• E-mail
• Print
• Comment
• Font Size
• Digg
• del.icio.us
• Discuss article

Meeting Report: EMBO Workshop "Pathogenesis of Amoebiasis: from Genomics to Disease", Institut Pasteur, Paris, May 19-22, 2003

Posted on: Friday, 9 January 2004, 06:00 CST

Amoebiasis is a human intestinal infectious disease caused by the protozoan parasite Entamoeba histolytica. The parasite infects 500 million people, causes 50 million cases of dysentery and liver abscesses, and kills about 100,000 humans each year around the world (Hague et al. 2003; Stanley 2003) (see also website at http:// homepages.lshtm.ac.uk/ entamoeba/). This parasitic infection, like other infectious diseases, is disproportionately prevalent in the poorer regions of the world. Amoebiasis also exists to a lesser extent in Europe, where it appears as clinical cases in circumstances of poor hygienic conditions (psychiatric hospitals, prisons, refugees camps) as well as among travelers returning from endemic areas.

During invasive amoebiasis, highly motile parasites invade the intestinal epithelium causing extensive tissue damage characterized by acute inflammation and ulceration with necrosis and hemorrhage. The amoebae kill the host cells and eventually ingest them. Tissue destruction occurs as the parasites progress into the tissue. The extracellular matrix is extensively degraded by the activity of amoebic proteases. Production of toxic factors results in the arrival of cells from the immune system and establishment of inflammation. The most common outcome of amoebic metastatic spread are liver abscesses. In contrast to intestinal amoebiasis, invasion of the liver is characterized by the presence of E. histolytica trophozoites that cause an acute inflammatory reaction. Individual infection foci contain mostly dead hepatocytes, polymorphonuclear leukocytes, macrophages and parasites. Differences in pathology between amoebic colitis and amoebic liver abscess likely result from a variation of the E. histolytica virulence repertoire in the two organs and from different responses of these organs to amoeba infection. One of the key questions which is still unanswered is why do the majority of individuals infected with E. histolytica remain asymptomatic.

Significant progress has been achieved in recent years in our understanding of the biology and pathogenesis of this parasite. Genome sequencing of E. histolytica is in progress and is being performed by a collaborative program conducted by The Sanger Center (UK) - Wellcome Trust (http://www.sanger.ac.uk/ Projects/ E_histolytica/) and TIGR - NIH (USA) (http://www.tigr.org/tdb/e2k1/ eha1/). By the end of the year 2003 a large consensus of this genome will be available and the exploitation of genetic information will become a new challenge. The purpose of the EMBO workshop "Pathogenesis of amoebiasis: from genomics to disease" held at the Pasteur Institute in May 2003 was to enable those involved in E. histolytica research to share information obtained by genome analysis of this parasite and to anticipate functional analysis of major pathogenic factors leading to symptoms of disease. A total of 87 attendees from 18 countries participated in the workshop. There were 37 posters and 30 invited oral presentations during the conference. Scientific sessions comprising topics on: 'Genome organization and sequencing issues', 'Regulation of gene expression during pathogenesis' and 'Cell biology and signaling pathways were held'.

Genome Organization and Sequencing Issues

This session provided information on novel genes and genome organization of E. histolytica following the sequencing effort. B. Loftus (TIGR, Rockville, USA) and N. Hall (Sanger Institute, Cambridge, England) reported on the progress of the Entamoeba genome project. Up to now, as much as 9 coverage of the E. histolytica genome has been sequenced. First assemblies and gene content searches revealed contigs of up to 220 kb in size containing >99% of all amoeba genes. In addition to E. histolytica, 2-3 coverage of both the E. dispar and the E. invadens genome have been sequenced. The information of these two additional organisms will be of considerable help for studies on evolutionary and phylogenetic aspects of the parasite, the analysis of cyst development and, more important, the identification of molecules and of regulatory pathways involved in E. histolytica pathogenicity.

