The Cytoskeleton of Entamoeba histolytica: Structure, Function, and Regulation by Signaling Pathways

doi:10.1016/j.arcmed.2005.09.008

Archives of Medical Research

Volume 37, Issue 2, February 2006, Pages 234-243

https://doi.org/10.1016/j.arcmed.2005.09.008 Get rights and content

Pathogenesis in the parasite Entamoeba histolytica has been related to motility of the trophozoites. Motility is an important feature in amebas as they perform multiple motile functions during invasion of host tissues. As motility depends on the organization and regulation of the cytoskeleton elements, in particular of the actin cytoskeleton, the study of the molecular components of the machinery responsible for movement has been a key aspect to study in this parasite. Although many of the components have high homology in amino acid sequence and function to those characterized in higher eukaryotic cells, there are important differences to suggest that parasitic organisms may have developed adaptative differences that could be useful as targets to stop invasion. The purpose of this review is to evaluate current knowledge about the cytoskeleton of E. histolytica and the ways in which the parasite controls motility.

Introduction

A striking feature of Entamoeba histolytica trophozoites is their high motility manifested by fast locomotion, pleiomorphism, and continuous movement of intracellular components. In culture, amebas form surface projections that allow pinocytosis, phagocytosis, adhesion, and displacement. During infection of the human host, motility is thought to be a main determinant for the invasive behavior of trophozoites, as they become avid phagocytes ingesting damaged cells, extracellular matrix, and erythrocytes. Responses to chemotactic cues are thought to induce directed migration and tissue penetration.

The intense motility of amebas would require a very dynamic cytoskeleton making difficult the visualization of cytoskeletal polymers. It was only after introduction of modified microscopic techniques and specific markers, such as fluorescent compounds and antibodies directed to cytoskeleton proteins, that identification of filaments corresponding to actin polymers and nuclear microtubules in cultured trophozoites was achieved. Molecular approaches allowed identification of genes encoding several cytoskeletal proteins and the inference of their amino acid sequences and functional domains. These advances, together with elucidation of signaling pathways that regulate actin turnover and interactions and the use of specific drugs to block cytoskeleton functions, have provided important but still incomplete information about amebic functions in which the cytoskeleton could play a role. Monitoring cytoskeletal rearrangements occurring during cellular activities still represents a technical challenge and there is much to explore about the mechanisms that induce and regulate motility in the parasite. As motility has been implicated in pathogenicity, it will be very important to discern how the machinery for cell movement supports survival of the parasite as commensal in the human host but, at the same time, permits cell and tissue damage during invasion.

Section snippets

Actin

Actin was first identified in trophozoites by immunostaining utilizing heterologous antibodies prepared against human and rabbit muscle actins 1, 2. Expression of the protein was confirmed when a protein with similar molecular and functional characteristics to typical actins was purified from trophozoites. Amebic actin showed the capacity to form filaments in vitro that could be decorated with heavy meromyosin and to induce myosin ATPase activity with similar kinetics to that induced by rabbit

Myosins

Myosins are proteins that function as molecular motors being essential for cellular movement. Myosins move on actin filaments converting chemical energy into mechanical work by ATP hydrolysis, producing contraction of the filaments. In eukaryotic cells there are at least 18 different classes of myosins and specific functions have been assigned to only a few of them. In Entamoeba histolytica, only two myosins have been identified. Characterization of a myosin II heavy chain gene (Myo II) showed

Tubulins

Cytoplasmic microtubules have not been observed in trophozoites of E. histolytica, in contrast with microtubules inside dividing nuclei that could be identified utilizing several microscopy techniques. The intranuclear mitotic spindle is formed by a bundle of microtubules that do not converge at the poles in well-defined asters, although what could be a single microtubule organizing center (MTOC) has been described in one of the poles in the nucleus of dividing cells (43). Gicquaud described

Cell Signaling

E. histolytica, either as a commensal or as an invasive cell, requires constant interaction with its extracellular environment and with other cells to survive. These interactions lead to changes in cell physiology, cellular architecture, and gene expression. External signals are perceived by membrane-bound receptors, resulting in changes in their biochemical or physical states that initiate a cascade of signaling events within the cell (55). One of the main functions of the cytoskeleton is to

