Molecular mechanism of enterohemolysin (EHly) produced by enteropathogenic Escherichia coli

Figueirêdo*, P.; M.T, Furumura, M., Ribeiro, D., de Paula, E.  and Yano, T.

Depto. Microbiologia e Imunologia, IB, Universidade Estadual de Campinas, SP, Brazil

Some strains of enteropathogenic Escherichia coli produce a hemolysin known as Enterohemolysin (EHly) because its association with enteric diseases. EHly shows hemolytic activity towards washed erythrocytes from different animal species on blood agar plates. EHly activity has recently shown be inhibited by normal serum lipoproteins and by cholesterol in vitro suggesting a simples leaflet insertion model for binding and neutralization of EHly by cholesterol in surface of lipoprotein, mainly high density lipoprotein (HDL). Probably the molecular mechanism of Ehly activity is the same, a simple insertion in erytrhocytes membranes similar to detergent hemolytic activity. The strain  of E. coli C3888 was used to carry out the production of EHly. Bacteria was cultivated in TSB medium with EDDA and the cells were sonicated and purificate by chromatography. In order to detect hemolytic activity, the material was incubated for 1h at 37°C in microplates with 1% horse erythrocytes. The inhibition of EHly by liposomes membranes with or without cholesterol  was examined by mixing equals volumes of EHly with serially diluted liposomes membranes. Emission spectra were recorded in a SPEX spectrofluorimeter (excitation wavelenght 339nm for pyrene) verified the capacity of inhibition of hemolytic activity of EHly by liposomes varying amounts of liposomes and a constant EHly concentration. After incubation at 37°C for 60 min followed by another incubation at 4°C for 18h . Analysis of the erythrocyte morphology was performed by scanning electron microscopy. Measuremet of K efflux and osmotic protection experiments to the extent of haemolyis was determined spectrophotometrically.Our results showed that only liposomes with cholesterol inhibited the hemolytic activity of EHly on washed erythrocytes. The osmotic proetectors did not inhibit the hemolytic activity of Ehly and others lipids as phosphatidyl –choline did not inhibit the Ehly activity. Morphologycal analysis of erytrhocytes incubated in the absence of Ehly showed a majority of normal biconcave disc appearance cells, addition of EHly  led to a wide variety of minor and major spiny protuberances. Recently has been suggested that EHly alters and disrupt cell membranes by a detergent-like mechanism. Detergents, a special groups of lipids used to solubilize membrane act by forming thermodynamically stable colloidal aggregates or micelles that may be asymmetrically distributed between the two leaflets of the membrane lipid bilayer. The results obtained suggest that hemolytic activity of EHly is similar to detergent because was detected aleration in membrane composition and structure in liposomes membranes when incubated in the presence of EHly, and these alteration were not dependent of temperature or time, the alterations were dose-dependent and the presence of osmotic protectrs did not alters teh hemolytic activity of EHly. invitro. Enterohemolysin produced by Escherichia coli enteropathogenic is a detergent –like hemolysin.

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