Structure and content of the Entamoeba histolytica genome.

The intestinal parasite Entamoeba histolytica is one of the first protists for which a draft genome sequence has been published. Although the genome is still incomplete, it is unlikely that many genes are missing from the list of those already identified. In this chapter we summarise the features of the genome as they are currently understood and provide previously unpublished analyses of many of the genes.

The cell cycle of Entamoeba invadens during vegetative growth and differentiation.

The cell division cycle of Entamoeba invadens was studied during vegetative growth of trophozoites and during their differentiation into cysts. During vegetative growth of trophozoites, it was observed that DNA synthesis typically continued after one genome content had been duplicated. During encystation, DNA synthesis was arrested after 4n genome content had been synthesised. Using multi-parameter flow cytometry, the light scattering properties of cysts and trophozoites were studied. The cytoplasmic granularity, reflected by the side scatter of light, was proportional to DNA content of trophozoites, whereas cysts with similar DNA contents showed heterogeneity in their cytoplasmic granularity. Dynamic changes in the intracellular calcium pools were observed during differentiation of trophozoites to cysts. Comparison of E. invadens and Entamoeba histolytica cell cycles suggest that both organisms may have similar regulatory processes during cell division and differentiation. Since E. histolytica cannot be induced to encyst in axenic culture, analysis of the E. invadens cell cycle during encystation may be useful for identifying homologous processes in E.histolytica.

Molecular epidemiology of Entamoeba spp.: evidence of a bottleneck (Demographic sweep) and transcontinental spread of diploid parasites.

Entamoeba histolytica causes amebic colitis and liver abscess in developing countries such as Mexico and India. Entamoeba dispar is morphologically identical but is not associated with disease. Here we determined the ploidy of E. histolytica and developed PCR-based methods for distinguishing field isolates of E. histolytica or E. dispar. Fluorescence in situ hybridization showed that E. histolytica trophozoites are diploid for five "single-copy" probes tested. Intergenic sequences between superoxide dismutase and actin 3 genes of clinical isolates of E. histolytica from the New and Old Worlds were identical, as were those of E. dispar. These results suggest a bottleneck or demographic sweep in entamoebae which infect humans. In contrast, E. histolytica and E. dispar genes encoding repeat antigens on the surface of trophozoites (Ser-rich protein) or encysting parasites (chitinase) were highly polymorphic. chitinase alleles suggested that the early axenized strains of E. histolytica, HM-1 from Mexico City, Mexico, and NIH-200 from Calcutta, India, are still present and that similar E. dispar parasites can be identified in both the New and Old Worlds. Ser-rich protein alleles, which suggested the presence of the HM-1 strain in Mexico City, included some E. histolytica genes that predicted Ser-rich proteins with very few repeats. These results, which suggest diversifying selection at chitinase and Ser-rich protein loci, demonstrate the usefulness of these alleles for distinguishing clinical isolates of E. histolytica and E. dispar.

L-myo-Inositol 1-phosphate synthase from Entamoeba histolytica.

L-myo-Inositol 1-phosphate synthase (I-1-P synthase) catalyses the primary reaction for the synthesis of inositol in a variety of prokaryotes, eukaryotes and in the chloroplasts of algae and higher plants. Inositol is a precursor of essential macromolecules like membrane phospholipids, GPI anchor proteins and lipophosphoglycans, which play a determinant role in the pathogenesis of protozoan parasites such as Leishmania and Entamoeba. However, there is no report of I-1-P synthase or its gene from these organisms. The gene INO1 coding for this enzyme was first cloned from Saccharomyces cerevisiae and subsequently from several plants. Using molecular cloning techniques we have isolated and characterised the INO1 gene coding for the enzyme I-1-P synthase from Entamoeba histolytica. Simultaneously, we have purified and characterised the native enzyme from E. histolytica trophozoites and the cloned gene product from Escherichia coli. The gene product and the purified enzyme were both shown to be recognised by a heterologous anti-I-1-P synthase antibody from the phytoflagellate Euglena gracilis. Phylogenetic analysis of I-1-P synthase sequences from different eukaryotes suggest that it is highly conserved across species and the origin of this enzyme precedes the evolutionary divergence of modern eukaryotes.