Integrin-Type Mediated Signaling

During tissue invasion, amebas bind to and destroy different types of cells and extracellular matrix components. In eukaryotic cells, actin is nucleated and polymerized at multimolecular complexes formed at the cell membrane at sites of contact. These complexes facilitate attachment of cells to the substrate and consist of integrin-type receptors that can interact with the extracellular matrix and intracellularly associate with protein complexes containing actin, talin, vinculin, α actinin,

Rho Protein-Mediated Signaling

Signaling pathways involving the participation of Rho family monomeric GTPases in actin organization have been extensively studied in higher eukaryotic cells (78). The first identified rho gene homologue in E. histolytica was cloned from parasite genomic DNA using PCR and degenerate oligonulceotide primers to GTP-binding sequences I and II that are conserved in ras family proteins. It was postulated that EhRho1 protein could participate as a factor controlling growth and differentiation (79).

Ca²⁺ Regulation

Intracellular calcium plays a crucial role as a second messenger for the control of a variety of cell functions in eukaryotes, including contraction, secretion, cell division, differentiation, and sodium and potassium permeability (82). The uptake and release of the calcium ion (Ca²⁺) across the plasma membrane and intracellular organelles by the concerted operation of distinct calcium transporting systems control intracellular Ca²⁺ concentration. In E. histolytica a partial sequence for a

Acknowledgments

We thank enthusiastic collaborators and members in our laboratories for their important contributions at different stages of our research with Entamoeba histolytica, as they have made possible the current view on the parasite's cytoskeleton and its functions.

This work was supported by grants from Conacyt, Mexico #42724 to IM, #37270 and #28077-M to PTR, and #39511-A1 to MV.

