Biomedical subjects

D J Beecher

Publications and source records attributed to D J Beecher.

11 recordsLinked to original sources

PubMed2000

Evidence for contribution of tripartite hemolysin BL, phosphatidylcholine-preferring phospholipase C, and collagenase to virulence of Bacillus cereus endophthalmitis.

Bacillus cereus causes a highly fulminant endophthalmitis which usually results in blindness. We previously concluded that hemolysin BL (HBL), a tripartite necrotizing pore-forming toxin, is a probable endophthalmitis virulence factor because it is highly toxic to retinal tissue in vitro and in vivo. We also determined that B. cereus produces additional retinal toxins that might contribute to virulence. Here we fractionated crude B. cereus culture supernatant by anion-exchange chromatography and found that in vitro retinal toxicity was also associated with phosphatidylcholine-preferring phospholipase C (PC-PLC). The pure enzyme also caused retinal necrosis in vivo. We showed that phosphatidylinositol-specific PLC and sphingomyelinase were nontoxic and that two hemolysins, cereolysin O and a novel hemolysin designated hemolysin IV, were marginally toxic in vitro. The histopathology of experimental septic endophthalmitis in rabbits mimicked the pathology produced by pure HBL, and both HBL and PC-PLC were detected at toxic concentrations in infected vitreous fluid. Bacterial cells were first seen associated with the posterior margin of the lens and eventually were located throughout the lens cortex. Detection of collagenase in the vitreous humor suggested that infiltration was facilitated by the breakdown of the protective collagen lens capsule by that enzyme. This work supports our conclusion that HBL contributes to B. cereus virulence and implicates PC-PLC and collagenase as additional virulence factors.

Amino Acid Sequence↗

PubMed1997

Tripartite hemolysin BL from Bacillus cereus. Hemolytic analysis of component interactions and a model for its characteristic paradoxical zone phenomenon.

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 (L(1+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.

Bacillus cereus↗

PubMed1997

Botulinum neurotoxin type A: limited proteolysis by endoproteinase Glu-C and alpha-chymotrypsin enhanced following reduction; identification of the cleaved sites and fragments.

Botulinum neurotoxin (NT) serotype A is a approximately 150-kDa dichain protein. Posttranslational nicking of the single-chain NT (residues Pro 1-Leu 1295) by the protease(s) endogenous to Clostridium botulinum excises 10 residues, leaving Pro 1-Lys 437 and Ala 448-Leu 1295 in the approximately 50-kDa light (L) and approximately 100-kDa heavy (H) chains, respectively, connected by a Cys 429-Cys 453 disulfide and noncovalent bonds [Krieglstein et al. (1994), J. Protein Chem. 13, 49-57]. The L chain is a metalloprotease, while the amino- and carboxy-terminal halves of the H chain have channel-forming and receptor-binding activities, respectively [Montecucco and Schiavo (1995), Q. Rev. Biophys. 28, 423-472]. Endoproteinase Glu-C and alpha-chymotrypsin were used for controlled digestion at pH 7.4 of the approximately 150-kDa dichain NT and the isolated approximately 100-kDa H chain (i.e., freed from the L chain) in order to map the cleavage sites and isolate the proteolytic fragments. The dichain NT appeared more resistant to cleavage by endoproteinase Glu-C than the isolated H chain. In contrast, the NT with its disulfide(s) reduced showed rapid digestion of both chains, including a cleavage between Glu 251 and Met 252 (resulting in approximately 30- and approximately 20-kDa fragments of the L chain) which was not noted unless the NT was reduced. Interestingly, an adjacent bond, Tyr 249-Tyr 250, was noted earlier [DasGupta and Foley (1989), Biochimie 71, 1193-1200] to undergo "self-cleavage" following reductive separation of the L chain from the H chain. The site Tyr-Tyr-Glu-Met (residues 249-252) appears to become exposed following reduction of Cys 429-Cys 453 disulfide. Identification of Glu 669-Ile 670 and Tyr 683-Ile 684 as protease-susceptible sites demonstrated for the first time that at least two peptide bonds in the segment of the H chain (residues 659-684), part of which (residues 659-681) is thought to interact with the endosomal membranes and forms channels [Oblatt-Montal et al., (1995), Protein Sci, 4, 1490-1497], are exposed on the surface of the NT. Two of the fragments of the H chain we generated and purified by chromatography are suitable for structure-function studies; the approximately 85- and approximately 45-kDa fragments beginning at residue Leu 544 and Ser 884, respectively (both extend presumably to Leu 1295) contain the channel-forming segment and receptor-binding segments, respectively. In determining partial amino acid sequences of 10 fragments, a total of 149 amino acids in the 1275-residue NT were chemically identified.

