Entamoeba histolytica Encodes a Highly Divergent β-Tubulin

ABSTRACT. The microtubules of the amitochondrial parasite Entamoeba histolytica are atypical in certain respects. Consistent with this, we report that E. histolytica encodes the most divergent β -tubulin identified to date, with only 54% to 58% identity to β -tubulins from various species. A similarly divergent β -tubulin is encoded by the related Entamoeba invadens ; single gene copies appear to be present in both organisms. The Entamoeba sequences were compared with a database of 101 β -tubulins, including the highly divergent sequence from another amitochondrial protozoan, Trichomonas vaginalis. A total of 81 residues were universally conserved, and 76 residues varied only once. Correlations with previous studies indicate that microtubule function is altered when most, but not all, conserved residues are mutated.     

Entamoeba invadens: in vitro axenic encystation with a serum substitute

The current media for axenic cultivation of Entamoeba histolytica and Entamoeba invadens are supplemented with bovine or equine serum, which provides several essential nutrients to amoebas. Serum has also been considered an essential component in encystation media for E. invadens. A substitute of serum, PACSR has been described as an alternative for growth of E. histolytica and also maintains growth of E. invadens. When PACSR was used instead of serum for encystation of E. invadens the efficiency was the same as for serum. Our present data show that PACSR can support the growth and induction of encystation of E. invadens strain IP-1.     

Entamoeba histolytica extrachromosomal circular ribosomal DNA: analysis of clonal variation in a hypervariable region.

The ribosomal RNA genes of the protozoan parasite Entamoeba histolytica are highly repeated and display restriction fragment length polymorphism. Using a set of four DNA probes spanning the coding region and part of the flanking region of the E. histolytica ribosomal RNA genes, an analysis of the DNA bands generated by EcoRI digestion of Entamoeba DNA is presented. This analysis included five strains of E. histolytica, four strains of E. moshkovskii, and one strain each of E. invadens and E. terrapinae. No common bands were observed between E. histolytica and the other Entamoeba. Within E. histolytica, two bands were conserved in all strains while the others were polymorphic. Detailed analysis of DNA from independently isolated clones of the strain HM-1:IMSS of E. histolytica showed two bands to be highly polymorphic. Of these, the 4.4-kb band of clone 6 was further analyzed. Polymorphism in this band could even be demonstrated in cells of the same clone. Restriction enzyme analysis of this DNA band from two clones of HM-1:IMSS showed that the polymorphism may be due to variable numbers of DraI repeat units present in this DNA stretch.     

Effect of jasplakinolide on the growth, encystation, and actin cytoskeleton of Entamoeba histolytica and Entamoeba invadens

The effect of jasplakinolide. an actin-polymerizing and filament-stabilizing drug, on the growth, encystation, and actin cytoskeleton of Entamoeba histolytica and Entamoeba invadens was examined. Jasplakinolide inhibited the growth of E. histolytica strain HM-1:IMSS and E. invadens strain IP-1 in a concentration-dependent manner, the latter being more resistant to the drug. The inhibitory effect of jasplakinolide on the growth of E. histolytica trophozoites was reversed by removal of the drug after exposure to 1 microM for 1 day. Encystation of E. invadens as induced in vitro was also inhibited by jasplakinolide. Trophozoites exposed to jasplakinolide in encystation medium for 1 day did not encyst after removal of the drug, whereas those exposed to the drug in growth medium for 7 days did encyst without the drug. The process of cyst maturation was unaffected by jasplakinolide. Large round structures were formed in trophozoites of both amoebae grown with jasplakinolide; these were identified as F-actin aggregates by staining with fluorescent phalloidin. Accumulation in trophozoites of both amoebae of actin aggregates was observed after culture in jasplakinolide. Also, E. invadens cysts formed from trophozoites treated with jasplakinolide contained the actin aggregate. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis revealed that the jasplakinolide treatment led to an increase in the proportion of F-actin associated with formation of the aggregate. The results suggest that aggregates are formed from the cortical flow of F-actin filaments, and that these filaments would normally be depolymerized but are artificially stabilized by jasplakinolide binding.     