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To infect the human host, Entamoeba histolytica carries out processes requiring cytoskeleton remodeling, which involves reorganizing the actin fibers. However, little is known about the external influence factors, e.g., the pH, on the parasite's cytoskeleton remodeling or cell morphology. Such influence becomes relevant given the pH gradient that the amoeba cope with when going through the human colonic mucus during infection. Therefore, we analyzed the proliferation, the reorganization of the actin fibers, and other actin structures and cell shape during adhesion to fibronectin and erythrophagocytosis in trophozoites at different external pH conditions (6.0, 6.5, 6.8, 7.5, 8.0). We found that the best condition of external pH to perform such functions was 6.8. At acid pH, the trophozoites presented better-defined actin fibers that formed a more compact network, while at alkaline pH, the fibers reorganized, forming a looser and less defined network. Similarly, the number of actin dots also changed from acid to alkaline pH. In conclusion, the external pH alters the proliferation of the amoebas and promotes the dynamic restructuration of their cytoskeleton, allowing them to carry out their functions.
Naegleria's mitotic spindles are built from unique tubulins and highlight core spindle features
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Naegleria gruberi is a unicellular eukaryote whose evolutionary distance from animals and fungi has made it useful for developing hypotheses about the last common eukaryotic ancestor. Naegleria amoebae lack a cytoplasmic microtubule cytoskeleton and assemble microtubules only during mitosis and thus represent a unique system for studying the evolution and functional specificity of mitotic tubulins and the spindles they assemble. Previous studies show that Naegleria amoebae express a divergent α-tubulin during mitosis, and we now show that Naegleria amoebae express a second mitotic α- and two mitotic β-tubulins. The mitotic tubulins are evolutionarily divergent relative to typical α- and β-tubulins and contain residues that suggest distinct microtubule properties. These distinct residues are conserved in mitotic tubulin homologs of the “brain-eating amoeba” Naegleria fowleri, making them potential drug targets. Using quantitative light microscopy, we find that Naegleria’s mitotic spindle is a distinctive barrel-like structure built from a ring of microtubule bundles. Similar to those of other species, Naegleria’s spindle is twisted, and its length increases during mitosis, suggesting that these aspects of mitosis are ancestral features. Because bundle numbers change during metaphase, we hypothesize that the initial bundles represent kinetochore fibers and secondary bundles function as bridging fibers.
Characterization of low molecular weight protein tyrosine phosphatases of Entamoeba histolytica
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Citation Excerpt :
In the interaction event with host components, complex signal transduction systems are detected in E. histolytica trophozoites. Trophozoites interact with extracellular matrix (ECM) molecules [11–14], whereby adhesion, actin cytoskeleton remodeling, and migration events have been observed [15,16], this, in turn, is coordinated by phosphorylation and dephosphorylation of citoskeletal proteins [17–19]. Reversible tyrosine phosphorylation of proteins is a common signal transduction mechanism, associated in the regulation of numerous important processes of eukaryotic physiology and is catalyzed by kinases and phosphatases.
Entamoeba histolytica is an intestinal protozoan parasite of humans and is endemic in developing countries. E. histolytica has two low molecular weight protein tyrosine phosphatase (LMW-PTP) genes, EhLMW-PTP1 and EhLMW-PTP2, which are expressed in cultured trophozoites, clinical isolates, and cysts. The amino acid sequences of proteins EhLMW-PTP1 and EhLMW-PTP2 showed only one amino acid difference between them at position A85V, respectively. Both genes are expressed in cultured trophozoites, mainly EhLMW-PTP2, and in trophozoites recovered from amoebic liver abscess, the expression of EhLMW-PTP1 is downregulated. We cloned the two genes and purified the corresponding recombinant (rEhLMW-PTPs) proteins. Antibodies anti-rEhLMW-PTP2 showed that during red blood cells uptake by E. histolytica, the EhLMW-PTPs were found in the phagocytic cups based on analysis of fluorescence signals. On the other hand, rEhLMW-PTPs showed an optimum phosphatase activity at pH 6.0 with p-nitrophenyl phosphate as the substrate. They dephosphorylate phosphotyrosine and 3-O-methylfluorescein phosphate, but not phosphoserine or phosphothreonine, and the enzymatic activity is inhibited by orthovanadate. rEhLMW-PTP1 and rEhLMW-PTP2 exhibited optimum temperatures of activities at 60 °C and 58 °C, respectively, with high thermal stability at 50 °C. Also, the rEhLMW-PTPs showed high specific activities and specific km value with pNPP or OMFP as the substrates at the physiological temperature (37 °C).
Tyrosine phosphorylation/dephosphorylation of myosin II essential light chains of Entamoeba histolytica trophozoites regulates their motility
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Citation Excerpt :
The basic motility apparatus of E. histolytica trophozoites shares several similarities with those described in other eukaryotes. However, the main molecular components of contractile fibers of the motor system of these amebas show specific features [6]. Trophozoites express only one isoform of actin, although several genes for this protein have been identified.
Entamoeba histolytica trophozoites dwell in the human intestine as comensals although under still unclear circumstances become invasive and destroy the host tissues. For these activities, trophozoites relay on remarkable motility provided by the cytoskeleton organization. Amebic actin and some of its actin-associated proteins are well known, while components of the myosin II molecule, although predicted from the E. histolytica genome, need biochemical and functional characterization. Recently, an amebic essential light myosin II chain, named EhMLCI, was identified and reported to be phosphorylated in tyrosines. The phosphorylated form of the protein was associated with the soluble assembly incompetent conformation of the heavy myosin chains, while the non-phosphorylated protein was identified with filamentous heavy chains, organized in an assembly competent conformation. It was postulated that EhMLCI tyrosine phosphorylation could act as a negative regulator of myosin II activity by its phosphorylation/dephosphorylation cycles. To test this hypothesis, we constructed an expression vector containing an EhMLCI DNA sequence where two tyrosine residues, with strong probability of phosphorylation and fall within the single EF-hand domain that interacts with the N-terminus of myosin II heavy chains, were replaced by phenylalanines. Transfected trophozoites, expressing the mutant MutEhMLCI protein cannot process it, thereby not incorporated into the phosphorylation/dephosphorylation cycles required for myosin II activity, results in motility defective trophozoites.
Detection of beta-tubulin in the cytoplasm of the interphasic Entamoeba histolytica trophozoites
2016, Experimental Parasitology
It is known that the microtubules (MT) of Entamoeba histolytica trophozoites form an intranuclear mitotic spindle. However, electron microscopy studies and the employment of anti-beta-tubulin (β-tubulin) antibodies have not exhibited these cytoskeletal structures in the cytoplasm of these parasites. The purpose of this work was to detect β-tubulin in the cytoplasm of interphasic E. histolytica trophozoites.
Activated or non-activated HMI-IMSS-strain E. histolytica trophozoites were used and cultured for 72 h at 37 °C in TYI-S-33 medium, and then these were incubated with the anti-β-tubulin antibody of E. histolytica.
The anti-β-tubulin antibody reacted with the intranuclear mitotic spindle of E. histolytica-activated trophozoites as control. In contrast, in non-activated interphasic parasites, anti-β-tubulin antibody reacted with diverse puntiform structures in the cytoplasm and with ring-shaped structures localized in the cytoplasm, cellular membrane and endocytic stomas. In this work, for the first time, the presence of β-tubulin is shown in the cytoplasm of E. histolytica trophozoites.
Kaempferol inhibits Entamoeba histolytica growth by altering cytoskeletal functions
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The flavonoid kaempferol obtained from Helianthemum glomeratum, an endemic Mexican medicinal herb used to treat gastrointestinal disorders, has been shown to inhibit growth of Entamoeba histolytica trophozoites in vitro; however, the mechanisms associated with this activity have not been documented. Several works reported that kaempferol affects cytoskeleton in mammalian cells. In order to gain insights into the action mechanisms involved in the anti-amoebic effect of kaempferol, here we evaluated the effect of this compound on the pathogenic events driven by the cytoskeleton during E. histolytica infection. We also carried out a two dimensional gel-based proteomic analysis to evidence modulated proteins that could explain the phenotypical changes observed in trophozoites. Our results showed that kaempferol produces a dose-dependent effect on trophozoites growth and viability with optimal concentration being 27.7 μM.
Kaempferol also decreased adhesion, it increased migration and phagocytic activity, but it did not affect erythrocyte binding nor cytolytic capacity of E. histolytica. Congruently, proteomic analysis revealed that the cytoskeleton proteins actin, myosin II heavy chain and cortexillin II were up-regulated in response to kaempferol treatment. In conclusion, kaempferol anti-amoebic effects were associated with deregulation of proteins related with cytoskeleton, which altered invasion mechanisms.

