The calcium binding protein EhCaBP6 is a microtubular-end binding protein in Entamoeba histolytica.

The genome of Entamoeba histolytica encodes several calcium binding proteins and those characterized thus far have been shown to participate predominantly in phagocytosis and endocytosis. Our study showed that EhCaBP6 has two EF-hand domains EFI and EFIII; it can bind Ca(2+) in vitro and undergoes conformational transition on binding Ca(2+) suggesting that it can serve as a calcium signal sensor. EhCaBP6 is localized in the nucleus, cytoplasm and plasma membrane and is sensitive to heat stress. Unlike other Ca(2+) binding proteins that have been studied in E. histolytica, EhCaBP6 is found at microtubule ends and at the intercellular bridge with the microtubules during cytokinesis. Furthermore, increased expression of EhCaBP6 was correlated with a significant increase in the number of microtubular structures suggesting that this protein may regulate chromosome segregation and cytokinesis in E. histolytica.

Entamoeba histolytica sirtuin EhSir2a deacetylates tubulin and regulates the number of microtubular assemblies during the cell cycle.

We have discovered four sirtuin genes in Entamoeba histolytica, two of which are similar to eukaryotic sirtuins and two to bacterial and archaeal sirtuins. The eukaryotic sirtuin homologue, EhSir2a, showed NAD(+)-dependent deacetylase activity and was sensitive to class III HDAC inhibitors. Localization of EhSir2a at different cellular sites suggested that this deacetylase could have multiple targets. Using an E. histolytica cDNA library in the yeast two-hybrid genetic screen, we identified several proteins that bound to EhSir2a. These proteins included Eh alpha-tubulin, whose interaction with EhSir2a was validated in E. histolytica. We have shown that EhSir2a deacetylated tubulin and localized with microtubules in E. histolytica. Increased expression levels of EhSir2a in stable transformants led to reduced number of microtubular assemblies in serum synchronized cells. This effect was abrogated by mutations in the deacetylase domain of EhSir2a, showing that EhSir2a deacetylase activity affected the stability and number of microtubular assemblies during the cell cycle of E. histolytica. Our results suggest that epigenetic modification of tubulin by EhSir2a is one of the mechanisms that regulates microtubular assembly in E. histolytica.

The genome of the protist parasite Entamoeba histolytica.

Entamoeba histolytica is an intestinal parasite and the causative agent of amoebiasis, which is a significant source of morbidity and mortality in developing countries. Here we present the genome of E. histolytica, which reveals a variety of metabolic adaptations shared with two other amitochondrial protist pathogens: Giardia lamblia and Trichomonas vaginalis. These adaptations include reduction or elimination of most mitochondrial metabolic pathways and the use of oxidative stress enzymes generally associated with anaerobic prokaryotes. Phylogenomic analysis identifies evidence for lateral gene transfer of bacterial genes into the E. histolytica genome, the effects of which centre on expanding aspects of E. histolytica's metabolic repertoire. The presence of these genes and the potential for novel metabolic pathways in E. histolytica may allow for the development of new chemotherapeutic agents. The genome encodes a large number of novel receptor kinases and contains expansions of a variety of gene families, including those associated with virulence. Additional genome features include an abundance of tandemly repeated transfer-RNA-containing arrays, which may have a structural function in the genome. Analysis of the genome provides new insights into the workings and genome evolution of a major human pathogen.

A novel galacto-glycerolipid from Oxalis corniculata kills Entamoeba histolytica and Giardia lamblia.

