of alleles from one population to another population through

  10. A genomewide overexpression screen identifies genes involved in the phosphatidylinositol 3-kinase pathway in the human protozoan parasite Entamoeba histolytica.

    PubMed

    Koushik, Amrita B; Welter, Brenda H; Rock, Michelle L; Temesvari, Lesly A

    2014-03-01

    Entamoeba histolytica is a protozoan parasite that causes amoebic dysentery and liver abscess. E. histolytica relies on motility, phagocytosis, host cell adhesion, and proteolysis of extracellular matrix for virulence. In eukaryotic cells, these processes are mediated in part by phosphatidylinositol 3-kinase (PI3K) signaling. Thus, PI3K may be critical for virulence. We utilized a functional genomics approach to identify genes whose products may operate in the PI3K pathway in E. histolytica. We treated a population of trophozoites that were overexpressing genes from a cDNA library with a near-lethal dose of the PI3K inhibitor wortmannin. This screen was based on the rationale that survivors would be overexpressing gene products that directly or indirectly function in the PI3K pathway. We sequenced the overexpressed genes in survivors and identified a cDNA encoding a Rap GTPase, a protein previously shown to participate in the PI3K pathway. This supports the validity of our approach. Genes encoding a coactosin-like protein, EhCoactosin, and a serine-rich E. histolytica protein (SREHP) were also identified. Cells overexpressing EhCoactosin or SREHP were also less sensitive to a second PI3K inhibitor, LY294002. This corroborates the link between these proteins and PI3K. Finally, a mutant cell line with an increased level of phosphatidylinositol (3,4,5)-triphosphate, the product of PI3K activity, exhibited increased expression of SREHP and EhCoactosin. This further supports the functional connection between these proteins and PI3K in E. histolytica. To our knowledge, this is the first forward-genetics screen adapted to reveal genes participating in a signal transduction pathway in this pathogen.

  11. Analysis of U3 snoRNA and small subunit processome components in the parasitic protist Entamoeba histolytica.

    PubMed

    Srivastava, Ankita; Ahamad, Jamaluddin; Ray, Ashwini Kumar; Kaur, Devinder; Bhattacharya, Alok; Bhattacharya, Sudha

    2014-02-01

    In the early branching parasitic protist Entamoeba histolytica, pre-rRNA synthesis continues when cells are subjected to growth stress, but processing slows down and unprocessed pre-rRNA accumulates. To gain insight into the regulatory mechanisms leading to accumulation, it is necessary to define the pre-rRNA processing machinery in E. histolytica. We searched the E. histolytica genome sequence for homologs of the SSU processome, which contains the U3snoRNA, and 72 proteins in yeast. We could identify 57 of the proteins with high confidence. Of the rest, 6 were absent in human, and 4 were non-essential in yeast. The remaining 5 were absent in other parasite genomes as well. Analysis of U3snoRNA showed that the E. histolytica U3snoRNA adopted the same conserved secondary structure as seen in yeast and human. The predicted structure was verified by chemical modification followed by primer extension (SHAPE). Further we showed that the predicted interactions of Eh_U3snoRNA boxes A and A' with pre-18S rRNA were highly conserved both in position and sequence. The predicted interactions of 5'-hinge and 3'-hinge sequences of Eh_U3 snoRNA with the 5'-ETS sequences were conserved in position but not in sequence. Transcription of selected genes of SSU processome was tested by northern analysis, and transcripts of predicted sizes were obtained. During serum starvation, when unprocessed pre-RNA accumulated, the transcript levels of some of these genes declined. This is the first report on pre-rRNA processing machinery in E. histolytica, and shows that the components are well conserved with respect to yeast and human.

  12. Effect of the Leptin Receptor Q223R Polymorphism on the Host Transcriptome following Infection with Entamoeba histolytica

    PubMed Central

    Mackey-Lawrence, Nicole M.; Guo, Xiaoti; Sturdevant, Daniel E.; Virtaneva, Kimmo; Hernandez, Matthew M.; Houpt, Eric; Sher, Alan; Porcella, Stephen F.

    2013-01-01

    Resistance to amebiasis is associated with a polymorphism in the leptin receptor. Previous studies demonstrated that humans with the ancestral Q223 leptin receptor allele were nearly four times less likely to be infected with Entamoeba histolytica than those carrying the mutant R223 allele. We hypothesized that the Q223 allele protected against E. histolytica via STAT3-mediated transcription of genes required for mucosal immunity. To test this, mice containing the humanized LEPR Q or R allele at codon 223 were intracecally infected with E. histolytica. Susceptibility to amebiasis was assessed, and cecal tissues were analyzed for changes in gene expression. By 72 h postchallenge, all Q223 mice had cleared E. histolytica, whereas 39% of 223R mice were infected. Thirty-seven genes were differentially expressed in response to infection at 72 h, including proinflammatory genes (CXCL2, S100A8/9, PLA2G7, ITBG2, and MMP9) and functions pertaining to the movement and activity of immune cells. A comparison at 12 h postchallenge of infected Q223 versus R223 mice identified a subset of differentially expressed genes, many of which were closely linked to leptin signaling. Further analyses indicated that the Q223 gene expression pattern was consistent with a suppressed apoptotic response to infection, while 223R showed increased cellular proliferation and recruitment. These studies are the first to illuminate the downstream effects of leptin receptor polymorphisms on intestinal infection by E. histolytica. As such, they are important for the insight that they provide into this previously uncharacterized mechanism of mucosal immunity. PMID:23429533

  13. A novel Entamoeba histolytica inositol phosphate kinase catalyzes the formation of 5PP-Ins(1,2,3,4,6)P(5).

    PubMed

    Löser, Benjamin; Nalaskowski, Marcus M; Fanick, Werner; Lin, Hongying; Tannich, Egbert; Mayr, Georg W

    2012-01-01

    The parasitic protozoan Entamoeba histolytica is able to invade human tissues by secreting proteolytic enzymes. This secretion is regulated by inositol phosphate-mediated Ca(2+) release from internal stores. To further investigate the inositol phosphate metabolism of Entamoeba histolytica four putative inositol phosphate kinase genes (ehipk1-4) were identified and their expression analyzed by real-time quantitative PCR using RNA of trophozoites. Furthermore inositol phosphate kinase EhIPK1 was recombinantly expressed, purified and enzymatically characterized. Its main activity is the conversion of InsP(6) to 5PP-Ins(1,2,3,4,6)P(5), one of the main inositol phosphates found in Entamoeba histolytica. Remarkably, EhIPK1 possesses several additional enzymatic activities, e.g. the phosphorylation of the Ca(2+)-releasing second messenger Ins(1,4,5)P(3).We were able to identify several compounds with inhibitory potential against EhIPK1. Because of the important role of inositol phosphates in the invasion of human tissues by Entamoeba histolytica, inositol phosphate metabolizing enzymes are interesting targets for novel therapeutic approaches.

  14. Antiamoebic Activity of Adenophyllum aurantium (L.) Strother and Its Effect on the Actin Cytoskeleton of Entamoeba histolytica

    PubMed Central

    Herrera-Martínez, Mayra; Hernández-Ramírez, Verónica I.; Hernández-Carlos, Beatriz; Chávez-Munguía, Bibiana; Calderón-Oropeza, Mónica A.; Talamás-Rohana, Patricia

    2016-01-01

    In Mexico, the Adenophyllum aurantium (L.) Strother plant is consumed as an infusion to treat intestinal diseases such as amoebiasis, which is an endemic health problem in Mexico and other countries. However, the effect of A. aurantium on Entamoeba histolytica, the causative agent of amoebiasis, is unknown. An aerial part methanolic extract (AaMeA), a root methanolic extract (AaMeR) and a root ethyl acetate extract (AaEaR) were tested on E. histolytica trophozoites. AaMeA and AaMeR did not show antiproliferative activity; however, AaEaR exhibited an in vitro GI50 of 230 μg/ml, and it was able to inhibit the differentiation of Entamoeba invadens trophozoites into cysts. The intraperitoneal administration of AaEaR (2.5 or 5 mg) to hamsters that were infected with E. histolytica inhibited the development of amoebic liver abscesses in 48.5 or 89.0% of the animals, respectively. Adhesion to fibronectin and erythrophagocytosis were 28.7 and 37.5% inhibited by AaEaR, respectively. An ultrastructure analysis of AaEaR-treated trophozoites shows a decrease in the number of vacuoles but no apparent cell damage. Moreover, this extract affected the actin cytoskeleton structuration, and it prevented the formation of contractile rings by mechanism(s) that were independent of reactive oxygen species and RhoA activation pathways. 13C NMR data showed that the major compounds in the AaEaR extract are thiophenes. Our results suggest that AaEaR may be effective in treatments against amoebiasis, nevertheless, detailed toxicity studies on thiophenes, contained in AaEaR, are required to avoid misuse of this vegetal species. PMID:27445810

  15. Efficacy of a Gal-lectin subunit vaccine against experimental Entamoeba histolytica infection and colitis in baboons (Papio sp.).

    PubMed

    Abd Alla, Mohamed D; Wolf, Roman; White, Gary L; Kosanke, Stanley D; Cary, David; Verweij, Jaco J; Zhang, Mie-Jie; Ravdin, Jonathan I

    2012-04-26

    To determine the efficacy of a Gal-lectin based intranasal synthetic peptide vaccine, we developed a new experimental primate model of Entamoeba histolytica intestinal infection. Release of xenic E. histolytica trophozoites (5×10(6)) into the small bowel of baboons (Papio sp.) resulted in a rapid intestinal anti-amebic antibody response and a brief infection; however, release of trophozoites directly into the cecum (5 baboons) elicited a sustained E. histolytica infection, as determined by quantitative fecal PCR, and an ulcerative, inflammatory colitis observed on colonoscopy and histopathology. In three controlled experiments, baboons received four immunizations at seven day intervals of 1600 μg of the vaccine/nostril, with Cholera toxin, 20 μg/nostril as adjuvant; vaccinated (n=6) and control baboons (n=6) baboons were then challenged via colonoscopy with xenic trophozoites (5×10(6)). During 90 days of follow up, 250 of 415 (60.24%) fecal samples in control baboons had a (+) PCR for E. histolytica, compared to only 36 of 423 (8.51%) samples from vaccinated baboons (P<0.001). All 6 vaccinated baboons were free of infection by the 51st day after challenge, 5 of 6 controls positive had (+) fecal PCRs for up to 126 days post-challenge (P=0.019). Inflammatory colitis developed in 4 of 6 control baboons post-challenge, with invasive E. histolytica trophozoites present in 2 of the 4 on histopathology. There was no evidence of inflammatory colitis or parasite invasion in any of the vaccinated baboons; there was a strong inverse correlation between positive ELISA OD value indicating the presence of intestinal anti-peptide IgA antibodies and baboons having a positive fecal PCR CT value, P<0.001. In conclusion, we developed a novel primate model of E. histolytica intestinal infection and demonstrated that a Gal-lectin-based intranasal synthetic peptide vaccine was highly efficacious in preventing experimental E. histolytica infection and colitis in baboons.

  16. Recent insights in pre-mRNA 3'-end processing signals and proteins in the protozoan parasite Entamoeba histolytica.

    PubMed

    López-Camarillo, César; Hernández de la Cruz, Olga N; Vivas, Jessica García; Retana, Jorge Fernández; Valdez, Marisol Pezet; Rosas, Itzel Lopez; Alvarez-Sánchez, Elizbeth; Marchat, Laurence A

    2010-08-01

    The messenger RNA precursors (pre-mRNA) 3'-end processing occurs in a two-step co-transcriptional coupled reaction, denoted as cleavage and polyadenylation. Both processes depend on trans-acting factors interacting in a coordinated manner with cis-sequence motifs located at the 3' untranslated region of transcripts. In this paper, we reviewed mechanisms involved in pre-mRNA processing in eukaryotic organisms, including our own findings about sequences and proteins potentially involved in mRNA 3'-end formation in the protozoan parasite Entamoeba histolytica. Interestingly, protein sequence comparisons among E. histolytica, yeast, and human pre-mRNA processing machineries showed that amoeba pre-mRNA 3'-end processing machinery appears to be in an intermediate evolutionary position between mammals and yeast. In addition, the presence of non canonical poly(A) polymerases family recently identified in E. histolytica, adds more complexity to the mRNA 3'-end formation process in this ancient eukaryote.

  17. Protection of gerbils from amebic liver abscess by immunization with the galactose-specific adherence lectin of Entamoeba histolytica.

    PubMed Central

    Petri, W A; Ravdin, J I

    1991-01-01

    No protective antigens from Entamoeba histolytica have been previously defined. We tested the ability of the galactose-specific adherence lectin of E. histolytica to elicit a protective immune response in conjunction with Freund's incomplete and complete adjuvants. The gerbil (Meriones unguiculatus) model of an experimental amebic liver abscess was used. Gerbils were immunized intraperitoneally or subcutaneously with 10 micrograms of the affinity-purified lectin in complete Freund's adjuvant and then at 2 and 4 weeks with 10 micrograms of the lectin in incomplete Freund's adjuvant. All of the immunized animals developed antilectin antibody titers of greater than 1/1,024 as measured by a radioimmunoassay. The gerbil antilectin antibodies were shown by Western immunoblotting to be directed to the heavy subunit but not the light subunit of the lectin. Immune gerbil sera inhibited amebic adherence by 100% at a 1/10 dilution. Immune and control gerbils were challenged at 6 weeks by the intrahepatic injection of 5 x 10(5) E. histolytica trophozoites. Four independent trials demonstrated complete protection from amebic liver abscess formation in 67% of lectin-immunized gerbils. Unexpectedly, liver abscess weights were significantly higher in the gerbils that failed to become immune than in the control animals. Our results demonstrate that the galactose lectin is a protective antigen and provide an immune-animal model to study the mechanisms of protection and potential disease exacerbation conferred by the antilectin immune response. Images PMID:1987067

  18. Immunogenicity and protective efficacy of heparan sulphate binding proteins of Entamoeba histolytica in a guinea pig model of intestinal amoebiasis.

    PubMed

    Kaur, Upninder; Khurana, Sumeeta; Saikia, Uma Nahar; Dubey, M L

    2013-11-01

    Entamoeba histolytica infection is associated with considerable morbidity and mortality in the form of intestinal and extraintestinal amoebiasis. No vaccine is yet available for amoebiasis. Heparan Sulphate Binding Proteins (HSBPs) from E. histolytica were evaluated for immunogenicity and protective efficacy in a Guinea pig model. Animals were immunized subcutaneously with 30μg of HSBP by three weekly inoculations. The immunogenicity of HSBP was determined by antibody response (IgG, IgM and IgA), splenocyte proliferation assay and in vitro direct amoebicidal assay with splenic lymphocytes and monocytes from vaccinated and control animals. The efficacy of the vaccine was evaluated by challenge infection to vaccinated and control animals by intra-caecal inoculation of E. histolytica trophozoites and comparing gross and histopathological findings in caeca of these animals. HSBP was found to induce specific anti-amoebic response as seen by specific antibody production and direct amoebicidal activity of splenocytes. The vaccine also showed partial protection against challenge infection in vaccinated animals as shown by mild/absent lesions and histopathological findings.

  19. Diagnosis of amebic liver abscess and amebic colitis by detection of Entamoeba histolytica DNA in blood, urine, and saliva by a real-time PCR assay.

    PubMed

    Haque, Rashidul; Kabir, Mamun; Noor, Zannatun; Rahman, S M Mazidur; Mondal, Dinesh; Alam, Faisal; Rahman, Intekhab; Al Mahmood, Abdullh; Ahmed, Nooruddin; Petri, William A

    2010-08-01

    The noninvasive diagnosis of amebic liver abscess is challenging, as most patients at the time of diagnosis do not have a concurrent intestinal infection with Entamoeba histolytica. Fecal testing for E. histolytica parasite antigen or DNA is negative in most patients. A real-time PCR assay was evaluated for detection of E. histolytica DNA in blood, urine, and saliva samples from amebic liver abscess as well as amebic colitis patients in Bangladesh. A total of 98 amebic liver abscess and 28 amebic colitis patients and 43 control subjects were examined. The real-time PCR assay detected E. histolytica DNA in 49%, 77%, and 69% of blood, urine, and saliva specimens from the amebic liver abscess patients. For amebic colitis the sensitivity of the real-time PCR assay for detection of E. histolytica DNA in blood, urine, and saliva was 36%, 61%, and 64%, respectively. All blood, urine, and saliva samples from control subjects were negative by the real-time PCR assay for E. histolytica DNA. When the real-time PCR assay results of the urine and saliva specimens were taken together (positive either in urine or saliva), the real-time PCR assay was 97% and 89% sensitive for detection of E. histolytica DNA in liver abscess and intestinal infection, respectively. We conclude that the detection of E. histolytica DNA in saliva and urine could be used as a diagnostic tool for amebic liver abscess.

  20. Membrane structure and surface coat of Entamoeba histolytica. Topochemistry and dynamics of the cell surface: cap formation and microexudate.

    PubMed

    Silva, P P; Martínez-Palomo, A; Gonzalez-Robles, A

    1975-03-01

    Treatment of living entamoeba histolytica cells with low concentrations of concanavalin A (con A) and peroxidase results in redistribution of the plasma membrane con A receptors to one pole of the cell where a morphologically distinct region--the uroid--is formed. Capping of con A receptors is not accompanied by parallel accumulation of ruthenium red-stainable components. In capped cells, the pattern of distribution of acidic sites ionized at pH 1.8 (labeled by colloidal iron) at the outer surface and of membrane particles (integral membrane components revealed by freeze-fracture) is not altered over the uroid region. Cytochemistry of substrate-attached microexudate located in regions adjacent to E. histolytica cells demonstrates the presence of con A binding sites and ruthenium red- and alcian blue-stainable components and the absent of colloidal iron binding sites. In a previous report we demonstrated that glycerol-induced aggregation of the plasma membrane particles is accompanied by a discontinuous distribution of colloidal iron binding sites, while con A receptors and acidic sites ionized at pH 4.0 remain uniformly distributed over the cell surface. Taken together, our experiments show that, in E. histolytica cells, peripheral membrane components may move independently of integral components and, also, that certain surface determinants may redistribute independently of others. These results point to the complexity of the membrane structure-cell surface relationship in E. histolytica plasma membranes relative to the membrane of the erythrocyte ghost where integral components (the membrane-intercalated particles) contain all antigens, receptors, and anionic sites labeled so far. We conclude that fluidity of integral membrane components (integral membrane fluidity) cannot be inferred from the demonstration of the mobility of surface components nor, conversely, can the fluidity of peripheral membrane components (peripheral membrane fluidity) be assumed from

  1. The α-helical regions of KERP1 are important in Entamoeba histolytica adherence to human cells

    PubMed Central

    Perdomo, Doranda; Baron, Bruno; Rojo-Domínguez, Arturo; Raynal, Bertrand; England, Patrick; Guillén, Nancy

    2013-01-01

    The lysine and glutamic acid rich protein KERP1 is a unique surface adhesion factor associated with virulence in the human pathogen Entamoeba histolytica. Both the function and structure of this protein remain unknown to this date. Here, we used circular dichroism, analytical ultracentrifugation and bioinformatics modeling to characterize the structure of KERP1. Our findings revealed that it is an α-helical rich protein organized as a trimer, endowed with a very high thermal stability (Tm = 89.6°C). Bioinformatics sequence analyses and 3D-structural modeling indicates that KERP1 central segments could account for protein trimerization. Relevantly, expressing the central region of KERP1 in living parasites, impair their capacity to adhere to human cells. Our observations suggest a link between the inhibitory effect of the isolated central region and the structural features of KERP1. PMID:23378906

  2. Molecular and functional characterisation of a stress responsive cysteine protease, EhCP6 from Entamoeba histolytica.

    PubMed

    Ghosh, Anupama; Raha, Sanghamitra

    2015-05-01

    Entamoeba histolytica cysteine protease 6 (EhCP6) is a stress responsive cysteine protease that is upregulated in response to heat shock and during pathogen invasion of the host tissue. In the present study an attempt has been made to express and purify recombinant EhCP6 in order to gain insights into its biochemical properties. The recombinant and refolded protein has been shown to undergo autoproteolysis in the presence of DTT and SDS to give rise to ∼25kDa mature form. The mature form of the protein was found to exhibit a protease activity that is sensitive to E-64, a specific cysteine protease inhibitor. In silico homology modelling of EhCP6 revealed that the protein exhibits conservation of almost all the major structural features of cathepsin-L like cysteine proteases. Further in vivo studies are needed to decipher the function of the protein in response to different stressed conditions.

  3. The monocyte locomotion inhibitory factor produced by Entamoeba histolytica inhibits induced nitric oxide production in human leukocytes.

    PubMed

    Rico, G; Leandro, E; Rojas, S; Giménez, J A; Kretschmer, R R

    2003-07-01

    The monocyte locomotion inhibitory factor, an anti-inflammatory pentapeptide produced by Entamoeba histolytica, inhibits the in vitro production of nitric oxide induced by cytokines (INF-gamma, TNF-alpha) or PMA in human leukocytes. This can be added to the other previously reported functional effects of this factor, such as the inhibition of monocyte locomotion and the synthesis of reactive oxygen intermediates in both monocytes and neutrophils. The decreased nitric oxide production may interfere with the killing of amebas by neutrophils in the early invasive stages of amebiasis, when oxidative mechanisms are used [reactive oxygen and nitrogen intermediates either individually or synergistically via peroxynitrite (ONOO(-))], and in the advanced stages, when both non-oxidative and oxidative (including nitric oxide) mechanisms are employed by macrophages. Diminished nitric oxide production by leukocytes may also contribute to the paucity of late inflammatory components in amebic abscess of the liver and other amebic lesions.

  4. Diagnosis of amebic dysentery by detection of Entamoeba histolytica fecal antigen by an invasive strain-specific, monoclonal antibody-based enzyme-linked immunosorbent assay.

    PubMed Central

    Gonzalez-Ruiz, A; Haque, R; Rehman, T; Aguirre, A; Hall, A; Guhl, F; Warhurst, D C; Miles, M A

    1994-01-01

    An invasive strain-specific monoclonal antibody against Entamoeba histolytica has been used in a capture enzyme-linked immunosorbent assay (ELISA) for the detection of invasive E. histolytica fecal antigen in clinical specimens and for the diagnosis of amebic dysentery in patients from Bangladesh. The fecal antigen capture ELISA (FAC-ELISA) did not cross-react with other parasite species in the clinical specimens or with noninvasive E. histolytica present in those specimens and in experimentally seeded stools. The limit of detection of the assay for invasive E. histolytica crude antigen diluted in phosphate-buffered saline or in stools was 0.58 and 3.9 micrograms/ml, respectively, which is the equivalent of approximately 72 and 487 E. histolytica trophozoites per well, respectively. The sensitivity, specificity, and efficiency of the FAC-ELISA were 87, 100, and 98%, respectively, for the detection of invasive E. histolytica antigens and 100, 100, and 100%, respectively, for the diagnosis of amebic dysentery. The FAC-ELISA is a potential alternative for the field diagnosis of amebic dysentery and for epidemiological studies to define the distribution of invasive E. histolytica. PMID:8027351

  5. Crystallization and preliminary X-ray analysis of RabX3, a tandem GTPase from Entamoeba histolytica.

    PubMed

    Kumar Srivastava, Vijay; Chandra, Mintu; Datta, Sunando

    2014-07-01

    Ras superfamily GTPases regulate signalling pathways that control multiple biological processes by modulating the GTP/GDP cycle. Various Rab GTPases, which are the key regulators of vesicular trafficking pathways, play a vital role in the survival and virulence of the enteric parasite Entamoeba histolytica. The Rab GTPases act as binary molecular switches that utilize the conformational changes associated with the GTP/GDP cycle to elicit responses from target proteins and thereby regulate a broad spectrum of cellular processes including cell proliferation, cytoskeletal assembly, nuclear transport and intracellular membrane trafficking in eukaryotes. Entamoeba histolytica RabX3 (EhRabX3) is a unique GTPase in the amoebic genome, the only member in the eukaryotic Ras superfamily that harbours tandem G-domains and shares only 8-16% sequence identity with other GTPases. Recent studies suggested that EhRabX3 binds to a single guanine nucleotide through its N-terminal G-domain (NTD), while the C-terminal G-domain (CTD) plays a potential role in binding of the nucleotide to the NTD. Thus, understanding the intermolecular regulation between the two GTPase domains is expected to reveal valuable information on the overall action of EhRabX3. To provide structural insights into the inclusive action of this unique GTPase, EhRabX3 was crystallized by successive micro-seeding using the vapour-diffusion method. A complete data set was collected to 3.3 Å resolution using a single native EhRabX3 crystal at 100 K on BM14 at the ESRF, Grenoble, France. The crystal belonged to monoclinic space group C2, with unit-cell parameters a=198.6, b=119.3, c=89.2 Å, β=103.1°. Preliminary analysis of the data using the Matthews Probability Calculator suggested the presence of four to six molecules in the asymmetric unit.

  6. Crystal Structure of Calcium Binding Protein-5 from Entamoeba histolytica and Its Involvement in Initiation of Phagocytosis of Human Erythrocytes

    PubMed Central

    Mazumder, Mohit; Dahiya, Pradeep; Murmu, Aruna; Manjasetty, Babu A.; Zaidi, Rana; Bhattacharya, Alok; Gourinath, S.

    2014-01-01

    Entamoeba histolytica is the etiological agent of human amoebic colitis and liver abscess, and causes a high level of morbidity and mortality worldwide, particularly in developing countries. There are a number of studies that have shown a crucial role for Ca2+ and its binding protein in amoebic biology. EhCaBP5 is one of the EF hand calcium-binding proteins of E. histolytica. We have determined the crystal structure of EhCaBP5 at 1.9 Å resolution in the Ca2+-bound state, which shows an unconventional mode of Ca2+ binding involving coordination to a closed yet canonical EF-hand motif. Structurally, EhCaBP5 is more similar to the essential light chain of myosin than to Calmodulin despite its somewhat greater sequence identity with Calmodulin. This structure-based analysis suggests that EhCaBP5 could be a light chain of myosin. Surface plasmon resonance studies confirmed this hypothesis, and in particular showed that EhCaBP5 interacts with the IQ motif of myosin 1B in calcium independent manner. It also appears from modelling of the EhCaBP5-IQ motif complex that EhCaBP5 undergoes a structural change in order to bind the IQ motif of myosin. This specific interaction was further confirmed by the observation that EhCaBP5 and myosin 1B are colocalized in E. histolytica during phagocytic cup formation. Immunoprecipitation of EhCaBP5 from total E. histolytica cellular extract also pulls out myosin 1B and this interaction was confirmed to be Ca2+ independent. Confocal imaging of E. histolytica showed that EhCaBP5 and myosin 1B are part of phagosomes. Overexpression of EhCaBP5 increases slight rate (∼20%) of phagosome formation, while suppression reduces the rate drastically (∼55%). Taken together, these experiments indicate that EhCaBP5 is likely to be the light chain of myosin 1B. Interestingly, EhCaBP5 is not present in the phagosome after its formation suggesting EhCaBP5 may be playing a regulatory role. PMID:25502654

  7. Crystallization and preliminary X-ray analysis of l-methionine γ-lyase 1 from Entamoeba histolytica

    SciTech Connect

    Sato, Dan; Karaki, Tsuyoshi; Shimizu, Akira; Kamei, Kaeko; Harada, Shigeharu; Nozaki, Tomoyoshi

    2008-08-01

    l-Methionine γ-lyase 1, a key enzyme in sulfur-containing amino-acid degradation, from the protozoan parasite E. histolytica was crystallized in a form suitable for X-ray structure analysis. l-Methionine γ-lyase (MGL) is a pyridoxal phosphate-dependent enzyme that is involved in the degradation of sulfur-containing amino acids. MGL is an attractive drug target against amoebiasis because the mammalian host of its causative agent Entamoeba histolytica lacks MGL. For the development of anti-amoebic agents based on the structure of MGL, one of two MGL isoenzymes (EhMGL1) was crystallized in the monoclinic space group P2{sub 1}, with unit-cell parameters a = 99.12, b = 85.38, c = 115.37 Å, β = 101.82°. The crystals diffract to beyond 2.0 Å resolution. The presence of a tetramer in the asymmetric unit (4 × 42.4 kDa) gives a Matthews coefficient of 2.8 Å{sup 3} Da{sup −1} and a solvent content of 56%. The structure was solved by the molecular-replacement method and structure refinement is now in progress.

  8. Endoplasmic reticulum‐resident Rab8A GTPase is involved in phagocytosis in the protozoan parasite Entamoeba histolytica

    PubMed Central

    Hanadate, Yuki; Saito‐Nakano, Yumiko; Nakada‐Tsukui, Kumiko

    2016-01-01

    Summary Phagocytosis is indispensable for the pathogenesis of the intestinal protozoan parasite Entamoeba histolytica. Here, we showed that in E. histolytica Rab8A, which is generally involved in trafficking from the trans‐Golgi network to the plasma membrane in other organisms but was previously identified in phagosomes of the amoeba in the proteomic analysis, primarily resides in the endoplasmic reticulum (ER) and participates in phagocytosis. We demonstrated that down‐regulation of EhRab8A by small antisense RNA‐mediated transcriptional gene silencing remarkably reduced adherence and phagocytosis of erythrocytes, bacteria and carboxylated latex beads. Surface biotinylation followed by SDS‐PAGE analysis revealed that the surface expression of several proteins presumably involved in target recognition was reduced in the EhRab8A gene‐silenced strain. Further, overexpression of wild‐type EhRab8A augmented phagocytosis, whereas expression of the dominant‐negative form of EhRab8A resulted in reduced phagocytosis. These results indicated that EhRab8A regulates transport of surface receptor(s) for the prey from the ER to the plasma membrane. To our knowledge, this is the first report that the ER‐resident Rab GTPase is involved in phagocytosis through the regulation of trafficking of a surface receptor, supporting a premise of direct involvement of the ER in phagocytosis. PMID:26807810

  9. Tyrosine phosphorylation/dephosphorylation of myosin II essential light chains of Entamoeba histolytica trophozoites regulates their motility.

    PubMed

    Bonilla-Moreno, Raúl; Pérez-Yépez, Eloy-Andrés; Villegas-Sepúlveda, Nicolás; Morales, Fernando O; Meza, Isaura

    2016-08-01

    Entamoeba histolytica trophozoites dwell in the human intestine as comensals although under still unclear circumstances become invasive and destroy the host tissues. For these activities, trophozoites relay on remarkable motility provided by the cytoskeleton organization. Amebic actin and some of its actin-associated proteins are well known, while components of the myosin II molecule, although predicted from the E. histolytica genome, need biochemical and functional characterization. Recently, an amebic essential light myosin II chain, named EhMLCI, was identified and reported to be phosphorylated in tyrosines. The phosphorylated form of the protein was associated with the soluble assembly incompetent conformation of the heavy myosin chains, while the non-phosphorylated protein was identified with filamentous heavy chains, organized in an assembly competent conformation. It was postulated that EhMLCI tyrosine phosphorylation could act as a negative regulator of myosin II activity by its phosphorylation/dephosphorylation cycles. To test this hypothesis, we constructed an expression vector containing an EhMLCI DNA sequence where two tyrosine residues, with strong probability of phosphorylation and fall within the single EF-hand domain that interacts with the N-terminus of myosin II heavy chains, were replaced by phenylalanines. Transfected trophozoites, expressing the mutant MutEhMLCI protein cannot process it, thereby not incorporated into the phosphorylation/dephosphorylation cycles required for myosin II activity, results in motility defective trophozoites.

  10. Multinucleation and Polykaryon Formation is Promoted by the EhPC4 Transcription Factor in Entamoeba histolytica

    PubMed Central

    Cruz, Olga Hernández de la; Marchat, Laurence A.; Guillén, Nancy; Weber, Christian; Rosas, Itzel López; Díaz-Chávez, José; Herrera, Luis; Rojo-Domínguez, Arturo; Orozco, Esther; López-Camarillo, César

    2016-01-01

    Entamoeba histolytica is the intestinal parasite responsible for human amoebiasis that is a leading cause of death in developing countries. In this protozoan, heterogeneity in DNA content, polyploidy and genome plasticity have been associated to alterations in mechanisms controlling DNA replication and cell division. Studying the function of the transcription factor EhPC4, we unexpectedly found that it is functionally related to DNA replication, and multinucleation. Site-directed mutagenesis on the FRFPKG motif revealed that the K127 residue is required for efficient EhPC4 DNA-binding activity. Remarkably, overexpression of EhPC4 significantly increased cell proliferation, DNA replication and DNA content of trophozoites. A dramatically increase in cell size resulting in the formation of giant multinucleated trophozoites (polykaryon) was also found. Multinucleation event was associated to cytokinesis failure leading to abortion of ongoing cell division. Consistently, genome-wide profiling of EhPC4 overexpressing trophozoites revealed the up-regulation of genes involved in carbohydrates and nucleic acids metabolism, chromosome segregation and cytokinesis. Forced overexpression of one of these genes, EhNUDC (nuclear movement protein), led to alterations in cytokinesis and partially recapitulated the multinucleation phenotype. These data indicate for the first time that EhPC4 is associated with events related to polyploidy and genome stability in E. histolytica. PMID:26792358

  11. Recognition of the galactose- or N-acetylgalactosamine-binding lectin of Entamoeba histolytica by human immune sera.

    PubMed Central

    Petri, W A; Joyce, M P; Broman, J; Smith, R D; Murphy, C F; Ravdin, J I

    1987-01-01

    Cure of amebic liver abscess is associated with resistance to recurrent invasive amebiasis and the development of a humoral and cell-mediated immune response. We determined whether human immune sera contain blocking antibody for the 170-kilodalton (kDa) galactose or N-acetylgalactosamine (Gal/GalNAc)-binding lectin of Entamoeba histolytica. By Western blot (immunoblot) of whole amebae subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, all eight immune sera studied here prominently recognized a 170-kDa amebic protein. Western blot of the purified Gal/GalNAc lectin with pooled human immune sera (PHIS) confirmed that the 170-kDa band was the adherence lectin. Immunoprecipitation of [35S]methionine-metabolically-labeled amebae with the antilectin monoclonal antibody H8-5 and with PHIS demonstrated that the 170-kDa lectin was the major antigen recognized by PHIS. The in vitro adherence of E. histolytica trophozoites to CHO cells at 4 degrees C was inhibited by prior exposure of amebae to greater than or equal to 1.0% PHIS. The humoral response to the Gal/GalNAc-binding lectin of the parasite may contribute to the development of protective immunity against invasive amebiasis. Images PMID:2888730

  12. Cysteine protease-binding protein family 6 mediates the trafficking of amylases to phagosomes in the enteric protozoan Entamoeba histolytica.

    PubMed

    Furukawa, Atsushi; Nakada-Tsukui, Kumiko; Nozaki, Tomoyoshi

    2013-05-01

    Phagocytosis plays a pivotal role in nutrient acquisition and evasion from the host defense systems in Entamoeba histolytica, the intestinal protozoan parasite that causes amoebiasis. We previously reported that E. histolytica possesses a unique class of a hydrolase receptor family, designated the cysteine protease-binding protein family (CPBF), that is involved in trafficking of hydrolases to lysosomes and phagosomes, and we have also reported that CPBF1 and CPBF8 bind to cysteine proteases or β-hexosaminidase α-subunit and lysozymes, respectively. In this study, we showed by immunoprecipitation that CPBF6, one of the most highly expressed CPBF proteins, specifically binds to α-amylase and γ-amylase. We also found that CPBF6 is localized in lysosomes, based on immunofluorescence imaging. Immunoblot and proteome analyses of the isolated phagosomes showed that CPBF6 mediates transport of amylases to phagosomes. We also demonstrated that the carboxyl-terminal cytosolic region of CPBF6 is engaged in the regulation of the trafficking of CPBF6 to phagosomes. Our proteome analysis of phagosomes also revealed new potential phagosomal proteins.

  13. Extensive transcriptome analysis correlates the plasticity of Entamoeba histolytica pathogenesis to rapid phenotype changes depending on the environment

    PubMed Central

    Weber, Christian; Koutero, Mikael; Dillies, Marie-Agnes; Varet, Hugo; Lopez-Camarillo, Cesar; Coppée, Jean Yves; Hon, Chung-Chau; Guillén, Nancy

    2016-01-01

    Amoebiasis is a human infectious disease due to the amoeba parasite Entamoeba histolytica. The disease appears in only 20% of the infections. Diversity in phenotypes may occur within the same infectious strain in the gut; for instance, parasites can be commensal (in the intestinal lumen) or pathogenic (inside the tissue). The degree of pathogenesis of clinical isolates varies greatly. These findings raise the hypothesis that genetic derivation may account for amoebic diverse phenotypes. The main goal of this study was to analyse gene expression changes of a single virulent amoebic strain in different environmental contexts where it exhibit different degrees of virulence, namely isolated from humans and maintained through animal liver passages, in contact with the human colon and short or prolonged in vitro culture. The study reveals major transcriptome changes in virulent parasites upon contact with human colon explants, including genes related to sugar metabolism, cytoskeleton rearrangement, stress responses and DNA repair. Furthermore, in long-term cultured parasites, drastic changes in gene expression for proteins with functions for proteasome and tRNA activities were found. Globally we conclude that rapid changes in gene expression rather than genetic derivation can sustain the invasive phenotype of a single virulent isolate of E. histolytica. PMID:27767091

  14. Multinucleation and Polykaryon Formation is Promoted by the EhPC4 Transcription Factor in Entamoeba histolytica.

    PubMed

    Hernández de la Cruz, Olga; Marchat, Laurence A; Guillén, Nancy; Weber, Christian; López Rosas, Itzel; Díaz-Chávez, José; Herrera, Luis; Rojo-Domínguez, Arturo; Orozco, Esther; López-Camarillo, César

    2016-01-21

    Entamoeba histolytica is the intestinal parasite responsible for human amoebiasis that is a leading cause of death in developing countries. In this protozoan, heterogeneity in DNA content, polyploidy and genome plasticity have been associated to alterations in mechanisms controlling DNA replication and cell division. Studying the function of the transcription factor EhPC4, we unexpectedly found that it is functionally related to DNA replication, and multinucleation. Site-directed mutagenesis on the FRFPKG motif revealed that the K127 residue is required for efficient EhPC4 DNA-binding activity. Remarkably, overexpression of EhPC4 significantly increased cell proliferation, DNA replication and DNA content of trophozoites. A dramatically increase in cell size resulting in the formation of giant multinucleated trophozoites (polykaryon) was also found. Multinucleation event was associated to cytokinesis failure leading to abortion of ongoing cell division. Consistently, genome-wide profiling of EhPC4 overexpressing trophozoites revealed the up-regulation of genes involved in carbohydrates and nucleic acids metabolism, chromosome segregation and cytokinesis. Forced overexpression of one of these genes, EhNUDC (nuclear movement protein), led to alterations in cytokinesis and partially recapitulated the multinucleation phenotype. These data indicate for the first time that EhPC4 is associated with events related to polyploidy and genome stability in E. histolytica.

  15. Resveratrol Induces Apoptosis-Like Death and Prevents In Vitro and In Vivo Virulence of Entamoeba histolytica.

    PubMed

    Pais-Morales, Jonnatan; Betanzos, Abigail; García-Rivera, Guillermina; Chávez-Munguía, Bibiana; Shibayama, Mineko; Orozco, Esther

    2016-01-01

    Entamoeba histolytica causes amoebiasis, an infection that kills 100,000 individuals each year. Metronidazole and its derivatives are currently used against this protozoan, but these drugs present adverse effects on human health. Here, we investigated the effect of resveratrol (a natural compound) on E. histolytica trophozoites viability, as well as its influence on the parasite virulence. Trophozoites growth was arrested by 72 μM resveratrol and the IC50 was determined as 220 μM at 48 h. Cells appeared smaller, rounded and in clusters, with debris-containing vacuoles and with abnormally condensed chromatin. Resveratrol triggered reactive oxygen species production. It caused lipid peroxidation and produced phosphatidylserine externalization and DNA fragmentation this latter evidenced by TUNEL assays. It also provoked an increase of intracellular Ca2+ concentration, activated calpain and decreased superoxide dismutase activity, indicating that an apoptosis-like event occurred; however, autophagy was not detected. Cytopathic activity, phagocytosis, encystment and in vivo virulence were diminished dramatically by pre-incubation of trophozoites with resveratrol, evidencing that resveratrol attenuated the trophozoite virulence in vitro. Interestingly, after the inoculation of virulent trophozoites, animals treated with the drug did not develop or developed very small abscesses. Our findings propose that resveratrol could be an alternative to contend amoebiasis.

  16. Entamoeba histolytica P-glycoprotein (EhPgp) inhibition, induce trophozoite acidification and enhance programmed cell death.

    PubMed

    Medel Flores, Olivia; Gómez García, Consuelo; Sánchez Monroy, Virgina; Villalba Magadaleno, José D'Artagnan; Nader García, Elvira; Pérez Ishiwara, D Guillermo

    2013-11-01

    Programmed cell death (PCD) is induced in Entamoeba histolytica by a variety of stimuli in vitro and in vivo. In mammals, intracellular acidification serves as a global switch for inactivating cellular processes and initiates molecular mechanisms implicated in the destruction of the genome. In contrast, intracellular alkalinization produced by P-glycoprotein overexpression in multidrug-resistant cells has been related to apoptosis resistance. Our previous studies showed that overexpression of E. histolytica P-glycoprotein (PGP) altered chloride-dependent currents and triggered trophozoite swelling, the reverse process of cell shrinkage produced during PCD. Here we showed that antisense inhibition of PGP expression produced a synchronous death of trophozoites and the enhancement of biochemical and morphological characteristics of PCD induced by G418. The nucleus was contracted, and the nuclear membrane was disrupted. Moreover, chromatin was extensively fragmented. Ca(2+) concentration was increased, while the intracellular pH (ipH) was acidified. In contrast, PGP overexpression prevented intracellular acidification and circumvented the apoptotic effect of G418.

  17. Resveratrol Induces Apoptosis-Like Death and Prevents In Vitro and In Vivo Virulence of Entamoeba histolytica

    PubMed Central

    Pais-Morales, Jonnatan; Betanzos, Abigail; García-Rivera, Guillermina; Chávez-Munguía, Bibiana; Shibayama, Mineko; Orozco, Esther

    2016-01-01

    Entamoeba histolytica causes amoebiasis, an infection that kills 100,000 individuals each year. Metronidazole and its derivatives are currently used against this protozoan, but these drugs present adverse effects on human health. Here, we investigated the effect of resveratrol (a natural compound) on E. histolytica trophozoites viability, as well as its influence on the parasite virulence. Trophozoites growth was arrested by 72 μM resveratrol and the IC50 was determined as 220 μM at 48 h. Cells appeared smaller, rounded and in clusters, with debris-containing vacuoles and with abnormally condensed chromatin. Resveratrol triggered reactive oxygen species production. It caused lipid peroxidation and produced phosphatidylserine externalization and DNA fragmentation this latter evidenced by TUNEL assays. It also provoked an increase of intracellular Ca2+ concentration, activated calpain and decreased superoxide dismutase activity, indicating that an apoptosis-like event occurred; however, autophagy was not detected. Cytopathic activity, phagocytosis, encystment and in vivo virulence were diminished dramatically by pre-incubation of trophozoites with resveratrol, evidencing that resveratrol attenuated the trophozoite virulence in vitro. Interestingly, after the inoculation of virulent trophozoites, animals treated with the drug did not develop or developed very small abscesses. Our findings propose that resveratrol could be an alternative to contend amoebiasis. PMID:26731663

  18. Virtual Screening, Identification and In Vitro Testing of Novel Inhibitors of O-Acetyl-L-Serine Sulfhydrylase of Entamoeba histolytica

    PubMed Central

    Nagpal, Isha; Raj, Isha; Subbarao, Naidu; Gourinath, Samudrala

    2012-01-01

    The explosive epidemicity of amoebiasis caused by the facultative gastrointestinal protozoan parasite Entamoeba histolytica is a major public health problem in developing countries. Multidrug resistance and side effects of various available antiamoebic drugs necessitate the design of novel antiamobeic agents. The cysteine biosynthetic pathway is the critical target for drug design due to its significance in the growth, survival and other cellular activities of E. histolytica. Here, we have screened 0.15 million natural compounds from the ZINC database against the active site of the EhOASS enzyme (PDB ID. 3BM5, 2PQM), whose structure we previously determined to 2.4 Å and 1.86 Å resolution. For this purpose, the incremental construction algorithm of GLIDE and the genetic algorithm of GOLD were used. We analyzed docking results for top ranking compounds using a consensus scoring function of X-Score to calculate the binding affinity and using ligplot to measure protein-ligand interactions. Fifteen compounds that possess good inhibitory activity against EhOASS active site were identified that may act as potential high affinity inhibitors. In vitro screening of a few commercially available compounds established their biological activity. The first ranked compound ZINC08931589 had a binding affinity of ∼8.05 µM and inhibited about 73% activity at 0.1 mM concentration, indicating good correlation between in silico prediction and in vitro inhibition studies. This compound is thus a good starting point for further development of strong inhibitors. PMID:22355310

  19. Virtual screening, identification and in vitro testing of novel inhibitors of O-acetyl-L-serine sulfhydrylase of Entamoeba histolytica.

    PubMed

    Nagpal, Isha; Raj, Isha; Subbarao, Naidu; Gourinath, Samudrala

    2012-01-01

    The explosive epidemicity of amoebiasis caused by the facultative gastrointestinal protozoan parasite Entamoeba histolytica is a major public health problem in developing countries. Multidrug resistance and side effects of various available antiamoebic drugs necessitate the design of novel antiamobeic agents. The cysteine biosynthetic pathway is the critical target for drug design due to its significance in the growth, survival and other cellular activities of E. histolytica. Here, we have screened 0.15 million natural compounds from the ZINC database against the active site of the EhOASS enzyme (PDB ID. 3BM5, 2PQM), whose structure we previously determined to 2.4 Å and 1.86 Å resolution. For this purpose, the incremental construction algorithm of GLIDE and the genetic algorithm of GOLD were used. We analyzed docking results for top ranking compounds using a consensus scoring function of X-Score to calculate the binding affinity and using ligplot to measure protein-ligand interactions. Fifteen compounds that possess good inhibitory activity against EhOASS active site were identified that may act as potential high affinity inhibitors. In vitro screening of a few commercially available compounds established their biological activity. The first ranked compound ZINC08931589 had a binding affinity of ∼8.05 µM and inhibited about 73% activity at 0.1 mM concentration, indicating good correlation between in silico prediction and in vitro inhibition studies. This compound is thus a good starting point for further development of strong inhibitors.

  20. Crystal structure of the cysteine protease inhibitor 2 from Entamoeba histolytica: Functional convergence of a common protein fold

    SciTech Connect

    Casados-Vázquez, Luz E.; Lara-González, Samuel; Brieb, Luis G.

    2012-04-18

    Cysteine proteases (CP) are key pathogenesis and virulence determinants of protozoan parasites. Entamoeba histolytica contains at least 50 cysteine proteases; however, only three (EhCP1, EhCP2 and EhCP5) are responsible for approximately 90% of the cysteine protease activity in this parasite. CPs are expressed as inactive zymogens. Because the processed proteases are potentially cytotoxic, protozoan parasites have developed mechanisms to regulate their activity. Inhibitors of cysteine proteases (ICP) of the chagasin-like inhibitor family (MEROPS family I42) were recently identified in bacteria and protozoan parasites. E. histolytica contains two ICP-encoding genes of the chagasin-like inhibitor family. EhICP1 localizes to the cytosol, whereas EhICP2 is targeted to phagosomes. Herein, we report two crystal structures of EhICP2. The overall structure of EhICP2 consists of eight {beta}-strands and closely resembles the immunoglobulin fold. A comparison between the two crystal forms of EhICP2 indicates that the conserved BC, DE and FG loops form a flexible wedge that may block the active site of CPs. The positively charged surface of the wedge-forming loops in EhICP2 contrasts with the neutral surface of the wedge-forming loops in chagasin. We postulate that the flexibility and positive charge observed in the DE and FG loops of EhICP2 may be important to facilitate the initial binding of this inhibitor to the battery of CPs present in E. histolytica.

  1. Modulation of endogenous Cysteine Protease Inhibitor (ICP) 1 expression in Entamoeba histolytica affects amoebic adhesion to Extracellular Matrix proteins.