Heterogeneity of the ribosomal DNA episome in strains and species of Entamoeba

Ribosomal DNA sequences in several species of the genus Entamoeba are highly repeated and display restriction fragment-length polymorphism (RFLP), which has been used to identify species and differentiate strains. However, the continuous variability of the non-transcribed repeat sequences in the ribosomal episome hinders an accurate typification. Looking for more reliable markers, we used DNA probes containing conserved sequences in the ribosomal episome — coding regions for the 16S and 5.8S rRNAs and transcribed spacers flanking the rDNA sequences, and the coding region for the 3 end of the 26S rRNA — to analyse hybridization patterns from five cloned pathogenic strains of Entamoeba histoiytica, two strains of the also pathogenic Entamoeba invadens and the non-pathogenic Laredo strain of Entamoeba moshkovskii. Our results provide reliable bases for the differentation of clones, strains and species of Entamoeba and the reconstruction of E. histolytica episomes. Differences in the number and length of rDNA-containing DNA fragments, previously observed by other investigators and confirmed by us, can be better defined by the present analysis.     

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.     

Comparative genomic hybridizations of Entamoeba strains reveal unique genetic fingerprints that correlate with virulence

Variable phenotypes have been identified for Entamoeba species. Entamoeba histolytica is invasive and causes colitis and liver abscesses but only in approximately 10% of infected individuals; 90% remain asymptomatically colonized. Entamoeba dispar, a closely related species, is avirulent. To determine the extent of genetic diversity among Entamoeba isolates and potential genotype-phenotype correlations, we have developed an E. histolytica genomic DNA microarray and used it to genotype strains of E. histolytica and E. dispar. On the basis of the identification of divergent genetic loci, all strains had unique genetic fingerprints. Comparison of divergent genetic regions allowed us to distinguish between E. histolytica and E. dispar, identify novel genetic regions usable for strain and species typing, and identify a number of genes restricted to virulent strains. Among the four E. histolytica strains, a strain with attenuated virulence was the most divergent and phylogenetically distinct strain, raising the intriguing possibility that genetic subtypes of E. histolytica may be partially responsible for the observed variability in clinical outcomes. This microarray-based genotyping assay can readily be applied to the study of E. histolytica clinical isolates to determine genetic diversity and potential genotypic-phenotypic associations.     

Elucidation of the DNA Synthetic Cycle of Entamoeba Spp. Using Flow Cytometry and Mathematical Modeling

ABSTRACT. We developed a method to study the DNA synthetic cycles of Entamoeba histolytica and Entamoeba invadens by flow cytometry (FCM) based on a preparative procedure to reduce both high levels of natural fluorescence and non-specific adsorption of fluorochromes. We modeled G₁, S, and G₂ phases as a series of overlapping Gaussian curves. Both E. histolytica and E. invadens displayed G₁, S, and G₂ proportions that are consistent with eukaryotic cell populations in exponential or stationary growth phase. Exponential phase E. histolytica populations contained a hypodiploid subset with a mass of about 20% less than the diploid value which we estimate by FCM to be 24 × 10^-14 g DNA/cell. Exponential phase E. invadens populations contained a hypodiploid subset with a mass of about 6% less than the diploid value which we estimate by FCM to be 30 × 10^-14 g DNA/cell.     

Characterisation of Ascaris from human and pig hosts by nuclear ribosomal DNA sequences.

