Structure and Content of the Entamoeba Histolytica Genome

C.G. Clark*; U.C.M. Alsmark†; M. Tazreiter‡; Y. Saito‐Nakano§; V. Ali¶; S. Marion||,1; C. Weber||; C. Mukherjee#; I. Bruchhaus**; E. Tannich**; M. Leippe††; T. Sicheritz‐Ponten‡‡; P.G. Foster§§; J. Samuelson¶¶; C.J. Noël†; R.P. Hirt†; T.M. Embley†; C.A. Gilchrist||||; B.J. Mann||||; U. Singh##; J.P. Ackers*; S. Bhattacharyaa; A. Bhattacharyab; A. Lohia#; N. Guillén||; M. Duchêne‡; T. Nozaki¶; N. Hallc,2    * Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London WC1E 7HT, UK
† Division of Biology, Newcastle University, Newcastle NE1 7RU, UK
‡ Department of Specific Prophylaxis and Tropical Medicine, Center for Physiology and Pathophysiology, Medical University of Vienna, A‐1090 Vienna, Austria
§ Department of Parasitology, National Institute of Infectious Diseases, Tokyo, Japan
¶ Department of Parasitology, Gunma University Graduate School of Medicine, Maebashi, Japan
|| Institut Pasteur, Unité Biologie Cellulaire du Parasitisme and INSERM U786, F‐75015 Paris, France
# Department of Biochemistry, Bose Institute, Kolkata 700054, India
** Bernhard Nocht Institute for Tropical Medicine, D‐20359 Hamburg, Germany
†† Zoologisches Institut der Universität Kiel, D‐24098 Kiel, Germany
‡‡ Center for Biological Sequence Analysis, BioCentrum‐DTU, Technical University of Denmark, DK‐2800 Lyngby, Denmark
§§ Department of Zoology, Natural History Museum, London, SW7 5BD, UK
¶¶ Department of Molecular and Cell Biology, Boston University Goldman School of Dental Medicine, Boston, Massachusetts 02118
|||| Department of Medicine, Division of Infectious Diseases, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908
## Departments of Internal Medicine, Microbiology, and Immunology, Stanford University School of Medicine, Stanford, California 94305
a School of Environmental Sciences, Jawaharlal Nehru University, New Delhi 110067, India
b School of Life Sciences and Information Technology, Jawaharlal Nehru University, New Delhi 110067, India
c The Institute for Genomic Research, Rockville, Maryland 20850
1 Present address: Cell Biology and Biophysics Program, European Molecular Biology Laboratory, 69117 Heidelberg, Germany
2 Present address: School of Biological Sciences, University of Liverpool, Liverpool L69 7ZB, United Kingdom

1 Introduction

Entamoeba histolytica is one of the most widespread and clinically important parasites, causing both serious intestinal (amoebic colitis) and extraintestinal (amoebic liver abscess) diseases throughout the world. A recent World Health Organization estimate (WHO, 1998) places E. histolytica second after Plasmodium falciparum as causing the most deaths annually (70,000) among protistan parasites.

Recently a draft of the complete genome of E. histolytica was published (Loftus et al., 2005) making it one of the first protist genomes to be sequenced. The E. histolytica genome project was initiated in 2000 with funding from the Wellcome Trust and the National Institute of Allergy and Infectious Diseases to the Wellcome Trust Sanger Institute and The Institute for Genomic Research (TIGR) in the UK and the USA, respectively. The publication describing the draft sequence concentrated on the expanded gene families, metabolism and the role of horizontal gene transfer in the evolution of E. histolytica. In this chapter we summarise the structure and content of the E. histolytica genome in comparison to other sequenced parasitic eukaryotes, provide a description of the current assembly and annotation, place the inferred gene content in the context of what is known about the biology of the organism and discuss plans for completing the E. histolytica genome project and extending genome sequencing to other species of Entamoeba.

The fact that the genome sequence is still a draft has several important consequences. The first is that a few genes may be missing from the sequence data we have at present, although the number is likely to be small. For example, at least one gene (amoebapore B) is not present in the genome data despite it having been cloned, sequenced and the protein extensively characterised well before the start of the genome project. The second consequence is that the assembly contains a number of large duplicated regions that may be assembly artefacts, meaning that the number of gene copies is overestimated in several cases. These problems cannot as yet be resolved but should be eventually as more data becomes available. Nevertheless, it is important to remember these issues when reading the rest of this chapter.

As the number of genes in E. histolytica runs into several thousands, it is not possible to discuss all of them. However, we have generated a number of tables that identify many genes and link them to their entries in GenBank using the relevant protein identifier. Only a few tables are included in the text of this chapter, but the others are available online as supplementary material, http://pathema.tigr.org/pathema/entamoeba_resources.shtml. The E. histolytica genome project data are being ‘curated’ at the J. Craig Venter Institute (JCVI, formerly TIGR), and it is on that site that the most current version of the assembled genome will be found. The ‘Pathema’ database will hold the data and the annotation (http://pathema.tigr.org/). The gene tables are also linked to the appropriate entry in the Pathema database, and the links will be maintained as the genome structure is refined over time.

Reference is made throughout the text to other species of Entamoeba where data are available. Entamoeba dispar is the sister species to E. histolytica and infects humans without causing symptoms. Entamoeba invadens is a reptilian parasite that causes invasive disease, primarily in snakes and lizards, and is widely used as a model for E. histolytica in the study of encystation, although the two species are not very closely related (Clark et al., 2006b). Genome projects for both these species are under way at TIGR, and it is anticipated that high‐quality draft sequences will be produced for both in the near future. It is hoped that the E. dispar sequence will prove useful in identifying genomic differences linked to disease causation while that of E. invadens will be used to study patterns of gene expression during encystation. Small‐scale genome surveys have been performed for two other species: Entamoeba moshkovskii, which is primarily a free‐living species although it occasionally infects humans, and Entamoeba terrapinae, a reptilian commensal species, http://www.sanger.ac.uk/Projects/Comp_Entamoeba/

2 Genome Structure

2.1 The E. histolytica genome sequencing, assembly and annotation process

The first choice to be made in the genome project was perhaps the easiest—the identity of the strain to be used for sequencing. A significant majority of the existing sequence data prior to the genome project was derived from one strain: HM‐1:IMSS. This culture was established in 1967 from a rectal biopsy of a Mexican man with amoebic dysentery and axenised shortly thereafter. It has been used widely for virulence, immunology, cell biology and biochemistry in addition to genetic studies. In an attempt to minimise the effects of long‐term culture cryopreserved cells that had been frozen in the early 1970s were revived and this uncloned culture used to generate the DNA for sequencing.

Before undertaking a genome scale analysis, it is important to understand the quality and provenance of the underlying data. The E. histolytica genome was sequenced by whole genome shotgun approach with each centre generating roughly half of the reads. Several different DNA libraries containing inserts of different sizes were produced using DNA that had been randomly sheared and sequences were obtained from both ends of each cloned fragment. The Phusion assembler (Mullikin and Ning, 2003) was used to assemble the 450,000 short...