    PubMed

    Lee, Young Ah; Saito-Nakano, Yumiko; Kim, Kyeong Ah; Min, Arim; Nozaki, Tomoyoshi; Shin, Myeong Heon

    2015-02-01

    Entamoeba histolytica is an enteric tissue-invading protozoan parasite that causes amoebic colitis and occasionally liver abscess in humans. During tissue invasion, amoebic adhesion to host components is an important event for host cell death leading to successful invasion and infection. Among amoebic virulence factors, Gal/GalNAc lectin is known to be major adhesion factor to host cells. In this study, we investigated the role of amoebic secreted CP (Cysteine Proteases) in amoebic adhesion to extracellular matrix (ECM) protein using CP inhibitor and E. histolytica strains in which the endogenous inhibitor of cysteine protease (ICP) 1 gene was overexpressed (ICP1(+)) or repressed by antisense small RNA-mediated gene silencing (ICP1(-)). We found that pretreatment of wild-type amoebae with CP inhibitor E64, or thiol-group modifiers such as diamide and N-Ethylmaleimide resulted in a significant decrease in adhesion to laminin and collagen ECM proteins. Furthermore, ICP1(+) strain, with a reduction of secreted CP activity, exhibited reduced ability by 40% to adhere to laminin. In contrast, ICP1(-) strain, with a 1.9-fold increase of secreted CP activity, showed a two-fold increase in amoebic adherence to laminin compared to the control strain. In addition, total amount of secreted CP5 was decreased in ICP1(+) amoeba. Conversely, total amount of secreted CP1 and mature-form CP5 were increased in ICP1(-) amoeba. We also found that ICP1 was secreted into extracellular milieu. These results suggest that secreted CP activity by E. histolytica may be an important factor affecting adhesion to host proteins, and regulation of CP secretion by ICP plays a major role in pathogenesis. This study provides insight into the CP-mediated tissue pathogenesis in amoeba-invaded lesions during human amoebiasis.

  2. Entamoeba histolytica rhomboid protease 1 has a role in migration and motility as validated by two independent genetic approaches.

    PubMed

    Rastew, Elena; Morf, Laura; Singh, Upinder

    2015-07-01

    Rhomboid proteins represent a recently discovered family of intramembrane proteases present in a broad range of organisms and with increasing links to human diseases. The enteric parasite Entamoeba histolytica has evolved multiple mechanisms to adapt to the human host environment and establish infection. Our recent studies identified EhROM1 as a functional E. histolytica rhomboid protease with roles in adhesion to and phagocytosis of host cells. Since those studies were performed in a non-virulent strain, roles in parasite virulence could not be assessed. We focused this study on the comparison and validation of two genetic manipulation techniques: overexpression of a dominant-negative catalytic mutant of EhROM1 and knock down of EhROM1 using a RNAi-based silencing approach followed by functional studies of phenotypic analyses in virulent parasites. Both the EhROM1 catalytic mutant and parasites with EhROM1 downregulation were reduced in cytotoxicity, hemolytic activity, and directional and non-directional transwell migration. Importantly, the role for EhROM1 in cell migration mimics similar roles for rhomboid proteases from mammalian and apicomplexan systems. However, the EhROM1 catalytic mutant and EhROM1 downregulation parasites had different phenotypes for erythrophagocytosis, while complement resistance was not affected in either strain. In summary, in this study we genetically manipulated E. histolytica rhomboid protease EhROM1 by two different approaches and identified similarly attenuated phenotypes by both approaches, suggesting a novel role for EhROM1 in amebic motility.

  3. Prevalence of Dientamoeba fragilis, Giardia duodenalis, Entamoeba histolytica/dispar, and Cryptosporidium spp in Da Nang, Vietnam, detected by a multiplex real-time PCR.

    PubMed

    Ögren, Jessica; Van Nguyen, Song; Nguyen, Minh Khac; Dimberg, Jan; Matussek, Andreas

    2016-06-01

    We surveyed the prevalence of Dientamoeba fragilis, Giardia duodenalis, Entamoeba histolytica, Entamoeba dispar, and Cryptosporidium spp in individuals with and without gastrointestinal (GI) symptoms residing in and around Da Nang city, Vietnam. Fecal samples were collected from children (n = 100) and adults (n = 80) with GI symptoms and from healthy individuals (n = 88) reporting no GI symptoms. Parasite detection was performed by multiplex real-time PCR. Overall, except for G. duodenalis, we found a low prevalence (<5%) of D. fragilis and E. dispar and no detection of E. histolytica and C. spp in all participants with GI symptoms. Specifically for D. fragilis this contrasts with findings in European populations of children with GI symptoms showing prevalence up to 73%. Moreover, our results indicate that the prevalence of G. duodenalis is higher in patients with GI symptoms compared to asymptomatic individuals and this difference is most obvious in young patients.

  4. Evaluation of factors affecting real-time PCR performance for diagnosis of Entamoeba histolytica and Entamoeba dispar in clinical stool samples.

    PubMed

    Forsell, Joakim; Koskiniemi, Satu; Hedberg, Ida; Edebro, Helén; Evengård, Birgitta; Granlund, Margareta

    2015-09-01

    Although PCR offers the potential for sensitive detection of parasites there are several pitfalls for optimal performance, especially when DNA is extracted from a complex sample material such as stool. With the aid of a sensitive inhibitor control in a duplex real-time PCR (qPCR) for identification of Entamoeba histolytica and Entamoeba dispar we have evaluated factors that influenced the performance of the qPCR and have suggested a rationale to be used in the analysis of clinical samples. Pre-PCR processing was found to be of outmost importance for an optimal amplification since inhibitors caused false-negative results when higher amounts of sample were used. Stool sampling with a flocked swab (ESwab, Copan), yielding on average 173 mg, gave positive qPCR results in samples with cysts of E. dispar that were negative in serially diluted stool samples. The degree of inhibition found varied between samples and was not an on-off phenomenon. Even low-grade inhibition, shown as an increase of two cycles in the qPCR for the inhibitor control, could lead to false negativity in samples with low amounts of parasites. Lack of amplification in the qPCR due to inhibition could be overcome by dilution of the extracted DNA by 1/10-1/20. We also describe the use of guanidinium thiocyanate buffer for transport and storage of samples as well as a time-saving semi-automated DNA extraction method in an Arrow instrument (Nordiag) preceded by bead beating.

  5. A new in vitro model of Entamoeba histolytica adhesion, using the human colon carcinoma cell line Caco-2: scanning electron microscopic study.

    PubMed Central

    Rigothier, M C; Coconnier, M H; Servin, A L; Gayral, P

    1991-01-01

    The human colon carcinoma cell line Caco-2, which is widely used to study the adhesion and cytotoxicity of enterobacteria, was used to investigate the adhesion of the trophozoites of Entamoeba histolytica. We observed a high percentage of adhesion of amoebae to Caco-2 cells. Scanning electron microscopy showed that amoebial membrane structures were involved in adhesion and the cytolytic action. These differentiated cells should prove to be a useful model system for investigation of the pathogenic action of amoebae. Images PMID:1937772

  6. First report of Entamoeba histolytica infection from Timor-Leste--acute amoebic colitis and concurrent late development of amoebic liver abscess in returned travellers to Australia.

    PubMed

    Nourse, Clare B; Robson, Jennifer M; Whitby, Michael R; Francis, Josh R

    2016-02-01

    This communication reports invasive amoebic colitis and late onset amoebic liver abscess in three members of a group of 12 Australian travellers to Timor-Leste (TL). This is the first report of Entamoeba histolytica infection from TL. Clinicians in Australia need to consider amoebiasis in the differential diagnosis in travellers returning with colitis, abdominal pain and fever. Presentation with amoebic liver abscess months after exposure is rare but should be suspected in symptomatic individuals with a relevant history of travel.

  7. A novel C-terminal homologue of Aha1 co-chaperone binds to heat shock protein 90 and stimulates its ATPase activity in Entamoeba histolytica.

    PubMed

    Singh, Meetali; Shah, Varun; Tatu, Utpal

    2014-04-17

    Cytosolic heat shock protein 90 (Hsp90) has been shown to be essential for many infectious pathogens and is considered a potential target for drug development. In this study, we have carried out biochemical characterization of Hsp90 from a poorly studied protozoan parasite of clinical importance, Entamoeba histolytica. We have shown that Entamoeba Hsp90 can bind to both ATP and its pharmacological inhibitor, 17-AAG (17-allylamino-17-demethoxygeldanamycin), with Kd values of 365.2 and 10.77 μM, respectively, and it has a weak ATPase activity with a catalytic efficiency of 4.12×10(-4) min(-1) μM(-1). Using inhibitor 17-AAG, we have shown dependence of Entamoeba on Hsp90 for its growth and survival. Hsp90 function is regulated by various co-chaperones. Previous studies suggest a lack of several important co-chaperones in E. histolytica. In this study, we describe the presence of a novel homologue of co-chaperone Aha1 (activator of Hsp90 ATPase), EhAha1c, lacking a canonical Aha1 N-terminal domain. We also show that EhAha1c is capable of binding and stimulating ATPase activity of EhHsp90. In addition to highlighting the potential of Hsp90 inhibitors as drugs against amoebiasis, our study highlights the importance of E. histolytica in understanding the evolution of Hsp90 and its co-chaperone repertoire.

  8. Influence of bacteria upon cytopathic effect and erythrophagocytosis of different axenic strains of Entamoeba histolytica.

    PubMed

    Gomes, M A; Martins, M S; Costa, A O; Silva, E F

    1995-01-01

    At this moment, the duality of species suggested for E. histolytica is being considered for discussion. In order to contribute to settling this question, we investigated the possibility of conversion of avirulent ameba to virulent ones, as well as, the possibility of increasing virulence of virulent strains, by means of association with bacteria. Five strains of E. histolytica were employed, two of them regarded as avirulent and three virulent ones. Amebas were associated with the bacteria Escherichia coli 055 and 0115, previously demonstrated as capable to modify the pathogenic behavior of E. histolytica. Changes in virulence of amebas were assessed by cytopathic effect upon cultured mammal cells and erythrophagocytosis. The virulence of pathogenic strains was significantly increased after bacteria association in opposition to what was observed for nonpathogenic ones, which were not influenced by bacteria association.

  9. Proteomic Identification of Oxidized Proteins in Entamoeba histolytica by Resin-Assisted Capture: Insights into the Role of Arginase in Resistance to Oxidative Stress.

    PubMed

    Shahi, Preeti; Trebicz-Geffen, Meirav; Nagaraja, Shruti; Alterzon-Baumel, Sharon; Hertz, Rivka; Methling, Karen; Lalk, Michael; Ankri, Serge

    2016-01-01

    Entamoeba histolytica is an obligate protozoan parasite of humans, and amebiasis, an infectious disease which targets the intestine and/or liver, is the second most common cause of human death due to a protozoan after malaria. Although amebiasis is usually asymptomatic, E. histolytica has potent pathogenic potential. During host infection, the parasite is exposed to reactive oxygen species that are produced and released by cells of the innate immune system at the site of infection. The ability of the parasite to survive oxidative stress (OS) is essential for a successful invasion of the host. Although the effects of OS on the regulation of gene expression in E. histolytica and the characterization of some proteins whose function in the parasite's defense against OS have been previously studied, our knowledge of oxidized proteins in E. histolytica is lacking. In order to fill this knowledge gap, we performed a large-scale identification and quantification of the oxidized proteins in oxidatively stressed E. histolytica trophozoites using resin-assisted capture coupled to mass spectrometry. We detected 154 oxidized proteins (OXs) and the functions of some of these proteins were associated with antioxidant activity, maintaining the parasite's cytoskeleton, translation, catalysis, and transport. We also found that oxidation of the Gal/GalNAc impairs its function and contributes to the inhibition of E. histolytica adherence to host cells. We also provide evidence that arginase, an enzyme which converts L-arginine into L-ornithine and urea, is involved in the protection of the parasite against OS. Collectively, these results emphasize the importance of OS as a critical regulator of E. histolytica's functions and indicate a new role for arginase in E. histolytica's resistance to OS.

  10. Proteomic Identification of Oxidized Proteins in Entamoeba histolytica by Resin-Assisted Capture: Insights into the Role of Arginase in Resistance to Oxidative Stress

    PubMed Central

    Shahi, Preeti; Trebicz-Geffen, Meirav; Nagaraja, Shruti; Alterzon-Baumel, Sharon; Hertz, Rivka; Methling, Karen; Lalk, Michael; Ankri, Serge

    2016-01-01

    Entamoeba histolytica is an obligate protozoan parasite of humans, and amebiasis, an infectious disease which targets the intestine and/or liver, is the second most common cause of human death due to a protozoan after malaria. Although amebiasis is usually asymptomatic, E. histolytica has potent pathogenic potential. During host infection, the parasite is exposed to reactive oxygen species that are produced and released by cells of the innate immune system at the site of infection. The ability of the parasite to survive oxidative stress (OS) is essential for a successful invasion of the host. Although the effects of OS on the regulation of gene expression in E. histolytica and the characterization of some proteins whose function in the parasite's defense against OS have been previously studied, our knowledge of oxidized proteins in E. histolytica is lacking. In order to fill this knowledge gap, we performed a large-scale identification and quantification of the oxidized proteins in oxidatively stressed E. histolytica trophozoites using resin-assisted capture coupled to mass spectrometry. We detected 154 oxidized proteins (OXs) and the functions of some of these proteins were associated with antioxidant activity, maintaining the parasite's cytoskeleton, translation, catalysis, and transport. We also found that oxidation of the Gal/GalNAc impairs its function and contributes to the inhibition of E. histolytica adherence to host cells. We also provide evidence that arginase, an enzyme which converts L-arginine into L-ornithine and urea, is involved in the protection of the parasite against OS. Collectively, these results emphasize the importance of OS as a critical regulator of E. histolytica's functions and indicate a new role for arginase in E. histolytica's resistance to OS. PMID:26735309

  11. Biochemical and kinetic characterization of the recombinant ADP-forming acetyl coenzyme A synthetase from the amitochondriate protozoan Entamoeba histolytica.

    PubMed

    Jones, Cheryl P; Ingram-Smith, Cheryl

    2014-12-01

    Entamoeba histolytica, an amitochondriate protozoan parasite that relies on glycolysis as a key pathway for ATP generation, has developed a unique extended PPi-dependent glycolytic pathway in which ADP-forming acetyl-coenzyme A (CoA) synthetase (ACD; acetate:CoA ligase [ADP-forming]; EC 6.2.1.13) converts acetyl-CoA to acetate to produce additional ATP and recycle CoA. We characterized the recombinant E. histolytica ACD and found that the enzyme is bidirectional, allowing it to potentially play a role in ATP production or in utilization of acetate. In the acetate-forming direction, acetyl-CoA was the preferred substrate and propionyl-CoA was used with lower efficiency. In the acetyl-CoA-forming direction, acetate was the preferred substrate, with a lower efficiency observed with propionate. The enzyme can utilize both ADP/ATP and GDP/GTP in the respective directions of the reaction. ATP and PPi were found to inhibit the acetate-forming direction of the reaction, with 50% inhibitory concentrations of 0.81 ± 0.17 mM (mean ± standard deviation) and 0.75 ± 0.20 mM, respectively, which are both in the range of their physiological concentrations. ATP and PPi displayed mixed inhibition versus each of the three substrates, acetyl-CoA, ADP, and phosphate. This is the first example of regulation of ACD enzymatic activity, and possible roles for this regulation are discussed.

  12. Novel transmembrane receptor involved in phagosome transport of lysozymes and β-hexosaminidase in the enteric protozoan Entamoeba histolytica.

    PubMed

    Furukawa, Atsushi; Nakada-Tsukui, Kumiko; Nozaki, Tomoyoshi

    2012-02-01

    Lysozymes and hexosaminidases are ubiquitous hydrolases in bacteria and eukaryotes. In phagocytic lower eukaryotes and professional phagocytes from higher eukaryotes, they are involved in the degradation of ingested bacteria in phagosomes. In Entamoeba histolytica, which is the intestinal protozoan parasite that causes amoebiasis, phagocytosis plays a pivotal role in the nutrient acquisition and the evasion from the host defense systems. While the content of phagosomes and biochemical and physiological roles of the major phagosomal proteins have been established in E. histolytica, the mechanisms of trafficking of these phagosomal proteins, in general, remain largely unknown. In this study, we identified and characterized for the first time the putative receptor/carrier involved in the transport of the above-mentioned hydrolases to phagosomes. We have shown that the receptor, designated as cysteine protease binding protein family 8 (CPBF8), is localized in lysosomes and mediates transport of lysozymes and β-hexosaminidase α-subunit to phagosomes when the amoeba ingests mammalian cells or Gram-positive bacillus Clostridium perfringens. We have also shown that the binding of CPBF8 to the cargos is mediated by the serine-rich domain, more specifically three serine residues of the domain, which likely contains trifluoroacetic acid-sensitive O-phosphodiester-linked glycan modifications, of CPBF8. We further showed that the repression of CPBF8 by gene silencing reduced the lysozyme and β-hexosaminidase activity in phagosomes and delayed the degradation of C. perfringens. Repression of CPBF8 also resulted in decrease in the cytopathy against the mammalian cells, suggesting that CPBF8 may also be involved in, besides the degradation of ingested bacteria, the pathogenesis against the mammalian hosts. This work represents the first case of the identification of a transport receptor of hydrolytic enzymes responsible for the degradation of microorganisms in phagosomes.

  13. Molecular and structural characterization of NADPH-dependent d-glycerate dehydrogenase from the enteric parasitic protist Entamoeba histolytica.

    PubMed Central

    Ali, Vahab; Shigeta, Yasuo; Nozaki, Tomoyoshi

    2003-01-01

    Putative NADPH-dependent GDH (L-glycerate dehydrogenase) of the protozoan parasite Entamoeba histolytica (EhGDH) has been characterized. The EhGDH gene encodes a protein of 318 amino acids with a calculated isoelectric point of 6.29 and a molecular mass of 35.8 kDa. EhGDH showed highest identities with GDH from epsilon-proteobacteria. This close kinship was also supported by phylogenetic analyses, suggesting possible lateral transfer of the gene from epsilon-proteobacteria to E. histolytica. In contrast with the implications from protein alignment and phylogenetic analysis, kinetic studies revealed that the amoebic GDH showed biochemical properties similar to those of mammalian GDH, i.e. a preference for NADPH as cofactor and higher affinities towards NADPH and beta-hydroxypyruvate than towards NADP+ and L-glycerate. Whereas the amino acids involved in nucleotide binding and catalysis are totally conserved in EhGDH, substitution of a negatively charged amino acid with a non-charged hydroxy-group-containing amino acid is probably responsible for the observed high affinity of EhGDH for NADP+/NADPH. In addition, the amoebic GDH, dissimilar to the bacterial and mammalian GDHs, lacks glyoxylate reductase activity. Native and recombinant EhGDH showed comparable subunit structure, kinetic parameters and elution profiles on anion-exchange chromatography. We propose that the GDH enzyme is likely to be involved in regulation of the intracellular concentration of serine, and, thus, also in controlling cysteine biosynthesis located downstream of serine metabolic pathways in this protist. PMID:12877657

  14. RNAi pathway genes are resistant to small RNA mediated gene silencing in the protozoan parasite Entamoeba histolytica.

    PubMed

    Pompey, Justine M; Morf, Laura; Singh, Upinder

    2014-01-01

    The RNA interference pathway in the protist Entamoeba histolytica plays important roles in permanent gene silencing as well as in the regulation of virulence determinants. Recently, a novel RNA interference (RNAi)-based silencing technique was developed in this parasite that uses a gene endogenously silenced by small RNAs as a "trigger" to induce silencing of other genes that are fused to it. Fusion to a trigger gene induces the production of gene-specific antisense small RNAs, resulting in robust and permanent silencing of the cognate gene. This approach has silenced multiple genes including those involved in virulence and transcriptional regulation. We now demonstrate that all tested genes of the amebic RNAi pathway are unable to be silenced using the trigger approach, including Argonaute genes (Ago2-1, Ago2-2, and Ago2-3), RNaseIII, and RNA-dependent RNA polymerase (RdRP). In all situations (except for RdRP), fusion to a trigger successfully induces production of gene-specific antisense small RNAs to the cognate gene. These small RNAs are capable of silencing a target gene in trans, indicating that they are functional; despite this, however, they cannot silence the RNAi pathway genes. Interestingly, when a trigger is fused to RdRP, small RNA induction to RdRP does not occur, a unique phenotype hinting that either RdRP is highly resistant to being a target of small RNAs or that small RNA generation may be controlled by RdRP. The inability of the small RNA pathway to silence RNAi genes in E. histolytica, despite the generation of functional small RNAs to these loci suggest that epigenetic factors may protect certain genomic loci and thus determine susceptibility to small RNA mediated silencing.

  15. The Antiretroviral Lectin Cyanovirin-N Targets Well-Known and Novel Targets on the Surface of Entamoeba histolytica Trophozoites ▿ †

    PubMed Central

    Carpentieri, Andrea; Ratner, Daniel M.; Ghosh, Sudip K.; Banerjee, Sulagna; Bushkin, G. Guy; Cui, Jike; Lubrano, Michael; Steffen, Martin; Costello, Catherine E.; O'Keefe, Barry; Robbins, Phillips W.; Samuelson, John

    2010-01-01

    Entamoeba histolytica, the protist that causes amebic dysentery and liver abscess, has a truncated Asn-linked glycan (N-glycan) precursor composed of seven sugars (Man5GlcNAc2). Here, we show that glycoproteins with unmodified N-glycans are aggregated and capped on the surface of E. histolytica trophozoites by the antiretroviral lectin cyanovirin-N and then replenished from large intracellular pools. Cyanovirin-N cocaps the Gal/GalNAc adherence lectin, as well as glycoproteins containing O-phosphodiester-linked glycans recognized by an anti-proteophosphoglycan monoclonal antibody. Cyanovirin-N inhibits phagocytosis by E. histolytica trophozoites of mucin-coated beads, a surrogate assay for amebic virulence. For technical reasons, we used the plant lectin concanavalin A rather than cyanovirin-N to enrich secreted and membrane proteins for mass spectrometric identification. E. histolytica glycoproteins with occupied N-glycan sites include Gal/GalNAc lectins, proteases, and 17 previously hypothetical proteins. The latter glycoproteins, as well as 50 previously hypothetical proteins enriched by concanavalin A, may be vaccine targets as they are abundant and unique. In summary, the antiretroviral lectin cyanovirin-N binds to well-known and novel targets on the surface of E. histolytica that are rapidly replenished from large intracellular pools. PMID:20852023

  16. Association between allelic variation due to short tandem repeats in tRNA gene of Entamoeba histolytica and clinical phenotypes of amoebiasis.

    PubMed

    Jaiswal, Virendra; Ghoshal, Ujjala; Mittal, Balraj; Dhole, Tapan N; Ghoshal, Uday C

    2014-05-01

    Genotypes of Entamoeba histolytica (E. histolytica) may contribute clinical phenotypes of amoebiasis such as amoebic liver abscess (ALA), dysentery and asymptomatic cyst passers state. Hence, we evaluated allelic variation due to short tandem repeats (STRs) in tRNA gene of E. histolytica and clinical phenotypes of amoebiasis. Asymptomatic cyst passers (n=24), patients with dysentery (n=56) and ALA (n=107) were included. Extracted DNA from stool (dysentery, asymptomatic cyst passers) and liver aspirate was amplified using 6 E. histolytica specific tRNA-linked STRs (D-A, A-L, N-K2, R-R, S-Q, and S(TGA)-D) primers. PCR products were subjected to sequencing. Association between allelic variation and clinical phenotypes was analyzed. A total of 9 allelic variations were found in D-A, 8 in A-L, 4 in N-K2, 5 in R-R, 10 in S(TAG)-D and 7 in S-Q loci. A significant association was found between allelic variants and clinical phenotypes of amoebiasis. This study reveals that allelic variation due to short tandem repeats (STRs) in tRNA gene of E. histolytica is associated different clinical outcome of amoebiasis.

  17. Influence of parasite density and sample storage time on the reliability of Entamoeba histolytica-specific PCR from formalin-fixed and paraffin-embedded tissues.

    PubMed

    Frickmann, Hagen; Tenner-Racz, Klara; Eggert, Petra; Schwarz, Norbert G; Poppert, Sven; Tannich, Egbert; Hagen, Ralf M

    2013-12-01

    We report on the reliability of polymerase chain reaction (PCR) for the detection of Entamoeba histolytica from formalin-fixed, paraffin-embedded tissue in comparison with microscopy and have determined predictors that may influence PCR results. E. histolytica-specific and Entamoeba dispar-specific real-time PCR and microscopy from adjacent histologic sections were performed using a collection of formalin-fixed, paraffin-embedded tissue specimens obtained from patients with invasive amebiasis. Specimens had been collected during the previous 4 decades. Association of sample age, parasite density, and reliability of PCR was analyzed. E. histolytica PCR was positive in 20 of 34 biopsies (58.8%); 2 of these 20 were microscopically negative for amebae in neighboring tissue sections. PCR was negative in 9 samples with visible amebae in neighboring sections and in 5 samples without visible parasites in neighboring sections. PCR was negative in all specimens that were older than 3 decades. Low parasite counts and sample ages older than 20 years were predictors for false-negative PCR results. All samples were negative for E. dispar DNA. PCR is suitable for the detection of E. histolytica in formalin-fixed, paraffin-embedded tissue samples that are younger than 2 decades and that contain intermediate to high parasite numbers. Negative results in older samples were due to progressive degradation of DNA over time as indicated by control PCRs targeting the human 18S rRNA gene. Moreover, our findings support previous suggestions that only E. histolytica but not E. dispar is responsible for invasive amebiasis.

  18. Entamoeba histolytica Cysteine Proteinase 5 Evokes Mucin Exocytosis from Colonic Goblet Cells via αvβ3 Integrin

    PubMed Central

    Cornick, Steve; Moreau, France; Chadee, Kris

    2016-01-01

    Critical to the pathogenesis of intestinal amebiasis, Entamoeba histolytica (Eh) induces mucus hypersecretion and degrades the colonic mucus layer at the site of invasion. The parasite component(s) responsible for hypersecretion are poorly defined, as are regulators of mucin secretion within the host. In this study, we have identified the key virulence factor in live Eh that elicits the fast release of mucin by goblets cells as cysteine protease 5 (EhCP5) whereas, modest mucus secretion occurred with secreted soluble EhCP5 and recombinant CP5. Coupling of EhCP5-αvβ3 integrin on goblet cells facilitated outside-in signaling by activating SRC family kinases (SFK) and focal adhesion kinase that resulted in the activation/phosphorlyation of PI3K at the site of Eh contact and production of PIP3. PKCδ was activated at the EhCP5-αvβ3 integrin contact site that specifically regulated mucin secretion though the trafficking vesicle marker myristoylated alanine-rich C-kinase substrate (MARCKS). This study has identified that EhCP5 coupling with goblet cell αvβ3 receptors can initiate a signal cascade involving PI3K, PKCδ and MARCKS to drive mucin secretion from goblet cells critical in disease pathogenesis. PMID:27073869

  19. Amino acid residues Leu135 and Tyr236 are required for RNA binding activity of CFIm25 in Entamoeba histolytica.

    PubMed

    Ospina-Villa, Juan David; Zamorano-Carrillo, Absalom; Lopez-Camarillo, Cesar; Castañon-Sanchez, Carlos A; Soto-Sanchez, Jacqueline; Ramirez-Moreno, Esther; Marchat, Laurence A

    2015-08-01

    Pre-mRNA 3' end processing in the nucleus is essential for mRNA stability, efficient nuclear transport, and translation in eukaryotic cells. In Human, the cleavage/polyadenylation machinery contains the 25 kDa subunit of the Cleavage Factor Im (CFIm25), which specifically recognizes two UGUA elements and regulates the assembly of polyadenylation factors, poly(A) site selection and polyadenylation. In Entamoeba histolytica, the protozoan parasite responsible for human amoebiasis, EhCFIm25 has been reported as a RNA binding protein that interacts with the Poly(A) Polymerase. Here, we follow-up with the study of EhCFIm25 to characterize its interaction with RNA. Using in silico strategy, we identified Leu135 and Tyr236 in EhCFIm25 as conserved amino acids among CFIm25 homologues. We therefore generated mutant EhCFIm25 proteins to investigate the role of these residues for RNA interaction. Results showed that RNA binding activity was totally abrogated when Leu135 and Tyr236 were replaced with Ala residue, and Tyr236 was changed for Phe. In contrast, RNA binding activity was less affected when Leu135 was substituted by Thr. Our data revealed for the first time -until we know-the functional relevance of the conserved Leu135 and Tyr236 in EhCFIm25 for RNA binding activity. They also gave some insights about the possible chemical groups that could be interacting with the RNA molecule.

  20. Role of EhRab7A in phagocytosis of type 1 fimbriated E. coli by Entamoeba histolytica.

    PubMed

    Verma, Kuldeep; Nozaki, Tomoyoshi; Datta, Sunando

    2016-12-01

    Entamoeba histolytica, the causative agent of amoebic colitis and liver abscess in human, ingests the intestinal bacteria and variety of host cells. Phagocytosis of bacteria by the amebic trophozoite has been reported to be important for the virulence of the parasite. Here, we set out to characterize different stages of phagocytosis of type 1 E. coli and investigated the role of a set of amoebic Rab GTPases in the process. The localizations of the Rab GTPases during different stages of the phagocytosis were investigated using laser scanning confocal microscopy and their functional relevance were determined using fluorescence activated cell sorter based assay as well as colony forming unit assay. Our results demonstrate that EhRab7A is localized on the phagosomes and involved in both early and late stages of type 1 E. coli phagocytosis. We further showed that the E. coli or RBC containing phagosomes are distinct from the large endocytic vacuoles in the parasite which are exclusively used to transport human holotransferrin and low density lipoprotein. Remarkably, type 1 E. coli uptake was found to be insensitive to cytochalasin D treatment, suggesting that the initial stage of E. coli phagocytosis is independent of the formation of actin filaments.

  1. Molecular Detection of the Carriage Rate of Four Intestinal Protozoa with Real-Time Polymerase Chain Reaction: Possible Overdiagnosis of Entamoeba histolytica in Nigeria.

    PubMed

    Efunshile, Michael A; Ngwu, Bethrand A F; Kurtzhals, Jørgen A L; Sahar, Sumrin; König, Brigitte; Stensvold, Christen R

    2015-08-01

    Diarrhea remains the second largest killer of children worldwide, and Nigeria ranks number two on the list of global deaths attributable to diarrhea. Meanwhile, prevalence studies on potentially diarrheagenic protozoa in asymptomatic carriers using molecular detection methods remain scarce in sub-Saharan countries. To overcome sensitivity issues related to microscopic detection and identification of cysts in stool concentrates, real-time polymerase chain reaction (PCR) was used to analyze genomic DNAs extracted from stool samples from 199 healthy school children for Entamoeba histolytica, E. dispar, Giardia intestinalis, and Cryptosporidium. Questionnaires were administered for epidemiological data collection. E. histolytica was not detected in any of the samples, whereas Giardia (37.2%), E. dispar (18.6%), and Cryptosporidium (1%) were found. Most of the children sourced their drinking water from community wells (91%), while the majority disposed of feces in the bush (81.9%). Our study is the first to use real-time PCR to evaluate the epidemiology of E. histolytica, Giardia, and Cryptosporidium in Nigeria where previous studies using traditional diagnostic techniques have suggested higher and lower carriage rates of E. histolytica and Giardia, respectively. It is also the first study to accurately identify the prevalence of common potentially diarrheagenic protozoa in asymptomatic carriers in sub-Saharan Africa.

  2. Molecular Detection of the Carriage Rate of Four Intestinal Protozoa with Real-Time Polymerase Chain Reaction: Possible Overdiagnosis of Entamoeba histolytica in Nigeria

    PubMed Central

    Efunshile, Michael A.; Ngwu, Bethrand A. F.; Kurtzhals, Jørgen A. L.; Sahar, Sumrin; König, Brigitte; Stensvold, Christen R.

    2015-01-01

    Diarrhea remains the second largest killer of children worldwide, and Nigeria ranks number two on the list of global deaths attributable to diarrhea. Meanwhile, prevalence studies on potentially diarrheagenic protozoa in asymptomatic carriers using molecular detection methods remain scarce in sub-Saharan countries. To overcome sensitivity issues related to microscopic detection and identification of cysts in stool concentrates, real-time polymerase chain reaction (PCR) was used to analyze genomic DNAs extracted from stool samples from 199 healthy school children for Entamoeba histolytica, E. dispar, Giardia intestinalis, and Cryptosporidium. Questionnaires were administered for epidemiological data collection. E. histolytica was not detected in any of the samples, whereas Giardia (37.2%), E. dispar (18.6%), and Cryptosporidium (1%) were found. Most of the children sourced their drinking water from community wells (91%), while the majority disposed of feces in the bush (81.9%). Our study is the first to use real-time PCR to evaluate the epidemiology of E. histolytica, Giardia, and Cryptosporidium in Nigeria where previous studies using traditional diagnostic techniques have suggested higher and lower carriage rates of E. histolytica and Giardia, respectively. It is also the first study to accurately identify the prevalence of common potentially diarrheagenic protozoa in asymptomatic carriers in sub-Saharan Africa. PMID:26101274

  3. Entamoeba histolytica: Overexpression of the gal/galnac lectin, ehcp2 and ehcp5 genes in an in vivo model of amebiasis.

    PubMed

    Sánchez, Virginia; Serrano-Luna, Jesús; Ramírez-Moreno, Esther; Tsutsumi, Víctor; Shibayama, Mineko

    2016-12-01

    The parasite Entamoeba histolytica causes intestinal amebiasis and amebic liver abscess as its main extraintestinal manifestation. To study the in vivo events related to inflammation and the interactions between hosts and parasites during amebiasis, we designed a novel model of host-parasite interactions using cellulose membrane dialysis bags containing E. histolytica trophozoites. A bag is placed into the hamster peritoneal cavity, as has been reported in previous studies of programmed cell death (PCD) in E. histolytica trophozoites. To determine if virulence factors such as cysteine proteinases (EhCP2 and EhCP5) and Gal/GalNAc lectin could be involved in the host-parasite interaction using this model, we examined the relative expression of the ehcp2 and ehcp5 genes and the carbohydrate recognition domain (crd) of Gal/GalNAc lectin using real-time quantitative PCR (qRT-PCR). All analyzed genes were over-expressed 0.5h after the initiation of the host-parasite interaction and were then progressively down-regulated. However, Gal/GalNAc lectin had the greatest increase in gene expression 1.5h after host-parasite interaction; Gal/GalNAc lectin had a 250-fold increase with respect to the axenically grown trophozoites, which over-express Gal/GalNAc lectin in in vivo models. These results support the important role of these molecules in the initiation of cell damage by E. histolytica.

  4. N-acetyl ornithine deacetylase is a moonlighting protein and is involved in the adaptation of Entamoeba histolytica to nitrosative stress.

    PubMed

    Shahi, Preeti; Trebicz-Geffen, Meirav; Nagaraja, Shruti; Hertz, Rivka; Alterzon-Baumel, Sharon; Methling, Karen; Lalk, Michael; Mazumder, Mohit; Samudrala, Gourinath; Ankri, Serge

    2016-11-03

    Adaptation of the Entamoeba histolytica parasite to toxic levels of nitric oxide (NO) that are produced by phagocytes may be essential for the establishment of chronic amebiasis and the parasite's survival in its host. In order to obtain insight into the mechanism of E. histolytica's adaptation to NO, E. histolytica trophozoites were progressively adapted to increasing concentrations of the NO donor drug, S-nitrosoglutathione (GSNO) up to a concentration of 110 μM. The transcriptome of NO adapted trophozoites (NAT) was investigated by RNA sequencing (RNA-seq). N-acetyl ornithine deacetylase (NAOD) was among the 208 genes that were upregulated in NAT. NAOD catalyzes the deacetylation of N-acetyl-L-ornithine to yield ornithine and acetate. Here, we report that NAOD contributes to the better adaptation of the parasite to nitrosative stress (NS) and that this function does not depend on NAOD catalytic activity. We also demonstrated that glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is detrimental to E. histolytica exposed to NS and that this detrimental effect is neutralized by NAOD or by a catalytically inactive NAOD (mNAOD). These results establish NAOD as a moonlighting protein, and highlight the unexpected role of this metabolic enzyme in the adaptation of the parasite to NS.

  5. The presence of liver auto-antibodies induced by Entamoeba histolytica in the sera from both naturally infected humans and immunized rabbits.

    PubMed

    Faubert, G M; Meerovitch, E; McLaughlin, J

    1978-09-01

    Auto-antibodies against normal human liver have been detected in the sera of humans with highly positive indirect hemagglutination (IHA) amebiasis titers and with clinically-proven amebic liver abscess. Sera of amebiasis patients and rabbits immunized with killed Entamoeba histolytica were tested for anti-amebic antibodies by the IHA test and for auto-antibodies by the complement fixation test, using the antigens prepared from extracts of human liver and rabbit liver. A direct correlation was found to exist between high anti-Entamoeba antibody titers and the presence of anti-liver antibody in the serum. It is proposed that, in addition to direct parasite damage to host tissue, immunological damage could result from the attachment of circulating antigen to the cell surfaces of host tissues such as the liver.

  6. Catalysis by N-acetyl-D-glucosaminylphosphatidylinositol de-N-acetylase (PIG-L) from Entamoeba histolytica: new roles for conserved residues.

    PubMed

    Ashraf, Mohammad; Sreejith, Perinthottathil; Yadav, Usha; Komath, Sneha Sudha

    2013-03-15

    We showed previously that Entamoeba histolytica PIG-L exhibits a novel metal-independent albeit metal-stimulated activity. Using mutational and biochemical analysis, here we identify Asp-46 and His-140 of the enzyme as being important for catalysis. We show that these mutations neither affect the global conformational of the enzyme nor alter its metal binding affinity. The defect in catalysis, due to the mutations, is specifically due to an effect on V(max) and not due to altered substrate affinity (or K(m)). We propose a general acid-base pair mechanism to explain our results.

  7. Biochemical, Mutational and In Silico Structural Evidence for a Functional Dimeric Form of the Ornithine Decarboxylase from Entamoeba histolytica

    PubMed Central

    Preeti; Tapas, Satya; Kumar, Pravindra; Madhubala, Rentala; Tomar, Shailly

    2012-01-01

    Background Entamoeba histolytica is responsible for causing amoebiasis. Polyamine biosynthesis pathway enzymes are potential drug targets in parasitic protozoan diseases. The first and rate-limiting step of this pathway is catalyzed by ornithine decarboxylase (ODC). ODC enzyme functions as an obligate dimer. However, partially purified ODC from E. histolytica (EhODC) is reported to exist in a pentameric state. Methodology and Results In present study, the oligomeric state of EhODC was re-investigated. The enzyme was over-expressed in Escherichia coli and purified. Pure protein was used for determination of secondary structure content using circular dichroism spectroscopy. The percentages of α-helix, β-sheets and random coils in EhODC were estimated to be 39%, 25% and 36% respectively. Size-exclusion chromatography and mass spectrophotometry analysis revealed that EhODC enzyme exists in dimeric form. Further, computational model of EhODC dimer was generated. The homodimer contains two separate active sites at the dimer interface with Lys57 and Cys334 residues of opposite monomers contributing to each active site. Molecular dynamic simulations were performed and the dimeric structure was found to be very stable with RMSD value ∼0.327 nm. To gain insight into the functional role, the interface residues critical for dimerization and active site formation were identified and mutated. Mutation of Lys57Ala or Cys334Ala completely abolished enzyme activity. Interestingly, partial restoration of the enzyme activity was observed when inactive Lys57Ala and Cys334Ala mutants were mixed confirming that the dimer is the active form. Furthermore, Gly361Tyr and Lys157Ala mutations at the dimer interface were found to abolish the enzyme activity and destabilize the dimer. Conclusion To our knowledge, this is the first report which demonstrates that EhODC is functional in the dimeric form. These findings and availability of 3D structure model of EhODC dimer opens up

  8. In vitro studies on the sensitivity of local Entamoeba histolytica to anti-amoebic drugs.

    PubMed

    Chintana, T; Sucharit, P; Mahakittikun, V; Siripanth, C; Suphadtanaphongs, W

    1986-12-01

    The in vitro activity of drugs, namely dehydroemetine, ornidazole, metronidazole and tinidazole were determined against the locally isolated strains of E. histolytica in Thailand. The test was performed in liquid monophasic medium, i.e. liver marmite serum medium. In all, locally isolated strains from thirty hosts studied, the minimal inhibitory concentration (MIC) for dehydroemetine ranged from 0.125 to 1 microgram/ml, ornidazole ranged from 0.0625 to 0.25 microgram/ml, metronidazole ranged from 0.0625 to 0.125 microgram/ml, and tinidazole ranged from 0.0625 microgram/ml to 0.25 microgram/ml. The MIC of dehydroemetine was significantly different from ornidazole, metronidazole and tinidazole. Metronidazole was superior to that of dehydroemetine but was not significantly different among ornidazole, metronidazole and tinidazole.

  9. Post-transcriptional regulation of ribosomal protein genes during serum starvation in Entamoeba histolytica.

    PubMed

    Ahamad, Jamaluddin; Ojha, Sandeep; Srivastava, Ankita; Bhattacharya, Alok; Bhattacharya, Sudha

    2015-06-01

    Ribosome synthesis involves all three RNA polymerases which are co-ordinately regulated to produce equimolar amounts of rRNAs and ribosomal proteins (RPs). Unlike model organisms where transcription of rRNA and RP genes slows down during stress, in E. histolytica rDNA transcription continues but pre-rRNA processing slows down and unprocessed pre-rRNA accumulates during serum starvation. To investigate the regulation of RP genes under stress we measured transcription of six selected RP genes from the small- and large-ribosomal subunits (RPS6, RPS3, RPS19, RPL5, RPL26, RPL30) representing the early-, mid-, and late-stages of ribosomal assembly. Transcripts of these genes persisted in growth-stressed cells. Expression of luciferase reporter under the control of two RP genes (RPS19 and RPL30) was studied during serum starvation and upon serum replenishment. Although luciferase transcript levels remained unchanged during starvation, luciferase activity steadily declined to 7.8% and 15% of control cells, respectively. After serum replenishment the activity increased to normal levels, suggesting post-transcriptional regulation of these genes. Mutations in the sequence -2 to -9 upstream of AUG in the RPL30 gene resulted in the phenotype expected of post-transcriptional regulation. Transcription of luciferase reporter was unaffected in this mutant, and luciferase activity did not decline during serum starvation, showing that this sequence is required to repress translation of RPL30 mRNA, and mutations in this region relieve repression. Our data show that during serum starvation E. histolytica blocks ribosome biogenesis post-transcriptionally by inhibiting pre-rRNA processing on the one hand, and the translation of RP mRNAs on the other.

  10. Prevalence of Entamoeba histolytica -like cysts compared to E. histolytica antigens detected by ELISA in the stools of 600 patients from three socioeconomic communities in the Metropolitan City of Lahore, Pakistan.

    PubMed

    Alam, Muhammad Azhar; Maqbool, Azhar; Nazir, Muhammad Mudasser; Lateef, Muhammad; Khan, Muhammad Sarwar; Ahmed, Atif Nisar; Ziaullah, M; Lindsay, David S

    2015-04-01

    Amoebiasis, caused by Entamoeba histolytica , has a worldwide distribution and is of public health significance in many developing countries. It has a fecal-oral transmission cycle and is most prevalent in developing countries in regions where substandard sanitary conditions exist due to poverty. Little is known about the epidemiology of E. histolytica infection and its presence in different socioeconomic communities in developing countries. We undertook the present study in the city of Lahore, Pakistan, and our prediction was that the prevalence of E. histolytica -like cysts and E. histolytica stool antigen would be lower in patients from upper socioeconomic levels than in individuals from middle or lower socioeconomic levels. We investigated the prevalence of E. histolytica in humans from 3 socioeconomic communities in territories of Lahore, Pakistan. Six hundred fecal samples were collected and examined using both microscopy (triple fecal test) to detect cysts of E. histolytica -like amoeba and ELISA (stool antigen ELISA) to demonstrate diagnostic stool antigens of E. histolytica . Samples were from individuals living under conditions deemed to be upper socioeconomic class (n = 287), middle socioeconomic class (n = 172), and lower socioeconomic class (n = 141). The total prevalence of positive samples was 22.5% (135/600) by triple test and 16.8% (101/600) by stool antigen ELISA in the 600 fecal samples. Statistically, significant (P < 0.05) differences in prevalence were seen between the 3 socioeconomic class groups. Forty-four (15.3%) and 32 (11.1%) of 287 in the fecal samples from the upper socioeconomic class were positive by triple test and by antigen ELISA, respectively. Thirty-nine (22.6%) and 29 (16.8%) of 172 in the fecal samples from the middle socioeconomic class were positive by the triple test and by antigen ELISA, respectively. Fifty-two (36.8%) and 40 (28.3%) of 141 in the fecal samples from the lower socioeconomic class were positive by the triple

  11. Heterodimerization of the Entamoeba histolytica EhCPADH virulence complex through molecular dynamics and protein-protein docking.

    PubMed

    Montaño, Sarita; Orozco, Esther; Correa-Basurto, José; Bello, Martiniano; Chávez-Munguía, Bibiana; Betanzos, Abigail

    2017-02-01

    EhCPADH is a protein complex involved in the virulence of Entamoeba histolytica, the protozoan responsible for human amebiasis. It is formed by the EhCP112 cysteine protease and the EhADH adhesin. To explore the molecular basis of the complex formation, three-dimensional models were built for both proteins and molecular dynamics simulations (MDS) and docking calculations were performed. Results predicted that the pEhCP112 proenzyme and the mEhCP112 mature enzyme were globular and peripheral membrane proteins. Interestingly, in pEhCP112, the propeptide appeared hiding the catalytic site (C167, H329, N348); while in mEhCP112, this site was exposed and its residues were found structurally closer than in pEhCP112. EhADH emerged as an extended peripheral membrane protein with high fluctuation in Bro1 and V shape domains. 500 ns-long MDS and protein-protein docking predictions evidenced different heterodimeric complexes with the lowest free energy. pEhCP112 interacted with EhADH by the propeptide and C-terminal regions and mEhCP112 by the C-terminal through hydrogen bonds. In contrast, EhADH bound to mEhCP112 by 442-479 residues, adjacent to the target cell-adherence region (480-600 residues), and by the Bro1 domain (9-349 residues). Calculations of the effective binding free energy and per residue free energy decomposition showed that EhADH binds to mEhCP112 with a higher binding energy than to pEhCP112, mainly through van der Waals interactions and the nonpolar part of solvation energy. The EhADH and EhCP112 structural relationship was validated in trophozoites by immunofluorescence, TEM, and immunoprecipitation assays. Experimental findings fair agreed with in silico results.