First genome-wide searches for particular genes and gene families were reported by J. Samuelson (Wang et al. 2003, Boston University, Boston, USA) and I. Bruchhaus (Bruchhaus et al. 2003, BNI, Hamburg, Germany). These investigations revealed two interesting findings. First, E. histolytica contains an incredibly high number of more than 80 different membrane-associated receptor kinases, which suggests a highly differentiated signaling pathway. Second, 12 novel genes encoding cysteine proteases were identified bringing the total number of members of this gene family to up to 20. Cysteine proteases are considered to be responsible for parasite-induced tissue destruction. Interestingly, 7 of the 12 newly identified enzymes are unique in that they contain membrane attachment moieties. Many of the molecules may have rather specific functions (e.g. during the parasite life-cycle or during infection of the human host) as the majority of the enzymes are not expressed during in vitro cultivation.

Amoeba chromosomes show extensive length polymorphism due to variations in telomeric and subtelomeric regions. In many other protozoan parasites, these regions contain repetitive sequences. A. Bhattacharya (Bhattacharya et al. 2002, Jawaharlal Nehru University, New Delhi, India) has analyzed the various classes of repeats present within the E. histolytica genome. A rough estimate suggested that up to 50% of the genome consists of repetitive sequences. These comprise protein-coding genes (e.g. HSP100, BspA) as well as insertial and transposable elements. However, the most unusual feature is the organization of tRNA genes, which are clustered into long tandem arrays. The array repeat units contain up to 5 different tRNA genes and some also incorporate a 5S RNA gene. In each unit, the tRNA and 5S RNA genes are separated by short tandem repeats of varying complexity. Altogether about 25 distinct arrays have been identified but the reason for their existence remains to be determined. It was discussed that they may substitute for telomeres, as classical telomeric repeats appear to be absent in E. histolytica.

DNA comparisons between pathogenic E. histolytica and nonpathogenic E. dispar are expected to identify candidate genes involved in amoeba pathogenicity. The two organisms represent the closest relatives among all Entamoeba species known so far. Accordingly, rRNA sequences are 98.3% identical and the two organsims are highly syntenic. Using DNA microarrays containing E. histolytica random-sheared genomic DNA library, U. Singh (Stanford University, Stanford, USA) reported on up to 1.5% DNA differences between E. histolytica and E. dispar. However, the degree of inter- and intra-species variations remains to be determined by comparing a broader set of amoeba isolates. On the other hand, clear cut differences for a number of molecules present in E. histolytica but absent in E. dipar were presented by E. Tannich (Willhoeft et al. 2002, BNI, Hamburg, Germany). These comprise a membrane-associated cysteine protease (EhCPS), an asparagine-rich surface protein (Ariel) and a high copy number non-LTR retrotransposon-like element (ehapt2). A link of any of these molecules to amoeba pathogenicity was shown by the group of K. Chadee (Moncada et al. 2003, McGill University, Ste.-Anne-de-Bellevue, Canada). Using antisense technology, E. histolytica with reduced expression of EhCPS were generated, which revealed significant impairment in their ability to degrade colonic mucin.

Regulation of Gene Expression during Pathogenesis of E. histolytica

E. histolytica isolates from different sources are known to have different degrees of pathogenicity. A number of virulence factors have been characterized and progress was reported on the analysis of genetic regulation during pathogenesis with the aim to encourage new directions and perspectives for the study of major pathogenic factors.

