Amoebic PI3K and PKC Is Required for Jurkat T Cell Death Induced by Entamoeba histolytica

The enteric protozoan parasite Entamoeba histolytica is the causative agent of human amebiasis. During infection, adherence of E. histolytica through Gal/GalNAc lectin on the surface of the amoeba can induce caspase-3-dependent or -independent host cell death. Phosphorylinositol 3-kinase (PI3K) and protein kinase C (PKC) in E. histolytica play an important function in the adhesion, killing, or phagocytosis of target cells. In this study, we examined the role of amoebic PI3K and PKC in amoeba-induced apoptotic cell death in Jurkat T cells. When Jurkat T cells were incubated with E. histolytica trophozoites, phosphatidylserine (PS) externalization and DNA fragmentation in Jurkat cells were markedly increased compared to those of cells incubated with medium alone. However, when amoebae were pretreated with a PI3K inhibitor, wortmannin before being incubated with E. histolytica, E. histolytica-induced PS externalization and DNA fragmentation in Jurkat cells were significantly reduced compared to results for amoebae pretreated with DMSO. In addition, pretreatment of amoebae with a PKC inhibitor, staurosporine strongly inhibited Jurkat T cell death. However, E. histolytica-induced cleavage of caspase-3, -6, and -7 were not inhibited by pretreatment of amoebae with wortmannin or staurosporin. In addition, we found that amoebic PI3K and PKC have an important role on amoeba adhesion to host compartment. These results suggest that amebic PI3K and PKC activation may play an important role in caspase-independent cell death in Entamoeba-induced apoptosis.

Specific and reversible inhibition of entamoeba histolytica cysteine proteinase activities by Zn²+: Implications for adhersion and cell damage1

Background. Cysteine-proteinases are thought to play an important role in E. histolytica pathogenicity. Although effective blockage of proteolytic activities can be obtained with sereveral known inhibitors, the high cellular toxicity of most of the inhibitors precludes experimentation with live cells or animal models. Specific cysteine-proteinase inhibitors that could be utilized in studies of virulence are of great need in the field of amebiasis. Methods. Cysteine-proteinase activities were determined in trophozoit lysates by azocasein degradation and after PAGE and gelatin zymograms. Inhibition of the activities was assessed in the presence of 0.01-2.5 mM concentrations of fivalent cations of the IIB and VIII series such as Zn, Cd, Hg, Ni, and Co. Reversibility was induced with 25 mM L-cysteine or 50 mM L-histidine and by metal chelation with 5 mM phenantroline. The inhibitory effect of ZnCI2 was tested with live cells in fibronectin-biding and cytotoxicity assays. Results. ZnCI2 specifically inhibited cysteine-proteinase activities in trophozoite lysates in a concentration-dependent manner. Additionally, 1.0-2.5 mM ZnCI2 bloked proteolysis in more than 70 percent. This inhibition was completely reverted by L-cysteine, L-histidine, or phenantroline. Similar results were obtained by analyzing indivual cysteine-proteinase activities separated in gelatin zymograms. At these concentrations, ZnCI2 significanty interfered with trophozoit adhesion, thus making amebas deficient in substrate degradation and cell damage. Conclusions. ZnCI2 is effective inhibitor of amebic cysteine-proteinases. Its low toxicity at relative high concentrations, high solubility, and low cost make it adequate for live cell experimentation and animal models of amebic virulence

Isoenzyme and molecular characterization of Entamoeba histolytica and Entamoeba dispar isolates from symptomatic and asymptomatic subjects.

AIM: To correlate the clinical features of amebic infections with the characteristics of Entamoeba culture isolates of stools. METHODS: Isolates from seven irritable bowel syndrome (IBS) patients, four asymptomatic cyst passers (ACP) and five patients with invasive amebic disease were subjected to hexokinase polyacrylamide electrophoresis (HK-PAGE) and their DNA subjected to restriction fragment (RF) analysis of amplified polymerase chain reaction (PCR) products. These findings were correlated with anti-amebic serology. Two axenic pathogenic strains (HM1:IMSS, NIH:200) and one xenic nonpathogenic strain (SAW1734) were used as standards. RESULTS: All isolates from IBS patients as well as ACP had slow-moving (nonpathogenic) band pattern, whereas those from patients with invasive disease had fast-moving (pathogenic) band pattern on HK-PAGE. Serological data using EIA and RF patterns of PCR-amplified genome corroborated these results. CONCLUSIONS: Our results support the view that there are two species of Entamoeba infecting humans--E. histolytica(pathogenic) and E. dispar (nonpathogenic), and HK-PAGE of culture isolates can differentiate between them.