  12. A New Human 3D-Liver Model Unravels the Role of Galectins in Liver Infection by the Parasite Entamoeba histolytica

    PubMed Central

    Petropolis, Debora B.; Faust, Daniela M.; Deep Jhingan, Gagan; Guillen, Nancy

    2014-01-01

    Investigations of human parasitic diseases depend on the availability of appropriate in vivo animal models and ex vivo experimental systems, and are particularly difficult for pathogens whose exclusive natural hosts are humans, such as Entamoeba histolytica, the protozoan parasite responsible for amoebiasis. This common infectious human disease affects the intestine and liver. In the liver sinusoids E. histolytica crosses the endothelium and penetrates into the parenchyma, with the concomitant initiation of inflammatory foci and subsequent abscess formation. Studying factors responsible for human liver infection is hampered by the complexity of the hepatic environment and by the restrictions inherent to the use of human samples. Therefore, we built a human 3D-liver in vitro model composed of cultured liver sinusoidal endothelial cells and hepatocytes in a 3D collagen-I matrix sandwich. We determined the presence of important hepatic markers and demonstrated that the cell layers function as a biological barrier. E. histolytica invasion was assessed using wild-type strains and amoebae with altered virulence or different adhesive properties. We showed for the first time the dependence of endothelium crossing upon amoebic Gal/GalNAc lectin. The 3D-liver model enabled the molecular analysis of human cell responses, suggesting for the first time a crucial role of human galectins in parasite adhesion to the endothelial cells, which was confirmed by siRNA knockdown of galectin-1. Levels of several pro-inflammatory cytokines, including galectin-1 and -3, were highly increased upon contact of E. histolytica with the 3D-liver model. The presence of galectin-1 and -3 in the extracellular medium stimulated pro-inflammatory cytokine release, suggesting a further role for human galectins in the onset of the hepatic inflammatory response. These new findings are relevant for a better understanding of human liver infection by E. histolytica. PMID:25211477

  13. Autophosphorylation of Ser428 of EhC2PK Plays a Critical Role in Regulating Erythrophagocytosis in the Parasite Entamoeba histolytica*

    PubMed Central

    Somlata; Kamanna, Sathisha; Agrahari, Mridula; Babuta, Mrigya; Bhattacharya, Sudha; Bhattacharya, Alok

    2012-01-01

    The protozoan parasite Entamoeba histolytica can invade both intestinal and extra intestinal tissues resulting in amoebiasis. During the process of invasion E. histolytica ingests red blood and host cells using phagocytic processes. Though phagocytosis is considered to be a key virulence determinant, the mechanism is not very well understood in E. histolytica. We have recently demonstrated that a novel C2 domain-containing protein kinase, EhC2PK is involved in the initiation of erythrophagocytosis. Because cells overexpressing the kinase-dead mutant of EhC2PK displayed a reduction in erythrophagocytosis, it appears that kinase activity is necessary for initiation. Biochemical analysis showed that EhC2PK is an unusual Mn2+-dependent serine kinase. It has a trans-autophosphorylated site at Ser428 as revealed by mass spectrometric and biochemical analysis. The autophosphorylation defective mutants (S428A, KDΔC) showed a reduction in auto and substrate phosphorylation. Time kinetics of in vitro kinase activity suggested two phases, an initial short slow phase followed by a rapid phase for wild type protein, whereas mutations in the autophosphorylation sites that cause defect (S428A) or conferred phosphomimetic property (S428E) displayed no distinct phases, suggesting that autophosphorylation may be controlling kinase activity through an autocatalytic mechanism. A reduction and delay in erythrophagocytosis was observed in E. histolytica cells overexpressing S428A and KDΔC proteins. These results indicate that enrichment of EhC2PK at the site of phagocytosis enhances the rate of trans-autophosphorylation, thereby increasing kinase activity and regulating the initiation of erythrophagocytosis in E. histolytica. PMID:22753771

  14. Expression, purification and crystallization of l-methionine γ-lyase 2 from Entamoeba histolytica

    SciTech Connect

    Sato, Dan; Yamagata, Wataru; Kamei, Kaeko; Nozaki, Tomoyoshi; Harada, Shigeharu

    2006-10-01

    l-Methionine γ-lyase 2 from E. histolytica, a key enzyme in sulfur-containing amino-acid degradation in this protozoan parasite, has been crystallized in a form suitable for X-ray structure analysis. l-Methionine γ-lyase (MGL) is considered to be an attractive target for rational drug development because the enzyme is absent in mammalian hosts. To enable structure-based design of drugs targeting MGL, one of the two MGL isoenzymes (EhMGL2) was crystallized in the orthorhombic space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 88.89, b = 102.68, c = 169.87 Å. The crystal diffracted to a resolution of 2.0 Å. The presence of a tetramer in the asymmetric unit (4 × 43.1 kDa) gives a Matthews coefficient of 2.2 Å{sup 3} Da{sup −1}. The structure was solved by the molecular-replacement method and structure refinement is now in progress.

  15. Entamoeba histolytica and E. dispar Calreticulin: inhibition of classical complement pathway and differences in the level of expression in amoebic liver abscess.

    PubMed

    Ximénez, Cecilia; González, Enrique; Nieves, Miriam E; Silva-Olivares, Angélica; Shibayama, Mineko; Galindo-Gómez, Silvia; Escobar-Herrera, Jaime; García de León, Ma Del Carmen; Morán, Patricia; Valadez, Alicia; Rojas, Liliana; Hernández, Eric G; Partida, Oswaldo; Cerritos, René

    2014-01-01

    The role of calreticulin (CRT) in host-parasite interactions has recently become an important area of research. Information about the functions of calreticulin and its relevance to the physiology of Entamoeba parasites is limited. The present work demonstrates that CRT of both pathogenic E. histolytica and nonpathogenic E. dispar species specifically interacted with human C1q inhibiting the activation of the classical complement pathway. Using recombinant EhCRT protein, we demonstrate that CRT interaction site and human C1q is located at the N-terminal region of EhCRT. The immunofluorescence and confocal microscopy experiments show that CRT and human C1q colocalize in the cytoplasmic vesicles and near to the surface membrane of previously permeabilized trophozoites or are incubated with normal human serum which is known to destroy trophozoites. In the presence of peripheral mononuclear blood cells, the distribution of EhCRT and C1q is clearly over the surface membrane of trophozoites. Nevertheless, the level of expression of CRT in situ in lesions of amoebic liver abscess (ALA) in the hamster model is different in both Entamoeba species; this molecule is expressed in higher levels in E. histolytica than in E. dispar. This result suggests that EhCRT may modulate some functions during the early moments of the host-parasite relationship.

  16. Protection against Invasive Amebiasis by a Single Monoclonal Antibody Directed against a Lipophosphoglycan Antigen Localized on the Surface of Entamoeba histolytica

    PubMed Central

    Marinets, Alexandra; Zhang, Tonghai; Guillén, Nancy; Gounon, Pierre; Bohle, Barbara; Vollmann, Ute; Scheiner, Otto; Wiedermann, Gerhard; Stanley, Samuel L.; Duchêne, Michael

    1997-01-01

    A panel of monoclonal antibodies was raised from mice immunized with a membrane preparation from Entamoeba histolytica, the pathogenic species causing invasive amebiasis in humans. Antibody EH5 gave a polydisperse band in immunoblots from membrane preparations from different E. histolytica strains, and a much weaker signal from two strains of the nonpathogenic species Entamoeba dispar. Although the exact chemical structure of the EH5 antigen is not yet known, the ability of the antigen to be metabolically radiolabeled with [32P]phosphate or [3H]glucose, its sensitivity to digestion by mild acid and phosphatidylinositol-specific phospholipase C, and its specific extraction from E. histolytica trophozoites by a method used to prepare lipophosphoglycans from Leishmania showed that it could be classified as an amebal lipophosphoglycan. Confocal immunofluorescence and immunogold labeling of trophozoites localized the antigen on the outer face of the plasma membrane and on the inner face of internal vesicle membranes. Antibody EH5 strongly agglutinated amebas in a similar way to concanavalin A (Con A), and Con A bound to immunoaffinity-purified EH5 antigen. Therefore, surface lipophosphoglycans may play an important role in the preferential agglutination of pathogenic amebas by Con A. The protective ability of antibody EH5 was tested in a passive immunization experiment in a severe combined immunodeficient (SCID) mouse model. Intrahepatic challenge of animals after administration of an isotype-matched control antibody or without treatment led to the development of a liver abscess in all cases, whereas 11 out of 12 animals immunized with the EH5 antibody developed no liver abscess. Our results demonstrate the importance and, for the first time, the protective capacity of glycan antigens on the surface of the amebas. PMID:9348313

  17. A whole-genome RNAi screen uncovers a novel role for human potassium channels in cell killing by the parasite Entamoeba histolytica

    PubMed Central

    Marie, Chelsea; Verkerke, Hans P.; Theodorescu, Dan; Petri, William A.

    2015-01-01

    The parasite Entamoeba histolytica kills human cells resulting in ulceration, inflammation and invasion of the colonic epithelium. We used the cytotoxic properties of ameba to select a genome-wide RNAi library to reveal novel host factors that control susceptibility to amebic killing. We identified 281 candidate susceptibility genes and bioinformatics analyses revealed that ion transporters were significantly enriched among susceptibility genes. Potassium (K+) channels were the most common transporter identified. Their importance was further supported by colon biopsy of humans with amebiasis that demonstrated suppressed K+ channel expression. Inhibition of human K+ channels by genetic silencing, pharmacologic inhibitors and with excess K+ protected diverse cell types from E. histolytica-induced death. Contact with E. histolytica parasites triggered K+ channel activation and K+ efflux by intestinal epithelial cells, which preceded cell killing. Specific inhibition of Ca2+-dependent K+ channels was highly effective in preventing amebic cytotoxicity in intestinal epithelial cells and macrophages. Blockade of K+ efflux also inhibited caspase-1 activation, IL-1β secretion and pyroptotic death in THP-1 macrophages. We concluded that K+ channels are host mediators of amebic cytotoxicity in multiple cells types and of inflammasome activation in macrophages. PMID:26346926

  18. Immunization with a tetramer derivative of an anti-inflammatory pentapeptide produced by Entamoeba histolytica protects gerbils (Meriones unguiculatus) against experimental amoebic abscess of the liver.

    PubMed

    Giménez-Scherer, Juan Antonio; Cárdenas, Guadalupe; López-Osuna, Martha; Velázquez, Juan Raymundo; Rico, Guadalupe; Isibasi, Armando; Maldonado, María del Carmen; Morales, María Esther; Fernández-Diez, Jorge; Kretschmer, Roberto R

    2004-01-01

    Axenically grown Entamoeba histolytica produces a pentapeptide (Met-Gln-Cys-Asn-Ser) with several anti-inflammatory properties, including the inhibition of human monocyte locomotion (Monocyte Locomotion Inhibitory Factor (MLIF)). A construct displays the same effects as the native material. It remains to be seen if MLIF is used, or even produced in vivo by the tissue-invading parasite. If MLIF were to be relevant in invasive amoebiasis, immunizing against it could diminish this parasite advantage and prevent lesions. KLH-linked MLIF mixed with Freund's adjuvant was too aggressive an immunizing material to answer this question. However, immunization with a tetramer of MLIF (but not a scrambled version of MLIF) around a lysine core (MLIF-MAPS), that displays increased antigenicity, yet lacks excessive innate immunity activation, completely protects gerbils against amoebic abscess of the liver caused by the intraportal injection of virulent E. histolytica. Liver abscesses caused by Listeria monocytogenes were not prevented. Invasive E. histolytica may produce the parent protein of MLIF in vivo, and if appropriately cleaved, it may play a role in invasive amoebiasis. MLIF may join new vaccination strategies against amoebiasis.

  19. Acetaldehyde/alcohol dehydrogenase-2 (EhADH2) and clathrin are involved in internalization of human transferrin by Entamoeba histolytica.

    PubMed

    Reyes-López, Magda; Bermúdez-Cruz, Rosa María; Avila, Eva E; de la Garza, Mireya

    2011-01-01

    Transferrin (Tf) is a host glycoprotein capable of binding two ferric-iron ions to become holotransferrin (holoTf), which transports iron in to all cells. Entamoeba histolytica is a parasitic protozoan able to use holoTf as a sole iron source in vitro. The mechanism by which this parasite scavenges iron from holoTf is unknown. An E. histolytica holoTf-binding protein (EhTfbp) was purified by using an anti-human transferrin receptor (TfR) monoclonal antibody. EhTfbp was identified by MS/MS analysis and database searches as E. histolytica acetaldehyde/alcohol dehydrogenase-2 (EhADH2), an iron-dependent enzyme. Both EhTfbp and EhADH2 bound holoTf and were recognized by the anti-human TfR antibody, indicating that they correspond to the same protein. It was found that the amoebae internalized holoTf through clathrin-coated pits, suggesting that holoTf endocytosis could be important for the parasite during colonization and invasion of the intestinal mucosa and liver.

  20. Prostaglandin E2 Produced by Entamoeba histolytica Signals via EP4 Receptor and Alters Claudin-4 to Increase Ion Permeability of Tight Junctions

    PubMed Central

    Lejeune, Manigandan; Moreau, France; Chadee, Kris

    2011-01-01

    Entamoeba histolytica is a protozoan parasite that causes amebic dysentery characterized by severe watery diarrhea. Unfortunately, the parasitic factors involved in the pathogenesis of diarrhea are poorly defined. Prostaglandin E2 (PGE2) is a host lipid mediator associated with diarrheal diseases. Intriguingly, E. histolytica produces and secretes this inflammatory molecule. We investigated the mechanism whereby ameba-derived PGE2 induces the onset of diarrhea by altering ion permeability of paracellular tight junctions (TJs) in colonic epithelia. PGE2 decreased barrier integrity of TJs in a dose- and time-dependent manner, as measured by transepithelial resistance. PGE2 signals were selectively transduced via the EP4 receptor. Furthermore, PGE2 signaling decreased TJ integrity, as revealed by EP receptor-specific agonist and antagonist studies. Loss of mucosal barrier integrity corresponded with increased ion permeability across TJs. Subcellular fractionation and confocal microscopy studies highlighted a significant spatial alteration of an important TJ protein, claudin-4, that corresponded with increased sodium ion permeability through TJs toward the lumen. Moreover, PGE2-induced luminal chloride secretion was a prerequisite for alterations at TJs. Thus, the gradient of NaCl created across epithelia could serve as a trigger for osmotic water flow that leads to diarrhea. Our results highlight a pathological role for E. histolytica-derived PGE2 in the onset of diarrhea. PMID:21683675

  1. A whole-genome RNAi screen uncovers a novel role for human potassium channels in cell killing by the parasite Entamoeba histolytica.

    PubMed

    Marie, Chelsea; Verkerke, Hans P; Theodorescu, Dan; Petri, William A

    2015-09-08

    The parasite Entamoeba histolytica kills human cells resulting in ulceration, inflammation and invasion of the colonic epithelium. We used the cytotoxic properties of ameba to select a genome-wide RNAi library to reveal novel host factors that control susceptibility to amebic killing. We identified 281 candidate susceptibility genes and bioinformatics analyses revealed that ion transporters were significantly enriched among susceptibility genes. Potassium (K(+)) channels were the most common transporter identified. Their importance was further supported by colon biopsy of humans with amebiasis that demonstrated suppressed K(+) channel expression. Inhibition of human K(+) channels by genetic silencing, pharmacologic inhibitors and with excess K(+) protected diverse cell types from E. histolytica-induced death. Contact with E. histolytica parasites triggered K(+) channel activation and K(+) efflux by intestinal epithelial cells, which preceded cell killing. Specific inhibition of Ca(2+)-dependent K(+) channels was highly effective in preventing amebic cytotoxicity in intestinal epithelial cells and macrophages. Blockade of K(+) efflux also inhibited caspase-1 activation, IL-1β secretion and pyroptotic death in THP-1 macrophages. We concluded that K(+) channels are host mediators of amebic cytotoxicity in multiple cells types and of inflammasome activation in macrophages.

  2. N-acetyl ornithine deacetylase is a moonlighting protein and is involved in the adaptation of Entamoeba histolytica to nitrosative stress

    PubMed Central

    Shahi, Preeti; Trebicz-Geffen, Meirav; Nagaraja, Shruti; Hertz, Rivka; Alterzon-Baumel, Sharon; Methling, Karen; Lalk, Michael; Mazumder, Mohit; Samudrala, Gourinath; Ankri, Serge

    2016-01-01

    Adaptation of the Entamoeba histolytica parasite to toxic levels of nitric oxide (NO) that are produced by phagocytes may be essential for the establishment of chronic amebiasis and the parasite’s survival in its host. In order to obtain insight into the mechanism of E. histolytica’s adaptation to NO, E. histolytica trophozoites were progressively adapted to increasing concentrations of the NO donor drug, S-nitrosoglutathione (GSNO) up to a concentration of 110 μM. The transcriptome of NO adapted trophozoites (NAT) was investigated by RNA sequencing (RNA-seq). N-acetyl ornithine deacetylase (NAOD) was among the 208 genes that were upregulated in NAT. NAOD catalyzes the deacetylation of N-acetyl-L-ornithine to yield ornithine and acetate. Here, we report that NAOD contributes to the better adaptation of the parasite to nitrosative stress (NS) and that this function does not depend on NAOD catalytic activity. We also demonstrated that glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is detrimental to E. histolytica exposed to NS and that this detrimental effect is neutralized by NAOD or by a catalytically inactive NAOD (mNAOD). These results establish NAOD as a moonlighting protein, and highlight the unexpected role of this metabolic enzyme in the adaptation of the parasite to NS. PMID:27808157

  3. Entamoeba histolytica Dmc1 Catalyzes Homologous DNA Pairing and Strand Exchange That Is Stimulated by Calcium and Hop2-Mnd1

    PubMed Central

    Kelso, Andrew A.; Say, Amanda F.; Sharma, Deepti; Ledford, LeAnna L.; Turchick, Audrey; Saski, Christopher A.; King, Ada V.; Attaway, Christopher C.; Temesvari, Lesly A.; Sehorn, Michael G.

    2015-01-01

    Meiosis depends on homologous recombination (HR) in most sexually reproducing organisms. Efficient meiotic HR requires the activity of the meiosis-specific recombinase, Dmc1. Previous work shows Dmc1 is expressed in Entamoeba histolytica, a eukaryotic parasite responsible for amoebiasis throughout the world, suggesting this organism undergoes meiosis. Here, we demonstrate Dmc1 protein is expressed in E. histolytica. We show that purified ehDmc1 forms presynaptic filaments and catalyzes ATP-dependent homologous DNA pairing and DNA strand exchange over at least several thousand base pairs. The DNA pairing and strand exchange activities are enhanced by the presence of calcium and the meiosis-specific recombination accessory factor, Hop2-Mnd1. In combination, calcium and Hop2-Mnd1 dramatically increase the rate of DNA strand exchange activity of ehDmc1. The biochemical system described herein provides a basis on which to better understand the role of ehDmc1 and other HR proteins in E. histolytica. PMID:26422142

  4. Bioassay-Guided Fractionation of Extracts from Codiaeum variegatum against Entamoeba histolytica Discovers Compounds That Modify Expression of Ceramide Biosynthesis Related Genes

    PubMed Central

    Mfotie Njoya, Emmanuel; Weber, Christian; Hernandez-Cuevas, Nora Adriana; Hon, Chung-Chau; Janin, Yves; Kamini, Melanie F. G.; Moundipa, Paul F.; Guillén, Nancy

    2014-01-01

    Leaves of Codiaeum variegatum (“garden croton”) are used against bloody diarrhoea by local populations in Cameroon. This study aims to search for the active components from C. variegatum against Entamoeba histolytica, and thereby initiate the study of their mechanism of action. A bioassay-guided screening of the aqueous extracts from C. variegatum leaves and various fractions was carried out against trophozoites of E. histolytica axenic culture. We found that the anti-amoebic activity of extracts changed with respect to the collection criteria of leaves. Thereby, optimal conditions were defined for leaves' collection to maximise the anti-amoebic activity of the extracts. A fractionation process was performed, and we identified several sub-fractions (or isolated compounds) with significantly higher anti-amoebic activity compared to the unfractionated aqueous extract. Anti-amoebic activity of the most potent fraction was confirmed with the morphological characteristics of induced death in trophozoites, including cell rounding and lysis. Differential gene expression analysis using high-throughput RNA sequencing implies the potential mechanism of its anti-amoebic activity by targeting ceramide, a bioactive lipid involved in disturbance of biochemical processes within the cell membrane including differentiation, proliferation, cell growth arrest and apoptosis. Regulation of ceramide biosynthesis pathway as a target for anti-amoebic compounds is a novel finding which could be an alternative for drug development against E. histolytica. PMID:24416462

  5. Antiprotozoal activity against Entamoeba histolytica of plants used in northeast Mexican traditional medicine. Bioactive compounds from Lippia graveolens and Ruta chalepensis.

    PubMed

    Quintanilla-Licea, Ramiro; Mata-Cárdenas, Benito David; Vargas-Villarreal, Javier; Bazaldúa-Rodríguez, Aldo Fabio; Kavimngeles-Hernández, Isvar; Garza-González, Jesús Norberto; Hernández-García, Magda Elizabeth

    2014-12-15

    Amoebiasis caused by Entamoeba histolytica is associated with high morbidity and mortality is becoming a major public health problem worldwide, especially in developing countries. Because of the side-effects and the resistance that pathogenic protozoa build against the standard antiparasitic drugs, e.g., metronidazole, much recent attention has been paid to plants used in traditional medicine around the world in order to find new antiprotozoal agents. We collected 32 plants used in Northeast Mexican traditional medicine and the methanolic extracts of these species were screened for antiprotozoal activity against E. histolytica trophozoites using in vitro tests. Only 18 extracts showed a significant inhibiting activity and among them six plant extracts showed more than 80% growth inhibition against E. histolytica at a concentration of 150 µg/mL and the IC50 values of these extracts were determined. Lippia graveolens Kunth and Ruta chalepensis Pers. showed the more significant antiprotozoal activity (91.54% and 90.50% growth inhibition at a concentration of 150 µg/mL with IC50 values of 59.14 and 60.07 µg/mL, respectively). Bioassay-guided fractionation of the methanolic extracts from these two plants afforded carvacrol (1) and chalepensin (2), respectively, as bioactive compounds with antiprotozoal activity.

  6. Bioassay-guided fractionation of extracts from Codiaeum variegatum against Entamoeba histolytica discovers compounds that modify expression of ceramide biosynthesis related genes.

    PubMed

    Mfotie Njoya, Emmanuel; Weber, Christian; Hernandez-Cuevas, Nora Adriana; Hon, Chung-Chau; Janin, Yves; Kamini, Melanie F G; Moundipa, Paul F; Guillén, Nancy

    2014-01-01

    Leaves of Codiaeum variegatum ("garden croton") are used against bloody diarrhoea by local populations in Cameroon. This study aims to search for the active components from C. variegatum against Entamoeba histolytica, and thereby initiate the study of their mechanism of action. A bioassay-guided screening of the aqueous extracts from C. variegatum leaves and various fractions was carried out against trophozoites of E. histolytica axenic culture. We found that the anti-amoebic activity of extracts changed with respect to the collection criteria of leaves. Thereby, optimal conditions were defined for leaves' collection to maximise the anti-amoebic activity of the extracts. A fractionation process was performed, and we identified several sub-fractions (or isolated compounds) with significantly higher anti-amoebic activity compared to the unfractionated aqueous extract. Anti-amoebic activity of the most potent fraction was confirmed with the morphological characteristics of induced death in trophozoites, including cell rounding and lysis. Differential gene expression analysis using high-throughput RNA sequencing implies the potential mechanism of its anti-amoebic activity by targeting ceramide, a bioactive lipid involved in disturbance of biochemical processes within the cell membrane including differentiation, proliferation, cell growth arrest and apoptosis. Regulation of ceramide biosynthesis pathway as a target for anti-amoebic compounds is a novel finding which could be an alternative for drug development against E. histolytica.

  7. Replication initiates at multiple dispersed sites in the ribosomal DNA plasmid of the protozoan parasite Entamoeba histolytica.

    PubMed Central

    Dhar, S K; Choudhury, N R; Mittal, V; Bhattacharya, A; Bhattacharya, S

    1996-01-01

    In the protozoan parasite Entamoeba histolytica (which causes amoebiasis in humans), the rRNA genes (rDNA) in the nucleus are carried on an extrachromosomal circular plasmid. For strain HM-1:IMSS, the size of the rDNA plasmid is 24.5 kb, and 200 copies per genome are present. Each circle contains two rRNA transcription units as inverted repeats separated by upstream and downstream spacers. We have studied the replication of this molecule by neutral/neutral two-dimensional gel electrophoresis and by electron microscopy. All restriction fragments analyzed by two-dimensional gel electrophoresis gave signals corresponding to simple Y's and bubbles. This showed that replication initiated in this plasmid at multiple, dispersed locations spread throughout the plasmid. On the basis of the intensity of the bubble arcs, initiations from the rRNA transcription units seemed to occur more frequently than those from intergenic spacers. Multiple, dispersed initiation sites were also seen in the rDNA plasmid of strain HK-9 when it was analyzed by two-dimensional gel electrophoresis. Electron microscopic visualization of replicating plasmid molecules in strain HM-1:IMISS showed multiple replication bubbles in the same molecule. The location of bubbles on the rDNA circle was mapped by digesting with PvuI or BsaHI, which linearize the molecule, and with SacII, which cuts the circle twice. The distance of the bubbles from one end of the molecule was measured by electron microscopy. The data corroborated those from two-dimensional gels and showed that replication bubbles were distributed throughout the molecule and that they appeared more frequently in rRNA transcription units. The same interpretation was drawn from electron microscopic analysis of the HK-9 plasmid. Direct demonstration of more than one bubble in the same molecule is clear evidence that replication of this plasmid initiates at multiple sites. Potential replication origins are distributed throughout the plasmid. Such a

  8. Use of recombinant Entamoeba histolytica cysteine proteinase 1 to identify a potent inhibitor of amebic invasion in a human colonic model.

    PubMed

    Meléndez-López, Samuel G; Herdman, Scott; Hirata, Ken; Choi, Min-Ho; Choe, Youngchool; Craik, Charles; Caffrey, Conor R; Hansell, Elisabeth; Chávez-Munguía, Bibiana; Chen, Yen Ting; Roush, William R; McKerrow, James; Eckmann, Lars; Guo, Jianhua; Stanley, Samuel L; Reed, Sharon L

    2007-07-01

    Cysteine proteinases are key virulence factors of the protozoan parasite Entamoeba histolytica. We have shown that cysteine proteinases play a central role in tissue invasion and disruption of host defenses by digesting components of the extracellular matrix, immunoglobulins, complement, and cytokines. Analysis of the E. histolytica genome project has revealed more than 40 genes encoding cysteine proteinases. We have focused on E. histolytica cysteine proteinase 1 (EhCP1) because it is one of two cysteine proteinases unique to invasive E. histolytica and is highly expressed and released. Recombinant EhCP1 was expressed in Escherichia coli and refolded to an active enzyme with a pH optimum of 6.0. We used positional-scanning synthetic tetrapeptide combinatorial libraries to map the specificity of the P1 to P4 subsites of the active site cleft. Arginine was strongly preferred at P2, an unusual specificity among clan CA proteinases. A new vinyl sulfone inhibitor, WRR483, was synthesized based on this specificity to target EhCP1. Recombinant EhCP1 cleaved key components of the host immune system, C3, immunoglobulin G, and pro-interleukin-18, in a time- and dose-dependent manner. EhCP1 localized to large cytoplasmic vesicles, distinct from the sites of other proteinases. To gain insight into the role of secreted cysteine proteinases in amebic invasion, we tested the effect of the vinyl sulfone cysteine proteinase inhibitors K11777 and WRR483 on invasion of human colonic xenografts. The resultant dramatic inhibition of invasion by both inhibitors in this human colonic model of amebiasis strongly suggests a significant role of secreted amebic proteinases, such as EhCP1, in the pathogenesis of amebiasis.

  9. Prostaglandin E2 produced by Entamoeba histolytica binds to EP4 receptors and stimulates interleukin-8 production in human colonic cells.

    PubMed

    Dey, Indranil; Chadee, Kris

    2008-11-01

    Entamoeba histolytica pathogenesis in the colon occurs in a stepwise fashion. It begins with colonization of the mucin layer, which is followed by stimulation of a proinflammatory response that causes nonspecific tissue damage that may facilitate parasite invasion of the underlying colonic mucosa. Unfortunately, the parasite and/or host factors that stimulate a proinflammatory response in the gut are poorly understood. In this study, we found that live E. histolytica or secretory or proteins (SP) and soluble ameba components (SAP) can markedly increase interleukin-8 (IL-8) mRNA expression and protein production in colonic epithelial cells. The IL-8-stimulating molecule produced by live amebae was identified as prostaglandin E(2) (PGE(2)) as trophozoites treated with cyclooxygenase inhibitors inhibited the biosynthesis of PGE(2) and eliminated IL-8 production induced by live parasites or ameba components. Moreover, using specific prostaglandin EP2 and EP4 receptor agonists and antagonists, we found that PGE(2) binds exclusively through EP4 receptors in colonic epithelial cells to stimulate IL-8 production. Silencing of EP4 receptors with EP4 small interfering RNA completely eliminated SP- and SAP-induced IL-8 production. These studies identified bioactive PGE(2) as a one of the major virulence factors produced by E. histolytica that can stimulate the potent neutrophil chemokine and activator IL-8, which can trigger an acute host inflammatory response. Thus, the induction of IL-8 production in response to E. histolytica-derived PGE(2) may be a mechanism that explains the initiation and amplification of acute inflammation associated with intestinal amebiasis.

  10. Simultaneous detection of Entamoeba histolytica/dispar, Giardia duodenalis and cryptosporidia by immunochromatographic assay in stool samples from patients living in the Greater Cairo Region, Egypt.

    PubMed

    Banisch, Dagmar M; El-Badry, Ayman; Klinnert, Jorge V; Ignatius, Ralf; El-Dib, Nadia

    2015-08-01

    Gastrointestinal infection due to intestinal parasites is an enormous health problem in developing countries and its reliable diagnosis is demanding. Therefore, this study aimed at evaluating a commercially available immunochromatographic assay (ICA) for the detection of cryptosporidia, Giardia duodenalis, and Entamoeba histolytica/dispar for its usefulness in the Greater Cairo Region, Egypt. Stool samples of 104 patients who presented between October 2012 and March 2013 with gastrointestinal symptoms or for the exclusion of parasites at Kasr-Al-Ainy University Medical School were examined by light microscopy of wet mounts and the triple ICA. Microscopy revealed in 20% of the patients [95% confidence interval (CI), 13.5-29.0%] parasites with Hymenolepis nana, E. histolytica/dispar and Blastocystis hominis being the most frequent ones, but was not able to detect G. duodenalis and cryptosporidia, whereas ICA was positive in 21% (95% CI, 14.3-30.0%) and detected E. histolytica/dispar in 12.5% (95% CI, 7.3-20.4%), cryptosporidia in 6.7% (95% CI, 3.1-13.5%) and G. duodenalis in 15.4% (95% CI, 9.6-23.6%) of the patients. Detection of one or more pathogens was associated with access to water retrieved from a well or pump (p = 0.01). Patients between 20 and 29 years of age (p = 0.08) and patients with symptoms of 5 days or longer (p = 0.07) tended to have a higher risk to be infected than patients of other age groups or with shorter-lasting symptoms. In conclusion, the ICA was easy to perform and timesaving. Importantly, it enabled the detection of cryptosporidia, which cannot be found microscopically in unstained smears, demonstrated a higher sensitivity for the detection of G. duodenalis than microscopy, and was more specific for distinguishing E. histolytica/dispar from apathogenic amoeba.

  11. Identification of Four Entamoeba histolytica Organellar DNA Polymerases of the Family B and Cellular Localization of the Ehodp1 Gene and EhODP1 Protein

    PubMed Central

    Herrera-Aguirre, María Esther; Luna-Arias, Juan Pedro; Labra-Barrios, María Luisa; Orozco, Esther

    2010-01-01

    We report the identification of a family of four active genes (Ehodp1, Ehodp2, Ehodp3, and Ehodp4) encoding putative DNA polymerases in Entamoeba histolytica, the protozoan parasite responsible of human amoebiasis. The four Ehodp genes show similarity to DNA polymerases encoded in fungi and plant mitochondrial plasmids. EhODP polypeptides conserve the 3′-5′ exonuclease II and 5′-3′ polymerization domains, and they have the I, II, and III conserved boxes that characterize them as DNA polymerases of family B. Furthermore, we found in EhODP polymerases two novel A and B boxes, present also in DNA polymerases encoded in fungi mitochondrial plasmids. By in situ PCR, Ehodp1 gene was located in nuclei and in DNA-containing cytoplasmic structures. Additionally, using polyclonal antibodies against a recombinant rEhODP1-168 polypeptide, and confocal microscopy, EhODP1 was located in cytoplasmic DNA-containing structures. PMID:20300437

  12. Proteomic Identification of S-Nitrosylated Proteins in the Parasite Entamoeba histolytica by Resin-Assisted Capture: Insights into the Regulation of the Gal/GalNAc Lectin by Nitric Oxide

    PubMed Central

    Hertz, Rivka; Ben Lulu, Shani; Shahi, Preeti; Trebicz-Geffen, Meirav; Benhar, Moran; Ankri, Serge

    2014-01-01

    Entamoeba histolytica is a gastrointestinal protozoan parasite that causes amebiasis, a disease which has a worldwide distribution with substantial morbidity and mortality. Nitrosative stress, which is generated by innate immune cells, is one of the various environmental challenges that E. histolytica encounters during its life cycle. Although the effects of nitric oxide (NO) on the regulation of gene expression in this parasite have been previously investigated, our knowledge on S-nitrosylated proteins in E.histolytica is lacking. In order to fill this knowledge gap, we performed a large-scale detection of S-nitrosylated (SNO) proteins in E.histolytica trophozoites that were treated with the NO donor, S-nitrosocysteine by resin-assisted capture (RAC). We found that proteins involved in glycolysis, gluconeogenesis, translation, protein transport, and adherence to target cells such as the heavy subunit of Gal/GalNac lectin are among the S-nitrosylated proteins that were enriched by SNO-RAC. We also found that the S-nitrosylated cysteine residues in the carbohydrate recognition domain (CRD) of Gal/GalNAc lectin impairs its function and contributes to the inhibition of E.histolytica adherence to host cells. Collectively, these results advance our understanding of the mechanism of reduced E.histolytica adherence to mammalian cells by NO and emphasize the importance of NO as a regulator of key physiological functions in E.histolytica. PMID:24626316

  13. Degradation of the Transcription Factors NF-κB, STAT3, and STAT5 Is Involved in Entamoeba histolytica-Induced Cell Death in Caco-2 Colonic Epithelial Cells

    PubMed Central

    Kim, Kyeong Ah; Min, Arim; Lee, Young Ah

    2014-01-01

    Entamoeba histolytica is a tissue-invasive protozoan parasite causing dysentery in humans. During infection of colonic tissues, amoebic trophozoites are able to kill host cells via apoptosis or necrosis, both of which trigger IL-8-mediated acute inflammatory responses. However, the signaling pathways involved in host cell death induced by E. histolytica have not yet been fully defined. In this study, we examined whether calpain plays a role in the cleavage of pro-survival transcription factors during cell death of colonic epithelial cells, induced by live E. histolytica trophozoites. Incubation with amoebic trophozoites induced activation of m-calpain in a time- and dose-dependent manner. Moreover, incubation with amoebae resulted in marked degradation of STAT proteins (STAT3 and STAT5) and NF-κB (p65) in Caco-2 cells. However, IκB, an inhibitor of NF-κB, was not cleaved in Caco-2 cells following adherence of E. histolytica. Entamoeba-induced cleavage of STAT proteins and NF-κB was partially inhibited by pretreatment of cells with a cell-permeable calpain inhibitor, calpeptin. In contrast, E. histolytica did not induce cleavage of caspase-3 in Caco-2 cells. Furthermore, pretreatment of Caco-2 cells with a calpain inhibitor, calpeptin (but not the pan-caspase inhibitor, z-VAD-fmk) or m-calpain siRNA partially reduced Entamoeba-induced DNA fragmentation in Caco-2 cells. These results suggest that calpain plays an important role in E. histolytica-induced degradation of NF-κB and STATs in colonic epithelial cells, which ultimately accelerates cell death. PMID:25352693

  14. Degradation of the transcription factors NF-κB, STAT3, and STAT5 is involved in Entamoeba histolytica-induced cell death in Caco-2 colonic epithelial cells.

    PubMed

    Kim, Kyeong Ah; Min, Arim; Lee, Young Ah; Shin, Myeong Heon

    2014-10-01

    Entamoeba histolytica is a tissue-invasive protozoan parasite causing dysentery in humans. During infection of colonic tissues, amoebic trophozoites are able to kill host cells via apoptosis or necrosis, both of which trigger IL-8-mediated acute inflammatory responses. However, the signaling pathways involved in host cell death induced by E. histolytica have not yet been fully defined. In this study, we examined whether calpain plays a role in the cleavage of pro-survival transcription factors during cell death of colonic epithelial cells, induced by live E. histolytica trophozoites. Incubation with amoebic trophozoites induced activation of m-calpain in a time- and dose-dependent manner. Moreover, incubation with amoebae resulted in marked degradation of STAT proteins (STAT3 and STAT5) and NF-κB (p65) in Caco-2 cells. However, IκB, an inhibitor of NF-κB, was not cleaved in Caco-2 cells following adherence of E. histolytica. Entamoeba-induced cleavage of STAT proteins and NF-κB was partially inhibited by pretreatment of cells with a cell-permeable calpain inhibitor, calpeptin. In contrast, E. histolytica did not induce cleavage of caspase-3 in Caco-2 cells. Furthermore, pretreatment of Caco-2 cells with a calpain inhibitor, calpeptin (but not the pan-caspase inhibitor, z-VAD-fmk) or m-calpain siRNA partially reduced Entamoeba-induced DNA fragmentation in Caco-2 cells. These results suggest that calpain plays an important role in E. histolytica-induced degradation of NF-κB and STATs in colonic epithelial cells, which ultimately accelerates cell death.

  15. Pyrazolo[3,4-d]pyrimidines as novel inhibitors of O-acetyl-L-serine sulfhydrylase of Entamoeba histolytica: an in silico study.

    PubMed

    Yadava, Umesh; Shukla, Bindesh Kumar; Roychoudhury, Mihir; Kumar, Devesh

    2015-04-01

    Amoebiasis, a worldwide explosive epidemic, caused by the gastrointestinal anaerobic protozoan parasite Entamoeba histolytica, infects the large intestine and, in advance stages, liver, kidney, brain and lung. Metronidazole (MNZ)-the first line medicament against amoebiasis-is potentially carcinogenic to humans and shows significant side-effects. Pyrazolo[3,4-d]pyrimidine compounds have been reported to demonstrate antiamoebic activity. In silico molecular docking simulations on nine pyrazolo[3,4-d]pyrimidine molecules without linkers (molecules 1-9) and nine pyrazolo[3,4-d]pyrimidine molecules with a trimethylene linker (molecules 10-18) along with the reference drug metronidazole (MNZ) were conducted using the modules of the programs Glide-SP, Glide-XP and Autodock with O-acetyl-L-serine sulfhydrylase (OASS) enzyme-a promising target for inhibiting the growth of Entamoeba histolytica. Docking simulations using Glide-SP demonstrate good agreement with reported biological activities of molecules 1-9 and indicate that molecules 2 and 4 may act as potential high affinity inhibitors. Trimethylene linker molecules show improved binding affinities among which molecules 15 and 16 supersede. MD simulations on the best docked poses of molecules 2, 4, 15, 16 and MNZ were carried out for 20 ns using DESMOND. It was observed that the docking complexes of molecules 4, 15 and MNZ remain stable in aqueous conditions and do not undergo noticeable fluctuations during the course of the dynamics. Relative binding free energy calculations of the ligands with the enzyme were executed on the best docked poses using the molecular mechanics generalized Born surface area (MM-GBSA) approach, which show good agreement with the reported biological activities.

  16. A Study of Intestinal Protozoa Including Non-Pathogenic Entamoeba histolytica from Patients in a Group of Mental Hospitals

    PubMed Central

    Sargeaunt, Peter G.; Williams, John E.

    1982-01-01

    Twenty-five per cent of 174 patients residing in three mental hospitals were found to be infected with protozoa. Seven per cent of the patients were infected with Entameba histolytica. Isoenzyme characterization of the E. histolytica grown in culture showed that there were no pathogenic zymodemes of this organism in any of the specimens tested. (Am J Public Health 1982;72:178-180.) ImagesFIGURE 1 PMID:6275721

  17. Regulation of serum-responsive transmembrane kinase EhTMKB1-9 by an unsaturated lipid, oleic acid in protistan parasite Entamoeba histolytica.

    PubMed

    Saha, Arpita; Bhattacharya, Sudha; Bhattacharya, Alok

    2014-11-01

    Transmembrane kinases of Entamoeba histolytica are known to play a wide range of roles from virulence, phagocytosis, and proliferation to stress response. Transmembrane kinase EhTMKB1-9 is thought to be involved in early proliferative response and it was originally identified as a serum inducible gene. Ability to stimulate EhTMKB1 expression of serum starved cells resides in unsaturated fatty acids associated with albumin fraction of serum and the mechanism of stimulation follows activation of EhTMKB1-9 promoter. Gel shift assay showed the presence of proteins that bind to the specific site of EhTMKB1-9 upstream region and the concentration of these protein(s) go down on serum starvation, but level of binding protein(s) go up on serum or fatty acid replenishment. This increase in concentration of binding molecule(s) is due to new synthesis rather than activation of existing molecule(s) as a protein synthesis inhibitor blocked enhanced level of gel shifted material on replenishment. The stimulating activity resides in the fatty acyl chain, but not in the head group. Moreover, the fatty acid initiates signaling through class I PI3 kinases that result in activation of EhTMKB1-9 expression. These results suggest a novel mechanism of gene regulation in E. histolytica, and unsaturated fatty acids as potential new signaling molecules.

  18. Expression of EhRAD54, EhRAD51, and EhBLM proteins during DNA repair by homologous recombination in Entamoeba histolytica.

    PubMed

    Charcas-Lopez, Ma del Socorro; Garcia-Morales, Lorena; Pezet-Valdez, Marisol; Lopez-Camarillo, Cesar; Zamorano-Carrillo, Absalom; Marchat, Laurence A

    2014-01-01

    Entamoeba histolytica, the protozoan responsible for human amoebiasis, exhibits a great genome plasticity that is probably related to homologous recombination events. It contains the RAD52 epistasis group genes, including Ehrad51 and Ehrad54, and the Ehblm gene, which are key homologous recombination factors in other organisms. Ehrad51 and Ehrad54 genes are differentially transcribed in trophozoites when DNA double-strand breaks are induced by ultraviolet-C irradiation. Moreover, the EhRAD51 recombinase is overexpressed at 30 min in the nucleus. Here, we extend our analysis of the homologous recombination mechanism in E. histolytica by studying EhRAD51, EhRAD54, and EhBLM expression in response to DNA damage. Bioinformatic analyses show that EhRAD54 has the molecular features of homologous proteins, indicating that it may have similar functions. Western blot assays evidence the differential expression of EhRAD51, EhRAD54, and EhBLM at different times after DNA damage, suggesting their potential roles in the different steps of homologous recombination in this protozoan.

  19. Detection of the Endosomal Sorting Complex Required for Transport in Entamoeba histolytica and Characterization of the EhVps4 Protein

    PubMed Central

    López-Reyes, Israel; García-Rivera, Guillermina; Bañuelos, Cecilia; Herranz, Silvia; Vincent, Olivier; López-Camarillo, César; Marchat, Laurence A.; Orozco, Esther

    2010-01-01

    Eukaryotic endocytosis involves multivesicular bodies formation, which is driven by endosomal sorting complexes required for transport (ESCRT). Here, we showed the presence and expression of homologous ESCRT genes in Entamoeba histolytica. We cloned and expressed the Ehvps4 gene, an ESCRT member, to obtain the recombinant EhVps4 and generate specific antibodies, which immunodetected EhVps4 in cytoplasm of trophozoites. Bioinformatics and biochemical studies evidenced that rEhVps4 is an ATPase, whose activity depends on the conserved E211 residue. Next, we generated trophozoites overexpressing EhVps4 and mutant EhVps4-E211Q FLAG-tagged proteins. The EhVps4-FLAG was located in cytosol and at plasma membrane, whereas the EhVps4-E211Q-FLAG was detected as abundant cytoplasmic dots in trophozoites. Erythrophagocytosis, cytopathic activity, and hepatic damage in hamsters were not improved in trophozoites overexpressing EhVps4-FLAG. In contrast, EhVps4-E211Q-FLAG protein overexpression impaired these properties. The localization of EhVps4-FLAG around ingested erythrocytes, together with our previous results, strengthens the role for EhVps4 in E. histolytica phagocytosis and virulence. PMID:20508821

  20. Nano-yeast-scFv probes on screen-printed gold electrodes for detection of Entamoeba histolytica antigens in a biological matrix.

    PubMed

    Grewal, Yadveer S; Shiddiky, Muhammad J A; Spadafora, Lauren J; Cangelosi, Gerard A; Trau, Matt

    2014-05-15

    The time and costs associated with monoclonal antibody production limit the potential for portable diagnostic devices to penetrate the market. Replacing the antibody with a low-cost alternate affinity reagent would reduce the costs of diagnostic development and use, and lead to new portable diagnostic devices towards many diseases. Herein, we present low-cost affinity reagents, nano-yeast-scFv, on commercially available, inexpensive, and portable screen-printed electrodes for the label-free electrochemical detection of Entamoeba histolytica cyst antigens. The biosensor was able to detect antigen at concentrations down to 10 pg mL(-1) in buffer with an inter-assay reproducibility of (% RSD, n=3) 4.1%. The applicability of two differently engineered nano-yeast-scFv to each specifically detect their cognant E. histolytica cyst antigens was demonstrated in a biological matrix derived from human stool. Because of the simple, inexpensive, and sensitive nature of this methodology, it may offer a low-cost alternative to immunosensors based on antibody-target recognition.

  1. A Single RNaseIII Domain Protein from Entamoeba histolytica Has dsRNA Cleavage Activity and Can Help Mediate RNAi Gene Silencing in a Heterologous System.

    PubMed

    Pompey, Justine M; Foda, Bardees; Singh, Upinder

    2015-01-01

    Dicer enzymes process double-stranded RNA (dsRNA) into small RNAs that target gene silencing through the RNA interference (RNAi) pathway. Dicer enzymes are complex, multi-domain RNaseIII proteins, however structural minimalism of this protein has recently emerged in parasitic and fungal systems. The most minimal Dicer, Saccharomyces castellii Dicer1, has a single RNaseIII domain and two double stranded RNA binding domains. In the protozoan parasite Entamoeba histolytica 27nt small RNAs are abundant and mediate silencing, yet no canonical Dicer enzyme has been identified. Although EhRNaseIII does not exhibit robust dsRNA cleavage in vitro, it can process dsRNA in the RNAi-negative background of Saccharomyces cerevisiae, and in conjunction with S. castellii Argonaute1 can partially reconstitute the RNAi pathway. Thus, although EhRNaseIII lacks the domain architecture of canonical or minimal Dicer enzymes, it has dsRNA processing activity that contributes to gene silencing via RNAi. Our data advance the understanding of small RNA biogenesis in Entamoeba as well as broaden the spectrum of non-canonical Dicer enzymes that contribute to the RNAi pathway.

  2. A Single RNaseIII Domain Protein from Entamoeba histolytica Has dsRNA Cleavage Activity and Can Help Mediate RNAi Gene Silencing in a Heterologous System

    PubMed Central

    Singh, Upinder

    2015-01-01

    Dicer enzymes process double-stranded RNA (dsRNA) into small RNAs that target gene silencing through the RNA interference (RNAi) pathway. Dicer enzymes are complex, multi-domain RNaseIII proteins, however structural minimalism of this protein has recently emerged in parasitic and fungal systems. The most minimal Dicer, Saccharomyces castellii Dicer1, has a single RNaseIII domain and two double stranded RNA binding domains. In the protozoan parasite Entamoeba histolytica 27nt small RNAs are abundant and mediate silencing, yet no canonical Dicer enzyme has been identified. Although EhRNaseIII does not exhibit robust dsRNA cleavage in vitro, it can process dsRNA in the RNAi-negative background of Saccharomyces cerevisiae, and in conjunction with S. castellii Argonaute1 can partially reconstitute the RNAi pathway. Thus, although EhRNaseIII lacks the domain architecture of canonical or minimal Dicer enzymes, it has dsRNA processing activity that contributes to gene silencing via RNAi. Our data advance the understanding of small RNA biogenesis in Entamoeba as well as broaden the spectrum of non-canonical Dicer enzymes that contribute to the RNAi pathway. PMID:26230096

  3. Multicenter Evaluation of BD Max Enteric Parasite Real-Time PCR Assay for Detection of Giardia duodenalis, Cryptosporidium hominis, Cryptosporidium parvum, and Entamoeba histolytica.

    PubMed

    Madison-Antenucci, S; Relich, R F; Doyle, L; Espina, N; Fuller, D; Karchmer, T; Lainesse, A; Mortensen, J E; Pancholi, P; Veros, W; Harrington, S M

    2016-11-01

    Common causes of chronic diarrhea among travelers worldwide include protozoan parasites. The majority of parasitic infections are caused by Giardia duodenalis, Entamoeba histolytica, Cryptosporidium parvum, and Cryptosporidium hominis Similarly, these species cause the majority of parasitic diarrhea acquired in the United States. Detection of parasites by gold standard microscopic methods is time-consuming and requires considerable expertise; enzyme immunoassays and direct fluorescent-antibody (DFA) stains have lowered hands-on time for testing, but improvements in sensitivity and technical time may be possible with a PCR assay. We performed a clinical evaluation of a multiplex PCR panel, the enteric parasite panel (EPP), for the detection of these common parasites using the BD Max instrument, which performs automated extraction and amplification. A total of 2,495 compliant specimens were enrolled, including 2,104 (84%) specimens collected prospectively and 391 (16%) specimens collected retrospectively. Approximately equal numbers were received in 10% formalin (1,273 specimens) and unpreserved (1,222 specimens). The results from the EPP were compared to those from alternate PCR and bidirectional sequencing (APCR), as well as DFA (G. duodenalis and C. parvum or C. hominis) or trichrome stain (E. histolytica). The sensitivity and specificity for prospective and retrospective specimens combined were 98.2% and 99.5% for G. duodenalis, 95.5% and 99.6 for C. parvum or C. hominis, and 100% and 100% for E. histolytica, respectively. The performance of the FDA-approved BD Max EPP compared well to the reference methods and may be an appropriate substitute for microscopic examination or immunoassays.

