Purificação e caracterização de enterohemolisina produzida por Escherichia coli enteropatogenicas

AUTOR(ES)
DATA DE PUBLICAÇÃO

1999

RESUMO

Enterohaemolysins (EHly) were ftrst detected in classical enteropathogenic Escherichia coZi strains (EPEC), isolated from feces of infants with diarrhea, and also detected in strains isolated from animaIs. Differring from a-haemolysin, EHly is not spontaneously released to culture medium. Sonication was the most efficient method to extract EHly. In order to detect haemolitic activity, the material was incubated for lh at 37°C in microplates or in tubes, with 1 % sheep erytrocytes, washed with PBS buffer. E. coZi strain C3888 was used to carry out production and puriftcation trials. Bacteria was cultivated in TSB medium with EDDA iron chelant, for 22h at 37°C. The cells were sonicated and the pellet submitted to cromatography in DEAE Fast-Flow column. The fractions that showed 8;ctivity were applied to Superdex-200 and Mono Q HPLC columns. The fraction with haemolitic activity obtained from Mono Q cromatography showed just one 60kDa band in SDS-P AGE electrophoresis gel. Increases of 2800 times in specrnc activity and aproximately 100 times in relative activity were obtained. The activity remained stable for several months in sonicated material when it was kept at low temperatures (-20°C to -70°C). However, it was not possible. to maintain the activity of pure haemolysin in the final purification procedures. Neutralization tests with specific anti-EHly antiserum did not present satisfactory results, despite the three different inoculation methods we tested in rabbits. Titles obtained with pre- and post-immunized sera were the same. In tests with different cell cultures, semipurified EHly proved to be citotoxic to Hep-2, HeLa, Caco-2, Vero, MDBK and 3T3 cells, showing cell rounding and detachment of the cell layer. CHO cells presented a cytopathic effect, with elongation and apparent condensation of the nuclei. No lethality or toxicity were observed in tests carried out in mice.

ASSUNTO(S)

escherichia coli toxinas

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