Entamoeba histolytica and Entamoeba dispar: Morphological and Behavioral Differences Induced by Fibronectin through GTPases Activation and Actin‐Binding Proteins

Early steps of tissue invasion by Entamoeba histolytica are mediated by adhesion and migration through matrix components such as fibronectin with the participation of the actin cytoskeleton. Striking differences in their produced structures, movement, and migration were found. These observations suggest differential changes in their ability to organize the actin cytoskeleton and, therefore, to modify its morphology after adhesion to fibronectin. To understand these observations, we explore deeper the cytoskeleton pathway of E. histolytica compared to Entamoeba dispar, analyzing the activation and involvement of actin cytoskeleton regulatory proteins such as small GTPases (Rho, Rac1 and Cdc42), myosin IB, paxillin, alpha‐actinin, and ARP2/3 during interaction with fibronectin. Results showed a higher activation of Rac1 in E. histolytica compared to E. dispar, while Cdc42 and RhoA were equally activated in both amebae; besides, variations in the amount of myosin IB, paxillin, and ARP2/3 were detected among these species, coinciding and reflected in formation of lamellipodia in E. histolytica and filopodia in E. dispar. These could partially explain the higher invasive capacity of E. histolytica compared to E. dispar, due to its pleomorphic ability, high motility, migration, activation, and abundance of proteins involved in the cytoskeleton arrangement.     

Characterization ofEntamoeba histolytica-induced dephosphorylation in Jurkat cells

Entamoeba histolytica killing of host cells is contact dependent and mediated by a Gal/GalNAc lectin. Upon contact with amoeba a rapid and extensive dephosphorylation of tyrosine phosphorylated host cell proteins is observed. This effect is mediated by the Gal/GalNAc lectin. However, it requires intact cells, as purified lectin failed to induce dephosphorylation in Jurkat cells. The nonpathogenic, but morphologically identical amoeba,Entamoeba moshkovskii also did not induce dephosphorylation in target cells. Treatment of Jurkat cells with phosphotyrosine phosphatase inhibitors has shown that a host phosphatase is responsible for dephosphorylation. However, it was found that the CD45 phosphotase was not necessary for dephosphorylation of host cell proteins.     

EhRho1 regulates phagocytosis by modulating actin dynamics through EhFormin1 and EhProfilin1 in Entamoeba histolytica

The protist parasite Entamoeba histolytica causes amoebiasis, a major public health problem in developing countries and a major cause of morbidity and mortality. Invasive infection in amoebiasis mostly affects intestinal epithelial cell lining but can also involve other organs, such as liver, lungs, or brain. Phagocytosis is an essential mode of nutrition in amoeba and has often been associated with virulence behaviour of E. histolytica. E. histolytica possesses a highly dynamic and actin‐rich cytoskeleton that is thought to be involved in many processes, such as motility, pseudopod formation, and pathogenesis. Rho GTPases are known to be key regulators of the actin cytoskeleton and consequently influence the shape and movement of cells. Our study is mainly focused to understand the role of EhRho1 in the phagocytosis process of E. histolytica. EhRho1 got enriched in the phagocytic cups along with EhActin and remains attached with phagosomal membrane. However, there was no direct binding of EhRho1 with G‐ or F‐actin, though binding was observed with the actin nucleating proteins EhFormin1 and EhProfilin1. Overexpression of dominant negative mutant or lowering the expression by antisense RNA of EhRho1 in trophozoites caused delocalisation of EhFormin1 and EhProfilin1 from phagocytic cups, which results in impairment of phagocytic process and decrease in F‐actin content. The overall results show that EhRho1 regulates phagocytosis by modulating actin dynamics through recruitment of EhFormin1 and EhProfilin1 at the phagocytosis nucleation site in E. histolytica.     

The interaction between Entamoeba histolytica and enterobacteria shed light on an ancient antibacterial response

The amoeba parasite Entamoeba histolytica interacts with the microbiota within the intestine. Enterobacteria are the major source of energy for this parasite. Here, we highlight that the interplay between enterobacteria and E. histolytica is also important for parasite survival during inflammatory stresses and for the success of amoebic infection.     