The sequences of the nuclear ribosomal DNA region spanning the first internal transcribed spacer, the 5.8S rRNA gene and the second internal transcribed spacer were determined for Ascaris samples from pigs and humans from different geographical regions. The sequences of the 5.8S gene and the second internal transcribed spacer were the same for all samples examined, whereas all Ascaris samples from humans had six (1.3%) nucleotide differences in the first internal transcribed spacer compared with those from pigs. These differences provided some support for the existence of separate species of Ascaris or population variation within this genus. Using a nucleotide difference within a site for the restriction enzyme HaeIII, a PCR-linked restriction fragment length polymorphism method was established which allowed the delineation of the Ascaris samples from pigs and humans used herein. Exploiting the sequence differences in the first internal transcribed spacer, a PCR-based single-strand conformation polymorphism method was established for future analysis of the genetic structure of pig and human Ascaris populations in sympatric and allopatric zones.     

Entamoeba shows reversible variation in ploidy under different growth conditions and between life cycle phases

Under axenic growth conditions, trophozoites of Entamoeba histolytica contain heterogenous amounts of DNA due to the presence of both multiple nuclei and different amounts of DNA in individual nuclei. In order to establish if the DNA content and the observed heterogeneity is maintained during different growth conditions, we have compared E. histolytica cells growing in xenic and axenic cultures. Our results show that the nuclear DNA content of E. histolytica trophozoites growing in axenic cultures is at least 10 fold higher than in xenic cultures. Re-association of axenic cultures with their bacterial flora led to a reduction of DNA content to the original xenic values. Thus switching between xenic and axenic growth conditions was accompanied by significant changes in the nuclear DNA content of this parasite. Changes in DNA content during encystation-excystation were studied in the related reptilian parasite E. invadens. During excystation of E. invadens cysts, it was observed that the nuclear DNA content increased approximately 40 fold following emergence of trophozoites in axenic cultures. Based on the observed large changes in nuclear size and DNA content, and the minor differences in relative abundance of representative protein coding sequences, rDNA and tRNA sequences, it appears that gain or loss of whole genome copies may be occurring during changes in the growth conditions. Our studies demonstrate the inherent plasticity and dynamic nature of the Entamoeba genome in at least two species.     

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.     

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.     

Effect of dinitroaniline herbicides on the growth of Entamoeba histolytica

The effect of the dinitroaniline herbicides oryzalin and trifluralin on the growth of Entamoeba histolytica was examined. Oryzalin inhibited the growth of E. histolytica strain HM-1:IMSS. Trifluralin was less effective than oryzalin for this parasite. Entamoeba histolytica was more resistant to these dinitroanilines than other parasitic protozoa examined so far, including Leishmania spp., Trypanosoma brucei, Plasmodium falciparum, Toxoplasma gondii, and Cryptosporidium parvum. Colchicine, a potent microtubule inhibitor of animal cells, was much less effective for E. histolytica, even at very high concentrations. A reptilian parasite, Entamoeba invadens strain IP-1, examined for comparison, was more resistant to these dinitroanilines than E. histolytica. Accumulation of E. histolytica trophozoites in mitosis was observed after culture in 100 microM oryzalin. The inhibitory effect of oryzalin on the growth of E. histolytica trophozoites was abrogated by removal of the drug after exposure to 100 microM for 2 days. In parallel to the recovery of growth after removal of the drug, the percentage of trophozoites in mitosis was reduced to a normal level. The results indicate that treatment of trophozoites with oryzalin arrests mitosis and that its effect is reversible. Therefore, oryzalin is a useful tool for studies relating to the cell cycle of this parasite.     

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.     

Torulaspora maleeae sp. nov., a novel ascomycetous yeast species from Japan and Thailand