Significant progress was reported on the elucidation of the molecular mechanisms, which regulate gene expression in E. histolytica. Many investigators reported that they are using transfectants with a variety of plasmid vectors to investigate the role of genes of interest under different conditions as well as to up- or down-regulate their expression (Katz et al. 2003). Novel techniques for complete silencing of gene expression were also reported at the meeting. D. Mirelman and R. Bracha (Bracha et al. 2003, Weizmann I., Rehovot, Israel) described their recent findings on the transcriptional gene silencing of the amoebapore A gene. Epigenetic silencing was triggered following transfection of trophozoites with a plasmid containing the 5' flanking region of the amoebapore A gene which also included a segment of the retrotransposon-like element ehap2, previously reported by the group of E. Tannich (Willhoeft et al. 2002, BNI, Hamburg, Germany). Removal of the plasmid did not restore transcription. The cytosine residues in the 5' upstream region were found to be methylated. The silenced, virulence-attenuated trophozoites are being tested for their immunoprotective potential. S. Ankri (Technion, Haifa, Israel) reported on the identification of a novel DNA methyl transferase Ehmeth, which is si\milar to the human Dnmt2. L. Vayssie (Institut Pasteur, Paris, France) reported the first successful use of the RNA interference (RNAi) technique for the post-transcriptional inhibition of gene expression in Entamoeba. Significant degradation of the [gamma]-tubulin gene transcript was observed in trophozoites fed with bacteria containing a plasmid that produced dsRNA. Gene silencing was also observed following the passive absorption of small dsRNA molecules added to the growth medium. Inhibition of gene expression by RNA induced the disappearance of microtubules, indicating that [gamma]-tubulin functions as a nucleator of microtubules in the trophozoites. S. Bhattacharya (Ghosh et al. 2003, Nehru University, New Delhi, India) described the multiple replication origins as well as their map locations in the circular ribosomal DNA episomes previously identified in E. histolytica. Differences in the utilization of the different replication origins were discovered under normal or serumstarved growth conditions, indicating that activation or silencing of the origin of replication is context-dependent. C. Gilchrist (Gilchrist et al. 2003, U. Virginia, Charlottesville, USA) reported on the URE3 element which is involved in regulating the transcription of a number of E. histolytica genes. The URE3 protein was found to bind to the promoter regions in a calcium-dependent manner and mutations can abolish this effect. The regulon exerts its regulatory effect in a context-dependent manner as it showed negative regulation on the lectin (hgl5) gene and a positive effect on the ferredoxin gene. C. Lopez-Camarillo (Lopez-Camarillo et al. 2003, CINVESTAV, Mexico City, Mexico) described the sequences responsible for the emetine response in the promoer region of the MDR-like EhPgp5 gene and reported that emetine-resistant trophozoites over-express an mRNA that contains a longer poly(A) tail which appears to correlate with the longer half-life of the transcript. L. Marchat (Marchat et al. 2002, CINVESTAV, Mexico City, Mexico) identified enhancer binding proteins (C/EBP) in the promoter region of EhPgp1, another MDR gene, which appear to participate in its transcription regulation. A. Rojas (CINVESTAV, Mexico City, Mexico) has identified a novel guanine-nucleotide exchange factor, EhGEF1, which was localized in the pseudopods of trophozoites. EhGEF1 has the ability to promote the binding and activation of small GTPases, such as RacG, which are involved in cytoskeleton changes during cytokinesis, capping and uroid formation. T. Nozaki (Natl. Inst. of Infect. Dis., Tokyo, Japan) identified two small GT-Pases, Rab5 and Rab7, which participate in vesicular trafficking and are involved in phagocytosis. Mutational studies with Rab5 indicate that it is involved in the formation of the prevacuole and RBC ingestion. Rab7 functions both in the formation of the prevacuole and binds to other proteins, analogs of VPS 26 and VPS 35 that function in retrograde transport of the carbohydrate hydrolase receptor VPS10. Overexpression of Rab7 in trophozoites inhibited the transport of the amoebapore proteins to the phagosome and decreased phagocytosis. E. Orozco (Solis et al. 2002, CINVESTAV, Mexico City, Mexico) described the presence of DNA-containing small vesicles (termed EhkO) in the trophozoites which suggest they may be primitive mitochondria. J. Tovar (Royal Holloway University, Egham, UK) also presented evidence for the presence of mitochondrial remnant organelles or mitosomes, which are associated with distinct proteins of mitochondrial origin such as the chaperonin Cpn60 and ferredoxin. No co-localization was detected, however, between the mitosomes and the EhkOs.

Pathogenesis, Cell Biology and Signaling Pathways

One of the important issues of research in pathogenesis of Entamoeba has been the development of animal models of disease in which the behavior of the parasite could be investigated under conditions, which approximate those present in the host. Recent work in this area has included a C3H mouse model of intestinal amoebiasis, as discussed by W. Petri (U. Virginia, Charlottesville, USA), that has enabled examination of both the immunopathogenesis and immunoprophylaxis of amoebic colitis (Houpt et al. 2002). S. Stanley (Stanley 2003; Zhang et al. 2002, Washington University, St. Louis MO, USA) presented work from a model of intestinal amoebiasis produced by implanting fetal human intestine into SCID mice. Although it is not possible to study acquired immunity with this model, it does offer invaluable insights into the innate response to E. histolytica. Introduction of E. histolytica trophozoites into the intestine results in an inflammatory response associated with activation of NF-kappaB, release of inflammatory mediators, and activation and attraction of neutrophils and monocytes/macrophages into the intestine. Important host mediators of inflammation in this model included IL-1, IL-8 and COX-2.