Modeling of Entamoeba histolytica ferredoxin.

The E. histolytica ferredoxin (EHFXD) is an iron-sulphur protein and is important in the control of E. histolytica because it donates an electron to and activates metronidazole (4) the most effective anti-amoebic drug. The knowledge of the three-dimensional structure of EHFXD can help to assist in rational design of anti-amebic drugs. Homology modeling of EHFXD has been done by using the knowledge of crystal structure of homologous Ferredoxin structures. It has been shown that EHFXD is more likely to contain 2[4Fe-4S] clusters and has a pseudodiad symmetrical fold. The fold is stabilised by hydrophobic patches as well as by intramolecular hydrogen bonds. The importance of two Leu residues in the N- and C-termi in bridging the two clusters has been emphasised. The electrostatic properties of the surface of EHFXD were examined and compared with that of other ferredoxins. It was observed that large regions of negative as well as positive electrostatic potential is a common feature of the [4Fe-4S] containing ferredoxins which might explain their biological role as both electron acceptor and electron donor molecules.

The mcm17 mutation of yeast shows a size-dependent segregational defect of a mini-chromosome.

Mini-chromosome-maintenance (mcm) mutants were described earlier as yeast mutants which could not stably maintain mini-chromosomes. Out of these, the ARS-specific class has been more extensively studied and is found to lose chromosomes and mini-chromosomes due to a defect in the initiation of DNA replication at yeast ARSs. In the present study we have identified a number of mcm mutants which show size-dependent loss of mini-chromosomes. When the size of the mini-chromosome was increased, from about 15 kb to about 60 kb, there was a dramatic increase in its mitotic stability in these mutants, but not in the ARS-specific class of mutants. One mutant, mcm17, belonging to the size-dependent class was further characterized. In this mutant, cells carried mini-chromosomes in significantly elevated copy numbers, suggesting a defect in segregation. This defect was largely suppressed in the 60-kb mini-chromosome. A non-centromeric plasmid, the TRP1ARS1 circle, was not affected in its maintenance. This mutant also displayed enhanced chromosome-III loss during mitosis over the wild-type strain, without elevating mitotic recombination. Cloning and sequencing of MCM17 has shown it to be the same as CHL4, a gene required for chromosome stability. This gene is non-essential for growth, as its disruption or deletion from the chromosome did not affect the growth-rate of cells at 23 degrees C or 37 degrees C. This work suggests that centromere-directed segregation of a chromosome in yeast is strongly influenced by its length.

Unusual genome organisation in Entamoeba histolytica leads to two overlapping transcripts.

We have isolated homologs of the mini chromosome maintenance (MCM) gene family from the parasitic protozoan Entamoeba histolytica. The full length genomic and cDNA clones for the Eh MCM3 gene have been characterised. The Eh MCM3 gene is much smaller than the Saccharomyces cerevisiae MCM3 gene and other eukaryotic homologs of the MCM3/P1 family. The predicted Eh Mcm3 protein was 597 amino acids long and showed 37 and 46% positional identity with the Sc Mcm3 and the mouse P1 homologs respectively. While proceeding along the chromosome from the Eh MCM3 gene, we have identified a homolog (Eh PAK) of the murine p21 activated kinase (Rn KPAK), or S. cerevisiae STE20. Eh PAK lies 126 bp upstream of the Eh MCM3 gene. The predicted Eh p21 activated kinase protein was 459 amino-acids long and showed 33% positional identity with the murine p21 activated kinase and its yeast homolog Ste20. Analysis of cDNA and genomic sequences shows that the 3' untranslated region (UTR) of the Eh PAK mRNA and the 5' UTR of the Eh MCM3 mRNA are transcribed from a common 40 bp genomic segment. This is the first report of an amoeba gene being physically linked to a second gene such that their transcripts are overlapping and there is no non-transcribed intergenic region between the two genes. Primer extension studies have confirmed that unlike most E. histolytica genes, which have short 5' UTRs, the Eh MCM3 mRNA has a 126 bp long 5' UTR and the Eh PAK mRNA has a 265 bp long 5' UTR.

