Poster 202 - Indution of apoptosis in human epithelial cells by the low molecular weight hemolysin

Poster 202. Congresso da Sociedade Brasileira de Toxinologia, 8., Symposium of the Pan American Section of the International Society on Toxinology, 8., 2004, Angra dos Reis, Brasil. Abstracts... J. Venom. Anim. Toxins incl.Trop. Dis., 2004, 10, 3, p.561.

Indution of apoptosis in human epithelial cells by the low molecular weight hemolysin of Enterococcus faecalis

¹Furumura, M.T.;¹Ludovico, M.S.; ¹Figueirêdo, P. M. S., ¹Carbonell, G. V., ²Darini, A. L. ¹and Yano, T.

1 Departamento de Microbiologia e Imunologia, Instituto de Biologia, Universidade Estadual de Campinas, SP, Brazil. 2 Departamento de Análises Clínicas, Toxicológicas e Bromatológicas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP, Brazil.

Enterococci are Gram-positive cocci that inhabit the gastrointestinal tract of many organisms, including humans. Enterococci are increasingly appreciated as causative agents of infections of urinary tract, bloodstream, endocardium, abdomen, biliary tract and burn wounds but the pathogenetic mechanism of Enterococcusfaecalis are not well understood. Recently we observed that E. faecalis produces a low molecular weight hemolysin, lytic to horse and sheep erythrocytes (submitted manuscript). In the present study, to evaluate its role in the pathogenesis the cytotoxic activity of this hemolysin was investigated. The clinical isolate EF20 strain was partially purified from the culture supernatant by ultrfiltration PM-10 membrane (Amicon), followed by precipitation with ethanol. The hemolysin ethanol-soluble fraction was dried by evaporation and this material was then applied on a mBondapak C18 column. Different dilutions of semi purified cytolysin were added to the cultures HeLa and HEp-2 cells followed by a 12 h and 24 h incubation period and after incubation the cells were stained with toluidine blue. The percentage of apoptotic cells was determined by Annexin V-FITC Apoptosis Detection Kit 1 and analyzed by flow citometry. Cells treatment with semipurified hemolysin became spherical showed morphological features associated with apoptosis, including a loss of the arrangement, extensive surface blebbing and cell detachment. The demonstration that the hemolysin of E. faecalis triggers a variety of complexes and induces ephitelial cells death by apoptosis may contribute to the comprehension of the biological mechanisms involved in the pathogenesis of disease.