K. Chadee (Gaucher and Chadee 2003, McGill University, Montreal, Canada) has examined the macrophage innate immune response stimulated in vitro by the parasite Gal/GalNAc adherence lectin. The lectin stimulated a dose-dependent increase in TLR-2 and ICAM-1 and an inhibition of TLR-4 and -5 mRNA expression. It also induced the nuclear translocation of both the p50 and p65 subunits of NF-kappa B. Thus, at least part of the pro-inflammatory response seen with amoebiasis may be due to direct interaction of the Gal/GalNAc lectin with macrophages.

On a related front, E. Labruyere (Zimmer et al. 2002, Institut Pasteur, Paris, France) examined using a novel video microscopy tracking system which factors stimulate amoebic motility which is required for amoebic invasion into the intestine. Consistent with the observations of pro-inflammatory cytokine production in infected intestine, E. Labruyere demonstrated that trophozoites chemotax to TNF alpha. The effects of TNF alpha on the invasive characteristics of E. histolytica are now under study.

The parasite protects itself from oxidant attack by the innate immune system in part with a peroxiredoxin enzyme that detoxifies hydrogen peroxide. S. Reed (Hughes et al. 2003, University of California, San Diego, USA) demonstrated that the peroxiredoxin localized to the cytoplasmic and possibly also to the extracytoplasmic face of the plasma membrane. This localization may be due to the discovered interaction of the enzyme with the cytoplasmic tail of the cell surface Gal/GalNAc lectin (Hughes et al. 2003, B. Mann and M. Hughes, University of Virginia, Charlottesville VA, USA). The peroxiredoxin is present in higher amounts in pathogenic E. histolytica than in nonpathogenic E. dispar and levels of the enzyme correlate with resistance to hydrogen peroxide.

W. Petri (Haque et al. 2003, University of Virginia, Charlottesville VA, USA) gave an overview of the parasite Gal/ GalNAc lectin. Interest in this molecule has been heightened by the recent discovery that acquired immunity to amoebiasis in children is associated with an anti-Gal/GalNAc lectin mucosal IgA antibody response, and by the demonstration that immunization of mice with the lectin provides substantial protection from amoebic colitis. Engagement of the Gal/GalNAc lectin to the host intestinal epithelium results in cytoskeletal reorganization in the parasite. The parasite cytoskeleton regulates the extracellular adhesive activity of the lectin and recruits to the host-parasite interface factors required for parasite survival within the host. If the parasite lectin attaches to the host mucin glycoproteins lining the intestine, the result is commensal infection. In contrast, attachment of the lectin to a host cell surface glycoprotein leads to lectin-induced calcium transients, caspase activation and destruction of the host via apoptosis.

The role of Gal-GalNAc lectin in the in vivo invasive process was studied by N. Guillen (Rigothier et al. 2002, Institut Pasteur, Paris, France) by the use of two photon laser microscopy. Movement of the parasite within the liver of a hamster was extremely directional, and interference with the Gal/GalNAc lectin (through inducible expression of a dominant negative mutant) completely inhibited parasite penetration into the liver. This demonstrates a requirement for Gal/GalNAc lectin signaling to the cytoskeleton for motility of the parasite in the three-dimensional environment of the tissue. The role of cysteine proteinases in the survival of trophozoites within the hamster liver abscesses is under investigation by A. Olivos (Olivos-Garcia et al. 2003, National Autonomous University, Mexico City, Mexico).

The acquired immune response to amoebic liver abscess in humans is under study by V. Juarez-Javier (Ventura-Juarez et al. 2002, Universidad Autonoma de Aguascalientes, Mexico). Early in infection, he observed a transient reduction in CD4 T cells and an increase in CD8 T cells in peripheral blood. In biopsies of human liver abscess, only minimal inflammatory infiltrates were observed, suggesting a profound difference in the degree of immune inflammatory response to intestinal vs. hepatic disease. The lack of inflammation in the liver could be a clue as to the ability of the parasite to persist in the liver for long periods of time.