  4. Evaluation of Multiplex Tandem Real-Time PCR for Detection of Cryptosporidium spp., Dientamoeba fragilis, Entamoeba histolytica, and Giardia intestinalis in Clinical Stool Samples ▿

    PubMed Central

    Stark, D.; Al-Qassab, S. E.; Barratt, J. L. N.; Stanley, K.; Roberts, T.; Marriott, D.; Harkness, J.; Ellis, J. T.

    2011-01-01

    The aim of this study was to describe the first development and evaluation of a multiplex tandem PCR (MT-PCR) assay for the detection and identification of 4 common pathogenic protozoan parasites, Cryptosporidium spp., Dientamoeba fragilis, Entamoeba histolytica, and Giardia intestinalis, from human clinical samples. A total of 472 fecal samples submitted to the Department of Microbiology at St. Vincent's Hospital were included in the study. The MT-PCR assay was compared to four real-time PCR (RT-PCR) assays and microscopy by a traditional modified iron hematoxylin stain. The MT-PCR detected 28 G. intestinalis, 26 D. fragilis, 11 E. histolytica, and 9 Cryptosporidium sp. isolates. Detection and identification of the fecal protozoa by MT-PCR demonstrated 100% correlation with the RT-PCR results, and compared to RT-PCR, MT-PCR exhibited 100% sensitivity and specificity, while traditional microscopy of stained fixed fecal smears exhibited sensitivities and specificities of 56% and 100% for Cryptosporidium spp., 38% and 99% for D. fragilis, 47% and 97% for E. histolytica, and 50% and 100% for G. intestinalis. No cross-reactivity was detected in 100 stool samples containing various other bacterial, viral, and protozoan species. The MT-PCR assay was able to provide rapid, sensitive, and specific simultaneous detection and identification of the four most important diarrhea-causing protozoan parasites that infect humans. This study also highlights the lack of sensitivity demonstrated by microscopy, and thus, molecular methods such as MT-PCR must be considered the diagnostic methods of choice for enteric protozoan parasites. PMID:21048004

  5. Computational prediction and validation of C/D, H/ACA and Eh_U3 snoRNAs of Entamoeba histolytica

    PubMed Central

    2012-01-01

    Background Small nucleolar RNAs are a highly conserved group of small RNAs found in eukaryotic cells. Genes encoding these RNAs are diversely located throughout the genome. They are functionally conserved, performing post transcriptional modification (methylation and pseudouridylation) of rRNA and other nuclear RNAs. They belong to two major categories: the C/D box and H/ACA box containing snoRNAs. U3 snoRNA is an exceptional member of C/D box snoRNAs and is involved in early processing of pre-rRNA. An antisense sequence is present in each snoRNA which guides the modification or processing of target RNA. However, some snoRNAs lack this sequence and often they are called orphan snoRNAs. Results We have searched snoRNAs of Entamoeba histolytica from the genome sequence using computational programmes (snoscan and snoSeeker) and we obtained 99 snoRNAs (C/D and H/ACA box snoRNAs) along with 5 copies of Eh_U3 snoRNAs. These are located diversely in the genome, mostly in intergenic regions, while some are found in ORFs of protein coding genes, intron and UTRs. The computationally predicted snoRNAs were validated by RT-PCR and northern blotting. The expected sizes were in agreement with the observed sizes for all C/D box snoRNAs tested, while for some of the H/ACA box there was indication of processing to generate shorter products. Conclusion Our results showed the presence of snoRNAs in E. histolytica, an early branching eukaryote, and the structural features of E. histolytica snoRNAs were well conserved when compared with yeast and human snoRNAs. This study will help in understanding the evolution of these conserved RNAs in diverse phylogenetic groups. PMID:22892049

  6. The EhCPADH112 Complex of Entamoeba histolytica Interacts with Tight Junction Proteins Occludin and Claudin-1 to Produce Epithelial Damage

    PubMed Central

    Betanzos, Abigail; Javier-Reyna, Rosario; García-Rivera, Guillermina; Bañuelos, Cecilia; González-Mariscal, Lorenza; Schnoor, Michael; Orozco, Esther

    2013-01-01

    Entamoeba histolytica, the protozoan responsible for human amoebiasis, causes between 30,000 and 100,000 deaths per year worldwide. Amoebiasis is characterized by intestinal epithelial damage provoking severe diarrhea. However, the molecular mechanisms by which this protozoan causes epithelial damage are poorly understood. Here, we studied the initial molecular interactions between the E. histolytica EhCPADH112 virulence complex and epithelial MDCK and Caco-2 cells. By confocal microscopy, we discovered that after contact with trophozoites or trophozoite extracts (TE), EhCPADH112 and proteins forming this complex (EhCP112 and EhADH112) co-localize with occludin and claudin-1 at tight junctions (TJ). Immunoprecipitation assays revealed interaction between EhCPADH112 and occludin, claudin-1, ZO-1 and ZO-2. Overlay assays confirmed an interaction of EhCP112 and EhADH112 with occludin and claudin-1, whereas only EhADH112 interacted also with ZO-2. We observed degradation of all mentioned TJ proteins after incubation with TE. Importantly, inhibiting proteolytic activity or blocking the complex with a specific antibody not only prevented TJ protein degradation but also epithelial barrier disruption. Furthermore, we discovered that TE treatment induces autophagy and apoptosis in MDCK cells that could contribute to the observed barrier disruption. Our results suggest a model in which epithelial damage caused by E. histolytica is initiated by the interaction of EhCP112 and EhADH112 with TJ proteins followed by their degradation. Disruption of TJs then induces increased paracellular permeability, thus facilitating the entry of more proteases and other parasite molecules leading eventually to tissue destruction. PMID:23762290

  7. Multicenter Evaluation of BD Max Enteric Parasite Real-Time PCR Assay for Detection of Giardia duodenalis, Cryptosporidium hominis, Cryptosporidium parvum, and Entamoeba histolytica

    PubMed Central

    Relich, R. F.; Doyle, L.; Espina, N.; Fuller, D.; Karchmer, T.; Lainesse, A.; Mortensen, J. E.; Pancholi, P.; Veros, W.; Harrington, S. M.

    2016-01-01

    Common causes of chronic diarrhea among travelers worldwide include protozoan parasites. The majority of parasitic infections are caused by Giardia duodenalis, Entamoeba histolytica, Cryptosporidium parvum, and Cryptosporidium hominis. Similarly, these species cause the majority of parasitic diarrhea acquired in the United States. Detection of parasites by gold standard microscopic methods is time-consuming and requires considerable expertise; enzyme immunoassays and direct fluorescent-antibody (DFA) stains have lowered hands-on time for testing, but improvements in sensitivity and technical time may be possible with a PCR assay. We performed a clinical evaluation of a multiplex PCR panel, the enteric parasite panel (EPP), for the detection of these common parasites using the BD Max instrument, which performs automated extraction and amplification. A total of 2,495 compliant specimens were enrolled, including 2,104 (84%) specimens collected prospectively and 391 (16%) specimens collected retrospectively. Approximately equal numbers were received in 10% formalin (1,273 specimens) and unpreserved (1,222 specimens). The results from the EPP were compared to those from alternate PCR and bidirectional sequencing (APCR), as well as DFA (G. duodenalis and C. parvum or C. hominis) or trichrome stain (E. histolytica). The sensitivity and specificity for prospective and retrospective specimens combined were 98.2% and 99.5% for G. duodenalis, 95.5% and 99.6 for C. parvum or C. hominis, and 100% and 100% for E. histolytica, respectively. The performance of the FDA-approved BD Max EPP compared well to the reference methods and may be an appropriate substitute for microscopic examination or immunoassays. PMID:27535690

  8. Dimethylated H3K27 Is a Repressive Epigenetic Histone Mark in the Protist Entamoeba histolytica and Is Significantly Enriched in Genes Silenced via the RNAi Pathway*

    PubMed Central

    Foda, Bardees M.; Singh, Upinder

    2015-01-01

    RNA interference (RNAi) is a fundamental biological process that plays a crucial role in regulation of gene expression in many organisms. Transcriptional gene silencing (TGS) is one of the important nuclear roles of RNAi. Our previous data show that Entamoeba histolytica has a robust RNAi pathway that links to TGS via Argonaute 2-2 (Ago2-2) associated 27-nucleotide small RNAs with 5′-polyphosphate termini. Here, we report the first repressive histone mark to be identified in E. histolytica, dimethylation of H3K27 (H3K27Me2), and demonstrate that it is enriched at genes that are silenced by RNAi-mediated TGS. An RNAi-silencing trigger can induce H3K27Me2 deposits at both episomal and chromosomal loci, mediating gene silencing. Our data support two phases of RNAi-mediated TGS: an active silencing phase where the RNAi trigger is present and both H3K27Me2 and Ago2-2 concurrently enrich at chromosomal loci; and an established silencing phase in which the RNAi trigger is removed, but gene silencing with H3K27Me2 enrichment persist independently of Ago2-2 deposition. Importantly, some genes display resistance to chromosomal silencing despite induction of functional small RNAs. In those situations, the RNAi-triggering plasmid that is maintained episomally gets partially silenced and has H3K27Me2 enrichment, but the chromosomal copy displays no repressive histone enrichment. Our data are consistent with a model in which H3K27Me2 is a repressive histone modification, which is strongly associated with transcriptional repression. This is the first example of an epigenetic histone modification that functions to mediate RNAi-mediated TGS in the deep-branching eukaryote E. histolytica. PMID:26149683

  9. Dimethylated H3K27 Is a Repressive Epigenetic Histone Mark in the Protist Entamoeba histolytica and Is Significantly Enriched in Genes Silenced via the RNAi Pathway.

    PubMed

    Foda, Bardees M; Singh, Upinder

    2015-08-21

    RNA interference (RNAi) is a fundamental biological process that plays a crucial role in regulation of gene expression in many organisms. Transcriptional gene silencing (TGS) is one of the important nuclear roles of RNAi. Our previous data show that Entamoeba histolytica has a robust RNAi pathway that links to TGS via Argonaute 2-2 (Ago2-2) associated 27-nucleotide small RNAs with 5'-polyphosphate termini. Here, we report the first repressive histone mark to be identified in E. histolytica, dimethylation of H3K27 (H3K27Me2), and demonstrate that it is enriched at genes that are silenced by RNAi-mediated TGS. An RNAi-silencing trigger can induce H3K27Me2 deposits at both episomal and chromosomal loci, mediating gene silencing. Our data support two phases of RNAi-mediated TGS: an active silencing phase where the RNAi trigger is present and both H3K27Me2 and Ago2-2 concurrently enrich at chromosomal loci; and an established silencing phase in which the RNAi trigger is removed, but gene silencing with H3K27Me2 enrichment persist independently of Ago2-2 deposition. Importantly, some genes display resistance to chromosomal silencing despite induction of functional small RNAs. In those situations, the RNAi-triggering plasmid that is maintained episomally gets partially silenced and has H3K27Me2 enrichment, but the chromosomal copy displays no repressive histone enrichment. Our data are consistent with a model in which H3K27Me2 is a repressive histone modification, which is strongly associated with transcriptional repression. This is the first example of an epigenetic histone modification that functions to mediate RNAi-mediated TGS in the deep-branching eukaryote E. histolytica.

  10. Simultaneous detection and differentiation of Entamoeba histolytica, E. dispar, E. moshkovskii, Giardia lamblia and Cryptosporidium spp. in human fecal samples using multiplex PCR and qPCR-MCA.

    PubMed

    Zebardast, Nozhat; Yeganeh, Farshid; Gharavi, Mohammad Javad; Abadi, Alireza; Seyyed Tabaei, Seyyed Javad; Haghighi, Ali

    2016-10-01

    Entamoeba histolytica, Giardia lamblia and Cryptosporidium spp. are common causes of diarrheal and intestinal diseases all over the world. Microscopic methods are useful in the diagnosis of intestinal parasites (IPs), but their sensitivity was assessed approximately 60 percent. Recently, molecular techniques have been used increasingly for the identification and characterization of the parasites. Among those, in this study we have used multiplex PCR and Real-time PCR with melting curve analysis (qPCR-MCA) for simultaneous detection and differentiation of E. histolytica, E. dispar, E. moshkovskii, G. lamblia and Cryptosporidium spp. in human fecal samples. Twenty DNA samples from 12 E. histolytica and 8 E. dispar samples and twenty stool samples confirmed positive for G. lamblia and Cryptosporidium spp. were analyzed. After DNA extraction from the samples, multiplex PCR was done for detection and differentiation of above mentioned parasites. QPCR-MCA was also performed for the detection and differentiation of 11 isolates of above mentioned parasite in a cycle with a time and temperature. Multiplex PCR was able to simultaneous detect and differentiate of above mentioned parasite in a single reaction. QPCR-MCA was able to differentiate genus and species those five protozoa using melting temperature simultaneously at the same time and temperature programs. In total, qPCR-MCA diagnosed 7/11 isolation of E. histolytica, 6/8 isolation of E. dispar, 1/1 E. moshkovskii Laredo, 10/11 G. Lamblia and 6/11 Cryptosporidium spp. Application of multiplex PCR for detection of more than one species in a test in developing countries, at least in reference laboratories has accurate diagnosis and plays a critical role in differentiation of protozoan species. Multiplex PCR assay with a template and multi template had different results and it seems that using a set of primers with one template has higher diagnostic capability in compare with multi template. The results of this study

  11. Prevalence and characters of Entamoeba histolytica infection in Saudi infants and children admitted with diarrhea at 2 main hospitals at South Jeddah: a re-emerging serious infection with unusual presentation.

    PubMed

    Hegazi, Moustafa Abdelaal; Patel, Tabarek Ahmed; El-Deek, Basem Salama

    2013-01-01

    In this study, Entamoeba histolytica had high prevalence and unusual presentation by affecting high proportion of infants under 1 year; severe clinical manifestations, and laboratory findings that were known to be usually encountered in invasive amebiasis as significant leukocytosis for age, neutrophilic leukocytosis for age, and positive C-reactive protein were found among more than 50% of admitted Saudi infants and children with E. histolytica infection in our locality. E. histolytica can be a re-emerging serious infection when it finds favorable environmental conditions and host factors which are mainly attributed to inadequate breastfeeding in this study. This may occur in any other area of the world with the same risk factors, so we must be ready to tackle it with effective and more powerful preventive measures.

  12. Efficiency of Direct Microscopy of Stool Samples Using an Antigen-Specific Adhesin Test for Entamoeba Histolytica

    PubMed Central

    İrvem, Arzu; Özdil, Kamil; Çalışkan, Zuhal; Yücel, Muhterem

    2016-01-01

    Background: E. histolytica is among the common causes of acute gastroenteritis. The pathogenic species E. histolytica and the nonpathogenic species E. dispar cannot be morphologically differentiated, although correct identification of these protozoans is important for treatment and public health. In many laboratories, the screening of leukocytes, erythrocytes, amoebic cysts, trophozoites and parasite eggs is performed using Native-Lugol’s iodine for pre-diagnosis. Aims: In this study, we aimed to investigate the frequency of E. histolytica in stool samples collected from 788 patients residing in the Anatolian region of İstanbul who presented with gastrointestinal complaints. We used the information obtained to evaluate the effectiveness of microscopic examinations when used in combination with the E. histolytica adhesin antigen test. Study Design: Retrospective cross-sectional study Methods: Preparations of stool samples stained with Native-Lugol’s iodine were evaluated using the E. histolytica adhesin test and examined using standard light microscopy at ×40 magnification. Pearson’s Chi-square and Fisher’s exact tests were used for statistical analysis. Logistic regression analysis was used for multivariate analysis. Results: Of 788 samples, 38 (4.8%) were positive for E. histolytica adhesin antigens. When evaluated together with the presences of erythrocytes, leukocytes, cysts, and trophozoites, respectively, using logistic regression analysis, leukocyte positivity was significantly higher. The odds ratio of leukocyte positivity increased adhesin test-positivity by 2,530-fold (95% CI=1.01–6.330). Adhesin test-positivity was significant (p=0.047). Conclusion: In line with these findings, the consistency between the presence of cysts and erythrocytes and adhesin test-positivity was found to be highly significant, but that of higher levels of leukocytes was found to be discordant. It was concluded that leukocytes and trophozoites were easily misjudged

  13. Assessments of Immunomodulatory and Inflammatory effects against Induction of Entamoeba histolytica (HM1 IMS strain) crude extract Antigen in Complete Freund's Adjuvant Induced Rheumatoid Arthritis Female Wistar Rats.

    PubMed

    Bagde, Swati; Singh, Vinod

    2015-01-01

    Today it is well known about mechanisms of cell communication, how the cells that mediate immune response and tissue injury accumulate in tissues but the aetiology of rheumatoid arthritis (RA) is still unknown. This study was to evaluate immunomodulatory effects of crude Entamoeba histolytica (HM1 IMS strain) antigen in complete freund's adjuvant female wistar rats by studying the alterations in humoral and cell mediated immune responses and also the inflammatory effects by evaluating the changes in body weight, paw thickness, biochemical, serological, interleukin-6 (IL-6), IL-10 and tumor necrosis factor-α (TNF-α) and histopathology activities. Animals were randomly divided into six groups (n=6). CFA was induced in arthritic, drug and AA+CFA group whereas, 0.5ml amoebic antigen was induced subplantal in AA group while 0.5ml dose of amoebic antigen was given orally to AA+CFA group for 7-28th days. Indomethacin was used as a standard drug. Effects of amoebic antigen were associated with increased paw thickness and decreased body weight when compared to healthy control showed a significant difference. Oral administration of amoebic antigen has showed increased severe symptoms of arthritis in AA+CFA on comparison to healthy control rats. Significant increase in serum level of IL-6 and α TNF were found in AA group followed by AA+CFA group whereas, decrease in concentration of IL-10 was appear in AA+CFA group on comparison to arthritic and healthy control group (P<0.05). Histopathology of AA group showed severe signs of necrotic and degenerative changes on comparison to healthy control group. Thus the results demonstrated that E. histolytica alone or in combination with CFA increased bone damage, with alterations in antioxidant level in liver and kidney tissue homogenates as well as showed immunomodulatory arthritogenic properties which may contribute and raise joint inflammation.

  14. Evidence for Lateral Transfer of Genes Encoding Ferredoxins, Nitroreductases, NADH Oxidase, and Alcohol Dehydrogenase 3 from Anaerobic Prokaryotes to Giardia lamblia and Entamoeba histolytica

    PubMed Central

    Nixon, Julie E. J.; Wang, Amy; Field, Jessica; Morrison, Hilary G.; McArthur, Andrew G.; Sogin, Mitchell L.; Loftus, Brendan J.; Samuelson, John

    2002-01-01

    Giardia lamblia and Entamoeba histolytica are amitochondriate, microaerophilic protists which use fermentation enzymes like those of bacteria to survive anaerobic conditions within the intestinal lumen. Genes encoding fermentation enzymes and related electron transport peptides (e.g., ferredoxins) in giardia organisms and amebae are hypothesized to be derived from either an ancient anaerobic eukaryote (amitochondriate fossil hypothesis), a mitochondrial endosymbiont (hydrogen hypothesis), or anaerobic bacteria (lateral transfer hypothesis). The goals here were to complete the molecular characterization of giardial and amebic fermentation enzymes and to determine the origins of the genes encoding them, when possible. A putative giardia [2Fe-2S]ferredoxin which had a hypothetical organelle-targeting sequence at its N terminus showed similarity to mitochondrial ferredoxins and the hydrogenosomal ferredoxin of Trichomonas vaginalis (another luminal protist). However, phylogenetic trees were star shaped, with weak bootstrap support, so we were unable to confirm or rule out the endosymbiotic origin of the giardia [2Fe-2S]ferredoxin gene. Putative giardial and amebic 6-kDa ferredoxins, ferredoxin-nitroreductase fusion proteins, and oxygen-insensitive nitroreductases each tentatively supported the lateral transfer hypothesis. Although there were not enough sequences to perform meaningful phylogenetic analyses, the unique common occurrence of these peptides and enzymes in giardia organisms, amebae, and the few anaerobic prokaryotes suggests the possibility of lateral transfer. In contrast, there was more robust phylogenetic evidence for the lateral transfer of G. lamblia genes encoding an NADH oxidase from a gram-positive coccus and a microbial group 3 alcohol dehydrogenase from thermoanaerobic prokaryotes. In further support of lateral transfer, the G. lamblia NADH oxidase and adh3 genes appeared to have an evolutionary history distinct from those of E. histolytica. PMID

  15. Contact-dependent transfer of the galactose-specific lectin of Entamoeba histolytica to the lateral surface of enterocytes in culture.

    PubMed

    Leroy, A; De Bruyne, G; Mareel, M; Nokkaew, C; Bailey, G; Nelis, H

    1995-11-01

    In a study to investigate early interactions of Entamoeba histolytica with epithelial cell monolayers, we found that a monoclonal antibody (MAb), CD6, against an ameba surface antigen recognized the lateral surface of epithelial cells after coculture with trophozoites. Display of the CD6 antigen on the epithelial cells necessitated contact with active trophozoites. It was found neither at 4 degrees C, nor with prefixed trophozoites, nor with trophozoite-conditioned media, nor when a filter prevented direct contact. Monolayers exposed to amebic sonicates or detergent lysates showed random immunostaining. Access to the antigenic site was limited, as immunostaining occurred predominantly with subconfluent monolayers. CD6 epithelial cell binding was first observed after 5 min of coculture; trophozoite-mediated target cell lysis was not detected until 15 min following coculture. Epithelial cell immunostaining occurred with some other ameba-specific antibodies but not with MAbs raised against the 170-kDa subunit of the galactose-N-acetylgalactosamine (Gal/GalNAc)-specific lectin. The CD6 MAb as well as some other ameba-specific antibodies immunoprecipitated from trophozoite lysates the same bands as the MAbs against the cysteine-rich domain of the 170-kDa subunit of the Gal/GalNAc-specific lectin. Why the latter MAbs failed to stain epithelial cells in the vicinity of attached trophozoites is presently unknown. We concluded that E. histolytica trophozoites transferred the intact amebic Gal/GalNAc-specific lectin or a portion of it to the lateral surface of epithelial cells. This juxtacrine transfer preceded killing of target cells.

  16. Crystallization and preliminary crystallographic analysis of calcium-binding protein-2 from Entamoeba histolytica and its complexes with strontium and the IQ1 motif of myosin V

    SciTech Connect

    Gourinath, S. Padhan, Narendra; Alam, Neelima; Bhattacharya, Alok

    2005-04-01

    Calcium-binding protein-2 (EhCaBP2) crystals were grown using MPD as a precipitant. EhCaBP2 also crystallized in complex with strontium (replacing calcium) at similar conditions. Preliminary data for EhCaBP2 crystals in complex with an IQ motif are also reported. Calcium plays a pivotal role in the pathogenesis of amoebiasis, a major disease caused by Entamoeba histolytica. Two domains with four canonical EF-hand-containing calcium-binding proteins (CaBPs) have been identified from E. histolytica. Even though they have very high sequence similarity, these bind to different target proteins in a Ca{sup 2+}-dependent manner, leading to different functional pathways. Calcium-binding protein-2 (EhCaBP2) crystals were grown using MPD as a precipitant. The crystals belong to space group P2{sub 1}, with unit-cell parameters a = 111.74, b = 68.83, c = 113.25 Å, β = 116.7°. EhCaBP2 also crystallized in complex with strontium (replacing calcium) at similar conditions. The crystals belong to space group P2{sub 1}, with unit-cell parameters a = 69.18, b = 112.03, c = 93.42 Å, β = 92.8°. Preliminary data for EhCaBP2 crystals in complex with an IQ motif are also reported. This complex was crystallized with MPD and ethanol as precipitating agents. These crystals belong to space group P2{sub 1}, with unit-cell parameters a = 60.5, b = 69.86, c = 86.5 Å, β = 97.9°.

  17. Overexpression of Differentially Expressed Genes Identified in Non-pathogenic and Pathogenic Entamoeba histolytica Clones Allow Identification of New Pathogenicity Factors Involved in Amoebic Liver Abscess Formation

    PubMed Central

    Lorenzen, Stephan; Schuldt, Kathrin; Bernin, Hannah; Zaruba, Mareen; Lender, Corinna; Ittrich, Harald; Roeder, Thomas; Tannich, Egbert; Lotter, Hannelore; Bruchhaus, Iris

    2016-01-01

    We here compared pathogenic (p) and non-pathogenic (np) isolates of Entamoeba histolytica to identify molecules involved in the ability of this parasite to induce amoebic liver abscess (ALA)-like lesions in two rodent models for the disease. We performed a comprehensive analysis of 12 clones (A1–A12) derived from a non-pathogenic isolate HM-1:IMSS-A and 12 clones (B1–B12) derived from a pathogenic isolate HM-1:IMSS-B. “Non-pathogenicity” included the induction of small and quickly resolved lesions while “pathogenicity” comprised larger abscess development that overstayed day 7 post infection. All A-clones were designated as non-pathogenic, whereas 4 out of 12 B-clones lost their ability to induce ALAs in gerbils. No correlation between ALA formation and cysteine peptidase (CP) activity, haemolytic activity, erythrophagocytosis, motility or cytopathic activity was found. To identify the molecular framework underlying different pathogenic phenotypes, three clones were selected for in-depth transcriptome analyses. Comparison of a non-pathogenic clone A1np with pathogenic clone B2p revealed 76 differentially expressed genes, whereas comparison of a non-pathogenic clone B8np with B2p revealed only 19 differentially expressed genes. Only six genes were found to be similarly regulated in the two non-pathogenic clones A1np and B8np in comparison with the pathogenic clone B2p. Based on these analyses, we chose 20 candidate genes and evaluated their roles in ALA formation using the respective gene-overexpressing transfectants. We conclude that different mechanisms lead to loss of pathogenicity. In total, we identified eight proteins, comprising a metallopeptidase, C2 domain proteins, alcohol dehydrogenases and hypothetical proteins, that affect the pathogenicity of E. histolytica. PMID:27575775

  18. X-ray structures of thioredoxin and thioredoxin reductase from Entamoeba histolytica and prevailing hypothesis of the mechanism of Auranofin action.

    PubMed

    Parsonage, Derek; Sheng, Fang; Hirata, Ken; Debnath, Anjan; McKerrow, James H; Reed, Sharon L; Abagyan, Ruben; Poole, Leslie B; Podust, Larissa M

    2016-05-01

    The anti-arthritic gold-containing drug Auranofin is lethal to the protozoan intestinal parasite Entamoeba histolytica, the causative agent of human amebiasis, in both culture and animal models of the disease. A putative mechanism of Auranofin action proposes that monovalent gold, Au(I), released from the drug, can bind to the redox-active dithiol group of thioredoxin reductase (TrxR). Au(I) binding in the active site is expected to prevent electron transfer to the downstream substrate thioredoxin (Trx), thus interfering with redox homeostasis in the parasite. To clarify the molecular mechanism of Auranofin action in more detail, we determined a series of atomic resolution X-ray structures for E. histolytica thioredoxin (EhTrx) and thioredoxin reductase (EhTrxR), the latter with and without Auranofin. Only the disulfide-bonded form of the active site dithiol (Cys(140)-Cys(143)) was invariably observed in crystals of EhTrxR in spite of the addition of reductants in various crystallization trials, and no gold was found associated with these cysteines. Non-catalytic Cys(286) was identified as the only site of modification, but further mutagenesis studies using the C286Q mutant demonstrated that this site was not responsible for inhibition of EhTrxR by Auranofin. Interestingly, we obtained both of the catalytically-relevant conformations of this bacterial-like, low molecular weight TrxR in crystals without requiring an engineered disulfide linkage between Cys mutants of TrxR and Trx (as was originally done with Escherichia coli TrxR and Trx). We note that the -CXXC- catalytic motif, even if reduced, would likely not provide space sufficient to bind Au(I) by both cysteines of the dithiol group.

  19. A surface membrane protein of Entamoeba histolytica functions as a receptor for human chemokine IL-8: its role in the attraction of trophozoites to inflammation sites.

    PubMed

    Diaz-Valencia, J Daniel; Pérez-Yépez, Eloy Andrés; Ayala-Sumuano, Jorge Tonatiuh; Franco, Elizabeth; Meza, Isaura

    2015-12-01

    Entamoeba histolytica trophozoites respond to the presence of IL-8, moving by chemotaxis towards the source of the chemokine. IL-8 binds to the trophozoite membrane and triggers a response that activates signaling pathways that in turn regulate actin/myosin cytoskeleton organisation to initiate migration towards the chemokine, suggesting the presence of a receptor for IL-8 in the parasite. Antibodies directed to the human IL-8 receptor (CXCR1) specifically recognised a 29 kDa protein in trophozoite membrane fractions. The same protein was immunoprecipitated by this antibody from total amebic extracts. Peptide analysis of the immunoprecipitated protein revealed a sequence with high homology to a previously identified amebic outer membrane peroxiredoxin and a motif within the third loop of human CXCR1, which is an important site for IL-8 binding and activation of signaling processes. Immunodetection assays demonstrated that the anti-human CXCR1 antibody binds to the 29 kDa protein in a different but close site to where IL-8 binds to the trophozoite surface membrane, suggesting that human and amebic receptors for this chemokine share common epitopes. In the context of the human intestinal environment, a receptor for IL-8 could be a great advantage for E. histolytica trophozoite survival, as they could reach an inflammatory milieu containing abundant nutrients. In addition, it has been suggested that the high content of accessible thiol groups of the protein and its peroxidase activity could provide protection in the oxygen rich milieu of colonic lesions, allowing trophozoite invasion of other tissues and escape from the host immune response.

  20. Mass Spectrometric Analysis of l-Cysteine Metabolism: Physiological Role and Fate of l-Cysteine in the Enteric Protozoan Parasite Entamoeba histolytica

    PubMed Central

    Jeelani, Ghulam; Sato, Dan; Soga, Tomoyoshi; Watanabe, Haruo

    2014-01-01

    ABSTRACT l-Cysteine is essential for virtually all living organisms, from bacteria to higher eukaryotes. Besides having a role in the synthesis of virtually all proteins and of taurine, cysteamine, glutathione, and other redox-regulating proteins, l-cysteine has important functions under anaerobic/microaerophilic conditions. In anaerobic or microaerophilic protozoan parasites, such as Entamoeba histolytica, l-cysteine has been implicated in growth, attachment, survival, and protection from oxidative stress. However, a specific role of this amino acid or related metabolic intermediates is not well understood. In this study, using stable-isotope-labeled l-cysteine and capillary electrophoresis-time of flight mass spectrometry, we investigated the metabolism of l-cysteine in E. histolytica. [U-13C3, 15N]l-cysteine was rapidly metabolized into three unknown metabolites, besides l-cystine and l-alanine. These metabolites were identified as thiazolidine-4-carboxylic acid (T4C), 2-methyl thiazolidine-4-carboxylic acid (MT4C), and 2-ethyl-thiazolidine-4-carboxylic acid (ET4C), the condensation products of l-cysteine with aldehydes. We demonstrated that these 2-(R)-thiazolidine-4-carboxylic acids serve for storage of l-cysteine. Liberation of l-cysteine occurred when T4C was incubated with amebic lysates, suggesting enzymatic degradation of these l-cysteine derivatives. Furthermore, T4C and MT4C significantly enhanced trophozoite growth and reduced intracellular reactive oxygen species (ROS) levels when it was added to cultures, suggesting that 2-(R)-thiazolidine-4-carboxylic acids are involved in the defense against oxidative stress. PMID:25370494

  1. Toll-like Receptor Signaling Activation by Entamoeba histolytica Induces Beta Defensin 2 in Human Colonic Epithelial Cells: Its Possible Role as an Element of the Innate Immune Response

    PubMed Central

    Ayala-Sumuano, Jorge-Tonatiuh; Téllez-López, Victor M.; Domínguez-Robles, M. del Carmen; Shibayama-Salas, Mineko; Meza, Isaura

    2013-01-01

    Background Entamoeba histolytica, a protozoan parasite of humans, produces dysenteric diarrhea, intestinal mucosa damage and extraintestinal infection. It has been proposed that the intestinal microbiota composition could be an important regulatory factor of amebic virulence and tissue invasion, particularly if pathogenic bacteria are present. Recent in vitro studies have shown that Entamoeba histolytica trophozoites induced human colonic CaCo2 cells to synthesize TLR-2 and TLR-4 and proinflammatory cytokines after binding to the amebic Gal/GalNac lectin carbohydrate recognition domain. The magnitude of the inflammatory response induced by trophozoites and the subsequent cell damage were synergized when cells had previously been exposed to pathogenic bacteria. Methodology/Principal Findings We show here that E. histolytica activation of the classic TLR pathway in CaCo2 cells is required to induce β defensin-2 (HBD2) mRNA expression and production of a 5-kDa cationic peptide with similar properties to the antimicrobial HBD2 expressed by CaCo2 cells exposed to enterotoxigenic Escherichia coli. The induced peptide showed capacity to permeabilize membranes of bacteria and live trophozoites. This activity was abrogated by inhibition of TLR2/4-NFκB pathway or by neutralization with an anti-HBD2 antibody. Conclusions/Significance Entamoeba histolytica trophozoites bind to human intestinal cells and induce expression of HBD2; an antimicrobial molecule with capacity to destroy pathogenic bacteria and trophozoites. HDB2's possible role as a modulator of the course of intestinal infections, particularly in mixed ameba/bacteria infections, is discussed. PMID:23469306

  2. Interaction between Nbp35 and Cfd1 Proteins of Cytosolic Fe-S Cluster Assembly Reveals a Stable Complex Formation in Entamoeba histolytica

    PubMed Central

    Anwar, Shadab; Dikhit, Manas Ranjan; Singh, Krishn Pratap; Kar, Rajiv Kumar; Zaidi, Amir; Sahoo, Ganesh Chandra; Roy, Awadh Kishore; Nozaki, Tomoyoshi; Das, Pradeep; Ali, Vahab

    2014-01-01

    Iron-Sulfur (Fe-S) proteins are involved in many biological functions such as electron transport, photosynthesis, regulation of gene expression and enzymatic activities. Biosynthesis and transfer of Fe-S clusters depend on Fe-S clusters assembly processes such as ISC, SUF, NIF, and CIA systems. Unlike other eukaryotes which possess ISC and CIA systems, amitochondriate Entamoeba histolytica has retained NIF & CIA systems for Fe-S cluster assembly in the cytosol. In the present study, we have elucidated interaction between two proteins of E. histolytica CIA system, Cytosolic Fe-S cluster deficient 1 (Cfd1) protein and Nucleotide binding protein 35 (Nbp35). In-silico analysis showed that structural regions ranging from amino acid residues (P33-K35, G131-V135 and I147-E151) of Nbp35 and (G5-V6, M34-D39 and G46-A52) of Cfd1 are involved in the formation of protein-protein complex. Furthermore, Molecular dynamic (MD) simulations study suggested that hydrophobic forces surpass over hydrophilic forces between Nbp35 and Cfd1 and Van-der-Waal interaction plays crucial role in the formation of stable complex. Both proteins were separately cloned, expressed as recombinant fusion proteins in E. coli and purified to homogeneity by affinity column chromatography. Physical interaction between Nbp35 and Cfd1 proteins was confirmed in vitro by co-purification of recombinant Nbp35 with thrombin digested Cfd1 and in vivo by pull down assay and immunoprecipitation. The insilico, in vitro as well as in vivo results prove a stable interaction between these two proteins, supporting the possibility of its involvement in Fe-S cluster transfer to target apo-proteins through CIA machinery in E. histolytica. Our study indicates that initial synthesis of a Fe-S precursor in mitochondria is not necessary for the formation of Cfd1-Nbp35 complex. Thus, Cfd1 and Nbp35 with the help of cytosolic NifS and NifU proteins can participate in the maturation of non-mitosomal Fe-S proteins without any

  3. Functional correlation of medicinal chemistry and Entamoeba histolytica treatment: an emphasis on the past, present and future chemotherapy.

    PubMed

    Gundamaraju, Rohit; Singla, Rajeev K; Vemuri, Ravi Chandra; Sekaran, Shamala Devi

    2015-01-01

    Entamoeba being an internal parasite is the causative organism of many human and animal infections with lethal consequences. Chemotherapy against Entamoeba was well established by both synthetic and natural products. Vigorous attainments were made by different researchers all over the world for discovering better chemical entities and the potent formulations. Preexisting compounds were developed into non-cytotoxic and with an enhanced therapeutic compounds by usage of multiparametric optimization techniques like QSAR, and molecular docking. The present review is therefore an aerial perspective of importance of medicinal chemistry in anti-amoebic chemotherapy. The review elucidates the existing synthetic compounds, their analogues, natural products, in silico studies and some novel methods adopted for the drug design as anti-amoebic agents which would be of assured benefit in the future drug design.

  4. Natural infection of baboons by Entamoeba histolytica elicits anti- gal-lectin heavy subunit IgA and IgG antibodies with shared epitope specificity to that of humans.

    PubMed

    Abd-Alla, Mohamed D; Wolf, Roman F; White, Gary L; Kosanke, Stanley D; Carey, David W; Verweij, Jaco J; El-Dessouky, Yasser M M; Zhang, Mie-Jie; Ravdin, Jonathan I

    2013-12-01

    Non-human primates, such as baboons (Papio hamadryas anubis), are natural hosts for Entamoeba species; infections can be asymptomatic or result in invasive lethal disease. It was sought to determine whether following natural infection by Entamoeba. histolytica, baboon anti-amebic antibodies recognized native Gallectin, a recombinant portion of the lectin heavy subunit (designated LC3) and specific heavy subunit epitopes; we compared the specificity of anti-amebic antibodies from baboons to that of humans following asymptomatic E. histolytica infection or cure of amebic liver abscess (ALA). Female baboons (n=54), aged one to three years of age and living in captivity were screened for infection by real time PCR. E. histolytica infection was found in 37 baboons and was associated with serum anti-LC3 IgG (73%) and anti-LC3 IgA (46%) or intestinal anti-Gal-Lectin IgA antibody responses (49%), p<0.021 for each compared to that observed with baboons having an E. dispar infection (n=10) or uninfected baboons (n=7). The ELISA OD reading for anti-LC3 or anti-lectin antibodies correlated strongly with the presence of a PCR CT value indicative of E. histolytica infection. In humans with asymptomatic E. histolytica infection or those recently cured of ALA, 63% and 57% had serum anti- LC3 IgA and 65% and 57% had serum anti-LC3 IgG antibodies respectively. Epitope- specific synthetic peptides were used as capture antigens in ELISA; for baboons that possessed anti-LC3 and anti-lectin antibodies, 74% had anti-peptide IgG or IgA antibodies, compared to 86% of asymptomatic humans and 92% of ALA subjects(P>0.05).

  5. Entamoeba histolytica: expression and localization of Gal/GalNAc lectin in virulent and non-virulent variants from HM1:IMSS strain.

    PubMed

    López-Vancell, R; Arreguín Espinosa, R; González-Canto, A; Néquiz Avendaño, M; García de León, M C; Olivos-García, A; López-Vancell, D; Pérez-Tamayo, R

    2010-07-01

    We have purified Gal/GalNAc lectin from Entamoeba histolytica by electroelution. The purified protein was used to immunize rabbits and obtain polyclonal IgG's anti-lectin. These antibodies were used as tools to analyze the expression and localization of the amoebic lectin in both virulent (vEh) and non-virulent (nvEh) variants of axenically cultured HM1:IMSS strain. vEh is able to induce liver abscesses in hamsters, whereas nvEh has lost this ability. In vitro, amoebic trophozoites from both variants equally express this protein as shown by densitometric analysis of the corresponding band in Western blots from lysates. In both types of trophozoites, the pattern of distribution of the lectin was mainly on the surface. We have also compared by immunohistochemistry the presence and distribution of lectin in the in vivo liver lesions produced in hamsters. In order to prolong the survival of nvEh to analyze both variants in an in vivo model, hamsters inoculated with nvEh were treated with methyl prednisolone. Our results suggest that the Gal/GalNAc lectin is equally expressed in both nvEh and vEh.

  6. Insights into the GTP/GDP cycle of RabX3, a novel GTPase from Entamoeba histolytica with tandem G-domains.

    PubMed

    Chandra, Mintu; Mukherjee, Madhumita; Srivastava, Vijay Kumar; Saito-Nakano, Yumiko; Nozaki, Tomoyoshi; Datta, Sunando

    2014-02-25

    Members of the small GTPase Ras superfamily regulate a host of systems through their ability to catalyze the GTP/GDP cycle. All family members reported thus far possess a single GTPase domain with a P-loop containing a nucleoside triphosphate hydrolase fold. Here for the first time we report a novel member from Entamoeba histolytica, EhRabX3, which harbors two GTPase domains in tandem and exhibits unique biochemical properties. A combination of biochemical and microcalorimetric studies revealed that EhRabX3 binds to a single guanine nucleotide through its N-terminal domain. Unlike most of the members of the Ras superfamily, the dissociation of the nucleotide from EhRabX3 is independent of Mg(2+), perhaps indicating a novel mechanism of nucleotide exchange by this protein. We found that EhRabX3 is extremely sluggish in hydrolyzing GTP, and that could be attributed to its atypical nucleotide binding pocket. It harbors substitutions at two positions that confer oncogenicity to Ras because of impaired GTP hydrolysis. Engineering these residues into the conserved counterparts enhanced their GTPase activity by at least 20-fold. In contrast to most of the members of the Ras superfamily, EhRabX3 lacks the prenylation motif. Using indirect immunofluorescence and biochemical fractionation, we demonstrated that the protein is distributed all over the cytosol in amoebic trophozoites. Collectively, this unique ancient GTPase exhibits a striking evolutionary divergence from the other members of the superfamily.

  7. Biophysical studies on calcium and carbohydrate binding to carbohydrate recognition domain of Gal/GalNAc lectin from Entamoeba histolytica: insights into host cell adhesion.

    PubMed

    Yadav, Rupali; Verma, Kuldeep; Chandra, Mintu; Mukherjee, Madhumita; Datta, Sunando

    2016-09-01

    Entamoeba histolytica, an enteric parasite expresses a Gal/GalNAc-specific lectin that contributes to its virulence by establishing adhesion to host cell. In this study, carbohydrate recognition domain of Hgl (EhCRD) was purified and biophysical studies were conducted to understand the thermodynamic basis of its binding to carbohydrate and Ca(++) Here, we show that carbohydrate recognition domain (CRD) of the lectin binds to calcium through DPN motif. To decipher the role of calcium in carbohydrate binding and host cell adhesion, biophysical and cell-based studies were carried out. We demonstrated that the presence of the cation neither change the affinity of the lectin for carbohydrates nor alters its conformation. Mutation of the calcium-binding motif in EhCRD resulted in complete loss of ability to bind calcium but retained its affinity for carbohydrates. Purified EhCRD significantly diminished adhesion of the amebic trophozoites to Chinese Hamster Ovary (CHO) cells as well as triggered red blood cell agglutination. The calcium-binding defective mutant abrogated amebic adhesion to CHO cells similar to the wild-type protein, but it failed to agglutinate RBCs suggesting a differential role of the cation in these two processes. This study provides the first molecular description of the role of calcium in Gal/GalNAc mediated host cell adhesion.

  8. Autophosphorylation at Thr279 of Entamoeba histolytica atypical kinase EhAK1 is required for activity and regulation of erythrophagocytosis

    PubMed Central

    Mansuri, M Shahid; Babuta, Mrigya; Ali, Mohammad Sabir; Bharadwaj, Ravi; jhingan, Gagan Deep; Gourinath, Samudrala; Bhattacharya, Sudha; Bhattacharya, Alok

    2016-01-01

    Phagocytosis plays a key role in survival and pathogenicity of Entamoeba histolytica. We have recently demonstrated that an atypical kinase EhAK1 is involved in phagocytosis in this parasite. It is recruited to the phagocytic cups through interaction with EhCaBP1. EhAK1 manipulates actin dynamics by multiple mechanisms including phosphorylation of G-actin. Biochemical analysis showed that EhAK1 is a serine/threonine kinase with broad ion specificity and undergoes multiple trans-autophosphorylation. Three autophosphorylation sites were identified by mass spectrometry. Out of these Thr279 appears to be involved in both autophosphorylation as well as substrate phosphorylation. Over expression of the mutant Thr279A inhibited erythrophagocytosis showing dominant negative phenotype. Multiple alignments of different kinases including alpha kinases displayed conserved binding sites that are thought to be important for function of the protein. Mutation studies demonstrated the importance of some of these binding sites in kinase activity. Binding studies with fluorescent-ATP analogs supported our prediction regarding ATP binding site based on sequence alignment. In conclusion, EhAK1 has multiple regulatory features and enrichment of EhAK1 at the site of phagocytosis stimulates trans-autophosphorylation reaction that increases kinase activity resulting in enhanced actin dynamics and phagocytosis. Some of the properties of EhAK1 are similar to that seen in alpha kinases. PMID:26739245

  9. STUDIES ON THE CARBOHYDRATE METABOLISM OF A GRAM-NEGATIVE ANAEROBE (BACTEROIDES SYMBIOSUS) USED IN THE CULTURE OF ENTAMOEBA HISTOLYTICA1

    PubMed Central

    Bragg, P. D.; Reeves, Richard E.

    1962-01-01

    Bragg, P. D. (Louisiana State University, New Orleans) and R. E. Reeves. Studies on the carbohydrate metabolism of a gram-negative anaerobe (Bacteroides symbiosus) used in the culture of Entamoeba histolytica. J. Bacteriol. 83:76–84. 1962—Resting cells of Bacteroides symbiosus have been shown to utilize glucose and several other monosaccharides. The fermentation of the sugars is mediated by demonstrable kinases except in the case of mannitol. The main end products of metabolism of glucose are CO2, H2, ethanol, and acetic, butyric, succinic, and lactic acids. Changes in the thiol used in the growth media produce different enzyme complements in the cells. Thus, cells grown with cysteine as the thiol are unable to metabolize glucosamine, whereas those grown with thiomalate rapidly degrade the amino sugar. The results of the enzyme assay and the results from experiments with C14-labelled glucose suggest that glucose is metabolized by resting cells mainly by the Embden-Myerhof pathway. PMID:13872395

  10. Prevalence of Cryptosporidium parvum/hominis, Entamoeba histolytica and Giardia lamblia among Young Children with and without Diarrhea in Dar es Salaam, Tanzania

    PubMed Central

    Tellevik, Marit G.; Moyo, Sabrina J.; Blomberg, Bjørn; Hjøllo, Torunn; Maselle, Samuel Y.; Langeland, Nina; Hanevik, Kurt

    2015-01-01

    Background Although enteroparasites are common causes of diarrheal illness, few studies have been performed among children in Tanzania. This study aimed to investigate the prevalence of Cryptosporidium parvum/hominis, Entamoeba histolytica and Giardia lamblia among young children in Dar es Salaam, Tanzania, and identify risk factors for infection. Methodology/Principal Findings We performed an unmatched case-control study among children < 2 years of age in Dar es Salaam, recruited from August 2010 to July 2011. Detection and identification of protozoans were done by PCR techniques on DNA from stool specimens from 701 cases of children admitted due to diarrhea at the three study hospitals, and 558 controls of children with no history of diarrhea during the last month prior to enrollment. The prevalence of C. parvum/hominis was 10.4% (84.7% C. hominis), and that of G. lamblia 4.6%. E. histolytica was not detected. The prevalence of Cryptosporidium was significantly higher in cases (16.3%) than in controls (3.1%; P < 0.001; OR = 6.2; 95% CI: 3.7–10.4). G. lamblia was significantly more prevalent in controls (6.1%) than in cases (3.4%; P = 0.027; OR = 1.8; 95% CI: 1.1–3.1). Cryptosporidium infection was found more often in HIV-positive (24.2%) than in HIV-negative children (3.9%; P < 0.001; OR = 7.9; 95% CI: 3.1–20.5), and was also associated with rainfall (P < 0.001; OR = 2.41; 95% CI: 1.5–3.8). Among cases, stunted children had significantly higher risk of being infected with Cryptosporidium (P = 0.011; OR = 2.12; 95% CI: 1.2–3.8). G. lamblia infection was more prevalent in the cool season (P = 0.004; OR = 2.2; 95% CI: 1.3–3.8), and more frequent among cases aged > 12 months (P = 0.003; OR = 3.5; 95% CI: 1.5–7.8). Among children aged 7–12 months, those who were breastfed had lower prevalence of G. lamblia infection than those who had been weaned (P = 0.012). Conclusions Cryptosporidium infection is common among young Tanzanian children with diarrhea

  11. Entamoeba histolytica: increase of enterotoxicity and of 53- and 75-kDa cysteine proteinases in a clone of higher virulence.