Production and Characterization of Monoclonal Antibodies Against a Highly Immunogenic Fraction of Entamoeba histolytica (NIH:200) and Their Application in the Detection of Current Amoebic Infection

Six monoclonal antibodies (MAbs) were produced against a highly immunogenic fraction derived by the chromatographic separation of the soluble preparation of axenic Entamoeba histolytica (strain NIH:200) trophozoites. Isotype characterization of the six MAbs revealed that four belonged to the IgM class and one each to the IgG1 and the IgG2a subclasses. The immunoreactivity patterns and the specificity of the MAbs with homologous and heterologous antigens were analyzed by the enzyme-linked immunotransfer blot technique and by the enzyme-linked immunosorbent assay. The MAbs reacted intensely with isolates of E. histolytica (strain NIH:200 as well as a local isolate MX1) but showed no reactivity with Entamoeba coli, Iodamoeba butschlii, Endolimax nana, Entamoeba hartmanni, free-living amoeba (Acanthamoeba harticolus) and other enteric parasites. Using the IgG1 MAb as a detecting antibody, a polyclonal-monoclonal antibody-based enzyme-linked immunosorbent assay was developed for the detection of E. histolytica antigens in stool samples of infected patients. The detection limit of the assay was 8 ng of amoebic antigen. This test was found to be specific and sensitive and yielded 100% positive results in cases with amoebiasis but did not react with controls included in the evaluation. The MAb-based enzyme-linked immunosorbent assay developed in this study will be an important test for the diagnosis of E. histolytica in the feces of infected humans; however, the limitation of the test is the inability to discriminate the pathogenic status of the amoeba detected in the stool.     

Introns of Entamoeba histolytica and Entamoeba dispar

The genome of Entamoeba histolytica is considered to possess very few intervening sequences (introns), as only 5 intron-containing genes from this protozoan parasite have been reported so far. However, while sequencing a number of genomic contigs as well as three independent genes coding for ribosomal protein L27a, we have identified 9 additional intron-containing genes of E. histolytica and the closely related species Entamoeba dispar,indicating that introns are more common in these organisms than previously suggested. The various amoeba introns are relatively short comprising between 46 and 115 nucleotides only and have a higher AT-content compared to thecorresponding exon sequences. In contrast to higher eukaryotes, amoeba introns do not contain a well-conserved branch point consensus, and have extended donor and acceptor splice sites of the sequences GTTTGTT and TAG, respectively. Consistent with the close phylogenetic relationship of E. histolytica and E. dispar, the position and length of introns is conserved between the two species but the degree of sequence identity is reduced compared to orthologous coding regions.     

Motion and twisting of magnetic particles ingested by alveolar macrophages in the human lung: effect of smoking and disease

Background

Magnetic microparticles being ingested by alveolar macrophages can be used as a monitor for intracellular phagosome motions and cytoskeletal mechanical properties. These studies can be performed in the human lung after voluntary inhalation. The influence of cigarette smoking and lung diseases on cytoskeleton dependent functions was studied.

Methods

Spherical 1.3 μm diameter ferrimagnetic iron oxide particles were inhaled by 17 healthy volunteers (40 – 65 years), 15 patients with sarcoidosis (SAR), 12 patients with idiopathic pulmonary fibrosis (IPF), and 18 patients with chronic obstructive bronchitis (COB). The retained particles were magnetized and aligned in an external 100 mT magnetic field. All magnetized particles induce a weak magnetic field of the lung, which was detected by a sensitive SQUID (superconducting quantum interference device) sensor. Cytoskeletal reorganizations within macrophages and intracellular transport cause stochastic magnetic dipole rotations, which are reflected in a decay of the magnetic lung field, called relaxation. Directed phagosome motion was induced in a weak magnetic twisting field. The resistance of the cytoplasm to particle twisting was characterized by the viscosity and the stiffness (ratio between stress to strain) of the cytoskeleton.

Results

One week after particle inhalation and later macrophage motility (relaxation) and cytoskeletal stiffness was not influenced by cigarette smoking, neither in healthy subjects, nor in the patients. Patients with IPF showed in tendency a faster relaxation (p = 0.06). Particle twisting revealed a non-Newtonian viscosity with a pure viscous and a viscoelastic compartment. The viscous shear was dominant, and only 27% of the shear recoiled and reflected viscoelastic properties. In patients with IPF, the stiffness was reduced by 60% (p < 0.02). An analysis of the shear rate and stress dependence of particle twisting allows correlating the rheological compartments to cytoskeletal subunits, in which microtubules mediate the pure viscous (non-recoverable) shear and microfilaments mediate the viscoelastic (recoverable) behavior. The missing correlation between relaxation and particle twisting shows that both stochastic and directed phagosome motion reflect different cytoskeletal mechanisms.

Conclusion

Faster relaxation and a soft cytoskeleton in patients with IPF indicate alterations in cytoskeleton dependent functions of alveolar macrophages, which may cause dysfunction's in the alveolar defense, like a slower migration, a retarded phagocytosis, a disturbed phagosome lysosome fusion and an impaired clearance.

