Purification and partial biological characterization of the Aeromonas veronii sobria vacuolating cytotoxic factor. 

^{1Martins, L.M.; 1Catani, C.F.; 2Azzoni, A.R.; 1Falcón, R.M.; 1Carbonell, G.V.}; ¹Yano, T.

1 Depto. Microbiologia e Imunologia, IB, UNICAMP, Campinas – SP; 2 Depto. Centro de Biologia Molecular e Genética, IB, UNICAMP. Campinas – SP.

Aeromonas has lately been described as one of the most important human enteropathogens. Recently we described a cytotoxic vacuolating factor (FCV), produced mainly by A. sobria and A. veronii sobria isolates. The FCV has showed that it is termolabile, sensible to proteinase K and that the cellular vacuoles formed are acidic, occurring around nuclei. This study has the objectives of purificate this cytotoxin and determinate its intracellular mechanism. The FCV production and purification was done by growing the A. veronii sobria As-31 at 37°C in CYE medium, pH 8.0, by 18 h and the culture supernatant obtained by centrifugation at 12,000 x g by 30 min, followed by Amicon XM-50 concentration. Afterwards, the Cu⁺ ion immobilized affinity chromatography - IMAC (Qiagen-Germany) was performed on FPLC system (Amersham), and a single protein band, with molecular weight estimated of 60 KDa, was verified in SDS-PAGE under reducing conditions. The purified FCV induces initial vacuolating effects soon after 15 min of Vero cells exposition. The TUNEL and flow cytometry analysis were conducted and pointed out that the cytotoxin provokes cellular death by apoptosis. We are studying this apoptotic induction property of the FCV as a possible virulence factor of this bacterium.

Support : FAPESP.

Correspondence to: lucianomm_2000@yahoo.com