Amino Acid Sequence↗

PubMed1995

Enterotoxic activity of hemolysin BL from Bacillus cereus.

Bacillus cereus causes exotoxin-mediated diarrheal food poisoning. Hemolysin BL (HBL) is a well-characterized B. cereus toxin composed of three components (B, L1, and L2) that together possess hemolytic, cytotoxic, dermonecrotic, and vascular permeability activities. Here, we show that HBL causes fluid accumulation in ligated rabbit ileal loops at a dose of 5 micrograms of each component per loop. Maximal fluid responses occurred for combinations of all three components at > or = 25 micrograms of each component per loop. Individual components and binary combinations did not cause significant fluid accumulation at 25 micrograms of each component. Specific antisera to HBL components inhibited the fluid accumulation response of crude culture supernatant from B. cereus F837/76. These antisera were tested against an antiserum to a partially characterized multicomponent diarrheal toxin described previously by Thompson et al. (N. E. Thompson, M. J. Ketterhagen, M. S. Bergdoll, and E. J. Shantz, Infect. Immun. 43:887-894, 1984). Immunoblot and immunoprecipitation analyses indicate that HBL and that toxin are identical. These results confirm previous speculation that HBL is a tripartite enterotoxin that, as for all of its other known activities, requires all three components for maximal activity.

Animals↗

PubMed1995

Extracellular virulence factors in Bacillus cereus endophthalmitis: methods and implication of involvement of hemolysin BL.

Bacillus cereus is a common cause of highly fulminant posttraumatic and metastatic endophthalmitis. Exotoxins or enzymes likely contribute to the severity of the infection, but specific virulence factors have not been identified. We developed two methods for the identification of B. cereus ocular virulence factors. In an in vitro assay that allows screening of multiple samples, retinal toxicity was estimated by measuring the release of lactate dehydrogenase from retinal buttons treated with B. cereus toxins. The results from this assay were confirmed with a sterile endophthalmitis model in which the histopathologic effect of intravitreally injected toxins was assessed. We tested pure hemolysin BL (HBL), a tripartite dermonecrotic vascular permeability factor of B. cereus, and crude exotoxin (CET) preparations, consisting of concentrated, cell-free B. cereus culture supernatant. In the in vitro assay, both CET and HBL caused rapid release of lactate dehydrogenase and retinal disintegration. In vivo, the toxins caused endophthalmitis clinically characteristic of B. cereus within 4 h. Histological changes included rapid retinal necrosis and detachment, choroidal edema, detachment and disruption of the retinal pigment epithelium, and rapid infiltration of polymorphonuclear leukocytes. Neutralization of HBL in CET preparations inhibited toxicity in vitro by 54%, and pure HBL was less toxic than CET with equal HBL contents in both methods. The results suggest that B. cereus ocular virulence is multifactorial and that HBL contributes to virulence.

Animals↗

PubMed1994

Identification and analysis of the antigens detected by two commercial Bacillus cereus diarrheal enterotoxin immunoassay kits.

The usefulness of two commercial immunoassays for the detection of diarrheal enterotoxin of Bacillus cereus is unclear because the identity of the enterotoxin(s) has not been proven and the kits detect different proteins. We found that the Bacillus cereus Enterotoxin-Reversed Passive Latex Agglutination kit (Oxoid) detects the L2 component from hemolysin BL, and the Bacillus Diarrhoeal Enterotoxin Visual Immunoassay (Tecra) detects two apparently nontoxic proteins.

Agglutination Tests↗

PubMed1994

Identification of hemolysin BL-producing Bacillus cereus isolates by a discontinuous hemolytic pattern in blood agar.

Bacillus cereus causes distinct exotoxin-mediated diarrheal and emetic food poisoning syndromes and a variety of nongastrointestinal infections. Evidence is accumulating that hemolysin BL is a major B. cereus virulence factor. We describe two methods for detection of hemolysin BL in crude samples and on primary culture media. In the first method, the highly unusual discontinuous hemolysis pattern that is characteristic of pure hemolysin BL was produced in sheep and calf blood agar around wells filled with crude culture supernatant from hemolysin BL-producing strains. In the second method, the pattern was formed surrounding colonies of hemolysin BL-producing strains grown on media consisting of nutrient agar, 0.15 M NaCl, 2% calf serum, and sheep or calf blood. Hemolysin BL production was detected with these methods in 41 of 62 (66%) previously identified B. cereus isolates and in 46 of 136 (34%) presumptive B. cereus isolates from soil. All nine isolates tested that were associated with diarrhea or nongastrointestinal illness were positive for hemolysin BL. The methods presented here are specific, simple, inexpensive, and applicable to the screening of large numbers of samples or isolates.