     

The Calmodulin-like Calcium Binding Protein EhCaBP3 of Entamoeba histolytica Regulates Phagocytosis and Is Involved in Actin Dynamics

Phagocytosis is required for proliferation and pathogenesis of Entamoeba histolytica and erythrophagocytosis is considered to be a marker of invasive amoebiasis. Ca²⁺ has been found to play a central role in the process of phagocytosis. However, the molecular mechanisms and the signalling mediated by Ca²⁺ still remain largely unknown. Here we show that Calmodulin-like calcium binding protein EhCaBP3 of E. histolytica is directly involved in disease pathomechanism by its capacity to participate in cytoskeleton dynamics and scission machinery during erythrophagocytosis. Using imaging techniques EhCaBP3 was found in phagocytic cups and newly formed phagosomes along with actin and myosin IB. In vitro studies confirmed that EhCaBP3 directly binds actin, and affected both its polymerization and bundling activity. Moreover, it also binds myosin 1B in the presence of Ca²⁺. In cells where EhCaBP3 expression was down regulated by antisense RNA, the level of RBC uptake was reduced, myosin IB was found to be absent at the site of pseudopod cup closure and the time taken for phagocytosis increased, suggesting that EhCaBP3 along with myosin 1B mediate the closure of phagocytic cups. Experiments with EhCaBP3 mutant defective in Ca²⁺ -binding showed that Ca²⁺ binding is required for phagosome formation. Liposome binding assay revealed that EhCaBP3 recruitment and enrichment to membrane is independent of any cellular protein as it binds directly to phosphatidylserine. Taken together, our results suggest a novel pathway mediating phagocytosis in E. histolytica, and an unusual mechanism of modulation of cytoskeleton dynamics by two calcium binding proteins, EhCaBP1 and EhCaBP3 with mostly non-overlapping functions.     

Crystal Structure of Calcium Binding Protein-5 from Entamoeba histolytica and Its Involvement in Initiation of Phagocytosis of Human Erythrocytes

Entamoeba histolytica is the etiological agent of human amoebic colitis and liver abscess, and causes a high level of morbidity and mortality worldwide, particularly in developing countries. There are a number of studies that have shown a crucial role for Ca²⁺ and its binding protein in amoebic biology. EhCaBP5 is one of the EF hand calcium-binding proteins of E. histolytica. We have determined the crystal structure of EhCaBP5 at 1.9 Å resolution in the Ca²⁺-bound state, which shows an unconventional mode of Ca²⁺ binding involving coordination to a closed yet canonical EF-hand motif. Structurally, EhCaBP5 is more similar to the essential light chain of myosin than to Calmodulin despite its somewhat greater sequence identity with Calmodulin. This structure-based analysis suggests that EhCaBP5 could be a light chain of myosin. Surface plasmon resonance studies confirmed this hypothesis, and in particular showed that EhCaBP5 interacts with the IQ motif of myosin 1B in calcium independent manner. It also appears from modelling of the EhCaBP5-IQ motif complex that EhCaBP5 undergoes a structural change in order to bind the IQ motif of myosin. This specific interaction was further confirmed by the observation that EhCaBP5 and myosin 1B are colocalized in E. histolytica during phagocytic cup formation. Immunoprecipitation of EhCaBP5 from total E. histolytica cellular extract also pulls out myosin 1B and this interaction was confirmed to be Ca²⁺ independent. Confocal imaging of E. histolytica showed that EhCaBP5 and myosin 1B are part of phagosomes. Overexpression of EhCaBP5 increases slight rate (∼20%) of phagosome formation, while suppression reduces the rate drastically (∼55%). Taken together, these experiments indicate that EhCaBP5 is likely to be the light chain of myosin 1B. Interestingly, EhCaBP5 is not present in the phagosome after its formation suggesting EhCaBP5 may be playing a regulatory role.     

