Entamoeba histolytica: cyst-like structures in vitro induction.

The cyst of Entamoeba histolytica is responsible for amebiasis infection. However, no axenic in vitro system exists that promotes mass encystation for studying this process of this human-infecting parasite. Cyst-like structures of E. histolytica obtained in this work were induced using TYI-S-33 media in combination with enterobacterias Escherichia coli and Enterococcus faecalis conditioned media, high CO2 tension and histamine. Cyst-like structures showed the same characteristics of a typical E. histolytica cyst: aggregation, resistance to 0.15% sarcosyl for 10 min, high signal of fluorescence under UV light when stained with 10% calcofluor M2r and the surface topology showed a wrinkled wall. In addition these structures are multinucleated with condensed chromatin attached to nuclear membrane, contain big vacuoles and ribonucleoproteic helices in the cytoplasm and also present a thin cell wall. Last all characteristics are all the same as a typical of E. histolytica cyst.

Entamoeba invadens: in vitro axenic encystation with a serum substitute.

The current media for axenic cultivation of Entamoeba histolytica and Entamoeba invadens are supplemented with bovine or equine serum, which provides several essential nutrients to amoebas. Serum has also been considered an essential component in encystation media for E. invadens. A substitute of serum, PACSR has been described as an alternative for growth of E. histolytica and also maintains growth of E. invadens. When PACSR was used instead of serum for encystation of E. invadens the efficiency was the same as for serum. Our present data show that PACSR can support the growth and induction of encystation of E. invadens strain IP-1.

Auxotrophy to lipoproteins of Entamoeba histolytica cultivated under axenic conditions.

Entamoeba histolytica grows in media without serum but with a mixture of aminoacids, vitamins, lipoproteins, free cholesterol, phospholipids and fatty acids called PACSR. The ability of lipoproteins and free lipids to support growth of three E. histolytica strains (HK9, HMI:IMSS and HM3:IMSS) was analysed. Tubes containing 5 ml culture medium, amino acids, vitamins and either 120-1,200 microg lipoproteins/ml or 0.017-0.10 mg free lipids/ml (predissolved in absolute ethanol) were inoculated with 1x10(4) trophozoites/ml and incubated at 37 degrees C for 72 h. Amoebae died within 12 h in the presence of any free lipid combination, while those having 240-480 mg lipoproteins/ml reached densities similar to or higher than those of controls (depending on strain). The addition of ethanol (0.1%) to the media produced stable lipid solutions and did not show significant adverse effects. Accordingly, E. histolytica is auxotrophic to lipoproteins and unable to use free cholesterol, phospholipids or fatty acids.

Axenic cultivation of Trichomonas vaginalis in a serum-free medium.

Mammalian serum or bovine serum albumin are essential for Trichomonas vaginalis cultivated under axenic conditions. Unfortunately, these components inhibit several biological properties of these parasites. PACSR is a serum replacement, free of bovine serum albumin. used for axenic cultivation of Entamoeba histolytica. We show that PACSR is also useful for axenic cultivation of T. vaginalis. Tubes containing 5.5 ml PEHP, or TYI basal media, plus 8% PACSR (v/v), were inoculated with 10(3) trichomonads/ml from 3 strains (GT-3, GT-13, and GT-15) and incubated at 36.5 C for 72 hr. Depending on the strain, cultures grown in PEHP plus PACSR reached densities of 50% (GT-13), 63% (GT-3), or 82% (GT-15) as compared to controls grown in PEHPS. On the other hand, yields of GT-3, GT-13, and GT-15 maintained in TYI plus PACSR were, respectively, 53%, 57%, and 67% among those of cultures grown in TYI-S-33. In all experiments, PEHPS and TYI-S-33 contained 8% bovine serum. Yields reached in PEHPS were 2.07+/-0.275 to 4.83+/-4.41 x 10(6) trichomonads/ml, whereas in TYI-S-33, densities were 1.68+/-0.315 to 4.16+/-8.07 x 10(6) trichomonads/ml. In conclusion, PACSR added to PEHP or TYI media is useful for axenic cultivation of T. vaginalis in the absence of serum or bovine serum albumin. PACSR could be useful in performing analyses of biological properties that are inhibited by serum or any of its components.

Isolation of an Entamoeba histolytica intracellular alkaline phospholipase A2.

The major hemolytic activity of Entamoeba histolytica is located in a subcellular fraction called P30. Its maximal effect is observed at pH 8.0 and 1 mM Ca2+ and is due to a phospholipase A (PLA). In the present study a membrane-associated phospholipase A2 was purified from P30 to homogeneity. P30 was fractionated with ethyl ether and the insoluble fraction was extracted with 1 M KCl. The KCl-soluble material was diluted ten times with 0.1 M TRIS-HCl (pH 9.5) and passed through a chromatofocusing column with a 9-4 pH gradient. Four peaks with PLA2 activity were obtained. By affinity chromatography, peak II, the one with the highest specific activity, was resolved in three more PLA2 peaks. Peak II.2 had the highest PLA2 specific activity. When analyzed by sodium dodecyl sulfate-polyacrylamide slab-gel electrophoresis under nonreducing conditions, peak II.2 yielded a single band with an apparent molecular mass of 30 kDa. Under reducing conditions the protein dissociated into two 15-kDa monomers. The purified PLA II.2 displayed its activity at the same conditions under which the P30 hemolytic activity was maximal. The isoelectric point of PLA II.2 was 7.0. The purification procedure described above provides sufficient material for determination of the relative importance of the enzyme in the E. histolytica pathogenic mechanisms.