Oxalis corniculata is a naturally occurring weed that has been used in traditional medicine for the cure of dysentery and diarrhea in India. One of the common causes of dysentery is due to infection by the protist pathogen Entamoeba histolytica. Bioactivity profiling of extracts from O. corniculata identified several compounds that showed antiamoebic activity in axenic cultures of E. histolytica. These were characterized by nuclear magnetic resonance, infrared, and mass spectrometry as (i) Oc-1, a mixture of saturated fatty acids C24 to C28; (ii) Oc-2, a mixture of long-chain alcohols C18 to C28; and (iii) Oc-3, a single compound that was a galacto-glycerolipid (GGL). Of the different compounds that were obtained, the strongest antiamoebic activity was found in GGL. The addition of GGL to E. histolytica xenic cultures containing other microbial flora from the large intestine did not affect its antiamoebic activity. Amoebicidal concentrations of GGL had no effect on intestinal microbial flora or on the mammalian cell line HEK-293. GGL was also found to be equally effective in killing another protist pathogen, Giardia lamblia, that causes diarrhea in humans. The importance of this study is based on the identification of novel natural products and the possibility of developing these compounds as active agents to treat at least two pathogenic parasitic intestinal infections endemic to tropical regions.

Bipolar spindle frequency and genome content are inversely regulated by the activity of two N-type kinesins in Entamoeba histolytica.

Bipolar microtubular spindles are seen infrequently in Entamoeba histolytica trophozoites while monopolar or radial microtubular assemblies are common. Additionally, heterogeneity in nuclear DNA content and multi-nucleation is found in amoeba cells growing in axenic culture. Taken together these observations indicate that genome segregation is irregular in these cells. In order to identify proteins involved in regulating genome segregation, we have focused on studying E. histolytica homologues of kinesin motor proteins that are known to affect stability of bipolar mitotic spindles. We have demonstrated earlier that increased levels of the kinesin--Eh Klp5--led to increased frequency of bipolar spindles accompanied with a reduction in the heterogeneity of genome content, showing that bipolar spindle frequency was inversely linked to genome content in E. histolytica. In this study, we have investigated the role of E. histolytica kinesins (Eh KlpA1, 2-4) in regulating bipolar spindle frequency and genome content. While downregulation of Eh Klp3, 4 and A1 showed no effect, downregulation of Eh Klp2 led to increased frequency of bipolar spindles and homogenization of genome content, similar to the effect of increased expression of Eh Klp5. In addition to microtubules, Eh Klp2-4 associated with F-actin in the cytoplasm, suggesting that these kinesins are multi-functional.

Entamoeba histolytica encodes unique formins, a subset of which regulates DNA content and cell division.

The formin family of proteins mediates dynamic changes in actin assembly in eukaryotes, and therefore it is important to understand the function of these proteins in Entamoeba histolytica, where actin forms the major cytoskeletal network. In this study we have identified the formin homologs encoded in the E. histolytica genome based on sequence analysis. Using multiple tools, we have analyzed the primary sequences of the eight E. histolytica formins and discovered three subsets: (i) E. histolytica formin-1 to -3 (Ehformin-1 to -3), (ii) Ehformin-4, and (iii) Ehformin-5 to -8. Two of these subsets (Ehformin-1 to -3 and Ehformin-4) showed significant sequence differences from their closest homologs, while Ehformin-5 to -8 were unique among all known formins. Since Ehformin-1 to -3 showed important sequence differences from Diaphanous-related formins (DRFs), we have studied the functions of Ehformin-1 and -2 in E. histolytica transformants. Like other DRFs, Ehformin-1 and -2 associated with F-actin in response to serum factors, in pseudopodia, in pinocytic and phagocytic vesicles, and at cell division sites. Ehformin-1 and -2 also localized with the microtubular assembly in the nucleus, indicating their involvement in genome segregation. While increased expression of Ehformin-1 and -2 did not affect phagocytosis or motility, it clearly showed an increase in the number of binucleated cells, the number of nuclei in multinucleated cells, and the average DNA content of each nucleus, suggesting that these proteins regulate both mitosis and cytokinesis in E. histolytica.

Genome re-duplication and irregular segregation occur during the cell cycle of Entamoeba histolytica.

Heterogeneity of genome content is commonly observed in axenic cultures of Entamoeba histolytica. Cells with multiple nuclei and nuclei with heterogenous genome contents suggest that regulatory mechanisms that ensure alternation of DNA synthesis and mitosis are absent in this organism. Therefore, several endo-reduplicative cycles may occur without mitosis. The data also shows that unlike other endo-reduplicating organisms, E.histolytica does not undergo a precise number of endo-reduplicative cycles. We propose that irregular endo-reduplication and genome partitioning lead to heterogeneity in the genome content of E.histolytica trophozoites in their proliferative phase. The goal of future studies should be aimed at understanding the mechanisms that are involved in (a) accumulation of multiple genome contents in a single nucleus; (b) genome segregation in nuclei that contain multiple genome contents and (c) maintenance of genome fidelity in E. histolytica.

