Toxicity of Bordetella avium beta-cystathionase toward MC3T3-E1 osteogenic cells.
Bordetella avium is the etiological agent of an upper respiratory disease in birds which, symptomatically and pathologically, resembles bordetellosis in humans. Studies of the virulence of this organism revealed a novel cytotoxic protein, designated osteotoxin, that was lethal for MC3T3-E1 osteogenic cells, fetal bovine trabecular cells, UMR106-01(BSP) rat osteosarcoma cells, and embryonic bovine tracheal cells. The osteotoxin lacked dermonecrotic toxin activity, exhibited no cross-reactivity with antibody against B. avium dermonecrotic toxin, and was non-proteolytic. Osteotoxin (M(r) approximately 80,000 by gel filtration, pI 5.4) was purified to electrophoretic homogeneity from B. avium 197. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and spectrophotometric analyses showed that the native protein was a homodimer and that each of the non-covalently linked subunits (M(r) approximately 41,000) contained one molecule of pyridoxal 5'-phosphate. Microsequencing of the first 32 amino acids from the NH2 terminus allowed the synthesis of two oligonucleotide probes, which, together with polyclonal antibody to the purified protein, facilitated cloning, sequencing, and expression of the osteotoxin gene product in Escherichia coli. The open reading frame encodes a polypeptide of 396 amino acid residues (M(r) = 42,606, calculated pI 5.9), whose sequence exhibits approximately 38% identity (approximately 60% similarity) to pyridoxal 5'-phosphate-dependent beta-cystathionase(s) from E. coli, Salmonella typhimurium, and rat liver. The characteristic motif, TKYXXGHSD, associated with binding the cofactor in these enzymes is also present in osteotoxin. Physicochemical and enzymatic analyses established the coidentity of osteotoxin with beta-cystathionase. The region upstream of the beta-cystathionase (metC) gene in B. avium 197 lacked regulatory sequences ("Met boxes") described for metC in enteric species, and enzyme production was not repressed by methionine. Incubation of MC3T3-E1 osteogenic cells in medium containing L-[35S]cystine and purified beta-cystathionase resulted in 35S-labeling of the enzyme and at least one major MC3T3-E1 cell protein (M(r) approximately 50,000). cytotoxicity can be attributed to: 1) beta-cystathionase-catalyzed cleavage of L-cystine in the medium and formation of reactive sulfane-containing derivative(s), and 2) transfer of sulfane sulfur to metabolically sensitive or structurally important proteins in the osteogenic cells.
Gentry-Weeks
C R
CR
Laboratory of Microbial Ecology, National Institute of Dental Research, National Institutes of Health, Bethesda, Maryland 20892.
Keith
J M
JM
Thompson
J
J
D000595:Amino Acid Sequence; D000818:Animals; D001427:Bacterial Toxins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000302:isolation & purification / Q000633:toxicity*; D001483:Base Sequence; D015153:Blotting, Western; D001884:Bordetella / Q000201:enzymology*; D002417:Cattle; D016923:Cell Death; D002460:Cell Line; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004269:DNA, Bacterial; D004591:Electrophoresis, Polyacrylamide Gel; D006168:Guinea Pigs; D007526:Isoelectric Point; D007700:Kinetics; D008190:Lyases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000302:isolation & purification / Q000633:toxicity*; D008969:Molecular Sequence Data; D010006:Osteoblasts / Q000187:drug effects*; D011817:Rabbits; D051381:Rats; D015183:Restriction Mapping; D017386:Sequence Homology, Amino Acid; D013057:Spectrum Analysis; D013379:Substrate Specificity; D013552:Swine; D011503:Transglutaminases; D014422:Turkeys; D010566:Virulence Factors, Bordetella
D001427:Bacterial Toxins; D004269:DNA, Bacterial; D010566:Virulence Factors, Bordetella; C060969:dermonecrotic toxin, Bordetella; D011503:Transglutaminases; D008190:Lyases; C019965:cystathionine beta-lyase