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.

De novo synthesis of sphingolipids plays an important role during in vitro encystment of Entamoeba invadens.

Entamoeba invadens is a protozoan, which causes multiple damages in reptiles and is considered a prototype for the study of the Entamoeba encystment in vitro. Here we report for the first time the role of the de novo synthesis pathway of sphingolipids during the encystment of E. invadens. In silico analysis showed that this parasite has six putative genes coding for ceramide synthases (CerS), all of them coding for proteins containing the Lag1p motif, a region conserved in the ceramide synthases of multiple organisms, suggesting that they might be bona fide CerS. The six genes of E. invadens are differentially expressed at different time intervals in both stages trophozoite and cyst, based on the results obtained through qRT-PCR assays, the genes involved in the synthesis of sphingolipids with long-chain fatty acids CerS 2,3,4 (EIN_046610, EIN_097030, EIN_130350) have maximum points of relative expression in both stages of the E. invadens life cycle, which strongly suggest that the signaling exerted from the synthesis pathway of sphingolipids is essential for the encystment of E. invadens, since the generation of the more abundant sphingomyelin (SM) subspecies with long-chain fatty acids are fundamental for the parasite to reach its conversion from trophozoite to cyst. When myriocin was used as an inhibitor of serine palmitoyl CoA transferase (SPT), first enzyme in the de novo biosynthesis of sphingolipids, the trophozoites of E. invadens were unable to reach the encystment. Since the effect of myriocin was reversed with exogenous d-erythrosphingosine (DHS), it was demonstrated that the inhibition was specific and it was confirmed that the synthesis of sphingolipids play an essential role during the encystment process of E. invadens.

Entamoeba invadens: Identification of a SERCA protein and effect of SERCA inhibitors on encystation.

Calcium has an important role on signaling of different cellular processes, including growth and differentiation. Signaling by calcium also has an essential function in pathogenesis and differentiation of the protozoan parasites Entamoeba histolytica and Entamoeba invadens. However, the proteins of these parasites that regulate the cytoplasmic concentration of this ion are poorly studied. In eukaryotic cells, the calcium-ATPase of the SERCA type plays an important role in calcium homeostasis by catalyzing the active efflux of calcium from cytoplasm to endoplasmic reticulum. Here, we reported the identification of SERCA of E. invadens (EiSERCA). This protein contains a putative sequence for endoplasmic reticulum retention and all domains involved in calcium transport identified in mammalian SERCA. By immunofluorescence assays, an antibody against SERCA of E. histolytica detected EiSERCA in a vesicular network in the cytoplasm of E. invadens trophozoites, co-localizing with calreticulin. Interestingly, EiSERCA was redistributed close to plasma membrane during encystation, suggesting that this pump could participate in regulate the calcium concentration during this process. In addition, thapsigargin and cyclopiazonic acid, both specific inhibitors of SERCA, affected the number and structure of cysts, supporting the hypothesis that calcium flux mediated by SERCA has an important role in the life cycle of Entamoeba.

Entamoeba invadens: sphingolipids metabolic regulation is the main component of a PKC signaling pathway in controlling cell growth and proliferation.

The sphingolipids biosynthesis pathway generates bioactive molecules crucial to the regulation of physiological processes. We have recently reported that DAG (diacylglycerol) generated during sphingomyelin synthesis, plays an important role in PKC (protein kinase C) activation, necessary for the transit through the cell cycle (G1 to S transition) and cell proliferation (Cerbon and Lopez-Sanchez, 2003. Diacylglycerol generated during sphingomyelin synthesis is involved in protein kinase C activation and cell proliferation in Madin-Darby canine kidney cells. Biochem. J. 373, 917-924). Since pathogenic Entamoeba invadens synthesize the sphingolipids inositol-phosphate ceramide (IPC) and ethanolamine-phosphate ceramide (EPC) as well as sphingomyelin (SM), we decided to investigate when during growth initiation, the synthesis of sphingolipids takes place, DAG is generated and PKC is activated. We found that during the first 6h of incubation there was a significant increase in the synthesis of all three sphingolipids, accompanied by a progressive increment (up to 4-fold) in the level of DAG, and particulate PKC activity was increased 4-8 times. The enhanced DAG levels coincided with decrements in the levels of sphingoid bases, conditions adequate for the activation of PKC. Moreover, we found that inhibition of sphingolipid synthesis with myriocin, specific inhibitor of the synthesis of sphinganine, reduce DAG generation, PKC activation and cell proliferation. All these inhibitory processes were restored by metabolic complementation with exogenous D-erythrosphingosine, indicating that the DAG generated during sphingolipid synthesis was necessary for PKC activation and cell proliferation. Also, we show that PI (phosphatidylinositol), PE (phosphatidylethanolamine) and PC (phosphatidylcholine) are the precursors of their respective sphingolipids (IPC, EPC and SM), and therefore sources of DAG to activate PKC.

Entamoeba invadens: influence of 60 Hz magnetic fields on growth and differentiation.

Exposure to extremely low-frequency (ELF) electromagnetic fields appears to result in a number of important biological changes. In the present study, we evaluated the effects of 60 Hz sinusoidal magnetic fields (MF) at magnetic flux densities of 1.0, 1.5 and 2.0 mT on growth and differentiation of the protozoan Entamoeba invadens. We demonstrated an inhibitory growth effect when trophozoite cultures were exposed to 1.5 and 2.0 mT. Furthermore, we found that there was not a synergistic effect in cultures co-exposed to MF and Metronidazole, a cytotoxic drug against amoebic cells. In addition, MF exposure inhibited the encystation process of E. invadens.

