EL TOR HAEMOLYSIN FROM VIBRIO CHOLERAE INDUCES APOPTOSIS AND INCREASES INTRACELLULAR Ca2+ LEVELS IN HUMAN INTESTINAL CELLS

SAKA, HÉCTOR A ; BIDINOST, CARLA; BONACCI, GUSTAVO; CHIABRANDO, GUSTAVO ; JOSÉ LUIS BOCCO

Puerto Iguazú, Misiones

Congreso; XL Annual Meeting Argentine Society for Biochemistry and Molecular Biology - SAIB 2004; 2004

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular

V. cholerae O1 biotype El Tor and most of V. cholerae non-O1 /non-O139 (VCN) produce El Tor Haemolysin (ETH), a pore forming toxin encoded by the hlyA gene. In previous studies we have shown that ETH could be involved in the diarrhea caused by a VCN clinical isolate lacking cholera toxin (VCN CT-). Additionally, we demonstrated ETH-inducedapoptosis on Cos-7 cells. To explore the potential involvement of apoptosis in the pathogenesis caused by ETH on human intestinal cells, we exposed undifferentiated Caco-2 and differentiated C2BBe1 to sterile culture supernatants from VCN CT- or from its isogenic hlyA null mutant. At different times post-incubation, caspase-3 activation, internucleosomal DNA fragmentation and increased sub-G1 fraction of fragmented nuclei were detected by a Caspase-3 apoptosis detection kit, agarose gel electrophoresis/ ethidium bromide staining and flow cytometry/propidium iodide staining, respectively. Toinvestigate the role of second messengers involved in response to ETH, changes in Caco-2 intracellular Ca2+ levels were measured after exposition to ETH by flow cytometry using Fluo3-AM. After 2 and 4 h of exposition to 200 ng/mL of ETH, intracellular Ca2+ increased 22,0% and 36,6%, respectively. These results demonstrate that when added to human intestinal cells, ETH is able of causing cell death via apoptosis and that an intracellular increase in Ca2+ concentration occurs in response to this toxin.