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Cell Biology International (2004) 28, 243248 (Printed in Great Britain)
Involvement of protein kinases on the process of erythrophagocytis by Entamoeba histolytica
Evander de J.O Batista* and Wanderley de Souza
Laboratório de Ultraestrutura Celular Hertha Meyer, Instituto de Biofı́sica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, CCS-Bloco G, Ilha do Fundão, Rio de Janeiro RJ, 21941-900, Brazil
Erythrophagocytic capacity of trophozoites of Entamoeba histolytica is considered a factor in the virulence of this pathogenic protozoan. We present evidence showing that such activity resembles the ingestion of microorganisms by highly differentiated phagocytic cells, such as macrophages. Previous treatment of the trophozoites with genistein or tyrphostin, inhibitors of tyrosine protein kinases, with staurosporine, a protein kinase C inhibitor, and wortmannin, a fungal metabolite that inhibits phosphoinositide 3-OH kinase, significantly inhibited their erythrophagocytic capacity.
Keywords: Entamoeba histolytica, Erythrophagocytosis, Protein tyrosine kinase, Protein kinase C, Phosphoinositide 3-kinase.
*Corresponding author. Fax: +55-21-2260-23-64
Amebiasis is the infection of the human gastrointestinal tract by Entamoeba histolytica, a protozoan parasite capable of invading the intestinal mucosa and spreading to other organs, mainly the liver (Espinosa-Cantellano and Martı́nez-Palomo, 2000). It affects ∼12% of the world's population (World Health Organization, 1997).
E. histolytica destroys almost all tissues of the human body using macromolecules involved in mechanisms such as adhesion, contact-dependent cytolysis, proteases and phagocytic activity. Several surface proteins have been reported as involved in the trophozoite-targeted cell contact. Some authors have shown that antibodies against membrane proteins, e.g. 112 and 66 kDa adhesins, block the adherence of trophozoites to epithelial cells and erythrocytes (Chavez-Rueda et al., 2002; Garcia-Rivera et al., 1999). The ability of E. histolytica to ingest erythrocytes is in some way related to its pathogenic activity; and erythrophagocytic activity is known to be higher in more pathogenic strains(Bhattacharya et al., 2002).
Phosphorylation is a post-translational covalent modification used by prokaryotic and eukaryotic cells to control the properties of a wide variety of proteins, including enzymes, receptors, ion channels and regulatory or structural proteins (Girault, 1994). These processes are performed by phosphotransferases, known as protein kinases, which catalyze the transfer of the phosphate group of ATP to an amino acid side chain in the presence of Mg++. Protein kinases play a crucial role in signal transduction for a variety of cellular responses, including cell proliferation, differentiation, and phagocytosis (Nishizuka, 1986; Waalas and Greengard, 1991). Among several types of protein kinases, protein kinase C (PKC), a large family of serine/threonine kinases with multiple isoforms, protein tyrosine kinase (PTK), and phosphoinositide 3-kinase (PI-3K), some of the most relevant in cell-to-cell interaction processes, including parasitic protozoa (Girault, 1994; Laroca et al., 2002; Vieira et al., 1994).
We decided to analyze whether cell-signaling events, with the participation of protein kinases, takes place during the erythrophagocytic process presented by E. histolytica. Our observations using protein kinase inhibitors, as staurosporine, genistein, tyrphostin and wortmannin, indicate the participation of protein kinase C, tyrosine kinase and PI-3 kinase on this process.
2 Materials and methods
Trophozoites of the HM1-ISS strain of E. histolytica were axenically cultivated in TYI-S-33 medium(Diamond et al., 1978), supplemented with 10% (V/V) bovine serum and 3% (V/V) Diamond vitamin Tween 80 solution (JRH Biociences) for 24 and 48 h in glass screw cap tubes at 37 °C. After that, cells were placed on ice water for 7 min to release them from the glass tube surface, collected by centrifugation at 350 g for 5 min and washed three times in 0.1 M phosphate-buffered saline (PBS, pH 7.2) at 37 °C.
Type O human erythrocytes (Rh+) were freshly obtained in Alsever's solution, washed three times in the same solution to remove white blood cells and diluted to 108cells/ml.