    PubMed

    Navarro-García, F; Chávez-Dueñas, L; Tsutsumi, V; Posadas del Río, F; López-Revilla, R

    1995-05-01

    We compared the enterotoxicity and cysteine proteinases (CP) of the low-virulence Entamoeba histolytica HM1 strain with the highly virulent 1659 clone, derived from HM1 by hamster liver passages. Enterotoxicity of 50,000 freeze-thawed trophozoites was determined on 0.28-cm2 intestinal segments mounted in Ussing chambers; CP activity of Nonidet-P40 amebal lysates was assayed by gelatin-sodium dodecyl sulfate-polyacrylamide gel electrophoresis and carbobenzoxy-L-arginine-L-arginyl-p-nitroaniline, a CP-specific substrate. Treatment of gerbil cecum segments with amebal lysates caused an immediate fall of their electrophysiologic properties (potential difference, short-circuit current, and transmural resistance) whose decay rates were clearly faster with 1659 than with HM1 lysates. Nonimmune and immune antiamebic human sera and the CP-specific inhibitor E-64 (trans-epoxysuccinyl-L-leucylamido(4-guanidino)butane) prevented the fall of the electrophysiologic properties. Gelatinases, less active in HM1 than in 1659 trophozoites, were better preserved in lysates containing 10 mM p-hydroxymercuribenzoate (pHMB) to prevent autoproteolysis: in lysates without pHMB nearly no gelatinase bands were observed in HM1 samples, whereas intense 30K, 35K, 44K, and 75K bands were seen in 1659 samples; in lysates with pHMB only 53K and 75K bands were found that were much more intense in 1659 samples, 75K being barely visible in HM1 samples. The overall CP activity was 17 times higher in 1659 than in HM1 lysates, was inhibited by E-64 (mean inhibitory dose, 20 microM), was stimulated by 2-mercaptoethanol (ME) 3.7 times in HM1 and 2.4 times in 1659 lysates, and was reactivated by ME in lysates containing pHMB. Most of the CP activity in HM1 lysates sedimented at 15,600g but predominated in 1659 supernatants. The increase of E. histolytica virulence thus correlates with a remarkable increase both of in vitro enterotoxicity and of two CPs (53K and 75K), suggesting that these proteinases are

  12. The NLRP3 Inflammasome Is a Pathogen Sensor for Invasive Entamoeba histolytica via Activation of α5β1 Integrin at the Macrophage-Amebae Intercellular Junction

    PubMed Central

    Mortimer, Leanne; Moreau, France; Cornick, Steve; Chadee, Kris

    2015-01-01

    Entamoeba histolytica (Eh) is an extracellular protozoan parasite of humans that invades the colon to cause life-threatening intestinal and extra-intestinal amebiasis. Colonized Eh is asymptomatic, however, when trophozoites adhere to host cells there is a considerable inflammatory response that is critical in the pathogenesis of amebiasis. The host and/or parasite factors that trigger the inflammatory response to invading Eh are not well understood. We recently identified that Eh adherence to macrophages induces inflammasome activation and in the present study we sought to determine the molecular events upon contact that coordinates this response. Here we report that Eh contact-dependent activation of α5β1 integrin is critical for activation of the NLRP3 inflammasome. Eh-macrophage contact triggered recruitment of α5β1 integrin and NLRP3 into the intercellular junction, where α5β1 integrin underwent activation by an integrin-binding cysteine protease on the parasite surface, termed EhCP5. As a result of its activation, α5β1 integrin induced ATP release into the extracellular space through opening of pannexin-1 channels that signalled through P2X7 receptors to deliver a critical co-stimulatory signal that activated the NLRP3 inflammasome. Both the cysteine protease activity and integrin-binding domain of EhCP5 were required to trigger α5β1 integrin that led to ATP release and NLRP3 inflammasome activation. These findings reveal engagement of α5β1 integrin across the parasite-host junction is a key regulatory step that initiates robust inflammatory responses to Eh. We propose that α5β1 integrin distinguishes Eh direct contact and functions with NLRP3 as pathogenicity sensor for invasive Eh infection. PMID:25955828

  13. The NLRP3 Inflammasome Is a Pathogen Sensor for Invasive Entamoeba histolytica via Activation of α5β1 Integrin at the Macrophage-Amebae Intercellular Junction.

    PubMed

    Mortimer, Leanne; Moreau, France; Cornick, Steve; Chadee, Kris

    2015-05-01

    Entamoeba histolytica (Eh) is an extracellular protozoan parasite of humans that invades the colon to cause life-threatening intestinal and extra-intestinal amebiasis. Colonized Eh is asymptomatic, however, when trophozoites adhere to host cells there is a considerable inflammatory response that is critical in the pathogenesis of amebiasis. The host and/or parasite factors that trigger the inflammatory response to invading Eh are not well understood. We recently identified that Eh adherence to macrophages induces inflammasome activation and in the present study we sought to determine the molecular events upon contact that coordinates this response. Here we report that Eh contact-dependent activation of α5β1 integrin is critical for activation of the NLRP3 inflammasome. Eh-macrophage contact triggered recruitment of α5β1 integrin and NLRP3 into the intercellular junction, where α5β1 integrin underwent activation by an integrin-binding cysteine protease on the parasite surface, termed EhCP5. As a result of its activation, α5β1 integrin induced ATP release into the extracellular space through opening of pannexin-1 channels that signalled through P2X7 receptors to deliver a critical co-stimulatory signal that activated the NLRP3 inflammasome. Both the cysteine protease activity and integrin-binding domain of EhCP5 were required to trigger α5β1 integrin that led to ATP release and NLRP3 inflammasome activation. These findings reveal engagement of α5β1 integrin across the parasite-host junction is a key regulatory step that initiates robust inflammatory responses to Eh. We propose that α5β1 integrin distinguishes Eh direct contact and functions with NLRP3 as pathogenicity sensor for invasive Eh infection.

  14. Crystal Structure Analysis of Wild Type and Fast Hydrolyzing Mutant of EhRabX3, a Tandem Ras Superfamily GTPase from Entamoeba histolytica.

    PubMed

    Srivastava, Vijay Kumar; Chandra, Mintu; Saito-Nakano, Yumiko; Nozaki, Tomoyoshi; Datta, Sunando

    2016-01-16

    The enteric protozoan parasite, Entamoeba histolytica, is the causative agent of amoebic dysentery, liver abscess and colitis in human. Vesicular trafficking plays a key role in the survival and virulence of the protozoan and is regulated by various Rab GTPases. EhRabX3 is a catalytically inefficient amoebic Rab protein, which is unique among the eukaryotic Ras superfamily by virtue of its tandem domain organization. Here, we report the crystal structures of GDP-bound fast hydrolyzing mutant (V71A/K73Q) and GTP-bound wild type EhRabX3 at 3.1 and 2.8Å resolutions, respectively. Though both G-domains possess "phosphate binding loop containing nucleoside triphosphate hydrolases fold", only the N-terminal domain binds to guanine nucleotide. The relative orientation of the N-terminal domain and C-terminal domain is stabilized by numerous inter-domain interactions. Compared to other Ras superfamily members, both the GTPase domains displayed large deviation in switch II perhaps due to non-conservative substitutions in this region. As a result, entire switch II is restructured and moved away from the nucleotide binding pocket, providing a rationale for the diminished GTPase activity of EhRabX3. The N-terminal GTPase domain possesses unusually large number of cysteine residues. X-ray crystal structure of the fast hydrolyzing mutant of EhRabX3 revealed that C39 and C163 formed an intra-molecular disulfide bond. Subsequent mutational and biochemical studies suggest that C39 and C163 are critical for maintaining the structural integrity and function of EhRabX3. Structure-guided functional investigation of cysteine mutants could provide the physiological implications of the disulfide bond and could allow us to design potential inhibitors for the better treatment of intestinal amebiasis.

  15. Conformational changes induced by detergents during the refolding of chemically denatured cysteine protease ppEhCP-B9 from Entamoeba histolytica.

    PubMed

    Zamudio-Prieto, Olga; Benítez-Cardoza, Claudia; Arroyo, Rossana; Ortega-López, Jaime

    2014-07-01

    EhCP-B9, a cysteine protease (CP) involved in Entamoeba histolytica virulence, is a potential target for disease diagnosis and drug design. After purification from inclusion bodies produced in Escherichia coli, the recombinant EhCP-B9 precursor (ppEhCP-B9) can be refolded using detergents as artificial chaperones. However, the conformational changes that occur during ppEhCP-B9 refolding remain unknown. Here, we comprehensively describe conformational changes of ppEhCP-B9 that are induced by various chemical detergents acting as chaperones, including non-ionic, zwitterionic, cationic and anionic surfactants. We monitored the effect of detergent concentration and incubation time on the secondary and tertiary structures of ppEhCP-B9 using fluorescence and circular dichroism (CD) spectroscopy. In the presence of non-ionic and zwitterionic detergents, ppEhCP-B9 adopted a β-enriched structure (ppEhCP-B9(β1)) without proteolytic activity at all detergent concentrations and incubation times evaluated. ppEhCP-B9 also exhibits a β-rich structure in low concentrations of ionic detergents, but at concentrations above the critical micelle concentration (CMC), the protein acquires an α+β structure, similar to that of papain but without proteolytic activity (ppEhCP-B9(α+β1)). Interestingly, only within a narrow range of experimental conditions in which SDS concentrations were below the CMC, ppEhCP-B9 refolded into a β-sheet rich structure (ppEhCP-B9(β2)) that slowly transforms into a different type of α+β conformation that exhibited proteolytic activity (ppEhCP-B9(α+β2)) suggesting that enzymatic activity is gained as slow transformation occurs.

  16. Laboratory Diagnostic Techniques for Entamoeba Species

    PubMed Central

    Fotedar, R.; Stark, D.; Beebe, N.; Marriott, D.; Ellis, J.; Harkness, J.

    2007-01-01

    The genus Entamoeba contains many species, six of which (Entamoeba histolytica, Entamoeba dispar, Entamoeba moshkovskii, Entamoeba polecki, Entamoeba coli, and Entamoeba hartmanni) reside in the human intestinal lumen. Entamoeba histolytica is the causative agent of amebiasis and is considered a leading parasitic cause of death worldwide in humans. Although recent studies highlight the recovery of E. dispar and E. moshkovskii from patients with gastrointestinal symptoms, there is still no convincing evidence of a causal link between the presence of these two species and the symptoms of the host. New approaches to the identification of E. histolytica are based on detection of E. histolytica-specific antigen and DNA in stool and other clinical samples. Several molecular diagnostic tests, including conventional and real-time PCR, have been developed for the detection and differentiation of E. histolytica, E. dispar, and E. moshkovskii in clinical samples. The purpose of this review is to discuss different methods that exist for the identification of E. histolytica, E. dispar, and E. moshkovskii which are available to the clinical diagnostic laboratory. To address the need for a specific diagnostic test for amebiasis, a substantial amount of work has been carried out over the last decade in different parts of the world. The molecular diagnostic tests are increasingly being used for both clinical and research purposes. In order to minimize undue treatment of individuals infected with other species of Entamoeba such as E. dispar and E. moshkovskii, efforts have been made for specific diagnosis of E. histolytica infection and not to treat based simply on the microscopic examination of Entamoeba species in the stool. The incorporation of many new technologies into the diagnostic laboratory will lead to a better understanding of the public health problem and measures to control the disease. PMID:17630338

  17. EhNPC1 and EhNPC2 Proteins Participate in Trafficking of Exogenous Cholesterol in Entamoeba histolytica Trophozoites: Relevance for Phagocytosis

    PubMed Central

    Bolaños, Jeni; Betanzos, Abigail; Javier-Reyna, Rosario; García- Rivera, Guillermina; Huerta, Miriam; Pais-Morales, Jonnatan; González-Robles, Arturo; Rodríguez, Mario A.

    2016-01-01

    Entamoeba histolytica, the highly phagocytic protozoan causative of human amoebiasis lacks the machinery to synthesize cholesterol. Here, we investigated the presence of NPC1 and NPC2 proteins in this parasite, which are involved in cholesterol trafficking in mammals. Bioinformatics analysis revealed one Ehnpc1 and two Ehnpc2 genes. EhNPC1 appeared as a transmembrane protein and both EhNPC2 as peripheral membrane proteins. Molecular docking predicted that EhNPC1 and EhNPC2 bind cholesterol and interact with each other. Genes and proteins were identified in trophozoites. Serum pulse-chase and confocal microscopy assays unveiled that after trophozoites sensed the cholesterol source, EhNPC1 and EhNPC2 were organized around the plasma membrane in a punctuated pattern. Vesicles emerged and increased in number and size and some appeared full of cholesterol with EhNPC1 or EhNPC2 facing the extracellular space. Both proteins, but mostly EhNPC2, were found out of the cell associated with cholesterol. EhNPC1 and cholesterol formed networks from the plasma membrane to the nucleus. EhNPC2 appeared in erythrocytes that were being ingested by trophozoites, co-localizing with cholesterol of erythrocytes, whereas EhNPC1 surrounded the phagocytic cup. EhNPC1 and EhNPC2 co-localized with EhSERCA in the endoplasmic reticulum and with lysobisphosphatidic acid and EhADH (an Alix protein) in phagolysosomes. Immunoprecipitation assays confirmed the EhNPC1 and EhNPC2 association with cholesterol, EhRab7A and EhADH. Serum starved and blockage of cholesterol trafficking caused a low rate of phagocytosis and incapability of trophozoites to produce damage in the mouse colon. Ehnpc1 and Ehnpc2 knockdown provoked in trophozoites a lower intracellular cholesterol concentration and a diminished rate of phagocytosis; and Ehnpc1 silencing also produced a decrease of trophozoites movement. Trafficking of EhNPC1 and EhNPC2 during cholesterol uptake and phagocytosis as well as their association with

  18. [Prevalence and polyparasitism of intestinal protozoa and spatial distribution of Entamoeba histolytica, E. dispar and Giardia intestinalis from pupils in the rural zone of Man in Côte d'Ivoire].

    PubMed

    Ouattara, Mamadou; Silué, Kigbafori Dieudonné; N'Guéssan, Aya Nicaise; Yapi, Ahoua; Barbara, Matthys; Raso, Giovanna; Utzinger, Juerg; N'Goran, Eliezer

    2008-01-01

    Diseases caused by environmental contamination by micro-organisms, including intestinal helminths and protozoa, are prevalent in developing countries. According to some authors, their strong expansion in some zones of these countries is due primarily to favourable climatic conditions, combined with inadequate hygiene measures and cleaning and the generally low socio-economic level. Progress in disease control has resulted from new studies that improve our understanding of the epidemiology of helminthiases and from the availability of simple tools that are inexpensive and effective against these diseases (chemotherapy with albendazole and mebendazole). On the other hand, surprisingly few such studies have looked at intestinal protozoa, although the WHO reports that approximately 480 million individuals throughout the world are infested by amoebiasis caused by the protozoon Entamoeba histolytica and that 40,000-110,000 people die from it each year. Giardiasis, a cosmopolitan parasitosis, is due to another intestinal protozoon called Giardia intestinalis. To help develop a database on these parasites, we conducted a cross-sectional epidemiological survey in the Man region in western Côte d'Ivoire. Its objectives were to determine the prevalence of intestinal protozoa, to evaluate polyparasitism and to assess the spatial distribution of the pathogenic protozoal species, E. histolytica and G. intestinalis. Overall, 4466 stools samples taken from pupils aged 6 to 16 years of age at 57 different schools were analyzed under an optical microscope by the formol-ether stool concentration method, after preservation in sodium acetate-acetic acid-formalin (SAF). The most common protozoa species in this area were Endolimax nanus (83.8%) and E. coli (74.7%). The regional prevalence of G. intestinalis was 17.5% and of E. histolytica/E. dispar 11.3%. Both species were found in each of the 57 schools. The prevalence of E. histolytica/E. dispar exceeded 15% in six schools, and its

  19. Oral immunization with an attenuated vaccine strain of Salmonella typhimurium expressing the serine-rich Entamoeba histolytica protein induces an antiamebic immune response and protects gerbils from amebic liver abscess.

    PubMed Central

    Zhang, T; Stanley, S L

    1996-01-01

    Attenuated salmonellae represent attractive candidates for the delivery of foreign antigens by oral vaccination. In this report, we describe the high-level expression of a recombinant fusion protein containing the serine-rich Entamoeba histolytica protein (SREHP), a protective antigen derived from virulent amebae, and a bacterially derived maltose-binding protein (MBP) in an attenuated strain of Salmonella typhimurium. Mice and gerbils immunized with S. typhimurium expressing SREHP-MBP produced mucosal immunoglobulin A antiamebic antibodies and serum immunoglobulin G antiamebic antibodies. Gerbils vaccinated with S typhimurium SREHP-MBP were protected against amebic liver abscess, the most common extraintestinal complication of amebiasis. Our findings indicate that the induction of mucosal and immune responses to the amebic SREHP antigen is dependent on the level of SREHP-MBP expression in S. typhimurium and establish that oral vaccination with SREHP can produce protective immunity to invasive amebiasis. PMID:8613356

  20. Two Atypical l-Cysteine-regulated NADPH-dependent Oxidoreductases Involved in Redox Maintenance, l-Cystine and Iron Reduction, and Metronidazole Activation in the Enteric Protozoan Entamoeba histolytica*

    PubMed Central

    Jeelani, Ghulam; Husain, Afzal; Sato, Dan; Ali, Vahab; Suematsu, Makoto; Soga, Tomoyoshi; Nozaki, Tomoyoshi

    2010-01-01

    We discovered novel catalytic activities of two atypical NADPH-dependent oxidoreductases (EhNO1/2) from the enteric protozoan parasite Entamoeba histolytica. EhNO1/2 were previously annotated as the small subunit of glutamate synthase (glutamine:2-oxoglutarate amidotransferase) based on similarity to authentic bacterial homologs. As E. histolytica lacks the large subunit of glutamate synthase, EhNO1/2 were presumed to play an unknown role other than glutamine/glutamate conversion. Transcriptomic and quantitative reverse PCR analyses revealed that supplementation or deprivation of extracellular l-cysteine caused dramatic up- or down-regulation, respectively, of EhNO2, but not EhNO1 expression. Biochemical analysis showed that these FAD- and 2[4Fe-4S]-containing enzymes do not act as glutamate synthases, a conclusion which was supported by phylogenetic analyses. Rather, they catalyze the NADPH-dependent reduction of oxygen to hydrogen peroxide and l-cystine to l-cysteine and also function as ferric and ferredoxin-NADP+ reductases. EhNO1/2 showed notable differences in substrate specificity and catalytic efficiency; EhNO1 had lower Km and higher kcat/Km values for ferric ion and ferredoxin than EhNO2, whereas EhNO2 preferred l-cystine as a substrate. In accordance with these properties, only EhNO1 was observed to physically interact with intrinsic ferredoxin. Interestingly, EhNO1/2 also reduced metronidazole, and E. histolytica transformants overexpressing either of these proteins were more sensitive to metronidazole, suggesting that EhNO1/2 are targets of this anti-amebic drug. To date, this is the first report to demonstrate that small subunit-like proteins of glutamate synthase could play an important role in redox maintenance, l-cysteine/l-cystine homeostasis, iron reduction, and the activation of metronidazole. PMID:20592025

  1. Diagnostic performances of antigen detection compared to conventional and nucleic acid detection of Entamoeba histolytica in a non-endemic setting.

    PubMed

    Calderaro, Adriana; Piergianni, Maddalena; Piccolo, Giovanna; Rossi, Sabina; Montecchini, Sara; Buttrini, Mirko; Arcangeletti, Maria Cristina; Medici, Maria Cristina; Chezzi, Carlo; De Conto, Flora

    2016-04-01

    This study evaluated the immunochromatographic (IC) assay "TECHLAB(®) E. HISTOLYTICA QUIK CHEK™" analysing 36 faecal samples and 7 cultured strains. This assay was compared to the methods performed in our laboratory for the diagnosis of amoebiasis. The IC assay revealed a detection limit of 103 trophozoites/g faeces and no cross-reactivity with other parasites and failed to detect E. histolytica antigen in frozen faeces. In our laboratory located in a non-endemic setting this assay could not replace the methods currently used for the diagnosis of amoebiasis.

  2. Involvement of p21racA, phosphoinositide 3-kinase, and vacuolar ATPase in phagocytosis of bacteria and erythrocytes by Entamoeba histolytica: suggestive evidence for coincidental evolution of amebic invasiveness.

    PubMed Central

    Ghosh, S K; Samuelson, J

    1997-01-01

    Trophozoites of Entamoeba histolytica, the protozoan parasite that causes amebic dysentery, phagocytose bacteria in the colonic lumen and erythrocytes (RBC) in host tissues. Because tissue invasion is an evolutionary dead end, it is likely that amebic pathogenicity is coincidentally selected, i.e., the same methods used to kill bacteria in the colonic lumen are used by parasites to damage host cells and cause disease. In support of this idea, the amebic lectin and pore-forming peptide are involved in binding and killing, respectively, bacteria and host epithelial cells. Here amebic phagocytosis of bacteria, RBC, and mucin-coated beads was disrupted by overexpression of E. histolytica p21(racA-V12), a ras-family protein involved in selection of sites of actin polymerization, which had been mutated to eliminate its GTPase activity. p21(racA-V12) transformants were also defective in capping and cytokinesis, while pinocytosis of fluorescent dextrans was not affected. Wortmannin, a fungal inhibitor of phosphoinositide 3-kinase, markedly inhibited phagocytosis of bacteria, RBC, and mucin-coated beads by wild-type amebae. In contrast to p21(racA-V12) overexpression, wortmannin abolished amebic pinocytosis of dextrans but had no inhibitory effects on capping. Inhibition of amebic vacuolar acidification by bafilomycin also decreased bacterial and RBC uptake. These results, which demonstrate similarities between mechanisms of phagocytosis of bacteria and RBC by amebae and macrophages, support the idea of coincidental selection of amebic genes encoding proteins that mediate destruction of host cells. PMID:9317033

  3. Increases in Entamoeba histolytica Antibody–Positive Rates in Human Immunodeficiency Virus–Infected and Noninfected Patients in Japan: A 10-Year Hospital-Based Study of 3,514 Patients

    PubMed Central

    Yanagawa, Yasuaki; Nagata, Naoyoshi; Watanabe, Koji; Tsukada, Kunihisa; Teruya, Katsuji; Kikuchi, Yoshimi; Gatanaga, Hiroyuki; Akiyama, Junichi; Uemura, Naomi; Oka, Shinichi

    2016-01-01

    Serological evidence of the epidemiological trends in Entamoeba histolytica infection is scarce, especially in nonendemic countries. We aimed to determine the antibody-positive rates over a 10-year period, and compare the trends between human immunodeficiency virus (HIV)–infected and –noninfected patients. We reviewed 3,514 patients who underwent antibody testing during the study periods, which were divided into five annual categories: 2004–2005, 2006–2007, 2008–2009, 2010–2011, and 2012–2013. Anti-E. histolytica antibody was assessed by indirect immunofluorescence assay. The antibody-positive rate increased yearly from 2004–2005 to 2012–2013 (P < 0.001), although there was no increase in the annual number of antibody tests. This trend was seen among males (18.6–28.3%; P < 0.01), females (5.4–28.2%; P < 0.01), HIV-infected patients (18.4–26.9%; P < 0.001), and non-HIV-infected patients (14.6–36.8%; P < 0.001), and HIV-infected men who have sex with men (19.4–29.1%; P < 0.001). Among antibody-positive patients, there was a significant increase in the proportion of patients with high (≥ 1,600) titers (0.7–12.9%; P < 0.001), whereas this trend was not seen in patients with low (100) or intermediate (200–800) titers (P = 0.282 and 0.409, respectively). This large hospital-based study demonstrated that positive anti-E. histolytica antibody rates increased over 10 years, even though the annual number of antibody tests remained constant. Moreover, this trend was identified in non-high-risk patients (females and non-HIV-infected patients) as well as in high-risk patients. The proportion of patients with high antibody titers significantly increased among the antibody-positive patients. PMID:27296390

  4. Insights into endosomal maturation of human holo-transferrin in the enteric parasite Entamoeba histolytica: essential roles of Rab7A and Rab5 in biogenesis of giant early endocytic vacuoles.

    PubMed

    Verma, Kuldeep; Saito-Nakano, Yumiko; Nozaki, Tomoyoshi; Datta, Sunando

    2015-12-01

    The pathogenic amoeba Entamoeba histolytica is one of the causative agents of health hazards in tropical countries. It causes amoebic dysentery, colitis and liver abscesses in human. Iron is one of the essential nutritional resources for survival and chronic infection caused by the amoeba. The parasite has developed multiple ways to import, sequester and utilize iron from various iron-binding proteins from its host. In spite of its central role in pathogenesis, the mechanism of iron uptake by the parasite is largely unknown. Here, we carried out a systematic study to understand the role of some of the amoebic homologues of mammalian endocytic Rab GTPases (Rab5 and Rab21, Rab7A and Rab7B) in intracellular transport of human holo-transferrin by the parasite. Flow cytometry and quantitative microscopic image analysis revealed that Rab5 and Rab7A are required for the biogenesis of amoebic giant endocytic vacuoles (GEVs) and regulate the early phase of intracellular trafficking of transferrin. Rab7B is involved in the late phase, leading to the degradation of transferrin in the amoebic lysosome-like compartments. Using time-lapse fluorescence imaging in fixed trophozoites, we determined the kinetics of the vesicular transport of transferrin through Rab5-, Rab7A- and Rab7B-positive compartments. The involvement of Rab7A in the early phase of endocytosis by the parasite marks a significant divergence from its host in terms of spatiotemporal regulation by the Rab GTPases.

  5. Molecular identification of Entamoeba species in savanna woodland chimpanzees (Pan troglodytes schweinfurthii).

    PubMed

    Jirků-Pomajbíková, Kateřina; Čepička, Ivan; Kalousová, Barbora; Jirků, Milan; Stewart, Fiona; Levecke, Bruno; Modrý, David; Piel, Alex K; Petrželková, Klára J

    2016-05-01

    To address the molecular diversity and occurrence of pathogenic species of the genus Entamoeba spp. in wild non-human primates (NHP) we conducted molecular-phylogenetic analyses on Entamoeba from wild chimpanzees living in the Issa Valley, Tanzania. We compared the sensitivity of molecular [using a genus-specific polymerase chain reaction (PCR)] and coproscopic detection (merthiolate-iodine-formaldehyde concentration) of Entamoeba spp. We identified Entamoeba spp. in 72 chimpanzee fecal samples (79%) subjected to species-specific PCRs for six Entamoeba species/groups (Entamoeba histolytica, Entamoeba nuttalli, Entamoeba dispar, Entamoeba moshkovskii, Entamoeba coli and Entamoeba polecki ST2). We recorded three Entamoeba species: E. coli (47%), E. dispar (16%), Entamoeba hartmanni (51%). Coproscopically, we could only distinguish the cysts of complex E. histolytica/dispar/moshkovskii/nuttalli and E. coli. Molecular prevalence of entamoebas was higher than the prevalence based on the coproscopic examination. Our molecular phylogenies showed that sequences of E. dispar and E. coli from Issa chimpanzees are closely related to sequences from humans and other NHP from GenBank. The results showed that wild chimpanzees harbour Entamoeba species similar to those occurring in humans; however, no pathogenic species were detected. Molecular-phylogenetic methods are critical to improve diagnostics of entamoebas in wild NHP and for determining an accurate prevalence of Entamoeba species.

  6. Is real-time PCR-based diagnosis similar in performance to routine parasitological examination for the identification of Giardia intestinalis, Cryptosporidium parvum/Cryptosporidium hominis and Entamoeba histolytica from stool samples? Evaluation of a new commercial multiplex PCR assay and literature review.

    PubMed

    Laude, A; Valot, S; Desoubeaux, G; Argy, N; Nourrisson, C; Pomares, C; Machouart, M; Le Govic, Y; Dalle, F; Botterel, F; Bourgeois, N; Cateau, E; Leterrier, M; Le Pape, P; Morio, F

    2016-02-01

    Microscopy is the reference standard for routine laboratory diagnosis in faecal parasitology but there is growing interest in alternative methods to overcome the limitations of microscopic examination, which is time-consuming and highly dependent on an operator's skills and expertise. Compared with microscopy, DNA detection by PCR is simple and can offer a better turnaround time. However, PCR performances remain difficult to assess as most studies have been conducted on a limited number of positive clinical samples and used in-house PCR methods. Our aim was to evaluate a new multiplex PCR assay (G-DiaParaTrio; Diagenode Diagnostics), targeting Giardia intestinalis, Cryptosporidium parvum/Cryptosporidium hominis and Entamoeba histolytica. To minimize the turnaround time, PCR was coupled with automated DNA extraction (QiaSymphony; Qiagen). The PCR assay was evaluated using a reference panel of 185 samples established by routine microscopic examination using a standardized protocol including Ziehl-Neelsen staining and adhesin detection by ELISA (E. histolytica II; TechLab). This panel, collected from 12 French parasitology laboratories, included 135 positive samples for G. intestinalis (n = 38), C. parvum/C. hominis (n = 26), E. histolytica (n = 5), 21 other gastrointestinal parasites, together with 50 negative samples. In all, the G-DiaParaTrio multiplex PCR assay identified 38 G. intestinalis, 25 C. parvum/C. hominis and five E. histolytica leading to sensitivity/specificity of 92%/100%, 96%/100% and 100%/100% for G. intestinalis, C. parvum/C. hominis and E. histolytica, respectively. This new multiplex PCR assay offers fast and reliable results, similar to microscopy-driven diagnosis for the detection of these gastrointestinal protozoa, allowing its implementation in routine clinical practice.

  7. Surface properties of Entamoeba: increased rates of human erythrocyte phagocytosis in pathogenic strains

    PubMed Central

    1978-01-01

    The assertion that ingestion of human erythrocytes is restricted to invasive strains of Entamoeba histolytica has not been evaluated previously by comparative studies. In this report we describe the in vitro ingestion of human erythrocytes by pathogenic and nonpathogenic Entamoeba. Microscopic evaluation of erythrophagocytosis by eight different Entamoeba grown in culture revealed that strains of E. histolytica isolated from cases of human dysentery show a much higher rate of erythrocyte ingestion than nonpathogenic strains. However, all strains are able to phagocytize erythrocytes. The extremely high rate of phagocytic activity shown by pathogenic E. histolytica could be one of the properties related to the pathogenicity of this parasitic protozoan. PMID:722237

  8. Entamoeba dispar: Could it be pathogenic

    PubMed Central

    Oliveira, Fabrício Marcus Silva; Neumann, Elisabeth; Gomes, Maria Aparecida; Caliari, Marcelo Vidigal

    2015-01-01

    Amebiasis is a disease caused by the protozoan parasite Entamoeba histolytica. This ameba can colonize the human intestine and persist as a commensal parasite, similar to Entamoeba dispar, an ameba considered to be non-pathogenic. The similarities between E. histolytica and E. dispar make the latter an attractive model for studies aimed at clarifying the pathogenesis of amebiasis. However, in addition to being an interesting experimental model, this relative of E. histolytica remains poorly understood. In the 1990, it was believed that E. dispar was unable to produce significant experimental lesions. This scenario began to change in 1996, when E. dispar strains were isolated from symptomatic patients in Brazil. These strains were able to produce liver and intestinal lesions that were occasionally indistinguishable from those produced by E. histolytica. These and other findings, such as the detection of E. dispar DNA sequences in samples from patients with amebic liver abscess, have revived the possibility that this species can produce lesions in humans. The present paper presents a series of studies on E. dispar that begin to reveal a new facet of this protozoan. PMID:25709947

  9. Giardia, Entamoeba, and Trichomonas enzymes activate metronidazole (nitroreductases) and inactivate metronidazole (nitroimidazole reductases).

    PubMed

    Pal, Dibyarupa; Banerjee, Sulagna; Cui, Jike; Schwartz, Aaron; Ghosh, Sudip K; Samuelson, John

    2009-02-01

    Infections with Giardia lamblia, Entamoeba histolytica, and Trichomonas vaginalis, which cause diarrhea, dysentery, and vaginitis, respectively, are each treated with metronidazole. Here we show that Giardia, Entamoeba, and Trichomonas have oxygen-insensitive nitroreductase (ntr) genes which are homologous to those genes that have nonsense mutations in metronidazole-resistant Helicobacter pylori isolates. Entamoeba and Trichomonas also have nim genes which are homologous to those genes expressed in metronidazole-resistant Bacteroides fragilis isolates. Recombinant Giardia, Entamoeba, and Trichomonas nitroreductases used NADH rather than the NADPH used by Helicobacter, and two recombinant Entamoeba nitroreductases increased the metronidazole sensitivity of transformed Escherichia coli strains. Conversely, the recombinant nitroimidazole reductases (NIMs) of Entamoeba and Trichmonas conferred very strong metronidazole resistance to transformed bacteria. The Ehntr1 gene of the genome project HM-1:IMSS strain of Entamoeba histolytica had a nonsense mutation, and the same nonsense mutation was present in 3 of 22 clinical isolates of Entamoeba. While ntr and nim mRNAs were variably expressed by cultured Entamoeba and Trichomonas isolates, there was no relationship to metronidazole sensitivity. We conclude that microaerophilic protists have bacterium-like enzymes capable of activating metronidazole (nitroreductases) and inactivating metronidazole (NIMs). While Entamoeba and Trichomonas displayed some of the changes (nonsense mutations and gene overexpression) associated with metronidazole resistance in bacteria, these changes did not confer metronidazole resistance to the microaerophilic protists examined here.

  10. Molecular Epidemiology of Entamoeba: First Description of Entamoeba moshkovskii in a Rural Area from Central Colombia

    PubMed Central

    León, Cielo M.; Fonseca, Jairo; Reyes, Patricia; Moncada, Ligia; Olivera, Mario J.

    2015-01-01

    Background Entamoeba histolytica, E. dispar and E. moshkovskii are the most frequent species described in human infection where E. histolytica is the only true pathogen. The epidemiology of this infection is complex due to the absence of a routine exam that allows a correct discrimination of the Entamoeba species complex. Therefore, molecular methods appear as the unique epidemiological tool to accomplish the species discrimination. Herein, we conducted a cross-sectional study to determine the frequency of Entamoeba species infections in a group of asymptomatic individuals from a rural area in central Colombia. Methodology/Principal Findings A total of 181 fecal samples from asymptomatic children under 16 years old from the hamlet La Vírgen, Cundinamarca (Colombia) that voluntarily accepted to participate in the study were collected. The fecal samples were examined by light microscopy and DNA-extracted, subsequently submitted to molecular discrimination of E. dispar/E. histolytica/E. moshkovskii infection based on a multiplex PCR assay targeting the 18S rRNA fragment. To confirm the species description, twenty samples were randomly submitted to DNA sequencing of the aforementioned fragment. By direct microscopic examination, frequency of the complex E. histolytica/E. dispar/E. moshkovskii was 18.8% (34/181). PCR showed a frequency of 49.1% (89/181), discriminated as 23.2% (42/181) that were positive for E. dispar, 25.4% (46/181) for E. moshkovskii and 0.55% (1/ 181) for E. histolytica. Also, mixed infections were detected between E. dispar and E. moshkovskii at 4.42% (8/181) of the samples. Molecular barcoding confirmed the diagnosis depicted by the multiplex PCR assay. Conclusions/Significance This is the first description of E. moshkovskii in Colombia and the second report in South-America to our knowledge. Our results suggest the need to unravel the true epidemiology of Entamoeba infections around the world, including the real pathogenic role that E

  11. LUMINEX®: a new technology for the simultaneous identification of five Entamoeba spp. commonly found in human stools

    PubMed Central

    2013-01-01

    Background Six species of the genus Entamoeba, i.e., E. histolytica, E. dispar, E. moshkovskii, E. polecki, E. coli, and E. hartmanii can be found in human stools. Among these, only E. histolytica is considered to be pathogenic, causing intestinal and extra-intestinal disease, but it is morphologically identical to E. dispar and E. moshkovskii. In general, E. polecki, E. coli, and E. hartmanii can be differentiated morphologically from E. histolytica, but some of their diagnostic morphologic features may overlap creating issues for the differential diagnosis. Moreover, the previous inability to differentiate among Entamoeba species has limited epidemiologic information on E histolytica. The objective of this study was to develop a rapid, high-throughput screening method using Luminex technique for the simultaneous detection and differentiation of Entamoeba species. Methods PCR amplification was performed with biotinylated Entamoeba sp 18S rRNA gene primers, designed to amplify a fragment ranging from 382 to 429 bp of the Entamoeba spp studied. Regions of this fragment that could differentiate among E. histolytica, E. moshkovskii, E. dispar, E. hartmanii and E. coli were selected to design hybridization probes to link to Luminex beads. The assay was standardized with cloned DNA samples of each species and evaluated with 24 DNA extracts from samples obtained from individuals diagnosed with these amebas in their stools. Results Using this approach we were able to correctly identify E. histoltyica, E. dispar, E hartmanni, E. coli and E. moshkovskii in all specimens studied. From twenty four samples tested by microscopy, PCR/DNA Sequencing and real-time PCR, 100% agreed with PCR-Luminex assay for identification of E. dispar, E. moshkovskii, E. hartmanni, E. histolytica, and E. coli. Conclusion These results show that this method could be used in the diagnostic detection of Entamoeba spp in fecal samples. This diagnostic test was useful to clearly distinguish E

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

    PubMed Central

    Costa, C.A.X.; Brito, K.N.O.; Gomes, M.A.; Caliari, M.V.

    2010-01-01

    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. PMID:20819776

  13. Reduced protein tyrosine phosphatase (PTPase) activity of CD45 on peripheral blood lymphocytes in patients with systemic lupus erythematosus (SLE).

    PubMed

    Takeuchi, T; Pang, M; Amano, K; Koide, J; Abe, T

    1997-07-01

    To disclose the mechanism of aberrant function of peripheral blood lymphocytes (PBL) in SLE, we focused on the catalytic function of CD45, and determined the CD45 PTPase activity in SLE patients. The sample population consisted of 32 SLE patients with different disease activity. PTPase activity of cell lysates immunoprecipitated by anti-CD45 MoAb was assayed against phosphotyrosine analogue PNPP, followed by measuring the release of para-nitro phenol at 410 nm. CD45 PTPase activity of PBL was significantly decreased in SLE patients, compared with that of normal controls and patients with systemic sclerosis (964 +/- 265, 1202 +/- 172, 1210 +/- 125, respectively; SLE versus normal, P<0.05). It was correlated with SLE Disease Activity Index (SLEDAI) score (r = 0.597, P = 0.0006), but not with the dose of prednisolone (r = 0.214, P = 0.2657), indicating that CD45 PTPase activity became reduced when the disease was active, but it was not affected by prednisolone. Moreover, it was not corrected by in vitro culture with or without stimulation. The expression of CD45 on PBL was comparable between normal and SLE, raising a possibility that it may be due to aberrant regulation of catalytic function of CD45 in SLE. Given the evidence that tyrosine phosphorylation of cellular proteins by tyrosine kinases and phosphatases is one of the key biochemical events in the signal transduction pathway, the decreased CD45 PTPase activity in SLE may account for the defective signal transduction via TCR/CD3, leading to dysregulated effector function of the lymphocytes.

  14. Genotyping of Giardia lamblia and Entamoeba spp from river waters in Iran.

    PubMed

    Mahmoudi, Mohammad Reza; Nazemalhosseini-Mojarad, Ehsan; Karanis, Panagiotis

    2015-12-01

    In this study, DNA from 55 surface and river water samples, which were collected from some water sources of Tehran and the Guilan Province, Iran, were extracted and examined for Entamoeba spp. and Giardia lamblia by PCR and genotyping. Twenty-seven samples, which were concentrated using the immunomagnetic separation technology (IMS) method, were examined for Giardia alone. Twenty-eight samples, which were concentrated using the sucrose flotation (SF) method, were examined for both Giardia and Entamoeba species. The results showed that 27/55 (17/27 and 10/28) (49 %), 4 /28 (14.28 %) and 3/28 (10.7 %) of the samples were positive for Giardia lamblia, Entamoeba spp and mixed infections (Entamoeba spp. and Giardia spp.), respectively. Sixteen out of 55 samples were negative. Entamoeba genus-specific PCR primers in single-round PCR were used to differentiate between the Entamoeba spp. (E. histolytica, E. dispar and E. moshkovskii). With respect to the 7 samples that were positive for Entamoeba, (14.28 %) 4 out of 28 were positive for E. moshkovskii, (7.14 %), 2 out of 28 were positive for E. histolytica and (3.57 %) 1 out of 28 was positive for E. dispar. Genus-specific PCR primers in a semi-nested PCR assay was performed to genotype Giardia species. Of the 27 samples that were positive for Giardia, 10 samples were sequences. All 10 successfully sequenced samples contained assemblage B of Giardia lamblia.This is first study to investigate the G. lamblia genotypes in the water supply of the Tehran and Guilan provinces, and it is the first study to investigate Entamoeba species in the water supplies of Iran. The investigated river water supplies, which are used for agriculture, camping and animal farming, were heavily contaminated by the human pathogenic Entamoeba and Giardia parasites. There is a potential risk of waterborne outbreaks in humans and animals.

  15. Entamoeba moshkovskii Is Associated With Diarrhea in Infants and Causes Diarrhea and Colitis in Mice

    PubMed Central

    Shimokawa, Chikako; Kabir, Mamun; Taniuchi, Mami; Mondal, Dinesh; Kobayashi, Seiki; Ali, Ibne Karim M.; Sobuz, Shihab U.; Senba, Masachika; Houpt, Eric; Haque, Rashidul; Petri, William A.; Hamano, Shinjiro

    2012-01-01

    Background.Entamoeba moshkovskii is prevalent in developing countries and morphologically indistinguishable from pathogenic Entamoeba histolytica and nonpathogenic Entamoeba dispar. It is not known if E. moshkovskii is pathogenic. Methods.Mice were intracecally challenged with the trophozoites of each Entamoeba spp. to test the ability to cause diarrhea, and infants in Bangladesh were prospectively observed to see if newly acquired E. moshkovskii infection was associated with diarrhea. Results.E. moshkovskii and E. histolytica caused diarrhea and weight loss in susceptible mice. E. dispar infected none of the mouse strains tested. In Mirpur, Dhaka, Bangladesh, E. moshkovskii, E. histolytica, and E. dispar were identified in 42 (2.95%), 66 (4.63%), and 5 (0.35%), respectively, of 1426 diarrheal episodes in 385 children followed prospectively from birth to one year of age. Diarrhea occurred temporally with acquisition of a new E. moshkovskii infection: in the 2 months preceding E. moshkvskii-associated diarrhea, 86% (36 of 42) of monthly surveillance stool samples were negative for E. moshkovskii. Conclusions.E. moshkovskii was found to be pathogenic in mice. In children, the acquisition of E. moshkovskii infection was associated with diarrhea. These data are consistent with E. moshkovskii causing disease, indicating that it is important to reexamine its pathogenicity. PMID:22723640

  16. The Interplay between Entamoeba and Enteropathogenic Bacteria Modulates Epithelial Cell Damage

    PubMed Central

    Galván-Moroyoqui, José Manuel; del Carmen Domínguez-Robles, M.; Franco, Elizabeth; Meza, Isaura

    2008-01-01

    Background Mixed intestinal infections with Entamoeba histolytica, Entamoeba dispar and bacteria with exacerbated manifestations of disease are common in regions where amoebiasis is endemic. However, amoeba–bacteria interactions remain largely unexamined. Methodology Trophozoites of E. histolytica and E. dispar were co-cultured with enteropathogenic bacteria strains Escherichia coli (EPEC), Shigella dysenteriae and a commensal Escherichia coli. Amoebae that phagocytosed bacteria were tested for a cytopathic effect on epithelial cell monolayers. Cysteine proteinase activity, adhesion and cell surface concentration of Gal/GalNAc lectin were analyzed in amoebae showing increased virulence. Structural and functional changes and induction of IL-8 expression were determined in epithelial cells before and after exposure to bacteria. Chemotaxis of amoebae and neutrophils to human IL-8 and conditioned culture media from epithelial cells exposed to bacteria was quantified. Principal Findings E. histolytica digested phagocytosed bacteria, although S. dysenteriae retained 70% viability after ingestion. Phagocytosis of pathogenic bacteria augmented the cytopathic effect of E. histolytica and increased expression of Gal/GalNAc lectin on the amoebic surface and increased cysteine proteinase activity. E. dispar remained avirulent. Adhesion of amoebae and damage to cells exposed to bacteria were increased. Additional increases were observed if amoebae had phagocytosed bacteria. Co-culture of epithelial cells with enteropathogenic bacteria disrupted monolayer permeability and induced expression of IL-8. Media from these co-cultures and human recombinant IL-8 were similarly chemotactic for neutrophils and E. histolytica. Conclusions Epithelial monolayers exposed to enteropathogenic bacteria become more susceptible to E. histolytica damage. At the same time, phagocytosis of pathogenic bacteria by amoebae further increased epithelial cell damage. Significance The in vitro system

  17. Recent advances in Entamoeba biology: RNA interference, drug discovery, and gut microbiome

    PubMed Central

    Singh, Upinder

    2016-01-01

    In recent years, substantial progress has been made in understanding the molecular and cell biology of the human parasite Entamoeba histolytica, an important pathogen with significant global impact. This review outlines some recent advances in the Entamoeba field in the last five years, focusing on areas that have not recently been discussed in detail: (i) molecular mechanisms regulating parasite gene expression, (ii) new efforts at drug discovery using high-throughput drug screens, and (iii) the effect of gut microbiota on amoebiasis. PMID:27853522

  18. Differential Diagnosis of Entamoeba spp. in Clinical Stool Samples Using SYBR Green Real-Time Polymerase Chain Reaction

    PubMed Central

    Gomes, Thiago dos Santos; Garcia, Mariana Coimbra; de Souza Cunha, Flavia; Peralta, José Mauro; Peralta, Regina Helena Saramago

    2014-01-01

    Amoebiasis, a disease caused by Entamoeba histolytica, is usually diagnosed by microscopic examination, which does not differentiate the morphologically identical species of the E. histolytica/E. dispar complex. Furthermore, morphologically similar species such as Entamoeba hartmanni contribute to misidentification. Therefore, there is a need for more sensitive and specific methods. This study standardized a multiplex real-time PCR system for E. histolytica and E. dispar and a single real-time PCR for E. hartmanni. The multiplex protocol detected up to 0.0143 pg of E. histolytica DNA and 0.5156 pg of E. dispar DNA, and the average melting temperature (Tm) was 73°C and 70°C, respectively. For E. hartmanni, the Tm was 73°C and the amplification was successful down to 0.03 fg of plasmid DNA. Negative controls and other intestinal parasites presented no amplification. Among the 48 samples tested, E. dispar DNA was detected in 37; none exhibited E. histolytica DNA and 11 were negative in the multiplex protocol. In 4 of these 11 samples, however, E. hartmanni DNA was amplified. SYBR Green is demonstrated to be an interesting option and these combined PCR reactions can improve laboratory diagnosis of amoebiasis in developing countries. PMID:24693242

  19. Entamoeba Encystation: New Targets to Prevent the Transmission of Amebiasis

    PubMed Central

    Mi-ichi, Fumika; Yoshida, Hiroki; Hamano, Shinjiro

    2016-01-01

    Amebiasis is caused by Entamoeba histolytica infection and can produce a broad range of clinical signs, from asymptomatic cases to patients with obvious symptoms. The current epidemiological and clinical statuses of amebiasis make it a serious public health problem worldwide. The Entamoeba life cycle consists of the trophozoite, the causative agent for amebiasis, and the cyst, the form responsible for transmission. These two stages are connected by “encystation” and “excystation.” Hence, developing novel strategies to control encystation and excystation will potentially lead to new measures to block the transmission of amebiasis by interrupting the life cycle of the causative agent. Here, we highlight studies investigating encystation using inhibitory chemicals and categorize them based on the molecules inhibited. We also present a perspective on new strategies to prevent the transmission of amebiasis. PMID:27764256

  20. Use of Liposomes for Directed Drug Delivery Against Entamoeba Histolytica

    DTIC Science & Technology

    1990-01-20

    terminal residuecfa stimulatory glycosphingolipid eliminated activity; attachment of fucose to the penultimate sugar reduced activity) Glycans with a...respectively) essentially eliminated the enhancing activity of the these glycosphingolipids. Fucose , attached to the penultimate sugar (GlcNAc) of...internal Galpl-4 residue of that glycan. NeuAc attached to the Gal terminus of an otherwise stimulatory glycosphingolipid or fucose attached to the

  1. Entamoeba moshkovskii perspectives of a new agent to be considered in the diagnosis of amebiasis.

    PubMed

    Heredia, Rubén Darío; Fonseca, Jairo Andrés; López, Myriam Consuelo

    2012-09-01

    During the last decade Entamoeba moshkovskii has become relevant given its capacity to infect humans, especially when considering that it is morphologically indistinguishable from E. histolytica. For a long time, E. moshkovskii was considered as a free living amoeba, but in the last decade it has been demonstrated that E. moshkovskii can infect humans and can be found more frequently in regions where amebiasis shows high prevalence values, becoming a challenge to differentiate it from the E. histolytica/E. dispar complex. Recently there have been studies that raise the possibility that E. moshkovskii could be a pathogenic species, as there are reports in different countries that associated this infection with gastrointestinal symptoms even though others have described it as a non pathogenic species. For this reasons, both clinical and epidemiological studies are required.

