Sampling gene diversity across the supergroup Amoebozoa: large EST data sets from Acanthamoeba castellanii, Hartmannella vermiformis, Physarum polycephalum, Hyperamoeba dachnaya and Hyperamoeba sp

From comparative analysis of EST data for five taxa within the eukaryotic supergroup Amoebozoa, including two free-living amoebae (Acanthamoeba castellanii, Hartmannella vermiformis) and three slime molds (Physarum polycephalum, Hyperamoeba dachnaya and Hyperamoeba sp.), we obtained new broad-range perspectives on the evolution and biosynthetic capacity of this assemblage. Together with genome sequences for the amoebozoans Dictyostelium discoideum and Entamoeba histolytica, and including partial genome sequence available for A. castellanii, we used the EST data to identify genes that appear to be exclusive to the supergroup, and to specific clades therein. Many of these genes are likely involved in cell-cell communication or differentiation. In examining on a broad scale a number of characters that previously have been considered in simpler cross-species comparisons, typically between Dictyostelium and Entamoeba, we find that Amoebozoa as a whole exhibits striking variation in the number and distribution of biosynthetic pathways, for example, ones for certain critical stress-response molecules, including trehalose and mannitol. Finally, we report additional compelling cases of lateral gene transfer within Amoebozoa, further emphasizing that although this process has influenced genome evolution in all examined amoebozoan taxa, it has done so to a variable extent.     

Entamoeba histolytica: observations on metabolism based on the genome sequence

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.     

Studies on codon usage in Entamoeba histolytica

Codon usage bias of Entamoeba histolytica, a protozoan parasite, was investigated using the available DNA sequence data. Entamoeba histolytica having AT rich genome, is expected to have A and/or T at the third position of codons. Overall codon usage data analysis indicates that A and/or T ending codons are strongly biased in the coding region of this organism. However, multivariate statistical analysis suggests that there is a single major trend in codon usage variation among the genes. The genes which are supposed to be highly expressed are clustered at one end, while the majority of the putatively lowly expressed genes are clustered at the other end. The codon usage pattern is distinctly different in these two sets of genes. C ending codons are significantly higher in the putatively highly expressed genes suggesting that C ending codons are translationally optimal in this organism. In the putatively lowly expressed genes A and/or T ending codons are predominant, which suggests that compositional constraints are playing the major role in shaping codon usage variation among the lowly expressed genes. These results suggest that both mutational bias and translational selection are operational in the codon usage variation in this organism.     

Identification and cellular localization of the actin-binding protein ABP-120 from Entamoeba histolytica

Several actin-binding proteins participate in the morphological changes that occur during amoeboid movement. The gene encoding one of these proteins, the gelation factor ABP-120, was identified and characterized from trophozoites of Entamoeba histolytica . The sequence contains 2574 nucleotides, with an open reading frame of 858 amino acids, giving a protein of 93 kDa belonging to the spectrin family. The N-terminal domain of ABP-120 from E. histolytica revealed a consensus site for actin binding homologous to the actin-binding sites of ABP-120 of Dictyostelium discoideum , α-actinin and spectrin. Analysis of the central domain revealed the presence of four repeats of a 73-amino-acid motif constituting 31% of the protein. In addition, a stretch of 105 amino acids was highly divergent when compared with the C-terminal domain of D. discoideum ABP-120. This sequence showed short motifs that are homologous to microtubule-binding domains. We found that ABP-120 from E. histolytica binds to F-actin. In addition, upon motility of the parasite, this protein localized in the pseudopod and the uroid region, implying a role for ABP-120 in movement and capping of surface receptors in E. histolytica .     