Agar↗

PubMed1994

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 as well as a variety of mild to severe infections. A dermonecrotic vascular permeability (VP) factor has been implicated as a virulence factor in these illnesses. Hemolysin BL was previously identified as a unique 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, pI, and N-terminal sequence of each component, was developed. Milligram quantities of the B, L1, and L2 components were highly purified by a combination of anion-exchange and hydroxylapatite chromatographies. The combined components had VP activity at low doses and were necrotic at higher doses. The toxin exhibited an unusual dose-response zone phenomenon in turbidometric hemolysis assays. Activity increased at doses 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 provide an explanation for the unusual discontinuous pattern typical of hemolysin BL in gel diffusion assays. At high concentrations of one or two components, the presence of low amounts of the complementary component(s) resulted in full hemolytic activity. Erythrocytes were protected from lysis by Zn2+ at micromolar concentrations but not by Ca2+ and Mg2+ at concentrations up to 25 mM. These data provide guidelines for future work on this toxin and indicate that hemolysin BL is the dermonecrotic VP factor implicated as a B. cereus virulence factor.

Amino Acid Sequence↗

PubMed1993

Molecular cloning and characterization of the hblA gene encoding the B component of hemolysin BL from Bacillus cereus.

Previous evidence suggests that hemolysin BL, which consists of a binding component, B, and two lytic components, L1 and L2, is the enterotoxin responsible for the diarrheal form of gastroenteritis caused by food-borne strains of Bacillus cereus. To prove that hemolysin BL and the enterotoxin are the same requires large amounts of these components free of other B. cereus proteins. For this purpose, we sought to clone the gene encoding the B component and to express it in Escherichia coli. A partial genomic library was constructed and a 29-base, 1,152-fold-degenerate oligonucleotide probe, designed from the N-terminal amino acid sequence of the B component, was used to identify recombinant clones containing the gene. Detection of gene products reactive with a monoclonal antibody specific for the B component and analysis of the nucleotide sequence confirmed that isolated clones, reactive with the oligonucleotide probe, did contain the gene encoding the B component. The protein, expressed in E. coli, apparently from the B. cereus promoter, produces a ring-shaped zone of hemolysis when combined with purified L components from B. cereus, a reaction typical of hemolysin BL. Northern (RNA) blot analysis of B. cereus RNA showed a large (5.1-kb) transcript which hybridized with a 500-bp probe internal to the B-component-coding sequence, suggesting that the hblA gene encoding the B component may be transcribed as part of a polycistronic message, possibly including the structural genes for the two lytic components. Higher levels of expression and disruption of the hblA gene are being pursued to resolve whether hemolysin BL is indeed the enterotoxin.

Amino Acid Sequence↗

PubMed1991

Characterization of the components of hemolysin BL from Bacillus cereus.

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.

Animals↗

PubMed1990

A novel bicomponent hemolysin from Bacillus cereus.

A procedure combining isoelectric focusing (Sephadex IEF) and fast protein liquid chromatography (Superose 12; Mono-Q) removed hemolytic activity (presumably a contaminant) from partially purified preparations of the multicomponent diarrheal enterotoxin produced by Bacillus cereus. However, when the separated fractions were recombined, hemolytic activity was restored, suggesting that hemolysis is a property of the enterotoxin components. Combined fractions exhibited a unique ring pattern in gel diffusion assays in blood agar. During diffusion of the hemolysin from an agar well, the erythrocytes closest to the well were not lysed initially. After diffusion, hemolysis was observed as a sharp ring beginning several millimeters away from the edge of the well. With time the cells closer to the well were also lysed. This novel hemolysin consists of a protein (component B) which binds to or alters cells, allowing subsequent lysis by a second protein (component L). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, isoelectric focusing, and Western blot analysis showed that hemolysin BL has properties similar to those described previously for the enterotoxin and that both components are distinct from cereolysin and cereolysin AB.

Antibodies, Monoclonal↗