Not all children infected with E. histolytica develop disease. R. Haque (Ali et al. 2003, International Centre for Diarrhoeal Disease Research, Dhaka, Bangladesh) compared the genotype frequencies of Class II HLA antigens among a cohort of unrelated Bangladeshi children intensively followed for E. histolytica infection over a 3 year period. Individuals heterozygous for the DQB1*601/DRB1*1501 haplotype were 10.1 times (95% Cl: 2.02, 50.6) more likely to be free of amoebic infection. This association may offer insight into why amoebiasis does not occur in some children exposed to the parasite, and implicates class II restricted immune responses in protectio\n from E. histolytica infection. To elucidate the mechanism of protective immunity that is associated in mice and men with intestinal IgA antibodies against the Gal/GalNAc lectin, J. Ravdin (University of Minnesota, Minneapolis MN, USA) studied the epitope specificity of IgA antibodies in adults cured of amoebic liver abscess or having asymptomatic infection. IgA antibodies which blocked adherence of the parasite in vitro mapped to amino acids 868944 of the Gal/GalNAc lectin heavy subunit, a region previously shown to contain a binding domain for the lectin.

Conclusion

The recent EMBO Workshop in Paris was the first meeting in which the research community engaged in studying the biology and pathogenesis of E. histolytica began to benefit from the recent, almost complete sequencing of the parasite's genome. As we have seen happening before in several other microorganisms, this new blueprint of information is becoming extremely useful and will help us tremendously in tackling some of the difficult and outstanding questions which we face. As we learn more about the biology of E. histolytica and how it interacts with mammalian tissues and cells in animal models and in the host, we keep finding that this primitive eukaryotic cell has developed some unique molecular mechanisms, which enable it to thrive as a parasite in Man as well as to survive in the environment. The understanding of these mechanisms and the elucidation of their complex regulation pose an exciting challenge which will certainly contribute to the clarification of basic biological phenomena. It will also help us understand the different clinical manifestations of an infection with the parasite and will be extremely useful for the development of novel therapies and vaccines.

Acknowledgements

We wish to acknowledge the financial support granted to the Workshop by EMBO as well as the contributions of the Pasteur Institute in Paris for the organization and facilities.

References

Ali IKM, Hossain MB, Roy S, Ayeh-Kumi PF, Petri WAJ, Hague R, Clark CG (2003) Entamoeba moshkovskii infections in children in Bangladesh. Emerg Infect Dis 9: 580-584

Bhattacharya S, Bakre A, Bhattacharya A (2002) Mobile genetic elements in protozoan parasites. J Genet 81: 73-86

Bracha R, Nuchamowitz Y, Mirelman D (2003) Transcriptional silencing of an amoebapore gene in Entamoeba histolytica: Molecular analysis and effect on pathogenicity. Eukaryot Cell 2: 295-305

Bruchhaus I, Loftus BJ, Hall N, Tannich E (2003) The intestinal protozoan parasite Entamoeba histolytica contains 20 cysteine protease genes, of which only a small subset is expressed during in vitro cultivation. Eukaryot Cell 2: 501-509

Gaucher D, Chadee K (2003) Prospects for an Entamoeba histolytica Gal-lectin-based vaccine. Parasite lmmunol 25: 55-58

Ghosh S, Satish S, Tyagi S, Bhattacharya A, Bhattacharya S (2003) Differential use of multiple replication origins in the ribosomal DNA of the protozoan parasite Entamoeba histolytica. Nucleic Acids Res 31: 2035-2044

Gilchrist CA, Leo M, Line CG, Mann BJ, Petri WAJ (2003) Calcium modulates promoter occupancy by the Entamoeba histolytica Ca^sup 2+^- binding transcription factor URE3-BP. J Biol Chem 278: 4646-4653

Haque R, Huston CD, Hughes M, Houpt E, Petri WAJ (2003) Amebiasis. N Engl J Med 348: 1565-1573

Houpt ER, Glembocki DJ, Obrig TG, Moskaluk CA, Lockhart LA, Wright RL, Seaner RM, Keepers T, Wilkins TD, Petri WA Jr (2002) The mouse model of amebic colitis reveals mouse strain susceptibility to infection and exacerbation of disease by CD^sup 4+^ T cells. J Immunol 169: 4496-4503