Inter- and intra-strain variation in the 5.8S ribosomal RNA and internal transcribed spacer sequences of Entamoeba histolytica and comparison with Entamoeba dispar, Entamoeba moshkovskii and Entamoeba invadens

The ribosomal RNA genes in Entamoeba histolytica are located on circular DNA molecules in about 200 copies per genome equivalent. Nucleotide sequence analysis of the 5.8S rRNA gene and the flanking internal transcribed spacers was carried out to determine the degree of sequence divergence in the multiple rRNA gene copies of a given strain; amongst three different E. histolytica strains (HM-1:IMSS, Rahman and HK-9); and amongst four species of Entamoeba (Entamoeba histolytica, Entamoeba dispar, Entamoeba moshkovskii and Entamoeba invadens). The results show that all rRNA gene copies of a given strain are identical. Few nucleotide positions varied between strains of a species but the differences were very pronounced amongst species. In general, the internal transcribed spacer 2 sequence was more variable and may be useful for strain- and species-identification. The 5.8S rRNA gene and the internal transcribed spacer 2 of E. invadens were unusually small in size.     

Unexpected diversity and differential success of DNA transposons in four species of entamoeba protozoans

We report the first comprehensive analysis of transposable element content in the compact genomes (approximately 20 Mb) of four species of Entamoeba unicellular protozoans for which draft sequences are now available. Entamoeba histolytica and Entamoeba dispar, two human parasites, have many retrotransposons, but few DNA transposons. In contrast, the reptile parasite Entamoeba invadens and the free-living Entamoeba moshkovskii contain few long interspersed elements but harbor diverse and recently amplified populations of DNA transposons. Representatives of three DNA transposase superfamilies (hobo/Activator/Tam3, Mutator, and piggyBac) were identified for the first time in a protozoan species in addition to a variety of members of a fourth superfamily (Tc1/mariner), previously reported only from ciliates and Trichomonas vaginalis among protozoans. The diversity of DNA transposons and their differential amplification among closely related species with similar compact genomes are discussed in the context of the biology of Entamoeba protozoans.     

Iron hydrogenases and the evolution of anaerobic eukaryotes

Hydrogenases, oxygen-sensitive enzymes that can make hydrogen gas, are key to the function of hydrogen-producing organelles (hydrogenosomes), which occur in anaerobic protozoa scattered throughout the eukaryotic tree. Hydrogenases also play a central role in the hydrogen and syntrophic hypotheses for eukaryogenesis. Here, we show that sequences related to iron-only hydrogenases ([Fe] hydrogenases) are more widely distributed among eukaryotes than reports of hydrogen production have suggested. Genes encoding small proteins which contain conserved structural features unique to [Fe] hydrogenases were identified on all well-surveyed aerobic eukaryote genomes. Longer sequences encoding [Fe] hydrogenases also occur in the anaerobic eukaryotes Entamoeba histolytica and Spironucleus barkhanus, both of which lack hydrogenosomes. We also identified a new [Fe] hydrogenase sequence from Trichomonas vaginalis, bringing the total of [Fe] hydrogenases reported for this organism to three, all of which may function within its hydrogenosomes. Phylogenetic analysis and hypothesis testing using likelihood ratio tests and parametric bootstrapping suggest that the [Fe] hydrogenases in anaerobic eukaryotes are not monophyletic. Iron-only hydrogenases from Entamoeba, Spironucleus, and Trichomonas are plausibly monophyletic, consistent with the hypothesis that a gene for [Fe] hydrogenase was already present on the genome of the common, perhaps also anaerobic, ancestor of these phylogenetically distinct eukaryotes. Trees where the [Fe] hydrogenase from the hydrogenosomal ciliate Nyctotherus was constrained to be monophyletic with the other eukaryote sequences were rejected using a likelihood ratio test of monophyly. In most analyses, the Nyctotherus sequence formed a sister group with a [Fe] hydrogenase on the genome of the eubacterium Desulfovibrio vulgaris. Thus, it is possible that Nyctotherus obtained its hydrogenosomal [Fe] hydrogenase from a different source from Trichomonas for its hydrogenosomes. We find no support for the hypothesis that components of the Nyctotherus [Fe] hydrogenase fusion protein derive from the mitochondrial respiratory chain.     

