Bacillus cereus, a widely distributed bacterium commonly isolated from soil and food, is the causative agent of diarrheal and emetic types of food poisoning (23) as well as a number of opportunistic infections, including endophthalmitis (24). This paper deals with the enterotoxin responsible for the diarrheal syndrome, which has been incompletely characterized due to difficulties in completely separating possible components of the enterotoxin from each other and from other potentially toxigenic proteins produced by this organism.Previously, we showed that B. cereus produces hemolysin BL, a tricomponent hemolysin which exhibits a ring-shaped pattern of hemolysis on blood agar gels (3, 4). Hemolysin BL is comprised of a binding component, B (37.5 kDa), and two lytic components, L 1 (38.2 kDa) and L 2 (43.5 kDa). All three are required for maximal lysis of erythrocytes. Several properties of hemolysin BL, including molecular weights and isoelectric points, are similar to those of the multicomponent toxin which Thompson et al. (22) and others (7) postulated as the cause of the diarrheal syndrome. In addition, hemolysin BL induces a vascular permeability reaction in rabbit skin (4), which is considered a bioassay for the enterotoxin (23), and is toxic to Chinese hamster ovary cells (1). Beecher et al. (5) tested the components of hemolysin BL for the ability to cause fluid accumulation in rabbit ileal loops, which is the definitive test for diarrheal enterotoxins (6), and found that all three components were required for maximal activity. However, some fluid accumulation was also observed in loops containing only one or two of the hemolysin components. Testing of recombinant proteins in this assay will establish if the apparent toxicity of the individual proteins is real or is due to trace contamination with other proteins.The gene encoding the B component (hblA) of hemolysin BL was previously cloned and characterized in our laboratory (13), and a strategy to overproduce the protein was devised (12). Here, we report the molecular cloning and nucleotide sequencing of hblC and hblD, the genes encoding the L 2 and L 1 proteins, respectively, and the characterization of the recombinant proteins in Escherichia coli. Toxicity of the individual L 1 and L 2 components to E. coli hindered our attempts to separately clone the genes encoding these proteins into this bacterium; however, a single recombinant clone expressing both of these proteins was characterized. The L 1 and L 2 components are expressed as 38-and 43.5-kDa proteins. These two genes, separated by only 37 bases, are clustered in the genome adjacent to hblA, the gene encoding the B component of hemolysin BL, and to hblB, a gene which shows extensive homology with hblA but which has been only partially cloned and characterized. A 5.5-kb transcript was detected by hybridization with each of three probes designed from the coding regions of hblA, hblC, and hblD, suggesting that the genes encoding the complete hemolysin are transcribed as a polycistronic message. MATE...

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Previously we described the partial purification of a novel hemolysin from Bacillus cereus and showed that hemolytic activity required the combined action of at least two components, called B and L to signify their cell-binding and cell-lytic roles in this activity. On further purification, as described in the present article, a combination of anion-exchange chromatography and polyacrylamide gel electrophoresis separated three proteins, B, L1, and L2 (35, 36, and 45 kDa, respectively). Individually, these proteins were inactive in hemolytic and vascular permeability assays, and combinations of B and L1 or B and L2 were either inactive or slightly active. Combinations of all three moieties produced the unique ring-shaped zone of hemolysis, a previously described characteristic of hemolysin BL, as well as edema and bluing in the vascular permeability assay. Since the vascular permeability assay is known to correlate with enterotoxicity, these results suggest that hemolysin BL is enterotoxigenic. Furthermore, the molecular weights and isoelectric point values of the hemolysin BL components are consistent with those described by others for the multicomponent diarrheal enterotoxin of B. cereus. Immunofluorescent staining of B-treated erythrocytes confirmed that B binds to cells as an initial step required before the L components can act to cause cell lysis. Bacillus cereus, which is best known as the cause of two distinct food poisoning syndromes, has also been implicated in a wide variety of illnesses, including systemic infections and panophthalmitis (19). The virulence factors of this bacterium remain ill defined, partly because it produces a large number of proteins that potentially possess toxigenic activity and partly because these metabolites are difficult to isolate. Possible virulence factors include three phospholipases, a diarrheal enterotoxin, an emetic enterotoxin, a factor lethal to mice, and several hemolysins (19). Initially, B. cereus was described as the producer of two hemolysins, cereolysin (3, 14) and a poorly characterized "secondary hemolysin" (5, 15). Later, Ikezawa et al. (9) reported that the sphingomyelinase of B. cereus lyses ovine and bovine erythrocytes, and Gilmore et al. (6) found that this hemolytic activity is enhanced by the phosphatidylcholine-preferring phospholipase C of this organism. The sphingomyelinase-phospholipase C combination was designated cereolysin AB. More recently (1), we described hemolysin BL, which is also composed of more than one component, and showed that it is distinct from both cereolysin and cereolysin AB. This hemolysin exhibits a unique ring-shaped zone in the lytic clearing of erythrocytes in gel diffusion assays.

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