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Review ArticleCell BiologyThe Cytoskeleton of Entamoeba histolytica: Structure, Function, and Regulation by Signaling Pathways

Introduction

Section snippets

Actin

Myosins

Tubulins

Cell Signaling

Integrin-Type Mediated Signaling

Rho Protein-Mediated Signaling

Ca2+ Regulation

Acknowledgments

J Biol Chem

Biochem Biophys Res Commun

Exp Parasitol

Exp Parasitol

Exp Parasitol

Arch Med Res

Parasitol Today

Exp Parasitol

Cell Biol Int

Exp Parasitol

Mol Biochem Parasitol

Trends Cell Biol

Exp Parasitol

Mol Biochem Parasitol

Mol Biochem Parasitol

Arch Med Res

Gene

Mol Biochem Parasitol

Biochem Biophys Res Commun

Trends Microbiol

Exp Parasitol

Exp Parasitol

Exp Parasitol

Exp Parasitol

Comp Biochem Physiol

Mol Biochem Parasitol

Mol Biochem Parasitol

Lancet

Exp Parasitol

Curr Opin Cell Biol

Mol Biochem Parasitol

Curr Opin Microbiol

Mol Biochem Parasitol

FEBS Lett

J Biol Chem

Actin in Entamoeba histolytica trophozoites revealed by human actin antibodies

J Parasitol

Localización de actina en trofozoítos de Entamoeba histolytica HM1

Arch Invest Med (Mex)

Trypanosoma cruzi: distribution of fluorescently labeled tubulin and actin in epimastigotes

J Parasitol

Rapid polymerization of Entamoeba histolytica actin induced by interaction with target cells

J Exp Med

Stimulation by target cell membrane lipid of actin polymerization and phagocytosis by Entamoeba histolytica

Infect Immun

Characterization of a cytochalasin resistant mutant of Entamoeba histolytica

J Protozool

Review Article
Cell Biology
The Cytoskeleton of Entamoeba histolytica: Structure, Function, and Regulation by Signaling Pathways

Ca²⁺ Regulation