Choke point analysis of metabolic pathways in E.histolytica: a computational approach for drug target identification

With the Entamoeba genome essentially complete, the organism can be studied from a whole genome standpoint. The understanding of cellular mechanisms and interactions between cellular components is instrumental to the development of new effective drugs and vaccines. Metabolic pathway analysis is becoming increasingly important for assessing inherent network properties in reconstructed biochemical reaction networks. Metabolic pathways illustrate how proteins work in concert to produce cellular compounds or to transmit information at different levels. Identification of drug targets in E. histolytica through metabolic pathway analysis promises to be a novel approach in this direction. This article focuses on the identification of drug targets by subjecting the Entamoeba genome to BLAST with the e-value inclusion threshold set to 0.005 and choke point analysis. A total of 86.9 percent of proposed drug targets with biological evidence are chokepoint reactions in Entamoeba genome database.     

Patterns of evolution in the unique tRNA gene arrays of the genus Entamoeba

Genome sequencing of the protistan parasite Entamoeba histolytica HM-1:IMSS revealed that almost all the tRNA genes are organized into tandem arrays that make up over 10% of the genome. The 25 distinct array units contain up to 5 tRNA genes each and some also encode the 5S RNA. Between adjacent genes in array units are complex short tandem repeats (STRs) resembling microsatellites. To investigate the origins and evolution of this unique gene organization, we have undertaken a genome survey to determine the array unit organization in 4 other species of Entamoeba-Entamoeba dispar, Entamoeba moshkovskii, Entamoeba terrapinae, and Entamoeba invadens-and have explored the STR structure in other isolates of E. histolytica. The genome surveys revealed that E. dispar has the same array unit organization as E. histolytica, including the presence and numerical variation of STRs between adjacent genes. However, the individual repeat sequences are completely different to those in E. histolytica. All other species of Entamoeba studied also have tandem arrays of clustered tRNA genes, but the gene composition of the array units often differs from that in E. histolytica/E. dispar. None of the other species' arrays exhibit the complex STRs between adjacent genes although simple tandem duplications are occasionally seen. The degree of similarity in organization reflects the phylogenetic relationships among the species studied. Within individual isolates of E. histolytica most copies of the array unit are uniform in sequence with only minor variation in the number and organization of the STRs. Between isolates, however, substantial differences in STR number and organization can exist although the individual repeat sequences tend to be conserved. The origin of this unique gene organization in the genus Entamoeba clearly predates the common ancestor of the species investigated to date and their function remains unclear.     

The mitosome, a novel organelle related to mitochondria in the amitochondrial parasite Entamoeba histolytica

Ultrastructural analysis of Entamoeba histolytica reveals that this intestinal human pathogen lacks recognizable mitochondria, but the presence in its genome of genes encoding proteins of mitochondrial origin suggests the existence of a mitochondrially derived compartment. We have cloned the full-length E. histolytica gene encoding one such protein, chaperonin CPN60, and have characterized its structure and expression. Using an affinity-purified antibody raised against recombinant protein, we have localized native E. histolytica CPN60 to a previously undescribed organelle of putative mitochondrial origin, the mitosome. Most cells contain only one mitosome, as determined by immunofluorescence studies. Entamoeba histolytica CPN60 has an amino-terminal extension reminiscent of known mitochondrial and hydrogenosomal targeting signals. Deletion of the first 15 amino acids of CPN60 leads to an accumulation of the truncated protein in the cytoplasm. However, this mutant phenotype can be reversed by replacement of the deleted amino acids with a mitochondrial targeting signal from Trypanosoma cruzi HSP70. The observed functional conservation between mitochondrial import in trypanosomes and mitosome import in Entamoeba is strong evidence that the E. histolytica organelle housing chaperonin CPN60 represents a mitochondrial remnant.     