Identification of Entamoeba histolytica intracellular phospholipase A and lysophospholipase L1 activities.

Entamoeba histolytica phospholipase A and lysophospholipase activities from a vesicular subcellular fraction (P30) were analyzed. The products, obtained using specific substrates labeled with 14C or 3H, indicated the presence of phospholipase A1 and A2 as well as lysophospholipase L1 activities. The enzymes detected could participate in phospholipid metabolism and the alkaline phospholipase A2 may contribute to E. histolytica cytopathogenicity.

Entamoeba histolytica axenic growth improvement by ox bile.

Axenic Entamoeba histolytica cultures, grown in PEHPS medium, showed increasing yields and growth velocity when added with 0.03 to 0.6 g/l gallbladder ox bile, while higher doses were toxic for amebas. The highest density (505 +/- 29 x 10(3) trophozoites/ml) and shortest duplication time (Dt = 10.74 +/- 0.2 h) corresponded to cultures added with 0.24 g bile/l. Their yields were 1.74 and 3.34 times higher, respectively, than those which were reached by the non-added PEHPS controls and by growth of amebas in TYI-S-33 medium. Between 72 and 96 h of incubation a noticeable increase in trophozoite density was observed in cultures added with 0.24 g/1 bile among controls. At 72 h yields of bile-added cultures inoculated with 5, 10 and 20 x 10(3) amebas/ml increased in function of the inoculum. The improved growth of E. histolytica by adding 0.24 g/l ox bile to culture medium suggests that bile has compounds are essential for amebas.

Differential effects of the (+)- and (-)-gossypol enantiomers upon Entamoeba histolytica axenic cultures.

The in-vitro anti-amoebic effects of (+/-)-, (+)-, (-)-gossypol and emetine were tested against axenic trophozoites from five Entamoeba histolytica strains. The (-)-isomer was more active than the racemate and the (+)-isomer. These results indicate that the gossypol anti-amoebic activity is mainly due to its content of (-)-gossypol in all strains tested.

Entamoeba histolytica: inhibition of malic enzyme and alcohol dehydrogenase by (+/- )-, (+)-, and (-)- gossypol.

Gossypol, a natural racemic mixture with action on NADP- and NAD-oxidoreductases from diverse species, has been proposed as a possible antiamebic medication considering several of its pharmacological properties. In this study it was found that malic enzyme and alcohol dehydrogenase from Entamoeba histolytica are strongly inhibited by (+/-)-gossypol, and both (+)- and (-)- enantiomers. The inhibition was of the noncompetitive type among their respective substrates in all cases. The (+/-), (+)-, (-)-gossypol half-maximal inhibitory concentrations (IC50) for the malic enzyme were 3.71, 13.37 and 1.03 microM, and against the alcohol dehydrogenase 79.64, 124.43 and 42.56 microM, respectively. Therefore, the (-) enantiomer resulted 3.6 and 13.0 times more potent than the racemic mixture and (+)-gossypol, respectively, to inhibit the malic enzyme, and 1.9 times and 2.9 times more potent than the racemic mixture and (+)-gossypol, respectively, against the alcohol dehydrogenase. Accordingly, one possible mechanism of the antiamebic effect of gossypol could be the inhibition of vital NADP-dependent enzymes as those analyzed in this study.

Entamoeba histolytica cysts with a defective wall formed under axenic conditions.

Axenic HK9 Entamoeba histolytica strain amoebae, maintained in PEHS medium, displayed several cystic characteristics that involve an active process of cystic wall formation, cellular volume and density diminution, and one or two nuclear divisions. The differentiation process was asynchronic, beginning after the logarithmic growth phase. The axenic cysts, which were maintained in a 50 mOsm/kg medium at 4 degrees C for 72 h, produced growing trophozoites within 1-7 days of incubation at 36 degrees C in fresh medium. Negative results were obtained with trophozoites submitted to the above treatment, and with axenic cysts maintained in double-distilled water at 4 degrees C for 24 h, or in 0.1% sarkosyl, for 10 min at room temperature instead of 55 mosmol/kg medium. Thus, the HK9 E. histolytica strain, cultured in PEHPS, produced under axenic conditions a small proportion of mature, metabolically active cysts, but with an immature or abnormal wall.

Axenic massive cultivation of Entamoeba histolytica trophozoites in spinner flasks and in a microfermentor.

A medium, PEHPS, whose main components are extracts of ox liver, and ox and swine pancreas (EHP) was used to develop two alternative methods for axenic cultivation of trophozoites in suspension: with magnetic stirrer and with a 14 l capacity microfermentor, giving the first technic yields of 2.36 x 10(5) to 3.08 x 10(5) and the second in a 14 l capacity microfermentor, which produced 1.65 x 10(5) amebas/ml in one batch and 2.94 to 3.65 x 10(5) of such parasites/ml at semicontinuous flux.