Purification and Characterization of Heat‐Labile Toxin from Bordetella bronchiseptica

Abstract

The heat-labile toxin (HLT) of Bordetella bronchiseptica was purified successively from sonic extracts of phase I organisms grown in Stainer-Scholte medium, by partition in hydrophobic interaction, sucrose density gradient centrifugation, gel filtration through Sepharose 4B and 6B, isoelectric precipitation and isoelectric focusing. The purified HLT was homogeneous by disc polyacrylamide gel electrophoresis and the gel diffusion-test, and free of detectable hemagglutinin and endotoxin activity. A 386-fold purification over the crude extract was obtained at a yield of about 28%, and a minimum dose of 0.9 ng was dermonecrotizing with a lesion 5 mm in diameter in guinea pigs and induced splenoatrophy. The mouse LD50 was 200 ng (intraperitoneal) or 70 ng (intravenous). The HLT was found to be a simple protein with an isoelectric point of pI 6.9. It has a molecular weight of 102,000 estimated by Sepharose 6B gel filtration and was found to consist of two different types of polypeptide by SDS-polyacrylamide gel electrophoresis, their molecular weights being 30,000 and 20,000. Amino acid analysis showed 15 common amino acid residues, and methionine, cysteine and tryptophan were undetectable. The HLT crystallized by methylpentanediol showed a block form. The HLT was inactivated at 56 C when heated for 10 min, and at above pH 9 and below pH 4.

Assessment of Toxic Activity of Heat Labile Toxin from Bordetella pertussis Vaccine Strain 134

“…The results indicates that the growth of B. pertussis was presence in BG agar slants for sample NIS, and absence contamination was also observed, in case of HIS there was no growth was observed after 7 days incubation, it illustrated that the culture cells were heated at 56ºC for 30 min, all dermonecrotizing activity (HLT) was diminished. The similar study of Endoh et al [11] shown that after the B. pertussis organism were heated for 30 min at 56ºC, all dermo necrotizing activity and a large part of the lethal toxicity were destroyed in tested animals.…”

“…The study of Endoh et al [11] report evident suggested that our study, after the cells were heated for 30 min at 56ºC, all dermo necrotizing activity and a large part of the lethal toxicity were destroyed, and cell homogenates of B. parapertussis, B. bronchiseptica, and B. avium produce similar dermonecrotic lesions [11].…”

Heat‐Labile Toxin from Bordetella parapertussis Induces Contraction of Smooth Muscle Cells in Culture

“…The descriptions of the physico-chemical properties of the molecule vary among the different reports. While we do not yet know whether only one type of HLT is produced by all of the Bordetella species, HLT isolated from each of these species induces skin lesions when intracutaneously injected into shaved back skin of animals (2,10,12,13,15). We have reported that HLT from B. parapertussis or B. bronchiseptica induces contraction of vascular smooth muscle strips (3,4) and vascular smooth muscle cells (5), suggesting that HLT might produce ischemia in animal skin.…”

“…Previously, we reported that HLT isolated from B. bronchiseptica has a molecular weight of 102 kDa with two types of subunits (20 and 30 kDa). Recently purified HLT preparations were obtained from B. pertussis (17,18), B. parapertussis (6,7) and B. bronchiseptica (2,14,15). The descriptions of the physico-chemical properties of the molecule vary among the different reports.…”

Cytopathic Effect of Heat‐Labile Toxin of Bordetella parapertussis on Aortic Smooth Muscle Cells from Pigs or Guinea Pigs

“…HLT purified from B. parapertussis, strain 23054, by the method previously described (3,4,6), was used. It showed 0.8 ng of the minimum dermonecrotizing dose (MND) in the shaved back of guinea pigs, similar molecular weight and subunit structure, and vasocontractive activities on peripheral vessels, to those of B. bronchiseptica-HLT described previously (3).…”