Enzymeba, Procedimiento eficaz para estudiar la prevalencia de infección intestinal por entamoeba histolytica / ENZYMEBA: an effective method for analzing the prevalence of Entamoeba histolytica intestinal infection

Por la unidad epidemiológica que tiene el desarrollo de procedimientos que permitan estudios confiables de prevalencia de amebiosis intestinal se estudió la eficacia de ENZYMEBA, ensayo inmunoenzimático para la detección en heces de Entamoeba histolytica. ENZYMEBA, mediante el empleo de una sola muestra por individuo participante en el estudio, demostró infección por E. histolytica en 27 de los 686 casos estudiados. La observación microscópica de heces sólo igualó los resultados de ENZYMEBA cuando fueron analizados los resultados de ambos procedimientos sobre 3 muestras de cada uno de los individuos en el estudio. Por su alta sensibilidad, requerir de una sola muestra por persona encuestada y ser un inmunoensayo realizable en condiciones mínimas de laboratorio, consideramos que ENZYMEBA es una herramienta muy útil para estudios de prevalencia de amebiosis intestinal

Proteinase activity & virulence of Entamoeba histolytica on passage through hamster liver.

This study was undertaken to determine the enzymatic differences in the process of increasing the degree of virulence in E. histolytica successively passaged in hamster liver. Substrate gel electrophoresis was used to compare proteinase banding patterns under reducing conditions from whole cell lysates of one axenic E. histolytica (strain HM1:IMSS) and five xenic isolates of the same strain of E. histolytica passaged five times through hamster liver. Trophozoites successively passaged in hamster liver showed in supernatants, major bands in the 56-97 kDa region whereas only the axenic strain produced additional band at 34 KDa. Inoculation of amoebic trophozoites into hamster led to progressive increase in proteinase activity of supernatants as well as increased virulence of amoebae; proteinase activity of amoebae showed an excellent correlation with their virulence. All the infected animals died when activity of proteinase was 0.152 mg of protein.

Validación en Ciudad de La Habana, Cuba, de ENZYMEBA, inmunoensayo para la detección de Entamoeba histolytica en heces / Validation in havana City, Cuba, of the ENZYMEBA, an immunoassay for deyecting Entamoeba histolytica in faeces

Parasitólogos de diferentes instituciones médicas de Ciudad de La Habana realizaron la validación externa de ENZYMEBA, procedimiento diagnóstico de amebiosis intestinal en el Instituto de Medicina Tropical "Pedro Kourí". Para ello fueron colectadas muestras seriadas de heces de 212 individuos sobre las que se realizó la observación microscópica (prueba de referencia) y el inmunoensayo ENZYMEBA (prueba en validación). ENZYMEBA, comparada con el examen microscópico de heces, mostró satisfactorios índices de sensibilidad y especificidad. No se observaron reacciones cruzadas con muestras de heces en que estaban presentes otros parásitos. Además, si se tiene en cuenta que para el diagnóstico de amebiosis intestinal con ENZYMEBA es suficiente una sola muestra de heces por paciente, este procedimiento pudiera ser útil en estudios de eficacia terapéutica y de prevalencia

Ausência de amebíase invasiva em aidéticos homossexuais masculinos, no Recife / Absence of invasive amoebiasis in male homosexual AIDS patients in Recife

The purpose of this paper was to determine if among male homosexual AIDS [correction of SIDA] patients, the Entamoeba histolytica was able to product invasive illness. From the 77 investigated patients only one (1.3 per cent) has cysts of E. histolytica in his stools. The electrophoresis of the only isolated strain has shown it was from zimodeme I, non pathogenic. The research of antiamebic antibodies was negative in the serum of the totality of patients. Those results showed that even in immunocompromised patients with AIDS [correction of SIDA], E. histolytica strain found in Recife was not able to produce disease. The utilization of metronidazol is not indicated in amoebic cysts carriers.

The ecto-ATPases of endoparasites and of blood cells and vessels

The cells of blood vessel walls and the external surface of all blood cells have an ecto-ATPase which hydrolyzes ATP to ADP and also ADP to AMP. This enzyme has also been called apyrase or ATP-diphosphohydrolase. The enzyme hydrolyzes a broad range of tri-and diphosphate nucleosides such as UTP and UDP, GTP and GDP in additon to the adenine nucleotides and because of that it has also been called a nucleoside triphosphate hydrolase. The possible physiological roles for this ecto-ATPase involve the control of vascular tone by modulation of the levels of ATP and ADP binding to purino-receptors of the vasculature, the modulation of thrombogenesis by controlling the extracellular level of ADP which is known to activate platelet aggregation, and the protection from cytolytic effects of extracellular ATP. An ATP-diphosphohydrolase activity has been characterized on the external surface of Schistosoma mansoni, a parasite that lives in the circulation of the human host, and on the outer surface of Entamoeba histolytica, a parasite that may enter the circulation of the host through ulceration in the intestinal mucosa. The endoparasite Toxoplasma gondii also exhibits a nucleoside triphosphate hydrolase of high activity, although in this case the ecto-localization is still not documented. We raise the possibility that the endoparasites have evolved in a way to possibly mimic some of the conditions on the surface of cells normally present in the host circulation, thus escaping hemostatic defense responses of the host which require extracellular ADP or ATP.