  2. Horizontal Gene Transfers from Bacteria to Entamoeba Complex: A Strategy for Dating Events along Species Divergence

    PubMed Central

    Romero, Miguel; Ximenez, Cecilia

    2016-01-01

    Horizontal gene transfer has proved to be relevant in eukaryotic evolution, as it has been found more often than expected and related to adaptation to certain niches. A relatively large list of laterally transferred genes has been proposed and evaluated for the parasite Entamoeba histolytica. The goals of this work were to elucidate the importance of lateral gene transfer along the evolutionary history of some members of the genus Entamoeba, through identifying donor groups and estimating the divergence time of some of these events. In order to estimate the divergence time of some of the horizontal gene transfer events, the dating of some Entamoeba species was necessary, following an indirect dating strategy based on the fossil record of plausible hosts. The divergence between E. histolytica and E. nuttallii probably occurred 5.93 million years ago (Mya); this lineage diverged from E. dispar 9.97 Mya, while the ancestor of the latter separated from E. invadens 68.18 Mya. We estimated times for 22 transferences; the most recent occurred 31.45 Mya and the oldest 253.59 Mya. Indeed, the acquisition of genes through lateral transfer may have triggered a period of adaptive radiation, thus playing a major role in the evolution of the Entamoeba genus. PMID:27239333

  3. An Annotated Checklist of the Human and Animal Entamoeba (Amoebida: Endamoebidae) Species- A Review Article

    PubMed Central

    HOOSHYAR, Hossein; ROSTAMKHANI, Parvin; REZAEIAN, Mostafa

    2015-01-01

    Background: The number of valid of pathogen and non-pathogen species of Entamoeba has continuously increased in human and animals. This review is performed to provide an update list and some summarized information on Entamoeba species, which were identified up to the 2014. Methods: We evaluated the Entamoeba genus with a broad systematic review of the literature, books and electronic databases until February 2014. The synonyms, hosts, pathogenicity and geographical distribution of valid species were considered and recorded. Repeated and unrelated cases were excluded. Results: Totally 51 defined species of Entamoeba were found and arranged by the number of nuclei in mature cyst according to Levin’s grouping. Seven of these species within the 4 nucleate mature cysts group and 1 species with one nucleate mature cyst are pathogen. E. histolytica, E. invadence, E. rananrum and E. anatis causes lethal infection in human, reptiles, amphibians and brides respectively, four species causes non-lethal mild dysentery. The other species were non-pathogen and are important to differential diagnosis of amoebiasis. Conclusion: There are some unknown true species of Entamoeba that available information on the morphology, hosts, pathogenicity and distribution of them are still very limited and more considerable investigation will be needed in order to clarify the status of them. PMID:26246811

  4. A Novel Mitosomal β-Barrel Outer Membrane Protein in Entamoeba

    PubMed Central

    Santos, Herbert J.; Imai, Kenichiro; Makiuchi, Takashi; Tomii, Kentaro; Horton, Paul; Nozawa, Akira; Ibrahim, Mohamed; Tozawa, Yuzuru; Nozaki, Tomoyoshi

    2015-01-01

    Entamoeba possesses a highly divergent mitochondrion-related organelle known as the mitosome. Here, we report the discovery of a novel protein in Entamoeba, which we name Mitosomal β-barrel Outer Membrane Protein of 30 kDa (MBOMP30). Initially identified through in silico analysis, we experimentally confirmed that MBOMP30 is indeed a β-barrel protein. Circular dichroism analysis showed MBOMP30 has a predominant β-sheet structure. Localization to Entamoeba histolytica mitosomes was observed through Percoll-gradient fractionation and immunofluorescence assay. Mitosomal membrane integration was demonstrated by carbonate fractionation, proteinase K digestion, and immunoelectron microscopy. Interestingly, the deletion of the putative β-signal, a sequence believed to guide β-barrel outer membrane protein (BOMP) assembly, did not affect membrane integration, but abolished the formation of a ~240 kDa complex. MBOMP30 represents only the seventh subclass of eukaryotic BOMPs discovered to date and lacks detectable homologs outside Entamoeba, suggesting that it may be unique to Entamoeba mitosomes. PMID:25711150

  5. Zinc in Entamoeba invadens.

    NASA Technical Reports Server (NTRS)

    Morgan, R. S.; Sattilaro, R. F.

    1972-01-01

    Atomic absorption spectroscopy, electron microprobe analysis, and dithizone staining of trophozoites and cysts of Entamoeba invadens demonstrate that these cells have a high concentration of zinc (approximately one picogram per cell or 1% of their dry weight). In the cysts of this organism, the zinc is confined to the chromatoid bodies, which previous work has shown to contain crystals of ribosomes. The chemical state and function of this zinc are unknown.

  6. Occurrence of Giardia, Cryptosporidium, and Entamoeba in wild rhesus macaques (Macaca mulatta) living in urban and semi-rural North-West India.

    PubMed

    Debenham, John J; Tysnes, Kristoffer; Khunger, Sandhya; Robertson, Lucy J

    2017-04-01

    Giardia duodenalis, Cryptosporidium spp., and Entamoeba spp. are intestinal protozoa capable of infecting a range of host species, and are important causes of human morbidity and mortality. Understanding their epidemiology is important, both for public health and for the health of the animals they infect. This study investigated the occurrence of these protozoans in rhesus macaques (Macaca mulatta) in India, with the aim of providing preliminary information on the potential for transmission of these pathogens between macaques and humans. Faecal samples (n = 170) were collected from rhesus macaques from four districts of North-West India. Samples were analysed for Giardia/Cryptosporidium using a commercially available direct immunofluorescent antibody test after purification via immunomagnetic separation. Positive samples were characterised by sequencing of PCR products. Occurrence of Entamoeba was investigated first by using a genus-specific PCR, and positive samples further investigated via species-specific PCRs for Entamoeba coli, Entamoeba histolytica, Entamoeba dispar and Entamoeba moshkovskii. Giardia cysts were found in 31% of macaque samples, with all isolates belonging to Assemblage B. Cryptosporidium oocysts were found in 1 sample, however this sample did not result in amplification by PCR. Entamoeba spp. were found in 79% of samples, 49% of which were positive for E. coli. Multiplex PCR for E. histolytica, E. dispar and E. moshkovskii, did not result in amplification in any of the samples. Thus in 51% of the samples positive at the genus specific PCR, the Entamoeba species was not identified. This study provides baseline information on the potential for transmission of these zoonotic parasites at the wildlife-human interface.

  7. Clinical case of cerebral amebiasis caused by E. histolytica.

    PubMed

    Maldonado-Barrera, Cinthya A; Campos-Esparza, Maria Del Rosario; Muñoz-Fernández, Luis; Victoria-Hernández, Joaquin A; Campos-Rodríguez, Rafael; Talamás-Rohana, Patricia; Ventura-Juárez, Javier

    2012-03-01

    Although amebic brain abscess is a rare form of invasive amebiasis, when present, it is frequently lethal. This disorder always begins with the infection of the colon by Entamoeba histolytica trophozoites, which then travel to extra-intestinal tissues through the bloodstream. Amebic brain abscesses are produced when trophozoites invade the central nervous system. Computerized axial tomography scans can be used to diagnose the presence or absence of a brain abscess with a certainty of 100%. However, this diagnostic tool does not reveal the etiological agent of disease. By analyzing the clinical case of a patient that died due to untimely treatment of this malady, the present study aims to identify a diagnostic tool that can give a precise determination of the etiological agent and therefore permit adequate and opportune treatment. Currently, diagnosis of amebic brain abscess is often done by identification of the ameba in a biopsy or autopsy. By immunohistochemistry and immunofluorescence with specific antibodies, we identified the existence of E. histolytica, which presents proteins similar to Naegleria fowleri in its membrane.

  8. Ethanol and Isopropanol in Concentrations Present in Hand Sanitizers Sharply Reduce Excystation of Giardia and Entamoeba and Eliminate Oral Infectivity of Giardia Cysts in Gerbils

    PubMed Central

    Chatterjee, Aparajita; Bandini, Giulia; Motari, Edwin

    2015-01-01

    Enteric protozoan parasites, which are spread by the fecal-oral route, are important causes of diarrhea (Giardia duodenalis) and amebic dysentery (Entamoeba histolytica). Cyst walls of Giardia and Entamoeba have a single layer composed of fibrils of β-1,3-linked GalNAc and β-1,4-linked GlcNAc (chitin), respectively. The goal here was to determine whether hand sanitizers that contain ethanol or isopropanol as the active microbicide might reduce transmission of these parasites. We found that treatment with these alcohols with or without drying in a rotary evaporator (to model rapid evaporation of sanitizers on hands) kills 85 to 100% of cysts of G. duodenalis and 90 to 100% of cysts of Entamoeba invadens (a nonpathogenic model for E. histolytica), as shown by nuclear labeling with propidium iodide and failure to excyst in vitro. Alcohols with or without drying collapsed the cyst walls of Giardia but did not collapse the cyst walls of Entamoeba. To validate the in vitro results, we showed that treatment with alcohols eliminated oral infection of gerbils by 1,000 G. duodenalis cysts, while a commercial hand sanitizer (Purell) killed E. invadens cysts that were directly applied to the hands. These results suggest that expanded use of alcohol-based hand sanitizers might reduce the transmission of Giardia and Entamoeba. PMID:26282413

  9. Ethanol and isopropanol in concentrations present in hand sanitizers sharply reduce excystation of Giardia and Entamoeba and eliminate oral infectivity of Giardia cysts in gerbils.

    PubMed

    Chatterjee, Aparajita; Bandini, Giulia; Motari, Edwin; Samuelson, John

    2015-11-01

    Enteric protozoan parasites, which are spread by the fecal-oral route, are important causes of diarrhea (Giardia duodenalis) and amebic dysentery (Entamoeba histolytica). Cyst walls of Giardia and Entamoeba have a single layer composed of fibrils of β-1,3-linked GalNAc and β-1,4-linked GlcNAc (chitin), respectively. The goal here was to determine whether hand sanitizers that contain ethanol or isopropanol as the active microbicide might reduce transmission of these parasites. We found that treatment with these alcohols with or without drying in a rotary evaporator (to model rapid evaporation of sanitizers on hands) kills 85 to 100% of cysts of G. duodenalis and 90 to 100% of cysts of Entamoeba invadens (a nonpathogenic model for E. histolytica), as shown by nuclear labeling with propidium iodide and failure to excyst in vitro. Alcohols with or without drying collapsed the cyst walls of Giardia but did not collapse the cyst walls of Entamoeba. To validate the in vitro results, we showed that treatment with alcohols eliminated oral infection of gerbils by 1,000 G. duodenalis cysts, while a commercial hand sanitizer (Purell) killed E. invadens cysts that were directly applied to the hands. These results suggest that expanded use of alcohol-based hand sanitizers might reduce the transmission of Giardia and Entamoeba.

  10. The cyst wall of Entamoeba invadens contains chitosan (deacetylated chitin).

    PubMed

    Das, Suchismita; Van Dellen, Katrina; Bulik, Dorota; Magnelli, Paula; Cui, Jike; Head, James; Robbins, Phillips W; Samuelson, John

    2006-07-01

    The cyst wall of Entamoeba invadens (Ei), a model for the human pathogen Entamoeba histolytica, contains chitin, which is a homopolymer of beta-1, 4-linked N-acetyl-glucosamine (GlcNAc). In fungi and in bacteria that make nodulation factors, chitin deacetylases make chitosan, which is a mixture of GlcNAc and glucosamine and so has a positive charge. The activity of an Ei chitin deacetylase was revealed by a 3-4-fold increase in released GlcNAc when deproteinated cyst walls were chemically acetylated prior to treatment with a commerical chitinase. Because this chitinase releases GlcNAc but not GlcN, increases in released GlcNAc after acetylation suggested the presence of chitosan in Ei cyst walls. Five putative Ei and Eh chitin deacetylase genes resembled those of fungi and bacteria. A predicted Eh chitin deacetylase matched closely the three-dimensional structure of a Bacillus subtilis peptiodglycan deacetylase. A recombinant Eh chitin deacetylase, expressed in Saccharomyces cerevisiae, deacetylated chitooligosaccharides in vitro. These results are consistent with the idea that Ei chitin deacetylases modify chitin to produce chitosan in the Ei cyst wall.

  11. Development of RNA Interference Trigger-Mediated Gene Silencing in Entamoeba invadens

    PubMed Central

    Suresh, Susmitha; Ehrenkaufer, Gretchen; Zhang, Hanbang

    2016-01-01

    Entamoeba histolytica, a protozoan parasite, is an important human pathogen and a leading parasitic cause of death. The organism has two life cycle stages, trophozoites, which are responsible for tissue invasion, and cysts, which are involved in pathogen transmission. Entamoeba invadens is the model system to study Entamoeba developmental biology, as high-grade regulated encystation and excystation are readily achievable. However, the lack of gene-silencing tools in E. invadens has limited the molecular studies that can be performed. Using the endogenous RNA interference (RNAi) pathway in Entamoeba, we developed an RNAi-based trigger gene-silencing approach in E. invadens. We demonstrate that a gene's coding region that has abundant antisense small RNAs (sRNAs) can trigger silencing of a gene that is fused to it. The trigger fusion leads to the generation of abundant antisense sRNAs that map to the target gene, with silencing occurring independently of trigger location at the 5′ or 3′ end of a gene. Gene silencing is stably maintained during development, including encystation and excystation. We have used this approach to successfully silence two E. invadens genes: a putative rhomboid protease gene and a SHAQKY family Myb gene. The Myb gene is upregulated during oxidative stress and development, and its downregulation led, as predicted, to decreased viability under oxidative stress and decreased cyst formation. Thus, the RNAi trigger silencing method can be used to successfully investigate the molecular functions of genes in E. invadens. Dissection of the molecular basis of Entamoeba stage conversion is now possible, representing an important technical advance for the system. PMID:26787723

  12. Entamoeba lysyl-tRNA synthetase contains a cytokine-like domain with chemokine activity towards human endothelial cells.

    PubMed

    Castro de Moura, Manuel; Miro, Francesc; Han, Jung Min; Kim, Sunghoon; Celada, Antonio; Ribas de Pouplana, Lluís

    2011-11-01

    Immunological pressure encountered by protozoan parasites drives the selection of strategies to modulate or avoid the immune responses of their hosts. Here we show that the parasite Entamoeba histolytica has evolved a chemokine that mimics the sequence, structure, and function of the human cytokine HsEMAPII (Homo sapiens endothelial monocyte activating polypeptide II). This Entamoeba EMAPII-like polypeptide (EELP) is translated as a domain attached to two different aminoacyl-tRNA synthetases (aaRS) that are overexpressed when parasites are exposed to inflammatory signals. EELP is dispensable for the tRNA aminoacylation activity of the enzymes that harbor it, and it is cleaved from them by Entamoeba proteases to generate a standalone cytokine. Isolated EELP acts as a chemoattractant for human cells, but its cell specificity is different from that of HsEMAPII. We show that cell specificity differences between HsEMAPII and EELP can be swapped by site directed mutagenesis of only two residues in the cytokines' signal sequence. Thus, Entamoeba has evolved a functional mimic of an aaRS-associated human cytokine with modified cell specificity.

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

    PubMed

    Makioka, Asao; Kumagai, Masahiro; Hiranuka, Kazushi; Kobayashi, Seiki; Takeuchi, Tsutomu

    2011-01-01

    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

  14. Identification of EhTIF-IA: The putative E. histolytica orthologue of the human ribosomal RNA transcription initiation factor-IA.

    PubMed

    Srivastava, Ankita; Bhattacharya, Alok; Bhattacharya, Sudha; Jhingan, Gagan Deep

    2016-03-01

    Initiation of rDNA transcription requires the assembly of a specific multi-protein complex at the rDNA promoter containing the RNA Pol I with auxiliary factors. One of these factors is known as Rrn3P in yeast and Transcription Initiation Factor IA (TIF-IA) in mammals. Rrn3p/TIF-IA serves as a bridge between RNA Pol I and the pre-initiation complex at the promoter. It is phosphorylated at multiple sites and is involved in regulation of rDNA transcription in a growth-dependent manner. In the early branching parasitic protist Entamoeba histolytica, the rRNA genes are present exclusively on circular extra chromosomal plasmids. The protein factors involved in regulation of rDNA transcription in E. histolytica are not known. We have identified the E. histolytica equivalent of TIF-1A (EhTIF-IA) by homology search within the database and was further cloned and expressed. Immuno-localization studies showed that EhTIF-IA co-localized partially with fibrillarin in the peripherally localized nucleolus. EhTIF-IA was shown to interact with the RNA Pol I-specific subunit RPA12 both in vivo and in vitro. Mass spectroscopy data identified RNA Pol I-specific subunits and other nucleolar proteins to be the interacting partners of EhTIF-IA. Our study demonstrates for the first time a conserved putative RNA Pol I transcription factor TIF-IA in E. histolytica.

  15. Entamoeba histolyticaelectrondense granules secretion in vitro and in vivo: ultrastructural study.

    PubMed

    Chávez-Munguía, Bibiana; Castañón, Guadalupe; Hernández-Ramírez, Verónica; González-Lázaro, Mónica; Talamás-Rohana, Patricia; Martínez-Palomo, Adolfo

    2012-02-01

    Electron dense granules (EDGs) were identified by transmission electron microscopy in Entamoeba histolytica trophozoites recovered from hamster liver lesions. Abundant granules were present in trophozoites recovered after 15 min of liver inoculation. Variation in the size and morphology of these EDGs was also observed. Numerous granules were present in the plasma membrane when these parasites were incubated for 5 min with MDCK monolayers. Release of these EDGs was suggested by the presence of granules in contact with the surface of the target cell plasma membrane. Parasite phagocytic invaginations were observed after 10 min of parasite-monolayer interaction. In these structures, scarce granules were seen. Granules secretion was corroborated by obtaining of a pellet of these small structures from the incubation of trophozoites with collagen supernatant. Collagenase and gellatinase activity of this pellet was identified in SDS-PAGE gels. EDGs were also present in amebic hamster liver lesions. Our observations corroborate that these granules are secreted and suggest that may participate in the cytopathic effect of E. histolytica both in vitro and in vivo.

  16. Proteases from Entamoeba spp. and Pathogenic Free-Living Amoebae as Virulence Factors

    PubMed Central

    Serrano-Luna, Jesús; Piña-Vázquez, Carolina; Reyes-López, Magda; Ortiz-Estrada, Guillermo

    2013-01-01

    The standard reference for pathogenic and nonpathogenic amoebae is the human parasite Entamoeba histolytica; a direct correlation between virulence and protease expression has been demonstrated for this amoeba. Traditionally, proteases are considered virulence factors, including those that produce cytopathic effects in the host or that have been implicated in manipulating the immune response. Here, we expand the scope to other amoebae, including less-pathogenic Entamoeba species and highly pathogenic free-living amoebae. In this paper, proteases that affect mucin, extracellular matrix, immune system components, and diverse tissues and cells are included, based on studies in amoebic cultures and animal models. We also include proteases used by amoebae to degrade iron-containing proteins because iron scavenger capacity is currently considered a virulence factor for pathogens. In addition, proteases that have a role in adhesion and encystation, which are essential for establishing and transmitting infection, are discussed. The study of proteases and their specific inhibitors is relevant to the search for new therapeutic targets and to increase the power of drugs used to treat the diseases caused by these complex microorganisms. PMID:23476670

  17. Identification of Entamoeba moshkovskii in Treated Waste Water Used for Agriculture.

    PubMed

    Fonseca, Jairo Andres; Heredia, Rubén Darío; Ortiz, Carolina; Mazo, Martín; Clavijo-Ramírez, Carlos Arturo; Lopez, Myriam Consuelo

    2016-03-01

    We conducted an observational study to determine the prevalence of Entamoeba spp., in samples collected in a waste water treatment plant that provides water for agricultural irrigation. Samples were collected weekly over a period of 10 weeks at representative contamination stages from within the treatment plant. Protozoan identification was performed via light microscopy and culture. PCR amplification of small subunit rRNA gene sequences of E. histolytica/dispar/moshkovskii was performed in culture positive samples. Light microscopy revealed the presence of Entamoeba spp., in 70% (14/20) of the raw waste water samples and in 80% (8/10) of the treated water samples. PCR amplification after culture at both 24 and 37°C revealed that 100% (29/29) of the raw waste water samples and 78.6% (11/14) of the treated waste water were positive for E. moshkovskii. We report the first isolation of E. moshkovskii in Colombia, confirmed by PCR. Recent reports of E. moshkovskii pathogenic potential suggest this finding could constitute a public health risk for people exposed to this water.

  18. Efficacy of New 5-Nitroimidazoles against Metronidazole-Susceptible and -Resistant Giardia, Trichomonas, and Entamoeba spp.

    PubMed Central

    Upcroft, Jacqueline A.; Campbell, Raymond W.; Benakli, Kamel; Upcroft, Peter; Vanelle, Patrice

    1999-01-01

    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. PMID:9869568

  19. High Throughput Sequencing of Entamoeba 27nt Small RNA Population Reveals Role in Permanent Gene Silencing But No Effect on Regulating Gene Expression Changes during Stage Conversion, Oxidative, or Heat Shock Stress.

    PubMed

    Zhang, Hanbang; Ehrenkaufer, Gretchen M; Manna, Dipak; Hall, Neil; Singh, Upinder

    2015-01-01

    The human parasite Entamoeba histolytica has an active RNA interference (RNAi) pathway with an extensive repertoire of 27nt small RNAs that silence genes. However the role of this pathway in regulating amebic biology remains unknown. In this study, we address whether silencing via 27nt small RNAs may be a mechanism for controlling gene expression changes during conversion between the trophozoite and cyst stages of the parasite. We sequenced small RNA libraries generated from trophozoites, early cysts, mature cysts, and excysting cells and mapped them to the E. invadens genome. Our results show that, as in E. histolytica, small RNAs in E. invadens are largely ~27nt in length, have an unusual 5'-polyphosphate structure and mediate gene silencing. However, when comparing the libraries from each developmental time-point we found few changes in the composition of the small RNA populations. Furthermore, genes targeted by small RNAs were permanently silenced with no changes in transcript abundance during development. Thus, the E. invadens 27nt small RNA population does not mediate gene expression changes during development. In order to assess the generalizability of our observations, we examined whether small RNAs may be regulating gene expression changes during stress response in E. histolytica. Comparison of the 27nt small RNA populations from E. histolytica trophozoites from basal conditions, or after heat shock or exposure to oxidative stress showed few differences. Similar to data in E. invadens development, genes targeted by small RNAs were consistently silenced and did not change expression under tested stress conditions. Thus, the biological roles of the 27nt small RNA population in Entamoeba remain elusive. However, as the first characterization of the RNAi pathway in E. invadens these data serve as a useful resource for the study of Entamoeba development and open the door to the development of RNAi-based gene silencing tools in E. invadens.

  20. Entamoeba histolytica pleuropulmonary infection. Case report and review of the literature.

    PubMed

    Chalhoub, Sanaa; Kanafani, Zeina

    2012-01-01

    Pleuropulmonary amebiasis is the 2nd most common extraintestinal site of amebiasis after liver abscess. We describe a man with pleuropulmonary amebiasis presenting with pulmonary consolidation and pleural effusion. In patients with pneumonia coming from endemic countries such as Lebanon, pleuropulmonary amebiasis should be considered in the setting of chocolate-colored sputum, negative respiratory cultures, and failure of antibacterial therapy.

  1. An Experimental Animal Model for the Study of Immunity to Entamoeba Histolytica.

    DTIC Science & Technology

    1983-04-15

    populations from widely separate geographic areas. After injections of high molecular weight proteins from axenic amebae in complete Freund’s adjuvant...high molecular weight fraction of axenic antigen with CFA given i.m. (Ghadirian et al. 1980). Sepulveda et al. (1978) developed improved vaccines...Reacciones serol6gicas en la amibiasis invasora con la tecnica de anticuerpos fluorescentes: II. Pruebas utilizando como antfgeno material de absceso

  2. Phosphorylation of Eukaryotic Initiation Factor-2α during Stress and Encystation in Entamoeba Species

    PubMed Central

    Hendrick, Holland M.; Welter, Brenda H.; Sykes, Steven E.; Sullivan, William J.; Temesvari, Lesly A.

    2016-01-01

    Entamoeba histolytica is an enteric pathogen responsible for amoebic dysentery and liver abscess. It alternates between the host-restricted trophozoite form and the infective environmentally-stable cyst stage. Throughout its lifecycle E. histolytica experiences stress, in part, from host immune pressure. Conversion to cysts is presumed to be a stress-response. In other systems, stress induces phosphorylation of a serine residue on eukaryotic translation initiation factor-2α (eIF2α). This inhibits eIF2α activity resulting in a general decline in protein synthesis. Genomic data reveal that E. histolytica possesses eIF2α (EheIF2α) with a conserved phosphorylatable serine at position 59 (Ser59). Thus, this pathogen may have the machinery for stress-induced translational control. To test this, we exposed cells to different stress conditions and measured the level of total and phospho-EheIF2α. Long-term serum starvation, long-term heat shock, and oxidative stress induced an increase in the level of phospho-EheIF2α, while short-term serum starvation, short-term heat shock, or glucose deprivation did not. Long-term serum starvation also caused a decrease in polyribosome abundance, which is in accordance with the observation that this condition induces phosphorylation of EheIF2α. We generated transgenic cells that overexpress wildtype EheIF2α, a non-phosphorylatable variant of eIF2α in which Ser59 was mutated to alanine (EheIF2α-S59A), or a phosphomimetic variant of eIF2α in which Ser59 was mutated to aspartic acid (EheIF2α-S59D). Consistent with the known functions of eIF2α, cells expressing wildtype or EheIF2α-S59D exhibited increased or decreased translation, respectively. Surprisingly, cells expressing EheIF2α-S59A also exhibited reduced translation. Cells expressing EheIF2α-S59D were more resistant to long-term serum starvation underscoring the significance of EheIF2α phosphorylation in managing stress. Finally, phospho-eIF2α accumulated during

  3. Expanding the Entamoeba Universe: New Hosts Yield Novel Ribosomal Lineages.

    PubMed

    Jacob, Alison S; Busby, Eloise J; Levy, Abigail D; Komm, Natasha; Clark, C Graham

    2016-01-01

    Removing the requirement for cell culture has led to a substantial increase in the number of lineages of Entamoeba recognized as distinct. Surveying the range of potential host species for this parasite genus has barely been started and it is clear that additional sampling of the same host in different locations often identifies additional diversity. In this study, using small subunit ribosomal RNA gene sequencing, we identify four new lineages of Entamoeba, including the first report of Entamoeba from an elephant, and extend the host range of some previously described lineages. In addition, examination of microbiome data from a number of host animals suggests that substantial Entamoeba diversity remains to be uncovered.

  4. EhVps32 Is a Vacuole-Associated Protein Involved in Pinocytosis and Phagocytosis of Entamoeaba histolytica

    PubMed Central

    Avalos-Padilla, Yunuen; Betanzos, Abigail; Javier-Reyna, Rosario; García-Rivera, Guillermina; Chávez-Munguía, Bibiana; Lagunes-Guillén, Anel; Ortega, Jaime; Orozco, Esther

    2015-01-01

    Here, we investigated the role of EhVps32 protein (a member of the endosomal-sorting complex required for transport) in endocytosis of Entamoeba histolytica, a professional phagocyte. Confocal microscopy, TEM and cell fractionation revealed EhVps32 in cytoplasmic vesicles and also located adjacent to the plasma membrane. Between 5 to 30 min of phagocytosis, EhVps32 was detected on some erythrocytes-containing phagosomes of acidic nature, and at 60 min it returned to cytoplasmic vesicles and also appeared adjacent to the plasma membrane. TEM images revealed it in membranous structures in the vicinity of ingested erythrocytes. EhVps32, EhADH (an ALIX family member), Gal/GalNac lectin and actin co-localized in the phagocytic cup and in some erythrocytes-containing phagosomes, but EhVps32 was scarcely detected in late phagosomes. During dextran uptake, EhVps32, EhADH and Gal/GalNac lectin, but not actin, co-localized in pinosomes. EhVps32 recombinant protein formed oligomers composed by rings and filaments. Antibodies against EhVps32 monomers stained cytoplasmic vesicles but not erythrocytes-containing phagosomes, suggesting that in vivo oligomers are formed on phagosome membranes. The involvement of EhVps32 in phagocytosis was further study in pNeoEhvps32-HA-transfected trophozoites, which augmented almost twice their rate of erythrophagocytosis as well as the membranous concentric arrays built by filaments, spirals and tunnel-like structures. Some of these structures apparently connected phagosomes with the phagocytic cup. In concordance, the EhVps32-silenced G3 trophozoites ingested 80% less erythrocytes than the G3 strain. Our results suggest that EhVps32 participates in E. histolytica phagocytosis and pinocytosis. It forms oligomers on erythrocytes-containing phagosomes, probably as a part of the scission machinery involved in membrane invagination and intraluminal vesicles formation. PMID:26230715

  5. Entamoeba histolytica: solubilization and biochemical characterization of dolichol phosphate mannose synthase, an essential enzyme in glycoprotein biosynthesis.

    PubMed

    Villagómez-Castro, J C; Calvo-Méndez, C; Vargas-Rodríguez, L; Flores-Carreón, A; López-Romero, E

    1998-02-01

    Sequential treatment of trophozoite membranes with the nonionic detergents Brij 35 and Igepal CA-630 released a soluble fraction that efficiently catalyzed the transfer of mannose from GDP-Man into a mannolipid that was identified as dolichol phosphate mannose (Dol-P-Man) by several criteria. The transfer reaction occurred only in the presence of exogenously added dolichol monophosphate (Dol-P). Plots of enzyme velocity versus Dol-P and GDP-Man concentrations revealed sigmoidal and hyperbolic kinetics, respectively. Values of S0.5 for Dol-P and K(m) for GDP-Man were 15 micrograms/ml and 4.1 microM, respectively. The solubilized fraction failed to transfer the label into other products such as lipid-linked oligosaccharides and glycoproteins. The optimum pH was 7.5-8.0 in potassium phosphate or Tris/HCl buffers and the enzyme required either Mg2+ or Mn2+. The latter was more effective but in a narrower range of concentrations. The transferase was inhibited by a number of nucleotides the strongest being GMP, GDP, and GTP. When assayed in the reverse direction, however, the enzyme catalyzed the transfer of mannose from Dol-P-Man back into GDP-Man as a function of increasing concentrations of GDP. Mg2+ was a better activator of the reverse reaction than Mn2+, which reached up to 60% at 2 mM GDP. These results suggest that some of the enzyme catalytic properties may change depending on the direction of the transfer reaction.

  6. [Fatal transmural colitis caused by Entamoeba histolytica. Differential diagnosis of chronic inflammatory bowel disease and colon cancer].

    PubMed

    Hansen, C R; Louw, P

    1993-09-13

    Perforation of the colon is a rare but frequently fatal complication of amoebiasis. We report a case of a 53 year-old male, with no history of travel abroad, who was admitted to hospital with haematochezia. A tumor of the rectum was diagnosed clinically. Due to acute intestinal obstruction, laparotomy was performed, revealing multiple perforations of the large bowel and severe peritonitis leading to subtotal colectomy. The histological examinations revealed transmural amoebic colitis. The patient died due to multi-organ failure.

  7. Diphtheroid colitis in a Boa constrictor infected with amphibian Entamoeba sp.

    PubMed

    Richter, Barbara; Kübber-Heiss, Anna; Weissenböck, Herbert

    2008-05-06

    A female boa (Boa constrictor) from a zoological collection was submitted for necropsy after sudden death. Prominent pathological findings included a diphtheroid colitis, endoparasitism, focal pneumonia and inclusion bodies typical for inclusion body disease (IBD). In the colon entamoebae were identified, which differed in size and distribution from Entamoeba invadens. Gene sequence analysis of the 18S ribosomal RNA revealed 100% similarity with an Entamoeba species from the African bullfrog (Pyxicephalus adspersus), probably Entamoeba ranarum. The snake was possibly immunosuppressed, and the source of infection remains unclear. This is the first report of an infection with an amphibian Entamoeba species associated with colitis in a snake.

  8. Entamoeba marina n. sp.; a New Species of Entamoeba Isolated from Tidal Flat Sediment of Iriomote Island, Okinawa, Japan.

    PubMed

    Shiratori, Takashi; Ishida, Ken-Ichiro

    2016-05-01

    The genus Entamoeba includes anaerobic lobose amoebae, most of which are parasites of various vertebrates and invertebrates. We report a new Entamoeba species, E. marina n. sp. that was isolated from a sample of tidal flat sediment collected at Iriomote Island, Okinawa, Japan. Trophozoites of E. marina were 12.8-32.1 μm in length and 6.8-15.9 μm in width, whereas the cysts were 8.9-15.8 μm in diam. and contained four nuclei. The E. marina cells contained a rounded nucleus with a small centric karyosome and uniformly arranged peripheral chromatin. Although E. marina is morphologically indistinguishable from other tetranucleated cyst-forming Entamoeba species, E. marina can be distinguished from them based on the combination of molecular phylogenetic analyses using SSU rDNA gene and the difference of collection sites. Therefore, we propose E. marina as a new species of the genus Entamoeba.

  9. Entamoeba sp. (Sarcomastigophora: Endamoebidae) from ostriches (Struthio camelus) (Aves: Struthionidae).

    PubMed

    Martínez-Díaz, R A; Herrera, S; Castro, A; Ponce, F

    2000-10-01

    The first case of Entamoeba of the 1-nucleate mature cyst group in birds is described. Trophozoites and cysts of Entamoeba have been found in ostriches (Struthio camelus) from farms located all over Spain. The cysts are large (13.47microm mean diameter); they possess one nucleus when mature, with a large endosome and peripheral chromatine arranged in small granules; chromatoid bodies, when present, are large and elongated. Trophozoites are large (19. 88microm mean diameter), with a clear differentiation between ecto- and endoplasm, this containing numerous vacuoles; the nucleus is large and diffuse. The characteristics of this amoeba resembles but do not completely fulfill those of E. suis and E. chattoni; also, these species are from mammals.

  10. [Immune response induced by phosphofructokinase from E. histolytica in hamsters].

    PubMed

    Jiménez Cardoso, J M; Jiménez, E; Kumate, J

    1991-01-01

    The enzymatic activity of inorganic pyrophosphate (PPi) dependent phosphofructokinase became manifest in the supernatant obtained by centrifugation in a homogenate of E. histolytica strain HMI-IMSS at 700,000 g. Partial purification of the enzyme was achieved by column chromatography with Ultrogel AcA-34. Ten protein elution spikes were obtained: five showed enzymatic activity. Elution spikes I and II attained the highest values of specific enzymatic activity 6.45 and 6.98 U/mg of protein, respectively. Next were spikes X and III with similar values 2.55 and 2.63 U/mg of protein, and spike IV presented the lowest value of 0.86 U/mg of protein. The five spikes were used to immunize hamsters which were challenged intrahepatically, four weeks later, with 3 x 10(5) trophozoites of E. histolytica. A control group of animals not immunized underwent intrahepatic challenge with the same number of amebae. The proteins with enzymatic activity contained in elution spikes I and II conferred immunologic protection in 100% of the animals, while elution spikes X and III were protective in 50 to 63%, and spike IV gave the lowest value of 37%. It can be assumed that there is an antienzyme antibody responsible for the absence of hepatic abscesses in the immunized hamsters.

  11. [Immunochemical differences in the surface polysaccharides obtained from Entamoeba histolytica strain HM1:IMSS and its virulent (C-A) and non-virulent (L-6) clones].

    PubMed

    Isibasi, A; Blanco, F; Arreguín, C; Martínez, G; Pelayo, R; Orozco, E; Kumate, J

    1990-01-01

    There probably exist many different surface molecular polysaccharides with different compositions of their constituent sugars, in the different zymodemes. This fact might have a repercussion upon the resistance to the lysis of the complement. Seeking to prove this hypothesis, the ideal would be to obtain polysaccharide molecules of type LPFG of pathogenic and non-pathogenic zymodemes, demonstrating that there is a difference in the chemical composition of their polysaccharides. If this were true, a serologic classification, of the different zymodemes, would be possible in a fashion similar to gram-negative bacteria. Our results suggest the existence of structural differences between the polysaccharides of a virulent clone (C-A) and a non-virulent one (L-6).

  12. Alphabetical Index of Parasitic Diseases

    MedlinePlus

    ... Entamoeba histolytica Infection) American Trypanosomiasis (Chagas Disease) Ancylostomiasis (Hookworm) Angiostrongyliasis ( Angiostrongylus Infection) Anisakiasis ( Anisakis Infection, Pseudoterranova Infection) ...

  13. Invasive entamoebae in pronghorn (Antilocapra americana) from Wyoming.

    PubMed

    Kingston, N; Williams, E S; Thorne, E T

    1990-01-01

    Trophozoites and cysts of an amoeba resembling Entamoeba bovis were recovered from soft stools of captive pronghorn fawns (Antilocapra americana). Chronic or intermittent diarrhea was observed in most individuals in two groups of hand-raised 1- to 8-mo-old pronghorns. Ileocecal lymph nodes were mildly to moderately enlarged. Microscopic lesions were characterized by lymphoid hyperplasia, focal necrosis and pyogranulomatous inflammation in lymph nodes and focal necrosis and diffuse nonsuppurative enteritis in ileum and cecum. In 12 of 17 fawns, trophic stages of amoebae were observed in the submucosa of the cecum and/or the cortex of the ileocecal lymph node. This is the first report of E. bovis-like organisms invading and causing pathological changes in the tissues of their host.

  14. Evaluation of five commercial methods for the extraction and purification of DNA from human faecal samples for downstream molecular detection of the enteric protozoan parasites Cryptosporidium spp., Giardia duodenalis, and Entamoeba spp.

    PubMed

    Paulos, Silvia; Mateo, Marta; de Lucio, Aida; Hernández-de Mingo, Marta; Bailo, Begoña; Saugar, José M; Cardona, Guillermo A; Fuentes, Isabel; Mateo, María; Carmena, David

    2016-08-01

    High quality, pure DNA is required for ensuring reliable and reproducible results in molecular diagnosis applications. A number of in-house and commercial methods are available for the extraction and purification of genomic DNA from faecal material, each one offering a specific combination of performance, cost-effectiveness, and easiness of use that should be conveniently evaluated in function of the pathogen of interest. In this comparative study the marketed kits QIAamp DNA stool mini (Qiagen), SpeedTools DNA extraction (Biotools), DNAExtract-VK (Vacunek), PowerFecal DNA isolation (MoBio), and Wizard magnetic DNA purification system (Promega Corporation) were assessed for their efficacy in obtaining DNA of the most relevant enteric protozoan parasites associated to gastrointestinal disease globally. A panel of 113 stool specimens of clinically confirmed patients with cryptosporidiosis (n=29), giardiasis (n=47) and amoebiasis by Entamoeba histolytica (n=3) or E. dispar (n=10) and apparently healthy subjects (n=24) were used for this purpose. Stool samples were aliquoted in five sub-samples and individually processed by each extraction method evaluated. Purified DNA samples were subsequently tested in PCR-based assays routinely used in our laboratory. The five compared methods yielded amplifiable amounts of DNA of the pathogens tested, although performance differences were observed among them depending on the parasite and the infection burden. Methods combining chemical, enzymatic and/or mechanical lysis procedures at temperatures of at least 56°C were proven more efficient for the release of DNA from Cryptosporidium oocysts.

  15. Chitin synthetase in encysting Giardia lamblia and Entamoeba invadens

    SciTech Connect

    Das, S.; Gillin, F.D.

    1987-05-01

    Giardia lamblia (Gl) and Entamoeba invadens (Ei) are protozoan parasites with two morphologic stages in their life cycles. Motile trophozoites colonize the intestine of humans and reptiles respectively. Water resistant cysts, which can survive outside the host, transmit infection. In vitro cyst formation of Ei from trophozoites has been reported, and the authors have recently induced in vitro encystation of Gl. Although the cyst walls of both parasites contain chitin, it synthesis by encysting trophozoites has not been reported. The authors now show that encystation conditions greatly increase chitin synthetase (CS) specific activity (incorporation of /sup 3/H GlcNAc from UDP-GlcNAc into TCA-or alcohol-precipitable material). Extracts of encysting Gl incorporated 3.6 nmol/mg protein in 5 hr compared to < 0.005 in controls. Extracts of encysting Fi incorporated 4.8 n mol/mg protein, compared to 1.7 in the control. CS activity of both parasites requires preformed chitin. The Gl enzyme requires a reducing agent, is inhibited by digitonin and the CS inhibitors, polyoxin D and Nikkomycin, but not by tunicamycin. The product is digested by chitinase. Ei enzyme does not require a reducing agent and is stimulated by 1 mg/ml digitonin, but inhibited by higher concentrations. These studies demonstrate CS enzymes which may play important roles in encystation of Gl and Ei.

  16. Biological aspects of a Brazilian strain of Entamoeba dispar.

    PubMed

    Furst, C; Gomes, M A; Tafuri, W L; Silva, E F

    2002-02-01

    A strain of Entamoeba dispar was characterized by clinical diagnosis, serological and electrophoretical isoenzyme analysis and by the polymorphism of a 482 bp genomic fragment analysis. The pathogenesis and virulence of this strain was investigated considering the experimental infection in hamster livers in association with the original intestinal microbiota. Liver lesions were observed in hamsters experimentally infected with trophozoites from xenic cultures, but not from the monoxenic cultures. Moreover, clones obtained from re-isolated strain Wil1R1 showed a distinct biological behavior. In fact, animals inoculated with Wil1R1ClB3 showed an intense acute inflammatory reaction with destructive focal hepatic lesions. These lesions were characterized as amebic abscesses. The association between bacteria and ameba has been fairly well studied because it affects the pathogenicity of the amebas and has important therapeutic implications. In this study, we demonstrated that E. dispar in association with the original microbiota is able to produce lesions in hamster liver in spite of its having been considered to be non-pathogenic in the hamster model. Based on these results we suggest that diagnosis of amebiasis needs to be made with more care and that clinical and therapeutical procedures need to be revised.

  17. Detection of the amoeba Entamoeba gingivalis in periodontal pockets.

    PubMed

    Bonner, Mark; Amard, Véronique; Bar-Pinatel, Charlotte; Charpentier, Frédéric; Chatard, Jean-Michel; Desmuyck, Yvan; Ihler, Serge; Rochet, Jean-Pierre; Roux de La Tribouille, Véronique; Saladin, Luc; Verdy, Marion; Gironès, Núria; Fresno, Manuel; Santi-Rocca, Julien

    2014-01-01

    Periodontitis is a public health issue, being one of the most prevalent diseases worldwide. However, the aetiology of the disease is still unclear: genetics of patients cannot explain the dispersed or isolated localisation of gingival pockets, while bacteria-based models are insufficient to distinguish gingivitis and periodontitis. The possible role of parasites in the establishment of periodontitis has been poorly studied until now. The aim of this project was to study a potential link between colonisation of gingival crevices by the amoeba Entamoeba gingivalis and periodontitis. In eight different dental clinics in France, samples were taken in periodontal pockets (72) or healthy sites (33), and submitted to microscopic observation and molecular identification by PCR with a new set of primers designed to specifically detect E. gingivalis. This blind sample analysis showed the strong sensitivity of PCR compared with clinical diagnosis (58/72 = 81%), and microscopy (51/65 = 78%). The results of this work show that the parasites detected by microscopy mainly - if not exclusively - belong to the species E. gingivalis and that the presence of the parasite is correlated with periodontitis.

  18. Detection of the amoeba Entamoeba gingivalis in periodontal pockets

    PubMed Central

    Bonner, Mark; Amard, Véronique; Bar-Pinatel, Charlotte; Charpentier, Frédéric; Chatard, Jean-Michel; Desmuyck, Yvan; Ihler, Serge; Rochet, Jean-Pierre; Roux de La Tribouille, Véronique; Saladin, Luc; Verdy, Marion; Gironès, Núria; Fresno, Manuel; Santi-Rocca, Julien

    2014-01-01

    Periodontitis is a public health issue, being one of the most prevalent diseases worldwide. However, the aetiology of the disease is still unclear: genetics of patients cannot explain the dispersed or isolated localisation of gingival pockets, while bacteria-based models are insufficient to distinguish gingivitis and periodontitis. The possible role of parasites in the establishment of periodontitis has been poorly studied until now. The aim of this project was to study a potential link between colonisation of gingival crevices by the amoeba Entamoeba gingivalis and periodontitis. In eight different dental clinics in France, samples were taken in periodontal pockets (72) or healthy sites (33), and submitted to microscopic observation and molecular identification by PCR with a new set of primers designed to specifically detect E. gingivalis. This blind sample analysis showed the strong sensitivity of PCR compared with clinical diagnosis (58/72 = 81%), and microscopy (51/65 = 78%). The results of this work show that the parasites detected by microscopy mainly – if not exclusively – belong to the species E. gingivalis and that the presence of the parasite is correlated with periodontitis. PMID:24983705

  19. Entamoeba struthionis n.sp. (Sarcomastigophora: Endamoebidae) from ostriches (Struthio camelus).

    PubMed

    Ponce Gordo, F; Martínez Díaz, R A; Herrera, S

    2004-02-06

    In the present work we identify the species of Entamoeba from ostriches (Struthio camelus). The complete sequence of the small subunit ribosomal RNA gene from this organism has been compared with those published for other species of the genus and clear differences have been found. These results confirm previous data which showed differences on parasite morphology and class of host with the other Entamoeba species. Taking all these data together, it can be concluded that the organism from ostriches is a new species whose proposed name is Entamoeba struthionis n.sp. This species probably infects rheas (Rhea americana), but genetic analysis of isolates from this host should be performed to confirm morphological data. Also, comparison of gene sequences with data from other authors on cysts recovered from human stool samples showed the possibility that this amoeba may affect humans. Further studies are needed to determine the risk of transmission of this new species to humans.

  20. Detection of Cryptosporidium spp., Entamoeba spp. and Monocercomonas spp. in the gastrointestinal tract of snakes by in-situ hybridization.

    PubMed

    Richter, B; Kübber-Heiss, A; Weissenböck, H; Schmidt, P

    2008-01-01

    This report describes the development of a diagnostic method for protozoal infections of the gastrointestinal tract of captive snakes, based on chromogenic in-situ hybridization with probes designed for the detection of 18S rRNA genes from Cryptosporidium spp., Entamoeba spp., Entamoeba invadens and Monocercomonas spp. The specificity of the probes was confirmed with the help of parasitic cultures and gene sequence analysis. The probes gave clear positive signals. Of 182 snakes examined, seven were positive with the Cryptosporidium probe, 13 with the Entamoeba probe (of which nine were also positive with the E. invadens probe), and 34 with the Monocercomonas probe.