Virulence and Functions of Myosin II Are Inhibited by Overexpression of Light Meromyosin in Entamoeba histolytica

Several changes in cell morphology take place during the capping of surface receptors in Entamoeba histolytica. The amoebae develop the uroid, an appendage formed by membrane invaginations, which accumulates ligand–receptor complexes resulting from the capping process. Membrane shedding is particularly active in the uroid region and leads to the elimination of accumulated ligands. This appendage has been postulated to participate in parasitic defense mechanisms against the host immune response, because it eliminates complement and specific antibodies bound to the amoeba surface. The involvement of myosin II in the capping process of surface receptors has been suggested by experiments showing that drugs that affect myosin II heavy-chain phosphorylation prevent this activity. To understand the role of this mechanoenzyme in surface receptor capping, a myosin II dominant negative strain was constructed. This mutant is the first genetically engineered cytoskeleton-deficient strain of E. histolytica. It was obtained by overexpressing the light meromyosin domain, which is essential for myosin II filament formation. E. histolytica overexpressing light meromyosin domain displayed a myosin II null phenotype characterized by abnormal movement, failure to form the uroid, and failure to undergo the capping process after treatment with concanavalin A. In addition, the amoebic cytotoxic capacities of the transfectants on human colon cells was dramatically reduced, indicating a role for cytoskeleton in parasite pathogenicity.     

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.     

Evidence for a novel Entamoeba histolytica lectin activity that recognises carbohydrates present on ovalbumin

Entamoeba histolytica, an intestinal amoeba that causes dysentery and liver abscesses, acquires nutrients by engulfing bacteria in the colonic lumen and phagocytoses apoptotic cells during tissue invasion. In preliminary studies to identify ligands that stimulate amoebic phagocytosis, we used ovalbumin immobilized on latex particles as a potential negative control protein. Surprisingly, ovalbumin strongly stimulated E. histolytica particle uptake. Experiments using highly purified ovalbumin confirmed the specificity of this finding. The mechanism of particle uptake was actin-dependent, and the Entamoeba phagosome marker amoebapore A localised to ovalbumin-bead containing vacuoles. The most well described amoebic receptor is a Gal/GalNAc-specific lectin, but d-galactose had no effect on ovalbumin-stimulated phagocytosis. Ovalbumin has a single N-glycosylation site (Asn₂₉₂) and is modified with oligomannose and hybrid-type oligosaccharides. We used both trifluoromethanesulfonic acid and N-glycanase to deglycosylate ovalbumin and tested the effect. Both methods substantially reduced the stimulatory effect of ovalbumin. Biotinylated ovalbumin bound the surface of fixed E. histolytica trophozoites saturably; furthermore, denatured ovalbumin and native ovalbumin both specifically inhibited ovalbumin-biotin binding, but deglycosylated ovalbumin had no effect. Collectively, these data suggest that E. histolytica has a previously unrecognised surface lectin activity that binds to carbohydrates on ovalbumin and stimulates phagocytosis.     

Differentiation of Entamoeba histolytica: a possible role for enolase

The study of the encystation process of Entamoeba histolytica has been hampered by the lack of experimental means of inducing mature cysts in vitro. Previously we have found that cytoplasmic vesicles similar to the encystation vesicles of Entamoeba invadens are present in E. histolytica trophozoites only in amebas recovered from experimental amebic liver abscesses. Here we report that a monoclonal antibody (B4F2) that recognizes the cyst wall of E. invadens also identifies a 48 kDa protein in vesicles of E. histolytica trophozoites recovered from hepatic lesions. This protein is less expressed in trophozoites continuously cultured in axenical conditions. As previously reported for E. invadens, the B4F2 specific antigen was identified as enolase in liver-recovered E. histolytica, by two-dimensional electrophoresis, Western blot and mass spectrometry. In addition, the E. histolytica enolase mRNA was detected by RT PCR. The antigen was localized by immunoelectron microscopy in cytoplasmic vesicles of liver-recovered amebas. The B4F2 antibody also recognized the wall of mature E. histolytica cysts obtained from human samples. These results suggest that the enolase-containing vesicles are produced by E. histolytica amebas, when placed in the unfavorable liver environment that could be interpreted as an attempt to initiate the encystation process.     

Lipid requirements for axenic cultivation ofEntamoeba histolytica

Fatty acids, cholesterol and glucose present in axenic medium are utilized by growingEntamoeba histolytica but the amoeba is unable to synthesize cholesterol from [U^-14 C^- ] glucose although the label is incorporated into the fatty acids and non-saponifiable fractions of the organism. Exogenously-added sonicated dispersions of cholesterol, Β-sitosterol, lanosterol, lecithin and lauric, palmitic, linoleic and stearic acids are ingested by the amoebae with subsequent loss in amoeboid movement. After a few hours the movement is regained. Cholesterol, lecithin and the fatty acids stimulate amoebic multiplication but are unable to replace serum in the medium either singly or in combination. CDRI Communication No. 2516.     

