IMPROVED PURIFICATION AND CHARACTERIZATION OF HEMOLYSIN BL, A HEMOLYTIC DERMONECROTIC VASCULAR-PERMEABILITY FACTOR FROM BACILLUS-CEREUS

Bacillus cereus causes diarrheal and emetic food poisoning syndromes a s well as a variety of mild to severe infections. A dermonecrotic vasc ular permeability (VP) factor has been implicated as a virulence facto r in these illnesses. Hemolysin BL was previously identified as a uniq ue tripartite hemolysin possessing VP activity. In this study, a high- yield purification scheme, which allowed quantitative characterization of hemolysin BL activity and determination of the molecular weight, p I, and N-terminal sequence of each component, was developed. Milligram quantities of the B, L(1,) and L(2) components were highly purified b y a combination of anion-exchange and hydroxylapatite chromatographies . The combined components had VP activity at low doses and were necrot ic at higher doses. The toxin exhibited an unusual dose-response zone phenomenon in turbidometric hemolysis assays. Activity increased at do ses up to 200 ng/ml, then decreased at doses up to 350 ng/ml, and was constant at doses up to at least 2,500 ng/ml. This behavior may provid e an explanation for the unusual discontinuous pattern typical of hemo lysin BL in gel diffusion assays. At high concentrations of one or two components, the presence of low amounts of the complementary componen t(s) resulted in full hemolytic activity. Erythrocytes were protected from lysis by Zn2+ at micromolar concentrations but not by Ca2+ and Mg 2+ at concentrations up to 25 mM. These data provide guidelines for fu ture work on this toxin and indicate that hemolysin BL is the dermonec rotic VP factor implicated as a B. cereus virulence factor.