Cloning and characterization of a high-copy-number novel insertion sequence from chemolithotrophic Thiobacillus ferrooxidans.

Two distinct families of repetitive DNA elements (1.4 and 1.2 kb) were identified from S1 nuclease-treated genomic DNA of four strains of Thiobacillus ferrooxidans. The 1.4-kb fragment hybridized with IST2, an insertion sequence of T. ferrooxidans. The 1.2-kb fragment was cloned and sequenced. The sequence (IST445), 1219 bp in length, with features characteristic of an insertion element, has a terminal inverted repeat of 8 bp, which can be further extended to 23 or 48 bp with 9 and 26 mismatches, respectively. It displays 54.4% identity in 967 nucleotides of overlap with ISAE1 of Alcaligenes eutrophus. The IST445 contains three open reading frames which have codon usage almost similar to 56 different coding genes of T. ferrooxidans. In Southern blots of restricted genomic DNAs probed with IST445, each of the several strains of T. ferrooxidans gives a distinctive fingerprint. IST445 is present in the range of 10-20 copies per genome in the four strains studied.

Heterogeneity of DNA content and expression of cell cycle genes in axenically growing Entamoeba histolytica HM1:IMSS clone A.

The cell division cycle of Entamoeba histolytica was studied using multi-parametric flow cytometry in asynchronous and partially synchronised cells. Dynamic changes in the DNA synthesis and DNA content of axenically growing trophozoites were observed by using 5-bromo-2'-deoxyuridine (BrdU) uptake and DNA specific fluorochromes. It was observed that DNA synthesis in these cells continues beyond the typical S-phase stop point when DNA duplication is complete. Asynchronously growing E. histolytica cells could be synchronised by serum starvation followed by serum re-addition. BrdU incorporation in synchronised cells showed that cell synchrony is maintained for at least one generation time, in which the G1 phase lasts for 2-3 h and the S-phase lasts for 5-6 h. Analysis of our results revealed that E. histolytica trophozoites, growing in axenic medium, are made up of a heterogenous population of euploid and polyploid cells. The number of polyploid cells increases with age of the cells in culture. Expression of putative cell cycle and signal transduction markers was studied using specific antibodies and changes in their expression levels have been correlated with changes in the DNA content. Based upon our results we could identify G1, S and G2 phases of the cell cycle of E. histolytica and also predict the mechanism underlying the generation of polyploidy in these cells, which may have significant effects on its biology and pathogenesis.

Heterogeneity of Entamoeba histolytica rac genes encoding p21rac homologues.

Entamoeba histolytica (Eh), the parasite that causes amebic dysentery, is the only protozoan that phagocytoses bacteria, epithelial cells and red blood cells. Numerous low-molecular weight GTP-binding proteins, called p21rac, are implicated in signal transduction and actin polymerization during phagocytosis by macrophages and Dictyostelium discoideum (Dd). Here, molecular cloning techniques were used to obtain four Eh rac genes that encoded putative p21rac, as well as segments of two Eh rac pseudogenes. The predicted Eh p21rac, which share 55-81% amino acid (aa) identities with each other, include one that closely resembles the p21rac1 of man, Dd, Drosophila melanogaster and Caenorhabditis elegans; two that resemble the p21racC of Dd; and one that is unique. An alignment of the Eh rac ORF with other rac family proteins reveals multiple aa that distinguish p21rac1, p21racC and p21cdc42. We conclude that the Eh genes encoding amebic p21rac, which are the first identified from a protozoan parasite, are numerous and heterogeneous.