Entamoeba histolytica DNA methyltransferase (Ehmeth) is a nuclear matrix protein that binds EhMRS2, a DNA that includes a scaffold/matrix attachment region (S/MAR).

The protozoan parasite Entamoeba histolytica express a cytosine-5 DNA methyltransferase (Ehmeth) that belongs to the DNMT2 protein family. The biological function of members of this DNMT2 family is unknown. In the present study, we have demonstrated that Ehmeth is a nuclear matrix protein. Indeed, we showed by south-western analysis and yeast one-hybrid system that Ehmeth binds to EhMRS2, a DNA element which contains the eukaryotic consensus scaffold/matrix attachment regions (S/MAR) bipartite recognition sequences. S/MARs have been implicated in a variety of important functions, such as genome organization and gene expression. The methylation status of cytosine located within EhMRS2 was analyzed by bisulfite genomic sequencing. We observed the presence of methylated cytosine within the 3'-end of EhMRS2. These data provide the first evidence that a member of the DNMT2 family interacts with a S/MAR containing DNA element.

Molecular analysis of repetitive DNA elements from Entamoeba histolytica, which encode small RNAs and contain matrix/scaffold attachment recognition sequences.

We have isolated two DNA elements-Eh MRS1 and Eh MRS2-from Entamoeba histolytica, which contain the eukaryotic consensus Scaffold/Matrix Attachment Region (S/MAR) bipartite recognition sequences. Both these sequences bind to high salt extractable nuclear proteins and insoluble nuclear matrix proteins in E. histolytica HM1:IMSS, suggesting that the predicted S/MAR recognition sequences may indeed function as scaffold attachment regions in E. histolytica. Sequence analysis shows that Eh MRS1 and Eh MRS2 contain internal tandem repeats ranging from units of 8-11bp and are themselves present as independent arrays of tandemly repeating units of approximately 1100bp each. Eh MRS1 and Eh MRS2 are localised on different chromosomes in E. histolytica HM1:IMSS. Both Eh MRS1 and Eh MRS2 also code for small molecular weight RNAs of unknown function. Thus, two unique sequences-Eh MRS1 and Eh MRS2-demonstrate very similar properties, suggesting that they belong to a superfamily of genomic elements, which may function as scaffold or matrix attachment sites in Entamoeba.

Polyunsaturated fatty acids induce polarized submembranous F-actin aggregates and kill Entamoeba histolytica.

We have recently identified a novel galacto-glycerolipid (GGL) from the plant Oxalis corniculata that killed the human pathogen Entamoeba histolytica. In this study, we show that the anti-amoebic activity of GGL was due to the polyunsaturated fatty acid α-linolenic acid (C18:3 ) side chain. Treatment of α-linolenic acid to E. histolytica trophozoites disrupted the cytoskeletal network and led to polarization of F-actin at one end of the cells with prominent filopodial extensions. In addition, clustering of surface receptors and signaling molecules was also observed adjacent to the polarized actin similar to concanavalin-A-(Con-A) induced capping. But, in contrast to Con-A-induced capping, α-linolenic acid induced caps were not shed and showed accumulation of long and numerous filopodia at the cap site. We found that α-linolenic acid disrupts the actin cytoskeletal network, which led to the detachment of plasma membrane from the underlying cytoskeleton. A similar effect was observed with other dietary fatty acids such as linoleic acid (C18:2 ), arachidonic acid (C20:4 ), eicosapentaenoic acid (C20:5 ), and docosahexaenoic acid (C22:6 ). Our findings showed that dietary polyunsaturated fatty acids are powerful anti-amoebic agents that lead to disruption of the actin cytoskeleton.