Eukaryotic checkpoints are absent in the cell division cycle ofEntamoeba histolytica

Fidelity in transmission of genetic characters is ensured by the faithful duplication of the genome, followed by equal segregation of the genetic material in the progeny. Thus, alternation of DNA duplication (S-phase) and chromosome segregation during the M-phase are hallmarks of most well studied eukaryotes. Several rounds of genome reduplication before chromosome segregation upsets this cycle and leads to polyploidy. Polyploidy is often witnessed in cells prior to differentiation, in embryonic cells or in diseases such as cancer. Studies on the protozoan parasite,Entamoeba histolytica suggest that in its proliferative phase, this organism may accumulate polyploid cells. It has also been shown that although this organism contains sequence homologs of genes which are known to control the cell cycle of most eukaryotes, these genes may be structurally altered and their equivalent function yet to be demonstrated in amoeba. The available information suggests that surveillance mechanisms or ‘checkpoints’ which are known to regulate the eukaryotic cell cycle may be absent or altered inE. histolytica.     

Species-Specific Immunity Induced by Infection with Entamoeba histolytica and Entamoeba moshkovskii in Mice

Entamoeba histolytica, the parasitic amoeba responsible for amoebiasis, causes approximately 100,000 deaths every year. There is currently no vaccine against this parasite. We have previously shown that intracecal inoculation of E. histolytica trophozoites leads to chronic and non-healing cecitis in mice. Entamoeba moshkovskii, a closely related amoeba, also causes diarrhea and other intestinal disorders in this model. Here, we investigated the effect of infection followed by drug-cure of these species on the induction of immunity against homologous or heterologous species challenge. Mice were infected with E. histolytica or E. moshkovskii and treated with metronidazole 14 days later. Re-challenge with E. histolytica or E. moshkovskii was conducted seven or 28 days following confirmation of the clearance of amoebae, and the degree of protection compared to non-exposed control mice was evaluated. We show that primary infection with these amoebae induces a species-specific immune response which protects against challenge with the homologous, but not a heterologous species. These findings pave the way, therefore, for the identification of novel amoebae antigens that may become the targets of vaccines and provide a useful platform to investigate host protective immunity to Entamoeba infections.     

Sequence specific 1H, 13C and 15N resonance assignments of a calmodulin-like calcium-binding protein from the protozoan parasite Entamoeba histolytica (EhCaM)

We report almost complete sequence specific ¹H, ¹³C and ¹⁵N NMR assignments of a 151-residue long calmodulin-like calcium-binding protein from Entamoeba histolytica (EhCaM).     

The Entamoeba histolytica,Arp2/3 Complex Is Recruited to Phagocytic Cups through an Atypical Kinase EhAK1

The parasite Entamoeba histolytica is the etiological agent of amoebiasis and phagocytosis plays a key role in virulence of this organism. Signaling pathways involved in activation of cytoskeletal dynamics required for phagocytosis remain to be elucidated. Phagocytosis is initiated with sequential recruitment of EhC2PK, EhCaBP1, EhCaBP3 and an atypical kinase EhAK1 after particle attachment. Here we show that EhARPC1, an essential subunit of the actin branching complex Arp 2/3 is recruited to the phagocytic initiation sites by EhAK1. Imaging, expression knockdown of different molecules and pull down experiments suggest that EhARPC1 interacts with EhAK1 and that it is required during initiation of phagocytosis and phagosome formation. Moreover, recruitment of EhARPC2 at the phagocytosis initiation by EhAK1 is also observed, indicating that the Arp 2/3 complex is recruited. In conclusion, these results suggests a novel mechanism of recruitment of Arp 2/3 complex during phagocytosis in E. histolytica.     

Intracellular traffic of the lysine and glutamic acid rich protein KERP1 reveals features of endomembrane organization in Entamoeba histolytica

The development of amoebiasis is influenced by the expression of the lysine and glutamic acid rich protein 1 (KERP1), a virulence factor involved in Entamoeba histolytica adherence to human cells. Up to date, it is unknown how the protein transits the parasite cytoplasm towards the plasma membrane, specially because this organism lacks a well‐defined endoplasmic reticulum (ER) and Golgi apparatus. In this work we demonstrate that KERP1 is present at the cell surface and in intracellular vesicles which traffic in a pathway that is independent of the ER–Golgi anterograde transport. The intracellular displacement of vesicles enriched in KERP1 relies on the actin‐rich cytoskeleton activities. KERP1 is also present in externalized vesicles deposited on the surface of human cells. We further report the interactome of KERP1 with its association to endomembrane components and lipids. The model for KERP1 traffic here proposed hints for the first time elements of the endocytic and exocytic paths of E. histolytica.