Phenotypic heterogeneity in the expression of a 30 kDa cysteine proteinase in axenic cultures of Entamoeba histolytica.

A polyclonal antibody raised against a purified approximately 30 kDa cysteine proteinase derived from extracts of axenically grown trophozoites of E. histolytica strain HM-1:IMSS was used to stain 72 h cultures of the same amebas by indirect immunofluorescence. Fluorescence was limited to the outer membrane of the parasite and was either uniformly distributed or more condensed on a segment, at times on a single point of the membrane. In relation to the intensity of fluorescence staining, three distinct amebic populations were present: negative, weakly stained and intensely stained. The relative numbers of these three groups remained quite constant for at least one year under the same culture conditions. Flow cytometry was used to quantitate simultaneous variations in amebic size and intensity of fluorescence at various times after different treatments. Amebic size was registered in three levels: small (< 7 microns), medium (7-20 microns), and large (> 20 microns). Staining intensity was measured in arbitrary units. Exposure to 100% fresh hamster serum, phagocytosis of erythrocytes, exposure to cysteine proteinase inhibitors E-64 and cystatin, and to calmodulin antagonist W-7, resulted in various modifications of the phenotype of amebas in very short time periods. We conclude that the expression of the membrane approximately 30 kDa cysteine proteinase in axenic amebic cultures is phenotypically heterogeneous, and that such heterogeneity is modulated and not constitutive.