E. histolytica trophozoites at a density of 106cells/ml were incubated in the presence of 100 or 200 μM of genistein, 1 or 2 μM of staurosporine, 10 or 20 μM of tyrphostine, 25 or 50 nM wortmannin for 20 min at 37 °C. The drugs were initially dissolved into dimethyl sulfoxide (DMSO) to make a stock solution. After previous treatments, the trophozoites were then washed in PBS pH 7.2 to remove drug excess and erythrocytes were added. Interaction was carried out for 15 min at 37 °C in TYI-S-33 without bovine serum using a 1:100 amoeba-erythrocytes ratio. In order to stop erythrophagocytosis, cells were fixed with 2.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.2, at 37 °C. Control experiments were done using TYI-S-33 and 0.01% DMSO. To better visualize adhered or internalized erythrocytes, after fixation the cells were incubated in a medium containing 2 mg/ml of diaminobenzidine (DAB) in 100 mM Tris–HCl buffer, pH 7.6 (Novikoff et al., 1972), followed by 30 min incubation at room temperature in the dark, and revealed with 0.3% hydrogen peroxide. For quantitative evaluation, the percentage of trophozoites containing adhered and ingested erythrocytes, and the average of erythrocytes per trophozoite were determined by counting 200 amoebas at random from at least three independent duplicate experiments. The Kruskal–Wallis and Whitney non-parametric statistics test were used for evaluation of experiments. Cell viability was tested using trypan blue exclusion test. In all experiments, including those using the various drugs test, cell viability was >90%.
3 Results and discussion
Light microscopic observation of cells stained using the Novikoff's procedure, which reveals the peroxidase activity of hemoglobin, showed that it is clearly possible to distinguish erythrocytes adherent to the cell surface from those within the cytoplasm of the trophozoites. Using these preparations the adhesion and endocytic indexes were determined. As shown previously (Mora-Galindo and Anaya-Velazquez, 1993), incubation of human erythrocytes in the presence of trophozoites leads to their attachment and subsequent ingestion. The adhesion and endocytic indexes varied according to the incubation time and the erythrocyte:trophozoite ratio. Based on preliminary experiments we decided to use a erythrocyte:trophozoite ratio of 100:1 and an incubation time of 15 min. Under these conditions, ∼90% of parasites showed adherent or internalized erythrocytes. The mean values for attached and internalized erythrocytes per trophozoite were 12 and 10, respectively.
PI 3-kinase is a family of enzymes which phosphorylate the D3 position of the phosphatidylinositol (PtdIns) ring, generating phosphorylated PtdIns species that serve as signaling molecules (Toker and Cantley, 1997). These enzymes are dimers, consisting of regulatory (85 kDa) and catalytic (110 kDa) subunits. PI 3-kinase has been described as a molecule involved in a widely variety of physiologic processes such as immune cell proliferation, survival, differentiation, chemotaxis, degranulation, respiratory burst, endocytosis and in multiple membrane traffic events (Booth et al., 2001; Fruman and Cantley, 2002). It is well established that PI 3-kinase plays an important role on the endocytic process. For instance, IgG-dependent phagocytosis is blocked in neutrophils, macrophages and a monocytic cell line when these cells are treated with LY294003 or wortmannin, well-characterized inhibitors of this enzyme.
Previous incubation of E. histolytica trophozoites in the presence of 25 or 50 nM wortmannin did not significantly inhibited the adhesion of the erythrocytes to the parasite surface (Fig. 1). However, erythrophagocytosis was inhibited by 90–95% (Fig. 1). Also, wortmanin-treated parasites were not able to internalize the erythrocytes, although previously treated parasites were able to emit lamelipodia (Fig. 2A) and form pseudopods (Fig. 2B). However, the endocytic process was not complete, as it was in untreated parasites (Fig. 3A and B). These observations are in agreement with previous studies carried out in other cells showing that PI 3-kinase is required for maximal pseudopod extension and closure of the phagocytic vacuole during phagocytosis, but it seems to be dispensable for the initial phases of actin polymerization (Araki et al., 1996; Cox et al., 1999). Wortmannin inhibits macropinocytosis by Dictyostelium discoideum amoeba (Rupper et al., 2001), FC? receptor signaling and phagocytosis by macrophages (Ninomiya et al., 1994), and phagocytosis of bacteria, mucin-coated beads and pinocytosis of FITC-dextran by E. histolytica (Ghosh and Samuelson, 1997).