Identificación por isoenzimas de cepas colombianas de Entamoeba histolítica / Identification by isoenzymes of Entamoeba histolytica Colombian strains

Se analizaron los patrones electroforéticos de 17 cepas de E. histolytica y 4 cepas de otras especies de amibas intestinales no patógenas. Los cultivos se hicieron en medio de Robinson a partir de materia fecal con quistes y/o trofozoítos observados en el examen directo. Se encontraron 2 zimodemas patógenos: II, XI alfa-, y 7 zimodemas no patógenos: I, III, XVII, y 3 aún no clasificados. el más frecuente es el XVII (29.4 por ciento). Los 2 zimodemas patógenos tienen una frecuencia de 5.88 por ciento cada uno, el zimodema XI alfa- por primera vez es descrito a partir de aislamiento de materia fecal de un paciente

Proteins of Entamoeba histolytica trophozoites involved in the adhesion to target cells

To identify the molecules involved in the adhesion of Entamoeba histolytica trophozoites to target cell we used monoclonal antibodies (MAbs) and adhesion-deficient mutants. Human red blood cell (RBcs) were also used as especific carriers to identify the ameba molecules with affinity to the target cell receptors. MAbs adh-1 and Adh-2 inhibited adhesion of RBCs to the trophozoites and recognized a 112-kDa surface protein that was present in the wild type strain, but was absent or modified in adhesion-deficient mutants. In other experiments, live trophozoites were incubated with fixed-RBCs and after lysis of the trophozoites, proteins attached to the RBCs surface were separated by PAGE, electrotransferred to nitrocellulose membranes and detected by polyclonal antibodies. The 112 kDa protein was found attached to the RBCs. Other molecules identified as proteins involved in the target cell-parasite contact were the 210, 160, 90, 70, 50 and 24 kDa proteins. The 112, 90 and 24kDa polypeptides were functionally altered in adhesion-deficient mutants

Partial purification of acid phosphomonoesterase in axenically grown Entamoeba histolytica NIH-200.

Entamoeba histolytica possesses significant acid phosphatase activity as compared to alkaline phosphatase activity. The acid phosphatase activity in the amoebic cells eluted at higher saline concentration as three distinct peaks at 200, 300 and 400 mM sodium chloride.

Variations in isoenzymes of cloned & uncloned axenic Entamoeba histolytica without bacterial association.

Five clones of axenic E. histolytica (HMI) grown as discrete colonies in semisolid agar medium were adapted in liquid medium and labelled as HMI-C121, HMI-C131, HMI-C143, HMI-C144 and HMI-C145. Isoenzymes of these 5 clones of E. histolytica (HMI) were investigated in starch gel electrophoresis. There were no differences in the electromobility of maleate NADP oxidoreductase and glucosephosphoisomerase amongst the five clones and uncloned cultures of axenic E. histolytica. The relative electromobility (rf) of a single phosphoglucomutase (PGM) band of uncloned Mexican E. histolytica (HMI) and Indian axenic E. histolytica (KCG: 0986: 11) cultures and cloned E. histolytica HMI-C121, HMI-C145 was 0.087 while a single PGM band of uncloned E. histolytica (NIH: 200) and cloned E. histolytica HMI-C131, HMI-C143 and HMI-C144 cultures had rf of 0.075. Isoenzyme characterization of four cloned HMI-C121, HMI-C131, HMI-C143, HMI-C144 cultures of axenic E. histolytica (HMI) revealed existence of three bands of hexokinase (HK). The additional third band of HK was located close to the place of application of lysate and had rf ranging from 0.11-0.14. The data indicated that parent axenic E. histolytica (HMI) consisted of several populations and each population expressed different isoenzyme pattern without an association of amoebic cultures with any bacterial species.

Specific features of phosphomonoesterase of Entamoeba histolytica (NIH-200).

Axenically grown E. histolytica possess significant acid phosphatase activity. The Km of the enzyme was found to be 7.1 x 10(-3) and was maximally active at pH 4.5. Acid phosphatase activity was significantly inhibited by Cu2+, cysteine and was activated by tartrate and fluoride. It was found that E. histolytica acid phosphatase differs in some properties as compared to the enzyme reported from other sources.