  1. Atg8 is involved in endosomal and phagosomal acidification in the parasitic protist E ntamoeba histolytica

    PubMed Central

    Picazarri, Karina; Nakada‐Tsukui, Kumiko; Tsuboi, Kumiko; Miyamoto, Eri; Watanabe, Naoko; Kawakami, Eiryo

    2015-01-01

    Summary Autophagy is one of two major bulk protein degradation systems and is conserved throughout eukaryotes. The protozoan E ntamoeba histolytica, which is a human intestinal parasite, possesses a restricted set of autophagy‐related (Atg) proteins compared with other eukaryotes and thus represents a suitable model organism for studying the minimal essential components and ancestral functions of autophagy. E. histolytica possesses two conjugation systems: Atg8 and Atg5/12, although a gene encoding Atg12 is missing in the genome. Atg8 is considered to be the central and authentic marker of autophagosomes, but recent studies have demonstrated that Atg8 is not exclusively involved in autophagy per se, but other fundamental mechanisms of vesicular traffic. To investigate this question in E . histolytica, we studied on Atg8 during the proliferative stage. Atg8 was constitutively expressed in both laboratory‐maintained and recently established clinical isolates and appeared to be lipid‐modified in logarithmic growth phase, suggesting a role of Atg8 in non‐stress and proliferative conditions. These findings are in contrast to those for E ntamoeba invadens, in which autophagy is markedly induced during an early phase of differentiation from the trophozoite into the cyst. The repression of Atg8 gene expression in En . histolytica by antisense small RNA‐mediated transcriptional gene silencing resulted in growth retardation, delayed endocytosis and reduced acidification of endosomes and phagosomes. Taken together, these results suggest that Atg8 and the Atg8 conjugation pathway have some roles in the biogenesis of endosomes and phagosomes in this primitive eukaryote. PMID:25923949

  2. Crithidia fasciculata induces encystation of Entamoeba invadens in a galactose-dependent manner.

    PubMed

    Cho, J; Eichinger, D

    1998-08-01

    The ability of the flagellate Crithidia fasciculata to induce encystation of the reptile pathogen, Entamoeba invadens, was studied in vitro. A specific ratio of flagellate to amoeba was required; both live and heat-killed C. fasciculata induced amoebic encystation. The interaction between the Crithidia and Entamoeba cells was found to be galactose-mediated because the addition of galactose to the culture medium, or the pretreatment of the flagellate with galactosidase, eliminated its ability to induce encystation. Galactose was also found to prevent the amoeba amoeba aggregation that normally occurs in axenic cultures of encystation-induced E. invadens. Both galactose and glcNAc completely inhibited cyst formation of these induced cultures, although the latter sugar did not prevent cell aggregation. These results indicate that a galactose-mediated interaction between E. invadens cells is an early step in the in vitro encystation pathway.

  3. Actinomycosis of Cecum Associated with Entamoeba Infection Mimicking Perforated Colon Cancer

    PubMed Central

    Böler, Deniz Eren; Uras, Cihan; Göksel, Süha; Karaarslan, Mehmet

    2013-01-01

    Actinomycosis is a granulomatous disease caused by Actinomyces that mimics other intra-abdominal pathologies especially neoplasms. Correct diagnosis can be rarely established before radical surgery. On the other hand Entamoeba infection affects a considerable number of people worldwide. To our knowledge only one case has been reported to be affected by both organisms. We report a man who has been operated for a mass in the cecum mimicking a perforated colon cancer. Abdominal CT revealed a mass with features of an invading neoplasm. After radical surgery, definitive pathology revealed that the mass was due to actinomycosis associated with Entamoeba infection. The postoperative period was uneventful and the patient was on long-course antibiotherapy. It is important to consider actinomycosis especially in patients with intra-abdominal masses with unusual aggressiveness to prevent unnecessary surgery. However, surgery can be unavoidable especially in the presence of complicated disease or high index of suspicion for malignancy. PMID:23738157

  4. Studies on Amoebiasis.

    DTIC Science & Technology

    1971-12-01

    sane way into the vacuoles for digestive purposes. In a senee these would then be serving as the lysosome-equivalent for Entamoeba histolytica ...now grow Entamoeba histolytica (strains 301, F-22, and K-9) in inonophasic medium. The basic preparation of this medium is described elsewhere...highly specialized synthetic system normally present in Entamoeba histolytica and modulated by the environment. p 32 IL Ii U: NC ES 1- Wittner, M1

  5. Mechanisms of Drug Resistance in Plasmodium falciparum

    DTIC Science & Technology

    1992-09-11

    parasites. With the collaboration of Dr. Esther Orozco, we cloned two mdr-like genes from Entamoeba histolytica and demonstrated an association of...Orozco (1990). " Entamoeba histolytica : "Physiology of multidrug resistance." Exp Parasitol. 71:169-175. Buschman, E., and P. Gros. (1991). "Functional...Ayala, E. Orozco, and D. Wirth. (1990). "Emetine-resistant mutants of Entamoeba histolytica overexpress mRNAs for multidrug resistance." Mol Biochem

  6. Amebic liver abscess

    MedlinePlus

    Hepatic amebiasis; Extraintestinal amebiasis; Abscess - amebic liver ... Amebic liver abscess is caused by Entamoeba histolytica. This parasite causes amebiasis , an intestinal infection that is also called ...

  7. The Effect of Nonsurgical Periodontal Therapy on Trichomonas Tenax and Entamoeba Gingivalis in Patients with Chronic Periodontitis

    PubMed Central

    Rashidi Maybodi, Fahimeh; Haerian Ardakani, Ahmad; Fattahi Bafghi, Ali; Haerian Ardakani, Alireza; Zafarbakhsh, Akram

    2016-01-01

    Statement of the Problem Trichomonas tenax and Entamoeba gingivalis are commensal protozoa which inhabit the human oral cavity. These parasites are found in patients with poor oral hygiene and might be a reason for progressive periodontal diseases. Purpose The aim of this study was to evaluate the effect of nonsurgical periodontal treatment on the frequency of these protozoa in saliva and plaque samples. Materials and Method In this clinical trial, samples of saliva and dental plaque were collected from 46 patients with moderate to severe chronic periodontitis before and after periodontal therapy. The samples were assessed for the frequency of parasites. Results The frequency of Entamoeba gingivalis was reduced in saliva (p= 0.007) and plaque (p= 0.027) three weeks after the treatment. Likewise, the frequency of Trichomonas tenax reduced in saliva (p= 0.030); however, the decrease was not significant in plaque (p= 0.913). Trichomonas tenax frequency in dental plaque directly related to the severity of periodontitis (r= 0.565, p≤ 0.000). In contrast, the number of Entamoeba gingivalis in both saliva (r= -0.405, p≤ 0.005) and plaque (r= -0.304, p= 0.040) was inversely related with the severity of the periodontal disease. Conclusion Nonsurgical periodontal treatment could reduce the number of Trichomonas Tenax and Entamoeba gingivalis in the oral environment of patients with chronic periodontitis. PMID:27602391

  8. Variation in Rural African Gut Microbiota Is Strongly Correlated with Colonization by Entamoeba and Subsistence

    PubMed Central

    Morton, Elise R.; Lynch, Joshua; Froment, Alain; Lafosse, Sophie; Heyer, Evelyne; Przeworski, Molly; Blekhman, Ran; Ségurel, Laure

    2015-01-01

    The human gut microbiota is impacted by host nutrition and health status and therefore represents a potentially adaptive phenotype influenced by metabolic and immune constraints. Previous studies contrasting rural populations in developing countries to urban industrialized ones have shown that industrialization is strongly correlated with patterns in human gut microbiota; however, we know little about the relative contribution of factors such as climate, diet, medicine, hygiene practices, host genetics, and parasitism. Here, we focus on fine-scale comparisons of African rural populations in order to (i) contrast the gut microbiota of populations inhabiting similar environments but having different traditional subsistence modes and either shared or distinct genetic ancestry, and (ii) examine the relationship between gut parasites and bacterial communities. Characterizing the fecal microbiota of Pygmy hunter-gatherers as well as Bantu individuals from both farming and fishing populations in Southwest Cameroon, we found that the gut parasite Entamoeba is significantly correlated with microbiome composition and diversity. We show that across populations, colonization by this protozoa can be predicted with 79% accuracy based on the composition of an individual's gut microbiota, and that several of the taxa most important for distinguishing Entamoeba absence or presence are signature taxa for autoimmune disorders. We also found gut communities to vary significantly with subsistence mode, notably with some taxa previously shown to be enriched in other hunter-gatherers groups (in Tanzania and Peru) also discriminating hunter-gatherers from neighboring farming or fishing populations in Cameroon. PMID:26619199

  9. First evidence of genetic intraspecific variability and occurrence of Entamoeba gingivalis in HIV(+)/AIDS.

    PubMed

    Cembranelli, Sibeli B S; Souto, Fernanda O; Ferreira-Paim, Kennio; Richinho, Túlio T; Nunes, Poliana L; Nascentes, Gabriel A N; Ferreira, Thatiana B; Correia, Dalmo; Lages-Silva, Eliane

    2013-01-01

    Entamoeba gingivalis is considered an oral commensal but demonstrates a pathogenic potential associated with periodontal disease in immunocompromised individuals. Therefore, this study evaluated the occurrence, opportunistic conditions, and intraspecific genetic variability of E. gingivalis in HIV(+)/AIDS patients. Entamoeba gingivalis was studied using fresh examination (FE), culture, and PCR from bacterial plaque samples collected from 82 HIV(+)/AIDS patients. Genetic characterization of the lower ribosomal subunit of region 18S (18S-SSU rRNA) was conducted in 9 positive samples using low-stringency single specific primer PCR (LSSP-PCR) and sequencing analysis. Entamoeba gingivalis was detected in 63.4% (52/82) of the samples. No association was detected between the presence of E. gingivalis and the CD4(+) lymphocyte count (≤200 cells/mm(3) (p = 0.912) or viral load (p = 0.429). The LSSP-PCR results helped group E. gingivalis populations into 2 polymorphic groups (68.3% similarity): group I, associated with 63.6% (7/11) of the samples, and group II, associated with 36.4% (4/11) of the samples, which shared 74% and 83.7% similarity and association with C and E isolates from HIV(-) individuals, respectively. Sequencing of 4 samples demonstrated 99% identity with the reference strain ATCC 30927 and also showed 2 divergent clusters, similar to those detected by LSSP-PCR. Opportunistic behavior of E. gingivalis was not detected, which may be related to the use of highly active antiretroviral therapy by all HIV(+)/AIDS patients. The high occurrence of E. gingivalis in these patients can be influenced by multifactorial components not directly related to the CD4(+) lymphocyte counts, such as cholesterol and the oral microbiota host, which could mask the potential opportunistic ability of E. gingivalis. The identification of the 18S SSU-rRNA polymorphism by LSSP-PCR and sequencing analysis provides the first evidence of genetic variability in E. gingivalis

  10. Role of Serum Amyloid A, Granulocyte-Macrophage Colony-Stimulating Factor, and Bone Marrow Granulocyte-Monocyte Precursor Expansion in Segmented Filamentous Bacterium-Mediated Protection from Entamoeba histolytica

    PubMed Central

    Burgess, Stacey L.; Saleh, Mahmoud; Cowardin, Carrie A.; Buonomo, Erica; Noor, Zannatun; Watanabe, Koji; Abhyankar, Mayuresh; Lajoie, Stephane; Wills-Karp, Marsha

    2016-01-01

    Intestinal segmented filamentous bacteria (SFB) protect from ameba infection, and protection is transferable with bone marrow dendritic cells (BMDCs). SFB cause an increase in serum amyloid A (SAA), suggesting that SAA might mediate SFB's effects on BMDCs. Here we further explored the role of bone marrow in SFB-mediated protection. Transient gut colonization with SFB or SAA administration alone transiently increased the H3K27 histone demethylase Jmjd3, persistently increased bone marrow Csf2ra expression and granulocyte monocyte precursors (GMPs), and protected from ameba infection. Pharmacologic inhibition of Jmjd3 H3K27 demethylase activity during SAA treatment or blockade of granulocyte-macrophage colony-stimulating factor (GM-CSF) signaling in SFB-colonized mice prevented GMP expansion, decreased gut neutrophils, and blocked protection from ameba infection. These results indicate that alteration of the microbiota and systemic exposure to SAA can influence myelopoiesis and susceptibility to amebiasis via epigenetic mechanisms. Gut microbiota-marrow communication is a previously unrecognized mechanism of innate protection from infection. PMID:27456830

  11. STUDY OF MICROBIOLOGIC FLORA OF THE REPRODUCTIVE TRACT OF THE CHIMPANZEE.

    DTIC Science & Technology

    parasite species were identified in the feces with Entamoeba coli, Balantidium coli and Entamoeba histolytica occurring most frequently. No...plasmodia or microfilariae were observed in blood smears. Entamoeba coli, observed on one occasion, was the only parasite species demonstrated in the vagina

  12. Molecular Epidemiology of Amebiasis

    PubMed Central

    Ali, Ibne Karim M.; Clark, C. Graham; Petri, William A.

    2008-01-01

    Entamoeba histolytica, the causative agent of human amebiasis, remains a significant cause of morbidity and mortality in developing countries and is responsible for up to 100,000 deaths worldwide each year. Entamoeba dispar, morphologically indistinguishable from E. histolytica, is more common in humans in many parts of the world. Similarly Entamoeba moshkovskii, which was long considered to be a free-living ameba, is also morphologically identical to E. histolytica and E. dispar, and is highly prevalent in some E. histolytica endemic countries. However, the only species to cause disease in humans is E. histolytica. Most old epidemiological data on E. histolytica are unusable as the techniques employed do not differentiate between the above three Entamoeba species. Molecular tools are now available not only to diagnose these species accurately but also to study intra-species genetic diversity. Recent studies suggest that only a minority of all E. histolytica infections progress to development of clinical symptoms in the host and there exist population level differences between the E. histolytica strains isolated from the asymptomatic and symptomatic individuals. Nevertheless the underlying factors responsible for variable clinical outcome of infection by E. histolytica remain largely unknown. We anticipate that the recently completed E. histolytica genome sequence and new molecular techniques will rapidly advance our understanding of the epidemiology and pathogenicity of amebiasis. PMID:18571478

  13. Role of PTPase LAR in EGF Receptor in the Mammary Gland

    DTIC Science & Technology

    2003-06-01

    development and tumorigenesis through negative modulation of EGFR signal transduction. In year 2, we demonstrated that LAR expression is regulated by...blocks the cell cycle. The central role of Akt regulation in this pathway is confirmed by recapitulating the effect with an adenovirus-mediated...epithelium. The experimental question to be asked is: Does LAR regulate pregnancy-dependent mammary gland maturation? This is very relevant to the hypothesis

  14. Enteric protozoal diseases.

    PubMed

    Lappin, Michael R

    2005-01-01

    The most common protozoal agents infecting the gastrointestinal tract of cats are Giardia spp, Cryptosporidium spp, Cystoisospora spp, Sarcocystis spp, Besnoitia spp, Hammondia spp, Toxoplasma gondii, Entamoeba histolytica, and Tritrichomonas fetus.

  15. Prevalence of intestinal protozoan infections among Orang Asli schoolchildren in Pos Senderut, Pahang, Malaysia.

    PubMed

    Al-Harazi, Talal; Ghani, Mohamed Kamel Abd; Othman, Hidayatulfathi

    2013-12-01

    The current study determined the prevalence of intestinal protozoan infections among the Orang Asli schoolchildren in Pos Senderut, Pahang, Malaysia. The overall intestinal protozoan infection rate was 85% (261 out of 307). The highest prevalence rates were due to Entamoeba coli (24.4%), Giardia lamblia (21.8%), Blastocystis hominis (21.2%) and Entamoeba histolytica (15.0%). The prevalence of Iodamoeba butschlii was only 2.9%. Among the positive samples, mixed infection with B. hominis and E. histolytica was 3.3%, B. hominis and G. lamblia was 2.9%, G. lamblia and E. histolytica was 2.0% and triple infections (B. hominis, G. lamblia and E. histolytica) was 1.0 %. The prevalence of the infection was high in all age groups (6-14 years old). Thus, we can conclude that intestinal protozoan infections are still representing a serious public health problem in aboriginal communities, especially among children.

  16. Amoebic anal fistula: new insight into an old disease.

    PubMed

    Agrawal, Vivek; Garg, Pankaj Kumar; Jain, Bhupendra Kumar; Mishra, Kiran; Mohanty, Debajyoti

    2014-04-01

    A 67-year-old gentleman underwent fistulectomy for low trans-sphincteric anal fistula along with curettage for an associated abscess extending proximally for half a centimeter into the intersphincteric plane. The roof of the cavity became clearly visible after satisfactory culmination of the surgical procedure. Histopathological examination of the fistulous tract and the curetted granulation tissue revealed presence of multiple trophozoites of Entamoeba histolytica exhibiting erythrophagocytosis in the background of mixed inflammatory infiltrate. This case report provides the outlook that yields the novel insight into the possible role of Entamoeba histolytica in the pathogenesis and persistence of the fistulous tract.

  17. Metronidazole hydrazone conjugates: Design, synthesis, antiamoebic and molecular docking studies.

    PubMed

    Ansari, Mohammad Fawad; Siddiqui, Shadab Miyan; Agarwal, Subhash M; Vikramdeo, Kunwar Somesh; Mondal, Neelima; Azam, Amir

    2015-09-01

    Metronidazole hydrazone conjugates (2-13) were synthesized and screened in vitro for antiamoebic activity against HM1: IMSS strain of Entamoeba histolytica. Six compounds were found to be better inhibitors of E. histolytica than the reference drug metronidazole. These compounds showed greater than 50-60% viability against HeLa cervical cancer cell line after 72 h treatment. Also, molecular docking study was undertaken on E. histolytica thioredoxin reductase (EhTHRase) protein which showed significant binding affinity in the active site. Out of the six actives, some of the compounds showed lipophilic characteristics.

  18. Parasitiosis in handicapped children in an Egyptian blind asylum.

    PubMed

    El-Sherbini, Gehad T; Aboul Noor, Mohamad F; Hegazi, Mamdouh M

    2008-04-01

    Examination of visually handicapped children and their serving staff revealed in a descending order Enterobius vermicularis, Giardia lamblia, Entamoeba histolytica, Hymenolepis nana, Ascaris lumbricoides, Cryptospodium parvum, Tricocephalus trichura and Strongyloides stercoralis. Head lice were encountered. But, neither blood parasites nor protozoan infective agent in water for human consumption.

  19. Bruceantin, a potent amoebicide from a plant, Brucea antidysenterica.

    PubMed Central

    Gillin, F D; Reiner, D S; Suffness, M

    1982-01-01

    Bruceantin, purified from an Ethiopian plant used to treat dysentery, killed Entamoeba histolytica in vitro (IC50 [the concentration of drug which decreased the number of colonies to half that of controls] = 0.018 microgram/ml). Six related quassinoids of 17 tested were also amoebicidal. No relationship between quassinoid structure and amoebicidal activity was apparent. PMID:6100431

  20. Chlorine Disinfection in the Use of Individual Water Purification Devices

    DTIC Science & Technology

    2006-03-01

    Protozoan cysts such as Entamoeba histolytica and Giardia lamblia are highly resistant to chlorine disinfection and may require prolonged contact times at...Environmental Microbiology, 43:250-250. 27. Clark, R. M., Read, E. J., and Hoff, J. C., 1989. Analysis of Inactivation of Giardia lamblia by

  1. Disposal of Industrial and Domestic Wastes: Land and Sea Alternatives.

    DTIC Science & Technology

    1984-01-01

    Bacillus anthracis Reovirua Ascaria lumbricoides Clostridiu perfringens Parvovirus Enterobius vermicularis Yersinia Hepatitis A Strongyloides sp...Texpratures (OC) Microorganisms 50 55 60 65 70 Cyst of Entamoeba 5 histolytica Eggs of Ascaris 60 7 lumbr icoides Brucella abortus 60 3 Corynebacterium

  2. INTESTINAL PARASITES IN PEOPLES OF TAIWAN. A CURSORY SURVEY OF HUA-FAN-SHE VILLAGE (SUN MOON LAKE, NAN-TOU, HSIEN); A NEW LOCALITY FOR ENDEMIC CLONORCHIASIS.

    DTIC Science & Technology

    Tsou aborigines in the central part of the island has shown a sparseness of the protozoans commonly associated with man and the pathogen, Entamoeba ... histolytica , was not detected. Rates of infection for the common helminths of man fell within the rates of expectation and were commensurate with

  3. Amebiasis in the newborn.

    PubMed

    Güven, Ayla

    2003-05-01

    Infestation with Entamoeba histolytica is especially common in areas with low socioeconomic status. Extra intestinal invasive involvement is more frequent in young children with significant mortality. This disease is rarely reported in the newborns. This 19-day-old newborn who was infected with orally given surgar solution is presented. He was successfully treated with omidazole.

  4. In vitro antiprotozoal activity of the leaves of Artemisia ludoviciana.

    PubMed

    Said Fernández, Salvador; Ramos Guerra, Monica Celina; Mata Cárdenas, Benito David; Vargas Villarreal, Javier; Villarreal Treviño, Licet

    2005-07-01

    The inhabitants of Northeast of Mexico use an infusion of leaves from Artemisia ludoviciana as an antidiarrheal remedy. The aqueous, methanol, acetone and hexane leaf extracts from mature plants were found to be active in vitro against the parasitic protozoa Entamoeba histolytica and Giardia lamblia.

  5. Plasmids containing small subunit ribosomal RNA gene fragments from Babesia bovis and Babesia bigemina

    Technology Transfer Automated Retrieval System (TEKTRAN)

    BEI Resources was developed by NIAID as a centralized biological resource center for research reagents to the scientific community (http://www.beiresources.org/). They have a considerable amount of reagents and isolates for parasitologists working with Entamoeba histolytica, Giardia, Toxoplasma, and...

  6. Operation, Maintenance and Performance Evaluation of the Potomac Estuary Experimental Water Treatment Plant. Executive Summary.

    DTIC Science & Technology

    1983-09-01

    parasites were monitored regularly in the parasite group includingGiardia cysts, Entamoeba histolytica, Acanthamoeba Naegleria Gruberi cysts, Ascaris...chemical adsorption of TOC on granular carbon using the appropriate differential equations describing the kinetics and mass transfer processes in a carbon

  7. Operation, Maintenance and Performance Evaluation of the Potomac Estuary Experimental Water Treatment Plant. Appendix. Volume 2.

    DTIC Science & Technology

    1983-09-01

    histolytica N.D. Acanthasooba N.D. Naogloria erubari N.D. Ascaris ND. Hookworm .D Trichuris trichura N.D. Potomac River Estuary Sgnles AosaYedt 12...Parasite Name Number Observed Glardia N.D. Entamoeba histolytica Acanthamoeba N.D. Na~eleria oruberi N.D. Ascaris N.D. Hookworm N.D. Trichuris...gruberi N.D. Ascaris N.D. Hookworm N.D. Trichuris trichiura N.Do. -- ----------- ----- ---- ------ ------------ F-0-26 TABLE F-9 CHARACTERIZATION OF

  8. Amoeboid movement in protozoan pathogens.

    PubMed

    Dufour, Alexandre C; Olivo-Marin, Jean-Christophe; Guillen, Nancy

    2015-10-01

    Entamoeba histolytica, the causative agent of amoebiasis, is a protozoan parasite characterised by its amoeboid motility, which is essential to its survival and invasion of the human host. Elucidating the molecular mechanisms leading to invasion of human tissues by E. histolytica requires a quantitative understanding of how its cytoskeleton deforms and tailors its mode of migration to the local microenvironment. Here we review the wide range of methods available to extract biophysical information from amoeboid cells, from interventional techniques to computational modelling approaches, and discuss how recent developments in bioimaging and bioimage informatics can complement our understanding of cellular morphodynamics at the intracellular level.

  9. Synthesis, antiamoebic and molecular docking studies of furan-thiazolidinone hybrids.

    PubMed

    Ansari, Mohammad Fawad; Siddiqui, Shadab Miyan; Ahmad, Kamal; Avecilla, Fernando; Dharavath, Sudhaker; Gourinath, Samudrala; Azam, Amir

    2016-11-29

    In continuation of our previous work, a series of furan-thiazolidinone hybrids was prepared by Knoevenagel condensation of 3-(furan-2-ylmethyl)-2-(phenylimino)-1, 3-thiazolidin-4-one with different aryl aldehydes in presence of strong base. Some members of the series exhibited remarkable antiamoebic activity and cell viability. Three compounds (3, 6 and 11) showed excellent binding energy for Entamoeba histolytica O-acetyle-l-serine sulfohydrolase and Entamoeba histolytica thioredoxin reductase. These compounds demonstrated significant inhibition of O-acetyle-l-serine sulfohydrolase. The promising antiamoebic activity and enzymatic assay of 3, 6 and 11 make them promising molecules for further lead optimization in the development of novel antiamoebic agents.

  10. Cellular immune responses to amoebic liver abcess in the guinea-pig.

    PubMed Central

    Bray, R S; Harris, W G

    1977-01-01

    Guinea-pigs infected in the liver with the Biswas strain of Entamoeba histolytica showed no dermal hypersensitivity but showed positive lymphocyte transformation and macrophage-migration inhibition. The time sequence showed an activated response at 4 days after infection, a full response at 8 days when the liver abscesses were resolving and a waning response at 12 days when the abscesses had healed. PMID:891028

  11. [Medical practice in Indians of the Colombian Amazon. Epidemiology of intestinal parasitoses].

    PubMed

    Penot, C; Picot, H; Grillot, M L

    1978-01-01

    In spite of the presence of a physician, the real pathology of an indian population of Colombie is not very well known. Some of ill people are probably entrusted to the traditional "chaman". The western medicine is symptomatic, curative and not preventive. Intestinal nematodiasis and amoebiasis are the major problem. Prevalences are: ascaris 46%, trichuris 30%, ankylostoma 39%, strongyloides 40%, Entamoeba histolytica 30%. Among this slowly evoluting population parasitism is related with cultural factors.

  12. ent-Kaurene Glycosides from Ageratina cylindrica.

    PubMed

    Bustos-Brito, Celia; Sánchez-Castellanos, Mariano; Esquivel, Baldomero; Calderón, José S; Calzada, Fernando; Yépez-Mulia, Lilian; Joseph-Nathan, Pedro; Cuevas, Gabriel; Quijano, Leovigildo

    2015-11-25

    The aqueous extract of the leaves of Ageratina cylindrica afforded six new ent-kaurenoic acid glycosides together with the known diterpenoid paniculoside V, the flavonoid astragalin, chlorogenic acid, and L-chiro-inositol. The structures were elucidated mainly by NMR and MS methods, and the absolute configuration was established by vibrational circular dichroism spectroscopy. The new compounds showed moderate antiprotozoal activity against Entamoeba histolytica and Giardia lamblia trophozoites.

  13. Synthesis and Characterization with Antineoplastic, Biochemical, Cytotoxic, and Antimicrobial Studies of Schiff Base Cu(II) Ion Complexes

    PubMed Central

    Haque, M. M.; Kudrat-E-Zahan, Md.; Banu, Laila Arjuman; Islam, Md. Shariful; Islam, M. S.

    2015-01-01

    Copper(II) complexes containing two Schiff base ligands derived from 2-hydroxybenzaldehyde with 2-aminophenol and 3-aminophenol have been synthesized and characterized by means of analytical, magnetic, and spectroscopic methods. Bacteria, fungus, Entamoeba histolytica, and antineoplastic activities of the synthesized complexes have been determined by monitoring the parameters cell growth inhibition, survival time of tumour mice, time-body relation, causing of intraperitoneal cells and macrophages, alkaline phosphatase activity, hematological effect, and biopsy of tumour. PMID:26294901

  14. Analysis of the Bacterial Diversity in Liver Abscess: Differences between Pyogenic and Amebic Abscesses

    PubMed Central

    Reyna-Fabián, Miriam E.; Zermeño, Valeria; Ximénez, Cecilia; Flores, Janin; Romero, Miguel F.; Diaz, Daniel; Argueta, Jesús; Moran, Patricia; Valadez, Alicia; Cerritos, René

    2016-01-01

    Several recent studies have demonstrated that virulence in Entamoeba histolytica is triggered in the presence of both pathogenic and nonpathogenic bacteria species using in vitro and in vivo experimental animal models. In this study, we examined samples aspirated from abscess material obtained from patients who were clinically diagnosed with amebic liver abscess (ALA) or pyogenic liver abscess (PLA). To determine the diversity of bacterial species in the abscesses, we performed partial 16S rRNA gene sequencing. In addition, the E. histolytica and Entamoeba dispar species were genotyped using tRNA-linked short tandem repeats as specific molecular markers. The association between clinical data and bacterial and parasite genotypes were examined through a correspondence analysis. The results showed the presence of numerous bacterial groups. These taxonomic groups constitute common members of the gut microbiota, although all of the detected bacterial species have a close phylogenetic relationship with bacterial pathogens. Furthermore, some patients clinically diagnosed with PLA and ALA were coinfected with E. dispar or E. histolytica, which suggests that the virulence of these parasites increased in the presence of bacteria. However, no specific bacterial groups were associated with this effect. Together, our results suggest a nonspecific mechanism of virulence modulation by bacteria in Entamoeba. PMID:26572872

  15. Analysis of the Bacterial Diversity in Liver Abscess: Differences Between Pyogenic and Amebic Abscesses.

    PubMed

    Reyna-Fabián, Miriam E; Zermeño, Valeria; Ximénez, Cecilia; Flores, Janin; Romero, Miguel F; Diaz, Daniel; Argueta, Jesús; Moran, Patricia; Valadez, Alicia; Cerritos, René

    2016-01-01

    Several recent studies have demonstrated that virulence in Entamoeba histolytica is triggered in the presence of both pathogenic and nonpathogenic bacteria species using in vitro and in vivo experimental animal models. In this study, we examined samples aspirated from abscess material obtained from patients who were clinically diagnosed with amebic liver abscess (ALA) or pyogenic liver abscess (PLA). To determine the diversity of bacterial species in the abscesses, we performed partial 16S rRNA gene sequencing. In addition, the E. histolytica and Entamoeba dispar species were genotyped using tRNA-linked short tandem repeats as specific molecular markers. The association between clinical data and bacterial and parasite genotypes were examined through a correspondence analysis. The results showed the presence of numerous bacterial groups. These taxonomic groups constitute common members of the gut microbiota, although all of the detected bacterial species have a close phylogenetic relationship with bacterial pathogens. Furthermore, some patients clinically diagnosed with PLA and ALA were coinfected with E. dispar or E. histolytica, which suggests that the virulence of these parasites increased in the presence of bacteria. However, no specific bacterial groups were associated with this effect. Together, our results suggest a nonspecific mechanism of virulence modulation by bacteria in Entamoeba.

  16. Role of Eosinophils and Tumor Necrosis Factor Alpha in Interleukin-25-Mediated Protection from Amebic Colitis

    PubMed Central

    Noor, Zannatun; Watanabe, Koji; Abhyankar, Mayuresh M.; Burgess, Stacey L.; Buonomo, Erica L.

    2017-01-01

    ABSTRACT The parasite Entamoeba histolytica is a cause of diarrhea in infants in low-income countries. Previously, it was shown that tumor necrosis factor alpha (TNF-α) production was associated with increased risk of E. histolytica diarrhea in children. Interleukin-25 (IL-25) is a cytokine that is produced by intestinal epithelial cells that has a role in maintenance of gut barrier function and inhibition of TNF-α production. IL-25 expression was decreased in humans and in the mouse model of amebic colitis. Repletion of IL-25 blocked E. histolytica infection and barrier disruption in mice, increased gut eosinophils, and suppressed colonic TNF-α. Depletion of eosinophils with anti-Siglec-F antibody prevented IL-25-mediated protection. In contrast, depletion of TNF-α resulted in resistance to amebic infection. We concluded that IL-25 provides protection from amebiasis, which is dependent upon intestinal eosinophils and suppression of TNF-α. PMID:28246365

  17. Outbreak of amebiasis in Tbilisi, Republic of Georgia, 1998.

    PubMed

    Barwick, Rachel S; Uzicanin, Amra; Lareau, Susan; Malakmadze, Naile; Imnadze, Paata; Iosava, Merab; Ninashvili, Nana; Wilson, Marianna; Hightower, Allen W; Johnston, Stephanie; Bishop, Henry; Petri, William A; Juranek, Dennis D

    2002-12-01

    In 1998, we investigated a suspected outbreak of amebic liver abscesses caused by Entamoeba histolytica in the Republic of Georgia, using a case-control study. A questionnaire was administered and blood samples were obtained from cases and controls for serologic diagnosis. Medical records showed that E. histolytica infections were rarely diagnosed before 1998. However, from July through September 1998, 177 cases of suspected amebiasis were identified. Of 52 persons who had diagnosed liver abscesses, 37 (71%) were confirmed serologically to have antibodies against E. histolytica, compared with 11 of 53 persons (20.8%) diagnosed with intestinal amebiasis. In addition, 9-14% of asymptomatic controls were seropositive. Logistic regression identified the fact that interruptions in the water supply, decreases in water pressure, and increased water consumption were significantly associated with infection. The data support the hypothesis that drinking water was the source of infection, either because of inadequate municipal water treatment or contamination of municipal water in the distribution system.

  18. Antiprotozoal Constituents from Annona cherimola Miller, a Plant Used in Mexican Traditional Medicine for the Treatment of Diarrhea and Dysentery

    PubMed Central

    Calzada, Fernando; Correa-Basurto, Jose; Barbosa, Elizabeth; Mendez-Luna, David; Yepez-Mulia, Lilian

    2017-01-01

    Background: Annona cherimola Miller (Annonaceae) is a medicinal plant frequently recommended in Mexican traditional medicine for the treatment of gastrointestinal disorders such as diarrhea and dysentery. Objective: This work was undertaken to obtain information that support the traditional use of A. cherimola, on pharmacological basis using in vitro and computational experiments. Material and Methods: Bioassay-guided fractionation of the ethanol extract of the leaves of A. cherimola afforded five phenolic compounds: caffeic acid, quercetin, kaempferol, nicotinflorin, and rutin. Results: The in vitro antiprotozoal assay showed that kaempferol was the most potent antiamoebic and antigiardial compound with IC50 values of 7.9 μg/mL for Entamoeba histolytica and 8.7 μg/mL for Giardia lamblia. Computational molecular docking study showed that kaempferol interacted in a region different than metronidazole in the enzyme pyruvate: ferredoxin oxidoreductase (PFOR). Conclusion: Considering that PFOR is a target of metronidazole; kaempferol may be a lead compound for the development of novel antiprotozoal agent. Also, these findings give support to the use of A. cherimola in the traditional medicine from México for the treatment of diarrhea and dysentery. SUMMARY Bioassay-guided fractionation of the ethanol extract of the leaves of Annona cherimola afforded five phenolic compounds: caffeic acid, quercetin, kaempferol, nicotinflorin and rutin. The in vitro antiprotozoal assay showed that kaempferol was the most potent antiamoebic and antigiardial compound with IC50 values of 7.9 μg/mL for Entamoeba histolytica and 8.7 μg/mL for Giardia lamblia. Computational molecular docking study showed that kaempferol interacted in a region different that metronidazole in the enzyme pyruvate: ferredoxin oxidoreductase. Abbreviations used: PFOR:Pyruvate:ferredoxin oxidoreductase, G: lamblia: Giardia lamblia, E: histolytica: Entamoeba histolytica PMID:28216899

  19. Modulation of innate immune response by the vagus nerve in experimental hepatic amebiasis in rats.

    PubMed

    Martínez-Jaimes, Mercedes D; García-Lorenzana, Mario; Muñoz-Ortega, Martin H; Quintanar-Stephano, Andrés; Ávila-Blanco, Manuel E; García-Agueda, Carlos E; Ventura-Juárez, Javier

    2016-10-01

    The parasympathetic nervous system has a crucial role in immunomodulation of the vagus nerve, its structure provides a pathogen detection system, and a negative feedback to the immune system after the pathogenic agent has been eliminated. Amebiasis is a disease caused by the protozoan parasite Entamoeba histolytica, considered the third leading cause of death in the world. The rats are used as a natural resistance model to amoebic liver infection. The aim of this study is to analyze the interaction of Entamoeba histolytica with neutrophils, macrophages, and NK cells in livers of intact and vagotomized rats. Six groups were studied (n = 4): Intact (I), Intact + amoeba (IA), Sham (S), Sham + amoeba (SA), Vagotomized (V) and Vagotomized + amoeba (VA). Animals were sacrificed at 8 h post-inoculation of E. histolytica. Then, livers were obtained and fixed in 4% paraformaldehyde. Tissue liver slides were stained with H-E, PAS and Masson. The best development time for E. histolytica infection was at 8 h. Amoeba was identified with a monoclonal anti-220 kDa E. histolytica lectin. Neutrophils (N) were identified with rabbit anti-human neutrophil myeloperoxidase, macrophages (Mɸ) with anti-CD68 antibody and NK cells (NK) with anti-NK. Stomachs weight and liver glycogen were higher in V. Collagen increased in VA, whereas vascular and neutrophilic areas were decreased. There were fewer N, Mɸ, NK around the amoeba in the following order IA > SA > VA (p < 0.05 between IA and VA). In conclusion, these results suggest that the absence of parasympathetic innervation affects the participation of neutrophils, macrophages and NK cells in the innate immune response, apparently by parasympathetic inhibition on the cellular functions and probably for participation in sympathetic activity.

  20. Novelties on amoebiasis: a neglected tropical disease.

    PubMed

    Ximénez, Cecilia; Morán, Patricia; Rojas, Liliana; Valadez, Alicia; Gómez, Alejandro; Ramiro, Manuel; Cerritos, René; González, Enrique; Hernández, Eric; Oswaldo, Partida

    2011-04-01

    In accordance with the 1997 documents of the World Health Organization (WHO), amoebiasis is defined as the infection by the protozoan parasite Entamoeba histolytica with or without clinical manifestations. The only known natural host of E. histolytica is the human with the large intestine as major target organ. This parasite has a very simple life cycle in which the infective form is the cyst, considered a resistant form of parasite: The asymptomatic cyst passers and the intestinal amoebiasis patients are the transmitters; they excrete cysts in their feces, which can contaminate food and water sources. E. histolytica sensu stricto is the potentially pathogenic species and E. dispar is a commensal non-pathogenic Entamoeba. Both species are biochemical, immunological and genetically distinct. The knowledge of both species with different pathogenic phenotypes comes from a large scientific debate during the second half of the 20(th) century, which gave place to the rapid development of diagnostics technology based on molecular and immunological strategies. During the last ten years, knowledge of the new epidemiology of amoebiasis in different geographic endemic and non-endemic areas has been obtained by applying mostly molecular techniques. In the present work we highlight novelties on human infection and the disease that can help the general physician from both endemic and non-endemic countries in their medical practice, particularly, now that emigration is undoubtedly a global phenomenon that is modifying the previous geography of infectious diseases worldwide.

  1. Two or more enteropathogens are associated with diarrhoea in Mexican children

    PubMed Central

    Paniagua, Gloria Luz; Monroy, Eric; García-González, Octavio; Alonso, Javier; Negrete, Erasmo; Vaca, Sergio

    2007-01-01

    Background Diarrhoeal diseases constitute a major public health problem, particularly in the developing world, where the rate of mortality and morbidity is very high. The purpose of this study was to conduct a 2 years and 3 months study in order to determine the prevalence of five enteropathogen diarrheogenic agents in Mexico City. Methods Faecal samples were obtained from 300 Mexican children diagnosed as positive for diarrhoea, aged > 2 to < 12 years old, and from 80 children matched for age but with no symptoms of the disease (control group). Two multiplex PCR were used to detect Escherichia coli, Salmonella spp., and Shigella spp. In addition, the two protozoan parasites Entamoeba histolytica/Entamoeba dispar and Giardia intestinalis were detected by conventional methods. Results All diarrhoeal samples were positive for one or more enteropathogens. The most common enteropathogens in diarrhoeal samples were E. histolytica/E. dispar (70.3%), Salmonella (ohio 28.3%; typhimurium 16.3%; infantis 8%; anatum 0.6%; Newport 0.3%), G. intestinalis (33%), E. coli (ETEC 13.3%; EPEC 9.3%; VTEC 8.6%; EIEC 1%) and Shigella spp. (flexneri 1.6%, sonnei 1%). Infections by two (24%) three (16%) and four (12%) pathogens were observed. Conclusion This study revealed that 52% of the patients were infected by more than one enteropathogen, notably E. histolitica/E. dispar and Salmonella ohio. These results are useful for clinicians to improve the empiric treatment used in such cases. PMID:18162140

  2. Novelties on Amoebiasis: A Neglected Tropical Disease

    PubMed Central

    Ximénez, Cecilia; Morán, Patricia; Rojas, Liliana; Valadez, Alicia; Gómez, Alejandro; Ramiro, Manuel; Cerritos, René; González, Enrique; Hernández, Eric; Oswaldo, Partida

    2011-01-01

    In accordance with the 1997 documents of the World Health Organization (WHO), amoebiasis is defined as the infection by the protozoan parasite Entamoeba histolytica with or without clinical manifestations. The only known natural host of E. histolytica is the human with the large intestine as major target organ. This parasite has a very simple life cycle in which the infective form is the cyst, considered a resistant form of parasite: The asymptomatic cyst passers and the intestinal amoebiasis patients are the transmitters; they excrete cysts in their feces, which can contaminate food and water sources. E. histolytica sensu stricto is the potentially pathogenic species and E. dispar is a commensal non-pathogenic Entamoeba. Both species are biochemical, immunological and genetically distinct. The knowledge of both species with different pathogenic phenotypes comes from a large scientific debate during the second half of the 20th century, which gave place to the rapid development of diagnostics technology based on molecular and immunological strategies. During the last ten years, knowledge of the new epidemiology of amoebiasis in different geographic endemic and non-endemic areas has been obtained by applying mostly molecular techniques. In the present work we highlight novelties on human infection and the disease that can help the general physician from both endemic and non-endemic countries in their medical practice, particularly, now that emigration is undoubtedly a global phenomenon that is modifying the previous geography of infectious diseases worldwide. PMID:21731305

  3. Invasive amebiasis and ameboma formation presenting as a rectal mass: An uncommon case of malignant masquerade at a western medical center

    PubMed Central

    Hardin, Rosemarie E; Ferzli, George S; Zenilman, Michael E; Gadangi, Pratap K; Bowne, Wilbur B

    2007-01-01

    A 54-year-old man presented with rectal pain and bleeding secondary to ulcerated, necrotic rectal and cecal masses that resembled colorectal carcinoma upon colonoscopy. These masses were later determined to be benign amebomas caused by invasive Entamoeba histolytica, which regressed completely with medical therapy. In Western countries, the occurrence of invasive protozoan infection with formation of amebomas is very rare and can mistakenly masquerade as a neoplasm. Not surprisingly, there have been very few cases reported of this clinical entity within the United States. Moreover, we report a patient that had an extremely rare occurrence of two synchronous lesions, one involving the rectum and the other situated in the cecum. We review the current literature on the pathogenesis of invasive E. histolytica infection and ameboma formation, as well as management of this rare disease entity at a western medical center. PMID:17948943

  4. Undiagnosed amebic brain abscess.

    PubMed

    Viriyavejakul, Parnpen; Riganti, Mario

    2009-11-01

    We report a case of amebic brain abscess due to Entamoeba histolytica. The patient was a 31-year-old man who presented with amebic liver abscess. His clinical course deteriorated in spite of proper drainage and treatment. He developed delirium, lethargy and then expired. With a history of heroin addiction, withdrawal syndrome from heroin was suspected. At autopsy, amebic abscesses were detected in the liver, large intestine, meninges and brain. A 19 cm amebic liver abscess was found in the right lobe of the liver. A 4 cm amebic brain abscess was found in the right occipital lobe. Microscopically, the tissue sections from the affected organs were confirmed to have degenerated E. histolytica trophozoites. Involvement of the brain in amebic liver abscess should be suspected in patients with neurological signs and symptoms.

  5. Hymenolepis nana: a common cause of paediatric diarrhoea in urban slum dwellers in India.

    PubMed

    Mirdha, Bijay Ranjan; Samantray, Jyotish Chandra

    2002-12-01

    The prevalence of intestinal parasitic infections was studied for a period of 5 years (April 1996-April 2001) among urban slum dwellers. All age groups were represented in the study. Parasitological examinations were performed on 939 faecal specimens collected on a household basis. The total prevalence of pathogenic parasites was 33.6 per cent. No significant age and sex differences in pathogenic parasites were observed. The prevalence of intestinal helminths and pathogenic protozoa was as follows: Hymenolepis nana (9.9 per cent), Ascaris lumbricoides (8.5 per cent), Giardia lamblia (8.4 per cent) and Entamoeba histolyticaldispar (3.7 per cent). Thirty-four E. histolytica/dispar positive samples were cultured and speciation was done using polymerase chain reaction (PCR). The predominant isolate was E. dispar compared to E. histolytica. The notable finding of the present study was high prevalence of Hymenolepis nana compared with other parasitic infections in slum dwellers.

  6. Intestinal protozoa.

    PubMed

    Juckett, G

    1996-06-01

    Giardia is the best known cause of protozoal gastrointestinal disease in North America, producing significant but not life-threatening gastrointestinal distress and diarrhea. Although diagnosis of giardiasis may be challenging, treatment is usually successful. Entamoeba histolytica poses a rarer but far more difficult clinical challenge. Dysentery caused by E. histolytica may be the most feared intestinal protozoal infection, although Cryptosporidium parvum, Balantidium coli, Isospora belli, Sarcocystis species and other newly described protozoa also may cause diarrhea in healthy individuals and may result in intractable, life-threatening illness in patients with acquired immunodeficiency syndrome or other immunosuppressive diseases. Certain protozoa once considered relatively unimportant, such as Cryptosporidium, are now recognized as significant causes of morbidity even in the United States, since transmission readily occurs through contaminated water.

  7. Prevalence and seasonal variation of human intestinal parasites in patients attending hospital with abdominal symptoms in northern Jordan.

    PubMed

    Jaran, A S

    2017-01-23

    This study was carried out to determine the prevalence of intestinal parasites and their seasonal variation in northern Jordan. A total of 21 906 stool samples were collected over a period of 4 years (2009- 2013) from 5 government hospitals in 3 cities. Samples were processed and examined microscopically and by concentration methods. Parasitic infection was found in 9611 samples (44%). Giardia lamblia was the most prevalent parasite (41%) followed by Entamoeba histolytica (31%) and Ent. coli (13%); the least prevalent parasites were Ascaris lumbricoides (1%), Hymenolepis nana, Taenia sp., and Chilomastix mesnili (all < 1%). The prevalence of different parasites varied according to season, on average the summer months showed the highest incidence of parasitic infection (62%) compared with the winter months (16%). Giardia lamblia and Ent. histolytica were most prevalent in the summer months.

  8. Invasive amoebiasis is associated with the development of anti-neutrophil cytoplasmic antibody.

    PubMed Central

    Pudifin, D J; Duursma, J; Gathiram, V; Jackson, T F

    1994-01-01

    Features of tissue damage in invasive amoebiasis, in particular polymorphonuclear neutrophil (PMN) degranulation and vasculitis, bear resemblance to that seen in Wegener's granulomatosis, the latter being associated with the presence of anti-neutrophil cytoplasmic antibodies (ANCA). We therefore tested sera from patients with confirmed amoebic liver abscess (ALA) for the presence of ANCA by means of an indirect fluorescent antibody test using pure neutrophils as substrate. ANCA was detected in 97.4% of amoebic sera; the pattern of staining was cytoplasmic, homogeneous, without central accentuation (C-ANCA). A proteinase 3 (PR3) ELISA demonstrated PR3 specificity in 75% of C-ANCA-positive ALA sera. Possible explanations are (i) a cross-reacting antibody to a component of Entamoeba histolytica, or (ii) an antibody to PMN components released, and possibly modified, by the action of E. histolytica on PMN. It is possible that this antibody contributes to the pathogenesis of invasive amoebiasis. Images Fig. 1 PMID:8033420

  9. Invasive amoebiasis is associated with the development of anti-neutrophil cytoplasmic antibody.

    PubMed

    Pudifin, D J; Duursma, J; Gathiram, V; Jackson, T F

    1994-07-01

    Features of tissue damage in invasive amoebiasis, in particular polymorphonuclear neutrophil (PMN) degranulation and vasculitis, bear resemblance to that seen in Wegener's granulomatosis, the latter being associated with the presence of anti-neutrophil cytoplasmic antibodies (ANCA). We therefore tested sera from patients with confirmed amoebic liver abscess (ALA) for the presence of ANCA by means of an indirect fluorescent antibody test using pure neutrophils as substrate. ANCA was detected in 97.4% of amoebic sera; the pattern of staining was cytoplasmic, homogeneous, without central accentuation (C-ANCA). A proteinase 3 (PR3) ELISA demonstrated PR3 specificity in 75% of C-ANCA-positive ALA sera. Possible explanations are (i) a cross-reacting antibody to a component of Entamoeba histolytica, or (ii) an antibody to PMN components released, and possibly modified, by the action of E. histolytica on PMN. It is possible that this antibody contributes to the pathogenesis of invasive amoebiasis.