Congo red effect on cyst viability and cell wall structure of encysting entamoeba invadens

Background. The cell wall of Entamoeba invadens cysts is composed of chitin microfibrils as the main structural component. It has been demonstrated in yeast that the chitin cell wall assembly is altered by dyes such as Congo red (CR) and Calcofluor. Methods. The purpose of this work was to study the cell wall assambly under the effect of CR dye on encysting E. invadens by means of light and electron microscopy, after the ammebas were subjected to the effect of 100-2,000 µg CR/mL. Experiments were performed either in BI-S-33 or in mLG media. Results. Trophozoit growth was not inhibited by 100-1,000 µg/mL CR after 8 days of incubation in BI-S-33 medium. However, low levels of growth were observed with 2,000 µg/mL of dye. No significant differences in morphologically viable (hyaline) cyst production occurred after 24-48 hm when 100 µg CR/mL was used, while the highest concentration of CR (2,000 µg/mL) resulted in a significant decrease of hyaline cyst yield; dead cysts prevailed in cultures, particularly at 72 h of CR treatment. Differentiation of amebas incubated in the presence of 500-2,000 µg/mL CR produced abnormal chitin deposits, rendering irregulary thick or double cell walls, as shown by transmission and scanning electron microscopy. Cyst cultures obtained under 100 µg/mL CR produced as many trophozoites as did the control when they were incubated in BI-S-33, but only low numbers of trophozoites were found in culture cysts obtained under higher CR doses. Conclusion. Our results suggest that CR affects E. invadens encystment, alters the cell wall formation, and also affects the cyst viability

[Growth inhibition of Entamoeba histolytica and E. invadens produced by pomegranate root (Punica granatum L.)]. / Inhibición del crecimiento de Entamoeba histolytica y E. invadens producida por la raíz del granado (Punica granatum L.).

The purpose of the present work has been to evaluate the biological activity of alkaloids and tannins extracted from roots of Punica granatum L. on axenic cultures from Entamoeba histolytica and E. invadens, strains. Initially, the total aqueous extract and later some of its components obtained by thin layer chromatography were tested. These compounds were identified by chromatography as alkaloids or tannins. The density of amebic cultures was determined with a hemocytometer after 48 and 96 hours of incubation, which was calculated by the difference between number of trophozoites obtained at the times chosen and the number of amoebae inoculated. Two milliliters of aqueous extract had higher activity on cultures from E. histolytica than E. invadens strains, producing growth inhibitions of about 100 and 40 per cent respectively. Alkaloid concentrations of 1 mg/ml had no amebicide activity, however tannins at concentrations of 10 micrograms/ml for E. histolytica, and 100 micrograms/ml for E. invadens were sufficient to produce an growth inhibition about 100 per cent. Tannic acid was also tested on the cultures of E. histolytica observing an high inhibitory activity on the growth, this effect was produced at 0.01 mg/ml was similar to that observed with the tannins mixture.

[Trans-membrane potential in Entamoeba]. / Potencial transmembranal en Entamoeba.

The membrane potential in Entamoeba is an important driving force for the uptake of substrates. In Entamoeba invadens PZ a membrane potential of -36 mV was obtained when Nernst equation was applied to the distribution at equilibrium of 86Rb+ in the presence of valinomycin. This could explain the levels of accumulation of up to 4 times found for positively charged substrates. Membrane potential was diminished by depolarizing conditions (high external K+ concentration in the presence of valinomycin). Moreover, we recorded continuously the membrane potential of Entamoeba invadens PZ and Entamoeba histolytica HM1 using the fluorescent lipophilic cation diisopropylthiodicarbocyanine. It was found that the uptake of this cation by the amoebae was fast in both species, conditions that modify the membrane potential (hyperpolarization and depolarization) produced changes in the fluorescence of the dye in agreement with its reported capability to detect variations in membrane potential. It can be concluded that these microorganisms have a membrane potential negative inside them.

Ultraestructura de quistes de Entamoeba invadens obtenidos bajo el efecto de emetina, tinidazol y rifampicina / Ultraestructure of entamoeba invadens cysts obtained under the effect of emetine, tinidazole and rifampin

Se determinaron las concentraciones inhibitorias mínimas (CIM) de emetina, tinidazol y rifampicina para trofozoítos de E. invadens, así como la ultraestructura de quistes obtenidos después de 22 y 45 horas, en presencia de la CIM de los fármacos. Bajo el efecto de tinidazol o de rifampicina los quistes obtenidos mostraron numerosas vacuolas que contenían elementos membranosos y vesículas llenas de material amorfo electrodenso; algunas de éstas se observaron en las proximidades de la membrana plasmática. En los quistes obtenidos en presencia de emetina, el citoplasma presentó numerosas vesículas y cisternas aplanadas particularmente después de 22 horas de incubación en médio de enquistamiento, las que disminuyeron después de 45 horas de diferenciación; se observaron además vasículas con material electrodenso próximas a la membrana plasmática. Las paredes celulares de los quistes obtenido bajo el efecto de los fármacos mencionados fueron irregulares tanto en el arreglo fibrilar como en el espesor; adicionalmente, bajo los efectos de tinidazol y emetina las paredes de los quistes se perdieron parcial o totalmente, observándose acúmlos de éstas entre los quistes. Los resultados sugieren que la CIM de los fármacos empleados provocaron un retardo en el proceso de difereciación de E. invadens y, posiblemente, interfieran con el ensamble de la pared celular y con la adherencia de ésta a la membrana plasmática durante el enquistamiento