Untersuchungen zu Bacillus cereus Enterotoxin-Komplexen auf zellulärer Ebene
This paper deals with the in vitro activity of the B. cereus enterotoxin-complexes Hemolysin BL (HBL) and non-hemolytic enterotoxin (Nhe) on cells. For that purpose the cytotoxic and hemolytic activity of crude B. cereus culture supernatants was analyzed in cell culture assays and on blood agar plates. Specific monoclonal antibodies were used for the detection of single components. By using neutralisation assays and subtractive immoaffinity chromatography the inactivation of the specific toxin complexes could be achieved. When testing different cell lines (Vero-, Neuro2A-, Jurkat-, hybridoma cells) for their sensibility against culture supernatants of toxic B. cereus isolates, Vero cells proved to be the most sensitive. The sensitivity pattern of hybridoma cells differed from the other cell lines, as HBL containing supernatants showed higher cytotoxicity than supernatants of single Nhe-producers. Analyses of 18 B. cereus isolates revealed that the 5 - 40 % of the cytotoxic activity measurable in culture supernatants could be attributed to HBL. A strong correlation (r2 = 0,938) could be found between remaining cytotoxic activity and the detectable L2 concentrations. The results of the neutralisation assays could be verified by subtractive immunoaffinity chromatography of Nhe B and testing the Nhe B negative, HBL positive B. cereus culture supernatants (Nhe B-/HBL+). Furthermore it could be demonstrated for the first time that Nhe B plays a major part in the appearance of the discontinuous hemolytic pattern typical for HBL producers. After sensibilisation with tunicamycin Vero cells showed a substantial increased cytotoxic activity (63 – 132 %). The same effect could be observed in Fumonisin B pretreated cells for crude supernatants, but not for the Nhe B-/HBL+ preparation. Neither treatment with swainsonine, castanospermine nor enzymatic treatment with sialidase and N-glycosidase F resulted in an alteration of toxicity.
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