Hughes MA, Lee CW, Holm CF, Ghosh S, Mills A, Lockhart LA, Reed SL, Mann BJ (2003) Identificaion of Entamoeba histolytica thiol- specific antioxidant as a GalNAc lectin-associated protein. Mol Biochem Parasitol 127: 113-120

Katz U, Bracha R, Nuchamowitz Y, Milstein O, Mirelman D (2003) Comparison between constitutive and inducible plasmid vectors used for gene expression in Entamoeba histolytica. Mol Biochem Parasitol 128: 229-233

Lopez-Camarillo C, Luna-Arias JP, Marchat LA, Orozco E (2003) EhPgp5 mRNA stability is a regulatory event in the Entamoeba histolytica multidrug resistance phenotype. J Biol Chem 278: 11273- 11280

Marchat LA, Gomez C, Perez DG, Paz F, Mendoza L, Orozco E (2002) Two CCAAT/enhancer binding protein sites are cis-activator elements of the Entamoeba histolytica EhPgp1 (mdr-like) gene expression. Cell Microbiol 4: 725-737

Moncada D, Keller K, Chadee K (2003) Entamoeba histolytica cysteine proteinases disrupt the polymeric structure of colonic mucin and alter its protective function. Infect Immun 71: 838-844

Olivos-Garcia A, Gonzalez-Canto A, Lopez-Vancell R, De Leon M, Tello E (2003) Amebic cysteine proteinase 2 (EhCP2) plays either a minor or no role in tissue damage in acute experimental amebic liver abscess in hamsters. Parasitol Res 90: 212-220

Rigothier MC, Khun H, Tavares P, Cardona A, Huerre M, Guillen N (2002) Fate of Entamoeba histolytica during establishment of amoebic liver abscess analyzed by quantitative radioimaging and histology. Infect Immun 70: 3208-3215

Solis F, Orozco E, Cordova L, Rivera B, Luna-Arias JP, Gomez- Conde E, Rodriguez MA (2002) Entamoeba histolytica: DNA carrier vesicles in nuclei and kinetoplast-like organelles (EhkOs). Mol Genet Genomics 267: 622-628

Stanley SLJ (2003) Amoebiasis. Lancet 361: 1025-1034

Ventura-Juarez J, Campos-Rodriguez R, Tsutsumi V (2002) Early interactions of Entamoeba histolytica trophozoites with parenchymal and inflammatory cells in the hamster liver: an immunocytochemical study. Can J Microbiol 48: 123-131

Wang Z, Samuelson J, Clark CG, Eichinger D, Paul J, Van Dellen K, Hall N, Anderson I, Loftus B (2003) Gene discovery in the Entamoeba invadens genome. Mol Biochem Parasitol 129: 23-31

Willhoeft U, Buss H, Tannich E (2002) The abundant polyadenylated transcript 2 DNA sequence of the pathogenic protozoan parasite Entamoeba histolytica represents a nonautonomous non-long-terminal- repeat-retrotransposon-like element which is absent in the closely related nonpathogenic species Entamoeba dispar. Infect Immun 70: 6798-6804

Zhang Z, Duchene M, Stanley SLJ (2002) A monoclonal antibody to the amebic lipophosphoglycan-proteophosphoglycan antigens can prevent disease in human intestinal xenografts infected with Entamoeba histolytica. Infect Immun 70: 5873-5876

Zimmer C, Labruyere E, Meas-Yedid V, Guillen N, Olivo-Marin JC (2002) Segmentation and tracking of migrating cells in videomicroscopy with parametric active contours: a tool for cell- based drug testing. IEEE Trans Med Imaging 21: 1212-1221

Egbert Tannicha,1, David Mirelmanb, and William A. Petri Jr.c

a Bernhard-Nocht Institute for Tropical Medicine, Bernhard-Nocht- Str. 74, D-20359 Hamburg, Germany

b Department of Biological Chemistry, Weizman Institute of Science, Rehovot, Israel

c Department of Internal Medicine, University of Virginia, Charlottesville, Virginia, USA

1 Corresponding author;

fax 49 40 42818 512

e-mail tannich@bni-hamburg.de

Copyright Urban & Fischer Verlag Oct 2003

More News in this Category