  10. Structure and biochemical characterization of proliferating cellular nuclear antigen from a parasitic protozoon

    SciTech Connect

    Cardona-Felix, Cesar S.; Lara-Gonzalez, Samuel; Brieba, Luis G.

    2012-02-08

    Proliferating cellular nuclear antigen (PCNA) is a toroidal-shaped protein that is involved in cell-cycle control, DNA replication and DNA repair. Parasitic protozoa are early-diverged eukaryotes that are responsible for neglected diseases. In this work, a PCNA from a parasitic protozoon was identified, cloned and biochemically characterized and its crystal structure was determined. Structural and biochemical studies demonstrate that PCNA from Entamoeba histolytica assembles as a homotrimer that is able to interact with and stimulate the activity of a PCNA-interacting peptide-motif protein from E. histolytica, EhDNAligI. The data indicate a conservation of the biochemical mechanisms of PCNA-mediated interactions between metazoa, yeast and parasitic protozoa.

  11. Amebiasis.

    PubMed Central

    Bruckner, D A

    1992-01-01

    Entamoeba histolytica, the causative agent of amebiasis, was first described in 1875. Although a large number of people throughout the world are infected with this organism, only a small percentage will develop clinical symptoms. Morbidity and mortality due to E. histolytica vary from area to area and person to person. Recent findings have suggested that there are pathogenic and nonpathogenic strains of E. histolytica that can be differentiated by isoenzyme (zymodeme) analysis, monoclonal antibodies, and DNA probes. Whether pathogenicity is a genotypic trait or can be changed by environmental influences has not been resolved. Exchange of genetic material between strains of amebae can influence zymodeme patterns. Currently, detection of E. histolytica infections depends on examinations for ova and parasites and on serologic tests; however, the development of monoclonal antibodies and DNA probes specific for pathogenic zymodemes may be beneficial for clinical laboratory testing and therapeutic decisions when approved tests become available. A better understanding of the mechanisms of pathogenicity at the molecular level is evolving and should promote the development of vaccines and better target selection for therapeutic agents. PMID:1423215

  12. Development of a multiplex PCR assay to detect gastroenteric pathogens in the feces of Mexican children.

    PubMed

    Tolentino-Ruiz, R; Montoya-Varela, D; García-Espitia, M; Salas-Benito, M; Gutiérrez-Escolano, A; Gómez-García, C; Figueroa-Arredondo, P; Salas-Benito, J; De Nova-Ocampo, M

    2012-10-01

    Acute gastroenteritis (AGE) is a major cause of childhood morbidity and mortality worldwide; the etiology of AGE includes viruses, bacteria, and parasites. A multiplex PCR assay to simultaneously identify human Astrovirus (HAstV), Calicivirus (HuCVs), Entamoeba histolytica (E. histolytica), and enteroinvasive Escherichia coli (EIEC) in stool samples is described. A total of 103 samples were individually analyzed by ELISA (enzyme-linked immunosorbent assays) and RT-PCR/PCR. HAstV and HuCVs were detected in four out of 103 samples (3.8 %) by RT-PCR, but ELISAs found only one sample as positive for HuCVs (2.5 %). E. histolytica was identified in two out of 19 samples (10.5 %) and EIEC in 13 out of 20 samples (70 %) by PCR, and all PCR products were sequenced to verify their identities. Our multiplex PCR results demonstrate the simultaneous amplification of different pathogens such as HAstV, EIEC, and E. histolytica in the same reaction, though the HuCVs signal was weak in every replicate. Regardless, this multiplex PCR protocol represents a novel tool for the identification of distinct pathogens and may provide support for the diagnosis of AGE in children.

  13. Intestinal parasites in school aged children and the first case report on amoebiasis in urinary bladder in Tripoli, Libya.

    PubMed

    Ben Musa, Najla A

    2007-12-01

    A total of 50 fresh fecal samples were collected from children of either sex and examined by direct wet films and Telemann's concentration technique. The results were based on a single stool per person study of the total children ranging from 5-18 years of age, 8 different types of intestinal parasites were recorded. The incidence of infection with one or the other parasites was found to be 42%. Double or mixed infection was not uncommon. The infection rates of helminthes were Hymenolepis nana 6%, Taenia saginata 2%, Ascaris lumbricoids 20%, Trichuris trichura 14% and Enterobius vermicularis 4%. The rates of protozoa were Entamoeba histolytica 4%, Entamoeba coli 4% and Giardia lamblia 2%. The problem was created by parasitosis in endangering children's health are more increasing in Tripoli District.

  14. INTESTINAL PARASITES DETECTED DURING PRE-EMPLOYMENT STOOL EXAMINATION AT TERTIARY HEALTH CARE CENTER IN MAKKAH, KINGDOM OF SAUDI ARABIA.

    PubMed

    Ahmed, Mona A; Alam-Eldin, Yosra H; Eltaweel, Nagwa A; Elmorsy, Soha

    2015-08-01

    This retrospective study determined the intestinal parasites discovered in 2490 new employees in a tertiary health care center in Makah as regards nationalities, age groups and sex over the period from January 2010 to January 2014 to decrease introduction of communicable diseases. All the data were provided through the electronic hospital information system. The overall positive cases were 16%, significantly higher in females 57.4% than males 42.6%. Differences in distribution of positive records among different geographical areas were highly significant being highest in East Asia 59.8 %. Blastocystis hominis was the most common parasite. It occurred in 78,9% of positive cases followed by Entamoeba coli 9.3%, Giardia lamblia 5.8% and Entamoeba histolytica/dispar 5.3%. These findings should motivate any other country importing expatriate employees to have effective screening programs for intestinal parasites.

  15. Review: amebic liver abscess in children - the role of diagnostic imaging

    SciTech Connect

    Merten, D.F.; Kirks, D.R.

    1984-12-01

    Amebiasis, infection with the protozoan Entamoeba histolytica, affects at least 10% of the world's population, with an incidence exceeding 30% in tropical and subtropical regions. Amebic liver abscess (ALA) is the most common extraintestinal form of invasive amebiasis and is a serious life-threatening disease in children. Recent experience indicates the prognosis of ALA in childhood to be improved with early identification of abscesses and prompt institution of treatment. The authors incorporate experience with a case of ALA in infancy with a review of current literature (1974-1983) to delineate clinical and radiologic features of ALA in childhood and further define the role of hepatic imaging in the diagnosis and treatment.

  16. Human intestinal parasites in primary school children in Kampala, Uganda.

    PubMed

    Kabatereine, N B; Kemijumbi, J; Kazibwe, F; Onapa, A W

    1997-05-01

    A cross sectional survey on intestinal parasite infections was carried out in 5,313 pupils between the ages of ten and fifteen years in 98 primary schools in Kampala. The aim was to identify the types and distribution of intestinal parasites and to estimate the prevalence in school children. Trichuris trichiura (28%), Ascaris lumbricoides (17%) and hookworms (12.9%) were common infections among the children. Other less commonly found parasites were S.mansoni, Strongyloides stercolaris, Taenia sp, Enterobius vermicularis, Giardia lamblia, Entamoeba coli and E. histolytica. Refuse dumps are probably a significant source of transmission of intestinal helminthic infections in Kampala.

  17. Antiprotozoal activity of the constituents of Teloxys graveolens.

    PubMed

    Calzada, Fernando; Velázquez, Claudia; Cedillo-Rivera, Roberto; Esquivel, Baldomero

    2003-08-01

    Antiprotozoal activity-guided fractionation of a methanol extract of the aerial parts of Teloxys graveolens led to the isolation of one coumarinic acid derivative, melilotoside, and five flavonoids, pinocembrine, pinostrobin, chrysin, narcissin and rutin. Among them, melilotoside exhibited the most potent activity toward Entamoeba histolytica and Giardia lamblia (IC(50) 12.5 and 16.8 micro g/mL, respectively). Narcissin showed selectivity against E. histolytlica (IC(50) 17.2 micro g/mL). The results support data for the traditional use of T. graveolens in some gastrointestinal diseases.

  18. Surveillance for parasites in unaccompanied minor refugees migrating to Germany in 2015.

    PubMed

    Heudorf, Ursel; Karathana, Maria; Krackhardt, Bernhard; Huber, Meike; Raupp, Peter; Zinn, Christian

    2016-01-01

    In 2015, most of the refugees arriving in Germany originated from countries with poor hygienic and sanitary conditions. Stool samples of 1,230 minor refugees unaccompanied by adults were investigated for possible parasites. Giardia lamblia was by far the most frequently detected parasite (n=165); all other parasites were considerably less frequent and encountered in the following order: Hymenolepis nana (n=23), Entamoeba histolytica (n=17), Trichuris trichiura (n=8), and Blastocystis hominis (n=1). Ascaris lumbricoides was not detected among any of the screened refugees. Considerable differences in prevalence rates in refugees originating from different countries could be observed.

  19. Amebic infections in asymptomatic homosexual men, lack of evidence of invasive disease.

    PubMed Central

    Sorvillo, F J; Strassburg, M A; Seidel, J; Visvesvara, G S; Mori, K; Todd, A; Portigal, L; Finn, M; Agee, B A

    1986-01-01

    A survey for enteric infections in 140 asymptomatic homosexual men who attended a community clinic revealed a high prevalence of infection with Entamoeba histolytica (27.1 per cent) and Giardia lamblia (15.7 per cent). In contrast, the prevalence of elevated indirect hemagglutination (IHA) titers (greater than or equal to 1:128), which indicate invasive amebiasis, was low (5.7 per cent). Our findings suggest that only a limited amount of invasive amebic disease is occurring in this group of homosexual men. PMID:2874747

  20. Three-year prospective study of intestinal pathogens in Madrid, Spain.

    PubMed Central

    Velasco, A C; Mateos, M L; Más, G; Pedraza, A; Díez, M; Gutiérrez, A

    1984-01-01

    During the period July 1980 through June 1983, in a General Hospital in Madrid, the following organisms were detected from 6,970 patients with gastroenteritis: 710 Salmonella spp. 506 Campylobacter jejuni, 379 Shigella spp., 12 Yersinia enterocolitica, 1,466 rotavirus, 134 Giardia lamblia, and 4 Entamoeba histolytica. Chloramphenicol showed good activity against most tested strains of Salmonella spp., Shigella spp., and C. jejuni. The incidence of Salmonella spp. and Shigella spp. was very marked in the hot dry months of the year, rotavirus predominated during the cold months, and no seasonal variations of importance were seen for C. jejuni and G. lamblia. PMID:6092418

  1. Cockroaches and flies in mechanical transmission of medical important parasites in Khaldyia Village, El-Fayoum, Governorate, Egypt.

    PubMed

    El-Sherbini, Gehad T; Gneidy, Morsy Rateb

    2012-04-01

    The role of non-blood sucking insects in dissemination of human parasites was investigated in Khaldyia Village, Al-Fayoum Governorate over during the summer of 2011. A total of 278 American cockroach (Periplaneta americana), and 508 house flies Musca domestica var. vicina were collected. The insects were collected indoors and outdoors. Flies were abundant in defecation areas and around houses. The recovered zoonotic parasites identified were cysts of Entamoeba histolytica Cryptosporidium parvum and Balantidium coli, and eggs of Ascaris lumbricoides, Anchylostoma deodunale, Enterobius vermicularis, and Trichuris trichura as well as larvae of Strongyloides stercoralis.

  2. Surveillance for parasites in unaccompanied minor refugees migrating to Germany in 2015

    PubMed Central

    Heudorf, Ursel; Karathana, Maria; Krackhardt, Bernhard; Huber, Meike; Raupp, Peter; Zinn, Christian

    2016-01-01

    In 2015, most of the refugees arriving in Germany originated from countries with poor hygienic and sanitary conditions. Stool samples of 1,230 minor refugees unaccompanied by adults were investigated for possible parasites. Giardia lamblia was by far the most frequently detected parasite (n=165); all other parasites were considerably less frequent and encountered in the following order: Hymenolepis nana (n=23), Entamoeba histolytica (n=17), Trichuris trichiura (n=8), and Blastocystis hominis (n=1). Ascaris lumbricoides was not detected among any of the screened refugees. Considerable differences in prevalence rates in refugees originating from different countries could be observed. PMID:26958459

  3. Underestimated Amoebic Appendicitis among HIV-1-Infected Individuals in Japan

    PubMed Central

    Kobayashi, Taiichiro; Yano, Hideaki; Murata, Yukinori; Igari, Toru; Nakada-Tsukui, Kumiko; Yagita, Kenji; Nozaki, Tomoyoshi; Kaku, Mitsuo; Tsukada, Kunihisa; Gatanaga, Hiroyuki; Kikuchi, Yoshimi; Oka, Shinichi

    2016-01-01

    ABSTRACT Entamoeba histolytica is not a common causative agent of acute appendicitis. However, amoebic appendicitis can sometimes be severe and life threatening, mainly due to a lack of awareness. Also, its frequency, clinical features, and pathogenesis remain unclear. The study subjects were HIV-1-infected individuals who presented with acute appendicitis and later underwent appendectomy at our hospital between 1996 and 2014. Formalin-fixed paraffin-embedded preserved appendix specimens were reexamined by periodic acid-Schiff (PAS) staining and PCR to identify undiagnosed amoebic appendicitis. Appendectomies were performed in 57 patients with acute appendicitis. The seroprevalence of E. histolytica was 33% (14/43) from the available stored sera. Based on the medical records, only 3 cases were clinically diagnosed as amoebic appendicitis, including 2 diagnosed at the time of appendectomy and 1 case diagnosed by rereview of the appendix after the development of postoperative complications. Retrospective analyses using PAS staining and PCR identified 3 and 3 more cases, respectively. Thus, E. histolytica infection was confirmed in 9 cases (15.8%) in the present study. Apart from a significantly higher leukocyte count in E. histolytica-positive patients than in negative patients (median, 13,760 versus 10,385 cells/μl, respectively, P = 0.02), there were no other differences in the clinical features of the PCR-positive and -negative groups. In conclusion, E. histolytica infection was confirmed in 9 (15.8%) of the appendicitis cases. However, only 3, including one diagnosed after intestinal perforation, were diagnosed before the present analyses. These results strongly suggest there is frequently a failure to detect trophozoites in routine examination, resulting in an underestimation of the incidence of amoebic appendicitis. PMID:27847377

  4. Pulmonary Thromboembolism Complicating Amebic Liver Abscess: First Reported Case in the United States—Case Report and Literature Review

    PubMed Central

    McKenzie, Devon; Gale, Michael; Patel, Sunny; Kaluta, Grazyna

    2015-01-01

    Even in the absence of Amebic colitis, Amebic liver abscess (ALA) is the most common extraintestinal complication of Entamoeba histolytica infection. In the USA, it is most prevalent in middle aged immigrant males from endemic countries such as Africa, Mexico, and India. One of the complications of ALA is inferior vena cava (IVC) thrombosis, which is believed to result from the mechanical compression of the IVC and the consequent thrombogenic nidus elicited from the resultant inflammatory response. There are very few reported cases and even fewer in which the thrombus became a harbinger to pulmonary thromboembolism. We present the case of a 43-year-old male from West Africa who presented with the chief complaint of right upper quadrant abdominal pain for one week associated with persistent nonproductive cough. He had a positive serum Entamoeba histolytica antibody with CT scan findings of a hepatic abscess with thrombosis of the hepatic vein and inferior vena cava and numerous bilateral pulmonary emboli. This amebic liver abscess was successfully treated with metronidazole and paromomycin, whereas the pulmonary thromboembolism was managed with medical anticoagulation. Based on current knowledge, this is the first reported case in the USA. PMID:26294987

  5. Distribution and abundance of organic thiols

    NASA Technical Reports Server (NTRS)

    Fahey, R.

    1985-01-01

    The role of glutathione (GSH) in protecting against the toxicity of oxygen and oxygen by products is well established for all eukaryotes studied except Entamoeba histolytica which lacks mitochrondria, chloroplasts, and microtubules. The GSH is not universal among prokaryotes. Entamoeba histolytica does not produce GSH or key enzymes of GSH metabolism. A general method of thiol analysis based upon fluorescent labeling with monobromobimane and HPLC separation of the resulting thiol derivatives was developed to determine the occurrence of GSH and other low molecular weight thiols in bacteria. Glutathione is the major thiol in cyanobacteria and in most bacteria closely related to the purple photosynthetic bacteria, but GSH was not found in archaebacteria, green bacteria, or GRAM positive bacteria. It suggested that glutathione metabolism was incorporated into eukaryotes at the time that mitochondria and chloroplasts were acquired by endosymbiosis. In Gram positive aerobes, coenzyme A occurs at millimolar levels and CoA disulfide reductases are identified. The CoA, rather than glutathione, may function in the oxygen detoxification processes of these organisms.

  6. [Cryptosporidium sp infections and other intestinal parasites in food handlers from Zulia state, Venezuela].

    PubMed

    Freites, Azael; Colmenares, Deisy; Pérez, Marly; García, María; Díaz de Suárez, Odelis

    2009-03-01

    Cryptosporidiosis in food handlers from Venezuela is unknown, being this an important public health problem in immunosuppressed patients. To determine the prevalence of Cryptosporidium sp and other intestinal parasites in food handlers from Zulia State, one hundred nineteen fecal samples were evaluated by wet mount, concentrated according to Ritchie and modified Ziehl-Neelsen staining. Fourteen (11.8%) were positive for Cryptosporidium sp and associated with other protozoosis (P < 0.05), being most frequent Endolimax nana (42.9%). The general prevalence of the intestinal parasitism was 48.7%, emphasizing E. nana (41.2%), followed by Blastocystis hominis (38.7%) and Entamoeba coli (17.6%). The most frequent pathogenic protozoa was Giardia lamblia (13.4%), followed by the complex Entamoeba histolytica/E. dispar (9.2%). 4.1% were positive for intestinal helminthes. The infection by Cryptosporidium sp is frequent in food handlers from Zulia State. Given to the results of this investigation and the nonexistence of studies in this population, is necessary to deepen in the impact of this parasitism in food handlers and the consumers of their products.

  7. Progress towards development of a vaccine for amebiasis.

    PubMed Central

    Stanley, S L

    1997-01-01

    The application of molecular biologic techniques over the past decade has seen a tremendous growth in our knowledge of the biology of Entamoeba histolytica, the causative agent of amebic dysentery and amebic liver abscess. This approach has also led to the identification and structural characterization of three amebic antigens, the serine-rich Entamoeba histolytica protein (SREHP), the 170-kDa subunit of the Gal/GalNAc binding lectin, and the 29-kDa cysteine-rich protein, which all show promise as recombinant antigen-based vaccines to prevent amebiasis. In recent studies, an immunogenic dodecapeptide derived from the SREHP molecule has been genetically fused to the B subunit of cholera toxin, to create a recombinant protein capable of inducing both antiamebic and anti-cholera toxin antibodies when administered by the oral route. Continued progress in this area will bring us closer to the goal of a cost-effective oral combination "enteric pathogen" vaccine, capable of inducing protective mucosal immune responses to several clinically important enteric diseases, including amebiasis. PMID:9336666

  8. Molecular detection of common intestinal parasites: a performance evaluation of the BD Max™ Enteric Parasite Panel.

    PubMed

    Batra, R; Judd, E; Eling, J; Newsholme, W; Goldenberg, S D

    2016-11-01

    The purpose of this study was to evaluate the level of agreement of the BD Max™ Enteric Parasite Panel (EPP) with microscopy for the detection of Giardia duodenalis, Cryptosporidium spp. and Entamoeba histolytica in stool samples. A total of 372 stool samples (partly collected on the basis of positive microscopy and partly unselected, consecutive sample submitted for parasite investigation) were tested with EPP according to manufacturer's instructions and also using microscopy according to standard techniques. Discrepant samples were further tested using PCR by the National Parasitology reference laboratory. Levels of agreement and laboratory turnaround times were measured and compared. Overall, positive and negative percent agreement was high between the two methods. However, microscopy resulted in four false positives and one false negative for G. duodenalis and two false positives for Cryptosporidium. Additionally, microscopy could not differentiate between E. histolytica and Entamoeba dispar. Median laboratory turnaround time was 65 hours for microscopy; results from EPP could be available after four hours. Blastocycstis hominis was detected by microscopy in one sample and would have been missed if only EPP was performed. The EPP was a good alternative to microscopy, detecting a small number of additional positives that were missed by microscopy. The assay is significantly faster than microscopy and allows laboratory workflows to be streamlined. The risk of missing parasites that are not included in the EPP appears to be minimal in the studied population; however, there may be certain patient groups who would benefit from microscopic examination of stools.

  9. Amoebicidal Activity of Milk, Apo-lactoferrin, sIgA and Lysozyme

    PubMed Central

    León-Sicairos, Nidia; López-Soto, Fernando; Reyes-López, Magda; Godínez-Vargas, Delfino; Ordaz-Pichardo, Cynthia; de la Garza, Mireya

    2006-01-01

    Objectives: To identify amoebicidal components in human milk and the effect of iron on the amoebicidal activity. Design: Investigation in axenic cultures of Entamoeba histolytica trophozoites. Methods: Amoebas were treated with 5%–20% of human, bovine and swine milk, with 10% of human milk fractions (i.e., casein, proteins except casein and fat) or with 1 mg/ml of human milk apo-lactoferrin, human secretory immunoglobulin type A (sIgA) and chicken egg-white lysozyme (i.e., purified proteins). Milk proteins were detected using immunoblot. Confocal microscopy was used to define the interaction of milk proteins (100 μM each) and amoebas. Experiments were done at least three times in triplicate, and mean and standard deviations were calculated. Results: Human and bovine milk were amoebicidal showing a concentration-dependent effect. The amoebicidal effect was increased in the absence of iron. Milk protein fractions, with the exception of casein, were the components responsible for the amoebicidal activity found. Apo-lactoferrin, sIgA and lysozyme were identified in the amoebicidal milk protein fraction. Apo-lactoferrin showed the major amoebicidal effect. These proteins, either alone or in combination, showed a killing effect on the trophozoites. They bound to the amoebic membrane causing cell rounding, lipid disruption and damage. Conclusions: Milk proteins such as apo-lactoferrin, sIgA and lysozyme are able to kill Entamoeba histolytica trophozoites. This study confirms the importance of feeding breast milk to newborns. PMID:16809402

  10. Prevalence of intestinal parasites in young Quichua children in the highlands of rural Ecuador.

    PubMed

    Jacobsen, Kathryn H; Ribeiro, Priscila S; Quist, Bradley K; Rydbeck, Bruce V

    2007-12-01

    The prevalence of intestinal parasites in young Quichua children was assessed in 20 rural communities in the highlands of Ecuador in August 2005. The caregivers of 293 children aged 12-60 months were interviewed about the status of child health, household socioeconomic and environmental factors, and water-use practices and were requested to collect a faecal sample from the study child. Two hundred three (69.3%) of the 293 children provided faecal samples that were tested for parasites. The overall prevalences of infection for specific agents were Entamoeba histolytica or dispar 57.1%, Ascaris lumbricoides 35.5%, Entamoeba coli 34.0%, Giardia intestinalis (lamblia) 21.1%, Hymenolepis nana 11.3%, Cryptosporidium parvum 8.9%, Chilomastix mesnili 1.7%, Hymenolepis diminuta 1.0%, Strongyloides stercoralis 0.7%, and Trichuris trichiura 0.5%. The prevalence of parasites increased with age. Water storage, water treatment, consistent latrine-use, and participation in a community-based clean water project were not strongly associated with the prevalence of intestinal parasites, although having dirt floors was a risk factor for infection with E. histolytica or dispar and G. intestinalis.

  11. Intestinal parasites infection among immunocompromised patients in Riyadh, Saudi Arabia.

    PubMed

    Al-Megrin, Wafa A I

    2010-04-15

    This study was conducted to determine the prevalence of intestinal parasites among immunocompromised patients in Riyadh, Saudi Arabia. Fecal samples were collected from 136 immunocompromised patients (52 females and 84 males), aged 2 to 69 years. Specimens were fixed with 10% formalin for 30 min (2-3 g fecal(-1)) and then concentrated by a formalin-ether sedimentation technique. Samples were examined as wet saline mounts and in iodine preparation for detection of protozoan oocysts, cysts, helminthic eggs and larvae. Permanent stained smears were performed for intestinal coccidian parasites by the modified Ziehl-Neelsen technique and the modified trichrome stain. Intestinal parasites were detected in 54 (39.7%) most of them (25.7%) were with diarrhea. More specifically the following parasites were detected Cryptosporidium parvum 11(8.1%), Giardia lamblia 9 (6.6%), Cyclospora cayetanensis 8 (5.9%), Blastocystis hominis 7 (5.2%), Entamoeba histolytica 7 (5.2%), Entamoeba coli 5 (3.7%), Strongyloides stercoralis 3 (2.2%), Ascaris lumbricoides 1 (0.7%), Hymenolepis nana 1 (0.7%), Dicrocoelium dendriticum 1 (0.7%) and Hook worm 1 (0.7%). The most common parasite associated with diarrhea in patients, was C. parvum (p < 0.001), followed by G. lamblia, C. cayetanensis and E. histolytica. However, differences between different age groups were statistically non-significant. Similarly, no significant difference in the infection rate were found between male and female patients.

  12. Strategies To Discover the Structural Components of Cyst and Oocyst Walls

    PubMed Central

    Bushkin, G. Guy; Chatterjee, Aparajita; Robbins, Phillips W.

    2013-01-01

    Cysts of Giardia lamblia and Entamoeba histolytica and oocysts of Toxoplasma gondii and Cryptosporidium parvum are the infectious and sometimes diagnostic forms of these parasites. To discover the structural components of cyst and oocyst walls, we have developed strategies based upon a few simple assumptions. Briefly, the most abundant wall proteins are identified by monoclonal antibodies or mass spectrometry. Structural components include a sugar polysaccharide (chitin for Entamoeba, β-1,3-linked glucose for Toxoplasma, and β-1,3-linked GalNAc for Giardia) and/or acid-fast lipids (Toxoplasma and Cryptosporidium). Because Entamoeba cysts and Toxoplasma oocysts are difficult to obtain, studies of walls of nonhuman pathogens (E. invadens and Eimeria, respectively) accelerate discovery. Biochemical methods to dissect fungal walls work well for cyst and oocyst walls, although the results are often unexpected. For example, echinocandins, which inhibit glucan synthases and kill fungi, arrest the development of oocyst walls and block their release into the intestinal lumen. Candida walls are coated with mannans, while Entamoeba cysts are coated in a dextran-like glucose polymer. Models for cyst and oocyst walls derive from their structural components and organization within the wall. Cyst walls are composed of chitin fibrils and lectins that bind chitin (Entamoeba) or fibrils of the β-1,3-GalNAc polymer and lectins that bind the polymer (Giardia). Oocyst walls of Toxoplasma have two distinct layers that resemble those of fungi (β-1,3-glucan in the inner layer) or mycobacteria (acid-fast lipids in the outer layer). Oocyst walls of Cryptosporidium have a rigid bilayer of acid-fast lipids and inner layer of oocyst wall proteins. PMID:24096907

  13. Diagnosis of intestinal parasites in a rural community of Venezuela: Advantages and disadvantages of using microscopy or RT-PCR.

    PubMed

    Incani, Renzo Nino; Ferrer, Elizabeth; Hoek, Denise; Ramak, Robbert; Roelfsema, Jeroen; Mughini-Gras, Lapo; Kortbeek, Titia; Pinelli, Elena

    2017-03-01

    A cross-sectional study was carried out to determine the prevalence and diagnostic performance of microscopy and real time PCR (RT-PCR) for 14 intestinal parasites in a Venezuelan rural community with a long history of persistent intestinal parasitic infections despite the implementation of regular anthelminthic treatments. A total of 228 participants were included in this study. A multiplex RT-PCR was used for the detection of Dientamoeba fragilis, Giardia intestinalis, Cryptosporidium sp. and a monoplex RT-PCR for Entamoeba histolytica. Furthermore, a multiplex PCR was performed for detection of Ascaris lumbricoides, Strongyloides stercoralis, Necator americanus and Ancylostoma duodenale. Combined microscopy-PCR revealed prevalences of 49.3% for A. lumbricoides, 10.1% for N. americanus (no A. duodenale was detected), 2.0% for S. stercoralis, 40.4% for D. fragilis, 35.1% for G. intestinalis, and 7.9% for E. histolytica/dispar. Significant increases in prevalence at PCR vs. microscopy were found for A. lumbricoides, G. intestinalis and D. fragilis. Other parasites detected by microscopy alone were Trichuris trichiura (25.7%), Enterobius vermicularis (3.4%), Blastocystis sp. (65.8%), and the non-pathogenic Entamoeba coli (28.9%), Entamoeba hartmanni (12.3%), Endolimax nana (19.7%) and Iodamoeba bütschlii (7.5%). Age- but no gender-related differences in prevalences were found for A. lumbricoides, T. trichiura, G. intestinalis, and E. histolytica/dispar. The persistently high prevalences of intestinal helminths are probably related to the high faecal pollution as also evidenced by the high prevalences of non-pathogenic intestinal protozoans. These results highlight the importance of using sensitive diagnostic techniques in combination with microscopy to better estimate the prevalence of intestinal parasites, especially in the case of D. fragilis trophozoites, which deteriorate very rapidly and would be missed by microscopy. In addition, the differentiation between

  14. Parasitological diagnosis combining an internally controlled real-time PCR assay for the detection of four protozoa in stool samples with a testing algorithm for microscopy.

    PubMed

    Bruijnesteijn van Coppenraet, L E S; Wallinga, J A; Ruijs, G J H M; Bruins, M J; Verweij, J J

    2009-09-01

    Molecular detection of gastrointestinal protozoa is more sensitive and more specific than microscopy but, to date, has not routinely replaced time-consuming microscopic analysis. Two internally controlled real-time PCR assays for the combined detection of Entamoeba histolytica, Giardia lamblia, Cryptosporidium spp. and Dientamoeba fragilis in single faecal samples were compared with Triple Faeces Test (TFT) microscopy results from 397 patient samples. Additionally, an algorithm for complete parasitological diagnosis was created. Real-time PCR revealed 152 (38.3%) positive cases, 18 of which were double infections: one (0.3%) sample was positive for E. histolytica, 44 (11.1%) samples were positive for G. lamblia, 122 (30.7%) samples were positive for D. fragilis, and three (0.8%) samples were positive for Cryptosporidium. TFT microscopy yielded 96 (24.2%) positive cases, including five double infections: one sample was positive for E. histolytica/Entamoeba dispar, 29 (7.3%) samples were positive for G. lamblia, 69 (17.4%) samples were positive for D. fragilis, and two (0.5%) samples were positive for Cryptosporidium hominis/Cryptosporidium parvum. Retrospective analysis of the clinical patient information of 2887 TFT sets showed that eosinophilia, elevated IgE levels, adoption and travelling to (sub)tropical areas are predisposing factors for infection with non-protozoal gastrointestinal parasites. The proposed diagnostic algorithm includes application of real-time PCR to all samples, with the addition of microscopy on an unpreserved faecal sample in cases of a predisposing factor, or a repeat request for parasitological examination. Application of real-time PCR improved the diagnostic yield by 18%. A single stool sample is sufficient for complete parasitological diagnosis when an algorithm based on clinical information is applied.

  15. Human intestinal epithelial cells produce proinflammatory cytokines in response to infection in a SCID mouse-human intestinal xenograft model of amebiasis.

    PubMed Central

    Seydel, K B; Li, E; Swanson, P E; Stanley, S L

    1997-01-01

    The protozoan parasite Entamoeba histolytica causes amebic dysentery and amebic liver abscess, diseases associated with significant morbidity and mortality worldwide. E. histolytica infection appears to involve the initial attachment of amebic trophozoites to intestinal epithelial cells, followed by lysis of these cells and subsequent invasion into the submucosa. A recent in vitro study (L. Eckmann, S. L. Reed, J. R. Smith, and M. F. Kagnoff, J. Clin. Invest. 96:1269-1279, 1995) demonstrated that incubation of E. histolytica trophozoites with epithelial cell lines results in epithelial cell production of inflammatory cytokines, including interleukin-1 (IL-1) and IL-8, suggesting that intestinal epithelial cell production of cytokines might play a role in the inflammatory response and tissue damage seen in intestinal amebiasis. To determine whether intestinal epithelial cell production of IL-1 and IL-8 occurs in response to E. histolytica infection in vivo and as an approach to studying the specific interactions between amebic trophozoites and human intestine, we used a SCID mouse-human intestinal xenograft (SCID-HU-INT) model of disease, where human intestinal xenografts were infected with virulent E. histolytica trophozoites. Infection of xenografts with E. histolytica trophozoites resulted in extensive tissue damage, which was associated with the development of an early inflammatory response composed primarily of neutrophils. Using oligonucleotide primers that specifically amplify human IL-1beta and IL-8, we could demonstrate by reverse transcription PCR that mRNA for both IL-1beta and IL-8 is produced by human intestinal xenografts in response to amebic infection. The increase in human intestinal IL-1beta and IL-8 in response to invasive amebiasis was confirmed by enzyme-linked immunosorbent assays specific for human IL-1beta and IL-8. Using immunohistochemistry, we confirmed that human intestinal epithelial cells were the source of IL-8 in infected xenografts

  16. Intestinal parasitic infection among children and neonatus admitted to Ibn-Sina Hospital, Sirt, Libya.

    PubMed

    Kasssem, Hamed H; Zaed, Hana Abdalsalam; Sadaga, Gazala A

    2007-08-01

    A total of 350 stool samples from 196 males and 154 female children and neonatus admitted in Ibn-Sina hospital, Sirt, were examined from June 2001 to May 2002, to determine the prevalence of intestinal parasites. Intestinal parasitic infections were identified in 196 (56%) of children and neonates. No intestinal helminthic parasites were detected but 13 intestinal protozoan parasites were detected. The most prevalent protozoan was Entamoeba histolytica /E. dispar (36.57%); Blastocystis hominis (12.57%), Giardia lamblia (10.29%), Isospora belli (3.14%) and Balantidium coli (0.86%), the latter was detected in non-Libyan children. The non-pathogenic ones were Entamoeba coli (15.14%), Endolimax nana (13.71%), Entamoeba hartmanni (4.29%), Chilomastix mesnilli (4.29%), Retortamonas intestinalis (3.43%), Dientamoeba fragilis (2%), Iodamoeba butschlii (0.86%) and Trichomonas hominis (0.86%). The result showed a significant difference exists between the prevalence of pathogenic and non-pathogenic protozoan parasites (P < 0.05). High prevalence of E. histolytica/ E. dispar followed by E. coli, E. nana, B. hominis and G. lamblia in both sexes of children, while the prevalence of other intestinal parasites were low in both sexes, significantly different existed in the prevalence of intestinal parasites between males and females children (t = 24.68; P < 0.05). Age groups had no effect on the prevalence of intestinal parasites (F = 0.66; P < 0.05). Significant differences existed in the prevalence between single and multiple infections with pathogenic protozoa. The socio-economic status of children parents revealed that high prevalence in children from medium socio-economic status. The family size had no significant effect on the prevalence of the intestinal parasites.

  17. Comparison of polyvinyl alcohol fixative with three less hazardous fixatives for detection and identification of intestinal parasites.

    PubMed

    Jensen, B; Kepley, W; Guarner, J; Anderson, K; Anderson, D; Clairmont, J; De L'aune, W; Austin, E H; Austin, G E

    2000-04-01

    Polyvinyl alcohol (PVA) containing the fixative mercuric chloride is considered the "gold standard" for the fixation of ova and parasites in the preparation of permanently stained smears of stool specimens. However, mercuric chloride is potentially hazardous to laboratory personnel and presents disposal problems. We compared three new alternative, nontoxic fixatives with PVA, analyzing ease of sample preparation and quality of smears. Sixty-eight fresh stool specimens were divided into aliquots and placed in each of four different fixatives: PARASAFE (PS) (Scientific Devices Laboratory, Inc., Des Plaines, Ill.), ECOFIX (EC) (Meridian Diagnostics, Inc., Cincinnati, Ohio), Proto-Fix (PF) (Alpha-Tec Systems, Inc., Vancouver, Wash.), and low-viscosity PVA fixative (PVA) (Meridian). Specimens were processed and stained according to each manufacturer's directions. Parasites were found in 31 of 68 slide preparations with PVA, 31 with PF, 30 with EC, and 30 with PS. Blastocystis hominis and Iodamoeba bütschlii were preserved in a readily identifiable state by all methods of fixation. However, some parasites were more easily identified with some of the fixatives because of differences in parasite distortion. For example, Entamoeba histolytica (Entamoeba dispar) was detected in 13 stools fixed with PF, 7 with PVA, and 6 with EC but none with PS. Likewise, Chilomastix mesnili was identified in 13 specimens fixed with PF, 8 with EC, and 5 with PVA but only 1 with PS, while Entamoeba coli was seen much less frequently with PS than with the other three fixatives. A dirty background was observed in 41% of specimens prepared with PS, whereas background quality was acceptable with other fixatives. Sample preparation was most rapid with PS, although the EC method involved the fewest steps. In conclusion, PVA and PF produced the least parasite distortion, while PS proved unsatisfactory for the identification of E. histolytica, E. coli, and C. mesnili. Both PF and EC appear to be

  18. Comparison of Polyvinyl Alcohol Fixative with Three Less Hazardous Fixatives for Detection and Identification of Intestinal Parasites

    PubMed Central

    Jensen, B.; Kepley, W.; Guarner, J.; Anderson, K.; Anderson, D.; Clairmont, J.; De l'aune, William; Austin, E. H.; Austin, G. E.

    2000-01-01

    Polyvinyl alcohol (PVA) containing the fixative mercuric chloride is considered the “gold standard” for the fixation of ova and parasites in the preparation of permanently stained smears of stool specimens. However, mercuric chloride is potentially hazardous to laboratory personnel and presents disposal problems. We compared three new alternative, nontoxic fixatives with PVA, analyzing ease of sample preparation and quality of smears. Sixty-eight fresh stool specimens were divided into aliquots and placed in each of four different fixatives: PARASAFE (PS) (Scientific Devices Laboratory, Inc., Des Plaines, Ill.), ECOFIX (EC) (Meridian Diagnostics, Inc., Cincinnati, Ohio), Proto-Fix (PF) (Alpha-Tec Systems, Inc., Vancouver, Wash.), and low-viscosity PVA fixative (PVA) (Meridian). Specimens were processed and stained according to each manufacturer's directions. Parasites were found in 31 of 68 slide preparations with PVA, 31 with PF, 30 with EC, and 30 with PS. Blastocystis hominis and Iodamoeba bütschlii were preserved in a readily identifiable state by all methods of fixation. However, some parasites were more easily identified with some of the fixatives because of differences in parasite distortion. For example, Entamoeba histolytica (Entamoeba dispar) was detected in 13 stools fixed with PF, 7 with PVA, and 6 with EC but none with PS. Likewise, Chilomastix mesnili was identified in 13 specimens fixed with PF, 8 with EC, and 5 with PVA but only 1 with PS, while Entamoeba coli was seen much less frequently with PS than with the other three fixatives. A dirty background was observed in 41% of specimens prepared with PS, whereas background quality was acceptable with other fixatives. Sample preparation was most rapid with PS, although the EC method involved the fewest steps. In conclusion, PVA and PF produced the least parasite distortion, while PS proved unsatisfactory for the identification of E. histolytica, E. coli, and C. mesnili. Both PF and EC appear to be

  19. Household and personal factors are sources of heterogenity in intestinal parasite clearance among Mexican children 6-15 months of age supplemented with vitamin A and zinc.

    PubMed

    Srinivasan, Punitha; Lawa, Ha'i Raga; Rosado, Jorge L; Al Mamun, Abdullah; Khatun, Mohsina; Santos, José I; Utzinger, Jürg; Long, Kurt Z

    2016-04-01

    A randomised, double-blind, placebo-controlled trial was carried out among Mexico children aged 6-15 months to determine how household characteristics modify vitamin A and zinc supplementation efficacy on Ascaris lumbricoides, Giardia intestinalis and Entamoeba histolytica/E. dispar infection durations. Children assigned to receive vitamin A every 2 months, a daily zinc supplement, a combined vitamin A-zinc supplement or a placebo were followed for 1 year. Parametric hazard models were fit to infection durations stratified by personal and household factors. Children supplemented with vitamin A and zinc combined from households lacking piped water and children in all three treatment arms from households with dirt floors had longer G. intestinalis and A. lumbricoides infection durations than their counterparts, respectively. Shorter E. histolytica/E.dispar durations were found among zinc-supplemented children of mothers who had <6 years of education and no indoor bathrooms. Heterogeneity in supplementation efficacy among children may reflect differences in exposure risk and baseline immune responses.

  20. Dramatic decrease in prevalence of soil-transmitted helminths and new insights into intestinal protozoa in children living in the Chaco region, Bolivia.

    PubMed

    Macchioni, Fabio; Segundo, Higinio; Gabrielli, Simona; Totino, Valentina; Gonzales, Patricia Rojas; Salazar, Esteban; Bozo, Ricardo; Bartoloni, Alessandro; Cancrini, Gabriella

    2015-04-01

    We assessed the prevalence of intestinal parasites among 268 2-12-year-old children living in rural areas, small villages, and semi-urban areas of the Chaco region, south-eastern Bolivia. The overall parasitism was 69%. Only protozoa, helminths, or co-infections were observed in 89.2%, 5.9%, or 4.9% of the positive children, respectively. A significant progressive increase in overall parasite prevalence was found when passing from rural areas to small villages and semi-urban areas. The most commonly found species were Entamoeba coli (38.4%), Giardia intestinalis (37.7%), and Blastocystis spp. (16%). Hymenolepis nana was the most prevalent helminth (5.6%), followed by Ascaris lumbricoides and hookworms (1.5% and 0.4%) evidenced only in rural areas and in villages. Molecular diagnostics identified Blastocystis subtypes 9 and 2, and 5 infections by Entamoeba histolytica and 4 by Entamoeba dispar. The dramatic decrease in prevalence of soil-transmitted helminths with respect to that observed about 20 years ago (> 40%) evidences the success of the preventive chemotherapy intervention implemented in 1986. Health education and improved sanitation should be intensified to control protozoan infections.

  1. Parasitic Infections Based on 320 Clinical Samples Submitted to Hanyang University, Korea (2004-2011)

    PubMed Central

    Choi, Sung-Chul; Lee, Soo-Young; Song, Hyun-Ouk; Ryu, Jae-Sook

    2014-01-01

    We analyzed 320 clinical samples of parasitic infections submitted to the Department of Environmental Biology and Medical Parasitology, Hanyang University from January 2004 to June 2011. They consisted of 211 nematode infections, 64 trematode or cestode infections, 32 protozoan infections, and 13 infections with arthropods. The nematode infections included 67 cases of trichuriasis, 62 of anisakiasis (Anisakis sp. and Pseudoterranova decipiens), 40 of enterobiasis, and 24 of ascariasis, as well as other infections including strongyloidiasis, thelaziasis, loiasis, and hookworm infecions. Among the cestode or trematode infections, we observed 27 cases of diphyllobothriasis, 14 of sparganosis, 9 of clonorchiasis, and 5 of paragonimiasis together with a few cases of taeniasis saginata, cysticercosis cellulosae, hymenolepiasis, and echinostomiasis. The protozoan infections included 14 cases of malaria, 4 of cryptosporidiosis, and 3 of trichomoniasis, in addition to infections with Entamoeba histolytica, Entamoeba dispar, Entamoeba coli, Endolimax nana, Giardia lamblia, and Toxoplasma gondii. Among the arthropods, we detected 6 cases of Ixodes sp., 5 of Phthirus pubis, 1 of Sarcoptes scabiei, and 1 of fly larva. The results revealed that trichuriasis, anisakiasis, enterobiasis, and diphyllobothriasis were the most frequently found parasitosis among the clinical samples. PMID:24850969

  2. Assessment of gastrointestinal parasites in wild chimpanzees (Pan troglodytes troglodytes) in southeast Cameroon

    PubMed Central

    Bertout, Sébastien; Locatelli, Sabrina; Butel, Christelle; Pion, Sébastien; Mpoudi-Ngole, Eitel; Delaporte, Eric; Peeters, Martine; Mallié, Michèle

    2014-01-01

    We tested 114 faecal samples from wild simian immunodeficiency virus (SIV)-positive (n=43) and SIV-negative (n=71) chimpanzees (Pan troglodytes troglodytes) in southeast Cameroon for the presence of gastrointestinal parasites by direct smear. We observed cysts from different protozoa (Entamoeba coli and Entamoeba histolytica/Entamoeba dispar, Endolimax nana, Iodamoeba butschlii, Chilomastix mesnili, Balantidium coli and Blastocystis cells) and trophozoites from Troglodytella abrassarti and Balantidium coli. Eggs from different helminths (strongylids, Ascaris lumbricoides, Abbreviata caucasica, Trichuris sp., Capillaria sp., Enterobius anthropopeci, Bertiella sp., Hymenolepis diminuta and an undetermined fluke) were also observed. Finally, we observed eggs that could not be properly identified and classified. We did not observe any differences between the SIV+ and SIV− samples except for the unidentified eggs. The studied chimpanzees were highly parasitised by strongylid (85.1 % of prevalence), Troglodytella (43.8 %) and Blastocystis (2.9 %), and the frequency of the other parasites ranged from 0.9 to 8.8 %. These high levels of parasite infections could represent an additional burden in a population where there is a high rate of the SIV virus in circulation. PMID:24781023

  3. Diversity and prevalence of gastrointestinal parasites in seven non-human primates of the Taï National Park, Côte d’Ivoire

    PubMed Central

    Kouassi, Roland Yao Wa; McGraw, Scott William; Yao, Patrick Kouassi; Abou-Bacar, Ahmed; Brunet, Julie; Pesson, Bernard; Bonfoh, Bassirou; N’goran, Eliezer Kouakou; Candolfi, Ermanno

    2015-01-01

    Parasites and infectious diseases are well-known threats to primate populations. The main objective of this study was to provide baseline data on fecal parasites in the cercopithecid monkeys inhabiting Côte d’Ivoire’s Taï National Park. Seven of eight cercopithecid species present in the park were sampled: Cercopithecus diana, Cercopithecus campbelli, Cercopithecus petaurista, Procolobus badius, Procolobus verus, Colobus polykomos, and Cercocebus atys. We collected 3142 monkey stool samples between November 2009 and December 2010. Stool samples were processed by direct wet mount examination, formalin-ethyl acetate concentration, and MIF (merthiolate, iodine, formalin) concentration methods. Slides were examined under microscope and parasite identification was based on the morphology of cysts, eggs, and adult worms. A total of 23 species of parasites was recovered including 9 protozoa (Entamoeba coli, Entamoeba histolytica/dispar, Entamoeba hartmanni, Endolimax nana, Iodamoeba butschlii, Chilomastix mesnili, Giardia sp., Balantidium coli, and Blastocystis sp.), 13 nematodes (Oesophagostomum sp., Ancylostoma sp., Anatrichosoma sp., Capillariidae Gen. sp. 1, Capillariidae Gen. sp. 2, Chitwoodspirura sp., Subulura sp., spirurids [cf Protospirura muricola], Ternidens sp., Strongyloides sp., Trichostrongylus sp., and Trichuris sp.), and 1 trematode (Dicrocoelium sp.). Diversity indices and parasite richness were high for all monkey taxa, but C. diana, C. petaurista, C. atys, and C. campbelli exhibited a greater diversity of parasite species and a more equitable distribution. The parasitological data reported are the first available for these cercopithecid species within Taï National Park. PMID:25619957