Inter-cellular variation in DNA content of Entamoeba histolytica originates from temporal and spatial uncoupling of cytokinesis from the nuclear cycle.

Accumulation of multiple copies of the genome in a single nucleus and several nuclei in a single cell has previously been noted in Entamoeba histolytica, contributing to the genetic heterogeneity of this unicellular eukaryote. In this study, we demonstrate that this genetic heterogeneity is an inherent feature of the cell cycle of this organism. Chromosome segregation occurs on a variety of novel microtubular assemblies including multi-polar spindles. Cytokinesis in E. histolytica is completed by the mechanical severing of a thin cytoplasmic bridge, either independently or with the help of neighboring cells. Importantly, cytokinesis is uncoupled from the nuclear division cycle, both temporally and spatially, leading to the formation of unequal daughter cells. Sorting of euploid and polyploid cells showed that each of these sub-populations acquired heterogeneous DNA content upon further growth. Our study conclusively demonstrates that genetic heterogeneity originates from the unique mode of cell division events in this protist.

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.

Eh Klp5 is a divergent member of the kinesin 5 family that regulates genome content and microtubular assembly in Entamoeba histolytica.

Earlier studies have established two unusual features in the cell division cycle of Entamoeba histolytica. First, microtubules form a radial assembly instead of a bipolar mitotic spindle, and second, the genome content of E. histolytica cells varied from 1x to 6x or more. In this study, Eh Klp5 was identified as a divergent member of the BimC kinesin family that is known to regulate formation and stabilization of the mitotic spindle in other eukaryotes. In contrast to earlier studies, we show here that bipolar microtubular spindles were formed in E. histolytica but were visible only in 8-12% of the cells after treatment with taxol. The number of bipolar spindles was significantly increased in Eh Klp5 stable transformants (20-25%) whereas Eh Klp5 double-stranded RNA (dsRNA) transformants did not show any spindles (< 1%). The genome content of Eh Klp5 stable transformants was regulated between 1x and 2x unlike control cells. Binucleated cells accumulated in Eh Klp5 dsRNA transformants and after inhibition of Eh Klp5 with small molecule inhibitors in control cells, suggesting that cytokinesis was delayed in the absence of Eh Klp5. Taken together, our results indicate that Eh Klp5 regulates microtubular assembly, genome content and cell division in E. histolytica. Additionally, Eh Klp5 showed alterations in its drug-binding site compared with its human homologue, Hs Eg5 and this was reflected in its reduced sensitivity to Eg5 inhibitors - monastrol and HR22C16 analogues.

EhRho1, a RhoA-like GTPase of Entamoeba histolytica, is modified by clostridial glucosylating cytotoxins.

Clostridial glucosylating cytotoxins inactivate mammalian Rho GTPases by mono-O glucosylation of a conserved threonine residue located in the switch 1 region of the target protein. Here we report that EhRho1, a RhoA-like GTPase from the protozoan parasite Entamoeba histolytica, is glucosylated by clostridial cytotoxins. Recombinant glutathione S-transferase-EhRho1 and EhRho1 from cell lysate of Entamoeba histolytica were glucosylated by Clostridium difficile toxin B and Clostridium novyi alpha-toxin. In contrast, Clostridium difficile toxin A, which shares the same mammalian protein substrates with toxin B, did not modify EhRho1. Change of threonine 52 of EhRho1 to alanine prevented glucosylation by toxin B from Clostridium difficile and by alpha-toxin from Clostridium novyi, which suggests that the equivalent threonine residues are glucosylated in mammalian and Entamoeba Rho GTPases. Lethal toxin from Clostridium sordellii did not glucosylate EhRho1 but labeled several other substrate proteins in lysates from Entamoeba histolytica in the presence of UDP-[14C]glucose.

Constitutive association of Mcm2-3-5 proteins with chromatin in Entamoeba histolytica.