Effect of previous treatment of E. histolytica trophozoites with wortmannin (Wt) on the interaction with erythrocytes. Dark bars: relative adhesion index; striped bars: relative endocytic index. The data are representative of three different experiments that agreed within 5%.
High Resolution Field Emission Scanning Electron Microscopy (FESEM) showing E. histolytica trophozoites pre-treated with wortmannin. (A) Initial step of emission of lamelipodia. Adherence of human erythrocyte to the trophozoite surface (8000×). (B) Initial step of the internalization of erythrocytes (8000×).
Micrographs showing the internalization of untreated parasites. (A and B) Ingestion of the erythrocytes is evident (A=5.500×, B=3.000×).
Another molecule that participates in the signalling cascade is protein kinase C, which is a phospholipid-dependent serine/threonine kinase that plays a crucial role in signal transduction leading to a variety ofresponses. Involvement of PKC has been inferred by the use of specific activators, such as phorbol esters, and specific inhibitors, as staurosporine. In preliminary studies several authors have demonstrated that amoeba trophozoites require the integrity and functionality of the actin cytoskeleton for motility and adhesion, processes dependent on PKC activity (Carbajal et al., 1996; Guillén, 1996; Santiago et al., 1994; Weikel et al., 1988).
Previous incubation of amoeba trophozoites in the presence of staurosporine reduced by 10–20% the adhesion index, and by 90–95% erythrocyte internalization (Fig. 4). There is little information on the presence of PKC isozymes and their function in E. histolytica. A partial purified PKC preparation from this parasite was shown to phosphorylate histone in the presence of calcium, phospholipid and DAG. During phagocytosis, PKC activity is relocalized to the membrane (De Meester et al., 1990). Also, activation of PKC appears to induce vesicle exocytosis following Entamoeba–fibronectin interaction (Santiago et al., 1994; Weikelet al., 1988). PKC appears to participate also in the growth and encystation in E. invadens (Makioka et al., 2000).
Effect of previous treatment of E. histolytica trophozoites with staurosporine (St), genistein (Ge) and tyrphostin (Ty) on the interaction with erythrocytes. Dark bars: relative adhesion index; striped bars: relative endocytic index. The data are representative of three different experiments that agreed within 5%.
Previous studies have shown that E. histolytica expresses protein tyrosine kinase activity that seems to be involved in cytoskeleton reorganization following fibronectin receptor activation (Hermandez-Ramirezet al., 2000). We observed here that previous incubation of E. histolytica in the presence of protein tyrosine kinase inhibitors such as genistein, an isoflavone isolated from Pseudomonas sp. and thyphorstin, both working as highly specific inhibitors of PTK, slightly inhibited erythrocyte adhesion and markedly inhibited theerythropagocytic process of E. histolytica (Fig. 4).
In conclusion, pathways of signal transduction, though differing remarkably in their complexity and in the use of cellular components seem to obey certain principles which are evolutionarily well conserved and ubiquitously distributed amongst living organisms, indicating that despite the progressive modifications of organisms, the mechanisms which govern exchange of information in cellular process have been essentially conserved. Our present observations show that protein tyrosine kinase, protein kinase C and phosphoinositide 3-kinase plays a central role in signal transduction during trophoziote erythrophagocytosis and consequently might also influence the virulence of E. histolytica. It is important to point out that a putative serine/threonine protein kinase gene has been cloned (Que et al., 1993; Urban et al., 1996). In addition, at least three potential E. histolytica PI3-kinase orthologues have been found in searching for matches in the TIGR Institute database at http://www.tigr.org/tdb/e2k1/eha1. These amoeba PI3-kinase genes correspond to the TIGR entries EH04182, EH06212 and EH01481.
This work was supported by PRONEX (Programa de Núcleos de Excelência) from MCT/CNPq, Conselho Nacional de Desenvolvimento Cientı́fico e Tecnológico (CNPq), Coordenadoria de Aperfeiçoamento de Pessoal de Ensino Superior (CAPES) and Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ).
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Received 4 August 2003/25 November 2003; accepted 20 January 2004doi:10.1016/j.cellbi.2004.01.005