Gene discovery in the Acanthamoeba castellanii genome

Acanthamoeba castellanii is a free-living amoeba found in soil, freshwater, and marine environments and an important predator of bacteria. Acanthamoeba castellanii is also an opportunistic pathogen of clinical interest, responsible for several distinct diseases in humans. In order to provide a genomic platform for the study of this ubiquitous and important protist, we generated a sequence survey of approximately 0.5 x coverage of the genome. The data predict that A. castellanii exhibits a greater biosynthetic capacity than the free-living Dictyostelium discoideum and the parasite Entamoeba histolytica, providing an explanation for the ability of A. castellanii to inhabit a diversity of environments. Alginate lyase may provide access to bacteria within biofilms by breaking down the biofilm matrix, and polyhydroxybutyrate depolymerase may facilitate utilization of the bacterial storage compound polyhydroxybutyrate as a food source. Enzymes for the synthesis and breakdown of cellulose were identified, and they likely participate in encystation and excystation as in D. discoideum. Trehalose-6-phosphate synthase is present, suggesting that trehalose plays a role in stress adaptation. Detection and response to a number of stress conditions is likely accomplished with a large set of signal transduction histidine kinases and a set of putative receptor serine/threonine kinases similar to those found in E. histolytica. Serine, cysteine and metalloproteases were identified, some of which are likely involved in pathogenicity.     

Species- and strain-specific probes derived from repetitive DNA for distinguishing Entamoeba histolytica and Entamoeba dispar

Entamoeba histolytica and Entamoeba dispar are two morphologically indistinguishable species that are found in the human gut. Of the two, E. histolytica is considered to be pathogenic while E. dispar is nonpathogenic. To generate molecular probes to detect and distinguish between the two species, we utilized repeat sequences present in Entamoeba genome. We have developed probes and primers from rDNA episomes, and unidentified Entamoeba EST1 repeat for this purpose, and used them for dot blot hybridization and PCR amplification. To investigate the possible existence of invasive and noninvasive strains of E. histolytica, the ability to differentiate individual isolates is necessary. For this purpose, we have utilized a modification of the AFLP procedure called 'Transposon display,' which generates and displays large number of genomic bands associated with a transposon. We have used the abundant retrotransposon, EhSINE1, for this purpose,and demonstrated its potential as a marker to study strain variation in E. histolytica. This technique could suitably be employed in carrying out significant molecular epidemiological studies and large-scale typing of this parasite.     

Analysis of the protein kinome of Entamoeba histolytica

Protein kinases play important roles in almost all major signaling and regulatory pathways of eukaryotic organisms. Members in the family of protein kinases make up a substantial fraction of eukaryotic proteome. Analysis of the protein kinase repertoire (kinome) would help in the better understanding of the regulatory processes. In this article, we report the identification and analysis of the repertoire of protein kinases in the intracellular parasite Entamoeba histolytica. Using a combination of various sensitive sequence search methods and manual analysis, we have identified a set of 307 protein kinases in E. histolytica genome. We have classified these protein kinases into different subfamilies originally defined by Hanks and Hunter and studied these kinases further in the context of noncatalytic domains that are tethered to catalytic kinase domain. Compared to other eukaryotic organisms, protein kinases from E. histolytica vary in terms of their domain organization and displays features that may have a bearing in the unusual biology of this organism. Some of the parasitic kinases show high sequence similarity in the catalytic domain region with calmodulin/calcium dependent protein kinase subfamily. However, they are unlikely to act like typical calcium/calmodulin dependent kinases as they lack noncatalytic domains characteristic of such kinases in other organisms. Such kinases form the largest subfamily of kinases in E. histolytica. Interestingly, a PKA/PKG-like subfamily member is tethered to pleckstrin homology domain. Although potential cyclins and cyclin-dependent kinases could be identified in the genome the likely absence of other cell cycle proteins suggests unusual nature of cell cycle in E. histolytica. Some of the unusual features recognized in our analysis include the absence of MEK as a part of the Mitogen Activated Kinase signaling pathway and identification of transmembrane region containing Src kinase-like kinases. Sequences which could not be classified into known subfamilies of protein kinases have unusual domain architectures. Many such unclassified protein kinases are tethered to domains which are Cysteine-rich and to domains known to be involved in protein-protein interactions. Our kinome analysis of E. histolytica suggests that the organism possesses a complex protein phosphorylation network that involves many unusual kinases.     