Nine strains of a new Torulaspora species were isolated from natural samples collected in Japan and Thailand including one strain obtained from a leaf of Rhizophora stylosa (NBRC 11061T), one strain from soil (NBRC 11062), six strains from mosses (ST-14, ST-266, ST-510, ST-511, ST-513 and ST-581) and one strain from sediment in mangrove forest (RV-51). On the basis of morphological, biochemical, physiological and chemotaxonomic characteristics, and the sequence analyses of the D1/D2 domain of the large subunit (LSU) rRNA gene and the internal transcribed spacer (ITS) (ITS1-5.8S rRNA gene-ITS2) region, the nine strains were found to represent a single novel species of the genus Torulaspora, which were named Torulaspora maleeae sp. nov. The type strain is NBRC 11061T (BCC 25515T=CBS 10694T). In the phylogenetic trees based on the sequences of the D1/D2 domain of the LSU rRNA gene, T. maleeae showed a close relationship with the five recognized species of the genus Torulaspora, Torulaspora delbrueckii, Torulaspora franciscae, Torulaspora globosa, Torulaspora microellipsoides and Torulaspora pretoriensis. Torulaspora maleeae differed from the five recognized species of the genus Torulaspora by six to 12 nucleotide substitutions (1.1-2.1%) in the D1/D2 domain of the LSU rRNA gene and by 6.4-11.7% nucleotide substitutions in the ITS (ITS1-5.8S rRNA gene-ITS2) region.     

High-resolution electron microscopical study of cyst walls of Entamoeba spp

Knowledge of the fine structural organization, molecular composition and permeability properties of the cell surface of intestinal protozoan cysts is important to understand the biologic basis of their resistance. Recent studies on the biology of the cyst walls of Entamoeba histolytica and Entamoeba invadens have considerably advanced knowledge on the cellular processes involved in the transport and surface deposition of the main cyst wall components. Using transmission electron microscopy, cytochemistry, scanning electron microscopy and freeze-fracture techniques, we have obtained new information. In mature cysts the permeability of Entamoeba cysts is limited to small molecules not by the cyst wall, but by the plasma membrane, as demonstrated with the use of ruthenium red as an electron-dense tracer. Cell walls of E. histolytica cysts are made up of five to seven layers of unordered fibrils 7-8 nm thick. Alcian blue stains a regular mesh of fibrils approximately 4 nm thick, running perpendicularly to the cyst wall. In addition, abundant ionogenic groups are seen in cyst walls treated with cationized ferritin. In the mature cysts of E. histolytica and E. invadens small cytoplasmic vesicles with granular material were in close contact with the plasma membrane, suggesting a process of fusion and deposition of granular material to the cell wall. The plasma membrane of mature cysts is devoid of intramembrane particles when analyzed with the freeze-fracture technique. When viewed with scanning electron microscopy the surface of E. histolytica cysts clearly differs from that of Entamoeba coli and E. invadens.     

A role for a galactose lectin and its ligands during encystment of Entamoeba

In the life cycle of Entamoeba parasites alternate between the colon-dwelling trophozoite and the infectious cyst forms. The physiologic stimuli that trigger differentiation of trophozoites into cysts remain undefined. On the surface of the human-infecting Entamoeba, parasites express a galactose/N-acetylgatactosamine (gal/galNAc)-binding lectin, which plays demonstrated roles in contact-dependent lysis of target cells and resistance to host complement. Using a reptilian parasite, Entamoeba invadens, to study cyst formation in vitro, we found that efficient encystation was dependent on the presence of gal-terminated ligands in the induction medium. Precise concentration ranges of several gal-terminated ligands, such as asialofetuin, gal-bovine serum albumin (gal-BSA), and mucin, functioned in encystation medium to stimulate differentiation. Greater than 10 mM levels of free gal inhibited the amoeba aggregation that precedes encystation and prevented formation of mature cysts. Inhibitory levels of gal also prevented the up-regulation of genes which normally occurs at 24 h of encystation. The surface of Entamoeba invadens was found to express a gal lectin which has a heterodimeric structure similar to that of Entamoeba histolytica. The 30 kDa light subunit (LGL) of the E. invadens lectin is similar in overall size and sequence to the LGL of E. histolytica. The heavy subunits, however, differ in size, have an identical spacing of cysteines in their extracellular domains, and have highly conserved C-terminal transmembrane and cytoplasmic domains. These results suggest a new role for the Entamoeba gal lectins in monitoring the concentrations of gal ligands in the colon and contributing to stimuli that induce encystment.