MECHANISMS OF E HISTOLYTICA ADHERENCE AND CYTOLYSIS

点击翻译按钮获取中文摘要

Entamoeba histolytica is an invasive enteric pathogen which causes substantial worldwide morbidity and mortality. Present evidence indicates that amebic adherence and cytolytic events are crucial in pathogenesis of disease. The objective of this proposal is to define the biochemical and cellular basis of amebic adherence and cytolytic mechanisms. The amebic in vitro adherence event is mediated by an N-acetyl-D-galactosamine (GalNAc) inhibitable adhesin lectin. Adherence to target cells initiates a cytolytic event which requires amebic microfilament function, phospholipase A (PLA) activity, Ca2+ ion flux, and maintenance of an acid pH in intracellular vesicles. The specific aims and methods of this proposal are: 1) To characterize the cellular function and purify the amebic GalNAc inhibitable adhesin (lectin) molecule. Mechanisms of in vitro adherence of amebae to human colonic mucosa will be defined. Binding studies with I125-asialofetuin (ASF) will enumerate and characterize the GalNAc inhibitable surface adhesin molecules, including cellular control and its relation to virulence. Mouse monoclonal antibodies which bind to the adhesin molecule will be produced. The soluble GalNAc inhibitable lectin will be purified by affinity chromatography with ASF or monoclonal IgG. The purified lectin will be characterized by SDS-PAGE, immunoprecipitation with monoclonal IgG, and carbohydrate specificity by competitive binding assays with I125-ASF. The effect on amebic adherence of monoclonal or polyclonal IgG to purified lectin will be described. 2) To define the role of the GalNAc inhibitable lectin, Ca2+ ions, amebic PLA enzymes, and acid pH intracellular vesicles in the amebic cytolytic event. Cytotoxicity of the purified GalNAc inhibitable lectin will be described. Amebic cytoplasmic free Ca2+ will be measured and visualized by use of the calcium activated photoprotein, aequorin. The relation of amebic PLA to free Ca2+ and surface adhesin molecules will be defined. PLA activity will be measured in amebae during cytolysis of target cells. Amebic PLA enzymes will be purified by ion exchange chromatography and assayed for cytotoxicity. Effects of anti-PLA-polyclonal antibodies will be studied. The relation of vesicle pH to PLA activity, free Ca2+, surface adhesin molecules distribution, vesicle exocytosis, and incorporation of enzymes into vesicles will be defined. Our long term goal is to apply these findings to the development of an immunologic or pharmacologic means of prophylaxis against invasive amebiasis.