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.