Eukaryotic cells duplicate their genome once and only once per cell cycle. Our earlier studies with the protozoan parasite, Entamoeba histolytica, have shown that genome reduplication may occur several times without nuclear or cellular division. The Mcm2-7 protein complex is required for licensing of DNA replication. In an effort to understand whether genome reduplication occurs due to absence or failure of the DNA replication licensing system, we analysed the function of Mcm2-3-5 proteins in E. histolytica. In this study, we have cloned E. histolytica (Eh) MCM2 and Eh MCM5 genes, while Eh MCM3 was cloned earlier. The sequence of Eh MCM2-3-5 genes is well conserved with other eukaryotic homologues. We have shown that Eh Mcm2,3 proteins are functional in Saccharomyces cerevisiae. Our studies in E. histolytica showed that Eh Mcm2-3-5 proteins are associated with chromatin constitutively in cycling cells and during arrest of DNA synthesis induced by serum starvation. Alternation of genome duplication with mitosis is regulated by association-dissociation of Mcm2-7 proteins with chromatin in other eukaryotes. Our results suggest that constitutive association of Mcm proteins with chromatin could be one of the reasons why genome reduplication occurs in E. histolytica.

The cell cycle of Entamoeba histolytica.

Entamoeba histolytica, is a microaerophilic protist, which causes amoebic dysentery in humans. This unicellular organism proliferates in the human intestine as the motile trophozoite and survives the hostile environment outside the human host as the dormant quadri-nucleate cyst. Lack of organelles--such as mitochondria and Golgi bodies--and an unequal mode of cell division, led to the popular belief, that this organism preceded other eukaryotes during evolution. However, data from several laboratories have shown that, contrary to this belief, E. histolytica is remarkable in its divergence from other eukaryotes. This uniqueness is witnessed in many aspects of its biochemical pathways, cellular biology and genetic diversity. In this context, I have analysed the cell division cycle of this organism and compared it to that of other eukaryotes. Studies on E. histolytica, suggest that in its proliferative phase, this organism may accumulate polyploid cells. Thus 'checkpoints' regulating alternation of genome duplication and cell division appear to be absent in this unicellular protist. Sequence homologs of several cell cycle regulating proteins have been identified in amoeba, but their structural divergence suggests that they may not have equivalent function in this organism. The regulation of cell proliferation in E. histolytica, may be ideally suited to survival of a parasite in a complex host. Analysis of these molecular details may offer solutions for eradicating the pathogen by hitherto unknown methods.

Inhibition of gene expression with double strand RNA interference in Entamoeba histolytica.

In order to inhibit gene expression in Entamoeba histolytica, we have developed a method based on expressing double strand RNA interference constructs in stable transformants. The 5' end of Eh Dia was cloned head to head with an intervening non-specific stuffer fragment in the E. histolytica expression vector pJST4. This construct was transformed in E. histolytica HM1:IMSS trophozoites and stable transformants were selected with 20microg/ml G418. Our results show that expression of Eh Dia was completely inhibited in these transformants. These stable transformants could be maintained indefinitely without expression of Eh Dia. This method therefore provides an effective tool to study the phenotypic changes, which occur due to inhibition of gene expression in the absence of mutants and other microbiological manipulations in this protozoan parasite.

Sequence divergence of Entamoeba histolytica tubulin is responsible for its altered tertiary structure.

Atypical microtubular structures of the protozoan parasite Entamoeba histolytica (Eh) have been attributed to amino acid sequence divergence of Eh tubulin. To investigate if this sequence divergence leads to significant differences in the tertiary structure of the Eh alphabeta-tubulin heterodimer, we have modeled alphabeta-tubulin heterodimer of Eh based on the crystal structure of mammalian tubulin. The predicted 3D homology model exhibits an overall resemblance with the known crystal structure of mammalian tubulin except for the 16 residue long carboxy terminal region of Eh beta-tubulin. We propose that this C-terminal region may provide steric hindrance in the polymerization of Eh alphabeta-tubulin for microtubule formation. Using docking studies, we have identified the binding sites for different microtubule specific drugs on Eh beta-tubulin. Our model provides a rational framework, both for understanding the contribution of Eh beta-tubulin C-terminal region to alphabeta-tubulin polymerization and design of new anti-protozoan drugs in order to control amoebiasis.