Molecular cloning of an Entamoeba histolytica gene encoding a putative mos family serine/threonine-kinase.

Molecular cloning techniques were used to isolate an ameba gene encoding a putative 33-kDa serine/thyronine-kinase. The open reading frame of this gene, called Ehmfk1 for E. histolytica mos family kinase, shows a 24% positional identity with the human proto-oncogene mos, including 32 of 33 amino acids conserved in other serine/threonine-kinases.

Molecular cloning of ras and rap genes from Entamoeba histolytica.

To better understand growth regulation in the protozoan parasite Entamoeba histolytica, ameba genes homologous to the ras oncogene and rap (Krev-1) anti-oncogene were cloned. Two putative ameba ras genes (Ehras1 and Ehras2) were identified, which contain 205 and 203 amino acid (aa) open reading frames (ORFs), respectively. The Ehras1 ORF shows an 91% positional identity with that of Ehras2, a 55% identity with Dictyostelium discoideum (Dd) ras, and a 47% identity with human (Hs) ras. Two ameba rap genes (Ehrap1 and Ehrap2) were identified, both of which contain 184-aa ORFs. The Ehrap1 ORF shows a 93% positional identity with that of Ehrap2, a 60% identity with Dd rap, a 61% identity with Hs Krev-1, and a 45% identity with that of Ehras1. Conserved aa in each ameba ras and rap ORF include GTP-binding sites, effector site, site of ADP-ribosylation by Pseudomonas exoenzyme S, and COOH-terminus CAAX. As all Xs = Leu or Phe, ameba ras and rap proteins may be gerenylgerenylated and not farnesylated. Both ras and rap genes are transcribed by trophozoites. A single 21-kDa ameba ras protein reacts with the rat Y13-259 anti-ras monoclonal antibody, which is located on the cytosolic side of the plasma membrane. These are the first ras and rap genes identified from a protozoan parasite.

Cloning of the Eh cdc2 gene from Entamoeba histolytica encoding a protein kinase p34cdc2 homologue.

To understand better growth regulation in the protozoan parasite, Entamoeba histolytica (Eh), a homologue of the cdc2 gene encoding the yeast cyclin-dependent protein kinase, p34cdc2, has been cloned and sequenced. This gene, called Eh cdc2, contains a 79-bp intron located in the same place as the second of four introns in the Schizosaccharomyces pombe cdc2 gene. The sequence of an Eh cdc2 cDNA confirms the conserved eukaryotic splice donor (GT) and acceptor (AG) sites and shows that Eh is able to splice mRNAs. The spliced Eh cdc2 open reading frame is 291 amino acids (aa) long, encoding an M(r) 33,806 protein. The primary sequence of Eh cdc2 is most like those of cdc2 homologues Eg1 of Xenopus laevis and CDK2 of man (52% aa identity with each) and codes for (i) the serine (Ser), threonine (Thr), and tyrosine residues phosphorylated in p34cdc2 proteins, (ii) 32 of 33 aa conserved in other Ser/Thr protein kinases, and (iii) the sequence PVTSVRE instead of PSTAIRE found in most p34cdc2 proteins. This is the first cell-division-cycle regulatory protein homologue, as well as the first intron identified from Eh.

Molecular cloning of a rho family gene of Entamoeba histolytica.

An Entamoeba histolytica gene (Eh rho1) was cloned that encodes a putative low-molecular-mass GTP-binding protein, most similar to the ras homologue rho. The Eh rho1 open reading frame was 208 amino acids long and encoded a 23-kDa protein similar to Saccharomyces cerevisiae RHO1-RHO4 and CDC42 and human rhoA, rac1, and G25K gene products. This similarity was greatest at the NH2 terminus of Eh rho1 where two GTP-binding sites and a possible effector site were conserved. A cysteine residue at the COOH terminus of Eh rho1 was followed by eight hydrophobic amino acids rather than the three hydrophobic amino acids present in other ras family proteins.