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Isolation, purification, and partial characterization of an enterotoxin from extracts of Entamoeba histolytica
Abstract:
Soluble cell-free extracts of pathogenic Entamoeba histolytica, as well as serum-free minimal media in which trophozoites are incubated, contain substances that cause the rapid rounding up and detachment of tissue-cultured monolayers of mammalian cells (cytopathic activity) and induce fluid secretion in ligated intestinal loops of indomethacin-pretreated rats (enterotoxic activity). A semiquantitative assay for the determination of the cytopathic activity based on the rate of detachment of tissue-cultured baby hamster kidney cells was developed. Two peaks containing cytopathic activity were obtained upon gel filtration of the soluble extracts: peak I, with over 60% of the activity, emerged in the 30,000 to 50,000 molecular weight region, and peak II, containing the remaining activity, was in the 15,000 to 25,000 molecular weight region. The activity of peak I was found to be heat labile and inhibited by sialoglycoproteins such as fetuin and mucin (5 mg/ml), as well as by sialic acid. Protease inhibitors such as antitrypsin, pepstatin, phenylmethylsulfonyl fluoride, metaloprotease inhibitors, and bacitracin had no effect on the cytopathic activity. Marked inhibition of cytopathic activity was observed, however, with iodoacetamide and p-chloromercuribenzoate, which affect sulfhydryl groups. The toxic material in peak II was found to have ionophoric activity and was not inhibited by sialic acid-containing compounds. The materials from both peaks had enterotoxic activity in intestinal ligated loops. The active substance from peak I was further purified (200X) on an agarose-fetuin affinity column, yielding one major protein band with an apparent molecular weight of ca. 30,000 on sodium dodecyl sulfate. Amino acid analysis revealed that the protein was very poor in sulfur amino acids. The sialic acid-sensitive toxic activity was higher in known virulent strains such as HM-1:IMSS and could be markedly augmented after preincubation of the trophozoites with certain Escherichia coli strains.
Insights
Pathogenic Entamoeba histolytica releases toxins causing cell damage and intestinal fluid secretion. These toxins, with distinct molecular weights, are characterized by their heat lability and sensitivity to sulfhydryl group inhibitors.
Area of Science:
- Microbiology
- Cell Biology
- Toxicology
Background:
- Pathogenic Entamoeba histolytica causes amoebiasis, a significant human disease.
- The mechanisms underlying E. histolytica's cytopathic and enterotoxic activities are not fully understood.
Purpose of the Study:
- To characterize the substances responsible for the cytopathic and enterotoxic activities of E. histolytica.
- To develop an assay for quantifying cytopathic activity.
Main Methods:
- Gel filtration chromatography to separate active components.
- Tissue culture assays for cytopathic activity using baby hamster kidney cells.
- Ligated intestinal loop assays in rats for enterotoxic activity.
- Affinity chromatography and SDS-PAGE for protein purification and characterization.
- Amino acid analysis.
Main Results:
- Two distinct peaks of cytopathic activity were identified (MW 30-50 kDa and 15-25 kDa).
- Peak I activity (30-50 kDa) was heat labile, inhibited by sialic acid-containing compounds and sulfhydryl-reactive agents, and purified to a ~30 kDa protein.
- Peak II activity (15-25 kDa) exhibited ionophoric properties and was not affected by sialic acid.
- Both peaks demonstrated enterotoxic activity.
Conclusions:
- E. histolytica produces multiple toxic substances with distinct biochemical properties.
- A ~30 kDa protein is a major contributor to the sialic acid-sensitive cytopathic activity.
- These toxins play a role in the pathogenesis of amoebiasis.