Very little intron gain in Entamoeba histolytica genes laterally transferred from prokaryotes

The evolution of spliceosomal introns remains intensely debated. We studied 96 Entamoeba histolytica genes previously identified as having been laterally transferred from prokaryotes, which were presumably intronless at the time of transfer. Ninety out of the 96 are also present in the reptile parasite Entamoeba invadens, indicating lateral transfer before the species' divergence approximately 50 MYA. We find only 2 introns, both shared with E. invadens. Thus, no intron gains have occurred in approximately 50 Myr, implying a very low rate of intron gain of less than one gain per gene per approximately 4.5 billion years. Nine other predicted introns are due to annotation errors reflecting apparent mistakes in the E. histolytica genome assembly. These results underscore the massive differences in intron gain rates through evolution.     

Identification and gene expression analysis of a large family of transmembrane kinases related to the Gal/GalNAc lectin in Entamoeba histolytica

We identified in the Entamoeba histolytica genome a family of over 80 putative transmembrane kinases (TMKs). The TMK extracellular domains had significant similarity to the intermediate subunit (Igl) of the parasite Gal/GalNAc lectin. The closest homolog to the E. histolytica TMK kinase domain was a cytoplasmic dual-specificity kinase, SplA, from Dictyostelium discoideum. Sequence analysis of the TMK family demonstrated similarities to both serine/threonine and tyrosine kinases. TMK genes from each of six phylogenetic groups were expressed as mRNA in trophozoites, as assessed by spotted oligoarray and real-time PCR assays, suggesting nonredundant functions of the TMK groups for sensing and responding to extracellular stimuli. Additionally, we observed changes in the expression profile of the TMKs in continuous culture. Antisera produced against the conserved kinase domain identified proteins of the expected molecular masses of the expressed TMKs. Confocal microscopy with anti-TMK kinase antibodies revealed a focal distribution of the TMKs on the cytoplasmic face of the trophozoite plasma membrane. We conclude that E. histolytica expresses members of each subgroup of TMKs. The presence of multiple receptor kinases in the plasma membrane offers for the first time a potential explanation of the ability of the parasite to respond to the changing environment of the host.     

Entamoeba histolytica: construction and applications of subgenomic databases

Knowledge about the influence of environmental stress such as the action of chemotherapeutic agents on gene expression in Entamoeba histolytica is limited. We plan to use oligonucleotide microarray hybridization to approach these questions. As the basis for our array, sequence data from the genome project carried out by the Institute for Genomic Research (TIGR) and the Sanger Institute were used to annotate parts of the parasite genome. Three subgenomic databases containing enzymes, cytoskeleton genes, and stress genes were compiled with the help of the ExPASy proteomics website and the BLAST servers at the two genome project sites. The known sequences from reference species, mostly human and Escherichia coli, were searched against TIGR and Sanger E. histolytica sequence contigs and the homologs were copied into a Microsoft Access database. In a similar way, two additional databases of cytoskeletal genes and stress genes were generated. Metabolic pathways could be assembled from our enzyme database, but sometimes they were incomplete as is the case for the sterol biosynthesis pathway. The raw databases contained a significant number of duplicate entries which were merged to obtain curated non-redundant databases. This procedure revealed that some E. histolytica genes may have several putative functions. Representative examples such as the case of the delta-aminolevulinate synthase/serine palmitoyltransferase are discussed.