Purification and biochemical characterization of a novel cysteine protease of Entamoeba histolytica.

Cysteine proteases are important virulence factors of Entamoeba histolytica, the causative agent of amoebiasis. A novel cysteine protease from parasite extracts was purified 15-fold by a procedure including concanavalin A-Sepharose, hydroxylapatite and DEAE-Sepharose chromatography. The purification resulted in the obtainment of an homogeneous protein with a molecular mass of 66 kDa on native PAGE. In 10% SDS/PAGE, three bands of 60, 54 and 50 kDa were evident. Each of the three specific mouse antisera raised against these proteins showed cross-reactivity with the three bands obtained from the purified eluate. The N-terminal sequencing of the first 10 amino acids from the three proteins showed 100% identity. These results support the hypothesis of a common precursor for the 60, 54 and 50-kDa proteins. Protease activity of the purified enzyme was demonstrated by electrophoresis in a gelatine-acrylamide copolymerized gel. Its activity was quantified by cleaving a synthetic fluorogenic peptide substrate such as N-carbobenzyloxy-arginyl-arginyl-7-amido-4-methylcoumarin. The optimum pH for the protease activity was 6.5; however, enzymatic activity was observed between pH 5 and pH 7.5. Typical of cysteine proteases, the enzyme was inhibited by 4-[(2S, 3S)-carboxyoxiran-2-ylcarbonyl-L-leucylamido]butylg uanidine and iodoacetamide, and activated by free sulfhydryl groups. The cellular location of the enzyme was examined on trophozoites before and after contact with red blood cells using indirect immunofluorescence and cellular fractionation. The 60-kDa cysteine protease translocated to the amoebic surface upon the interaction of trophozoites with red blood cells. This result provided evidence for participation of the 60-kDa protease in erythrophagocytosis.

Inhibitory activity of saccharomyces yeasts on the adhesion of Entamoeba histolytica trophozoites to human erythrocytes in vitro.

Adhesion to target cells represents the first step in infection by Entamoeba histolytica. Binding of axenic amoeba (HMI strain) to human red cells in vitro was employed as a model of the adhesion process. The influence of precontact of trophozoites with suspensions of live Saccharomyces boulardii yeasts, their fractions (membranes and yeast-content supernatant before and after filtration to eliminate the membrane) or yeast culture medium before and after fermentation was investigated. N-Acetylgalactosamine (GalNAC) was employed as the reference inhibitory sugar. The percentage of amoebae bearing red cells after pretreatment of amoebae with the various suspensions and derivates was determined. Adhesion was also evaluated by scanning electron microscopy (SEM). Pretreatment of amoebae with the live yeast suspension led to a significant reduction in the percentage of adhesion [32% vs 70% in the phosphate-buffered saline (PBS) control]. Reduced adhesion was also observed with the filtered and unfiltered supernatant of the yeast suspension homogenate [32% and 34%, respectively, vs 69% in the PBS control], yeast culture medium at the end of fermentation [49% vs 76% in the PBS control] and GalNAC [32% vs 72% in the PBS control]. SEM showed a decrease in the number of amoebae bearing red cells and a reduction in the number of red cells adhering to amoebae. We conclude that substances produced by the yeasts compete with red cells for adhesion sites on amoebae.

Purification and functional characterization of the 112 kDa adhesin of Entamoeba histolytica.

The 112 kDa adhesin of E. histolytica is directly involved in the cytopathogenic activity of the parasite. We describe here the purification of the 112 kDa protein by electroelution and immunoaffinity chromatography using a monoclonal antibody against the adhesin. Two proteins of 70 and 50 kDa were eluted from the immunoaffinity column along with the 112 kDa adhesin. The three proteins were recognized by monospecific polyclonal antibodies against the adhesin. The same peptides (72 and 56 kDa) were also observed after incubation of the purified intact adhesin in diethylamine buffer. Proteins of 112 and 72 kDa were found to have protease activity, evidenced by their ability to degrade gelatin. Our results indicate that the 112 kDa adhesin was specifically broken down into two polypeptides of 50-56 and 70-72 kDa. The significance of this in vivo is as yet unclear. The adhesin has proteolytic activity, which is retained in the 70-72 kDa polypeptide but not in the 50-56 kDa one.

A new in vitro model of Entamoeba histolytica adhesion, using the human colon carcinoma cell line Caco-2: scanning electron microscopic study.

The human colon carcinoma cell line Caco-2, which is widely used to study the adhesion and cytotoxicity of enterobacteria, was used to investigate the adhesion of the trophozoites of Entamoeba histolytica. We observed a high percentage of adhesion of amoebae to Caco-2 cells. Scanning electron microscopy showed that amoebial membrane structures were involved in adhesion and the cytolytic action. These differentiated cells should prove to be a useful model system for investigation of the pathogenic action of amoebae.

[Effects of Saccharomyces boulardii yeast on trophozoites of Entamoeba histolytica in vitro and in cecal amebiasis in young rats]. / Effets des levures Saccharomyces boulardii sur les trophozoïtes d'Entamoeba histolytica in vitro et dans l'amibiase caecale du jeune rat.

Lyophilized and rehydrated Saccharomyces boulardii yeasts were administered to young rats previously inoculated into the cecum with Entamoeba histolytica. The numbers of diseased young rats and the severity of the infection, assessed from the appearance of the cecum and its content of mucus and amebae, were significantly reduced by the treatment. The lesions observed resembled those seen in the untreated controls, although healing was faster in the treated animals. Saccharomyces boulardii had no intrinsic amebicidal action in vitro.

[A new experimental model of cecal amebiasis in rats]. / Nouveau modèle expérimental d'amibiase caecale chez le rat.

The young rat was used as an experimental model of caecal amebiasis by implanting, without injury of the caecum, a suspension of E. histolytica trophozoites gnotoxenic strain Rahman of high virulence. The degree of pathogenicity is demonstrated by the macroscopic aspect of caecum, by the density of trophozoites and by an anatomopathologic examination. As a correlation exits between all these macroscopic parameters and histology, a degree of illness is fixed which include several macro- and microscopic criteria. This model enables to observe the different phases of cicatrization and of recovering of young rats and so to appreciate the activity of antiamebic agents.