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(Received for publication, June 25, 1996, and in revised form, October 11, 1996)
From the Food Research Institute, Department of Food Microbiology
and Toxicology, University of Wisconsin, Madison, Wisconsin 53706
Hemolysin BL (HBL) is a unique membrane-lytic
toxin from Bacillus cereus composed of three distinct
proteins, designated B, L1, and L2. HBL
produces a paradoxical zone phenomenon in gel diffusion assays in sheep
blood agar. Lysis does not begin immediately adjacent to the source of
diffusion; rather, it begins several millimeters away. Cells near the
source and at intersections of lysis zones remain intact longer. Here,
we developed a spectrophotometric hemolysis assay system that measures
the activities of the individual HBL components and used it to analyze
the mechanisms of hemolysis and the paradoxical zone phenomenon. The B
component was rate-limiting, and erythrocytes were slowly primed by B
at an optimal concentration of about 1.3 nM to rapid lytic
action by the combination of the L components (L1+2). All
of the individual components bound to cells independently, and
membrane-associated HBL components were neutralized by specific
antibodies, suggesting that lysis was caused by formation of a membrane
attack complex on the cell surface. Osmotic protection experiments
indicate a colloid osmotic lysis mechanism. Concentrations of the B
component above 1.3 nM caused inhibition of
L1-mediated lysis, and L1 inhibited the priming reaction of B over a similar concentration range. From analyses of
spectrophotometric and diffusion assays we constructed a basic model
for the interactions between HBL components and for the paradoxical
zone phenomenon in blood agar. In the latter, areas of slow lysis near
diffusion sources are caused primarily by the accumulation of
inhibitory levels of L1 reached before cells are primed by
B.
Volume 272, Number 1,
Issue of January 3, 1997
pp. 233-239
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.
HEMOLYTIC ANALYSIS OF COMPONENT INTERACTIONS AND A MODEL FOR ITS
CHARACTERISTIC PARADOXICAL ZONE PHENOMENON
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