TY - JOUR
T1 - Detection, isolation, and analysis of a released Bordetella pertussis product toxic to cultured tracheal cells
AU - Goldman, W. E.
AU - Klapper, D. G.
AU - Baseman, J. B.
PY - 1982
Y1 - 1982
N2 - Cultured hamster trachea epithelial cells were selected as an in vitro model system to study Bordetella pertussis pathogenesis in the respiratory tract. DNA synthesis by serum-stimulated tracheal cells, in contrast to other cell types tested, was inhibited by the supernatant from log-phase B. pertussis broth cultures. A sensitive microassay with these tracheal cells permitted the development of a chromatographic purification scheme based on aggregation of the biological activity under salt-free conditions. The active fraction from this first stage of purification caused a dose-dependent inhibition of DNA synthesis without a similar effect on RNA or protein synthesis. Organ cultures of hamster tracheal rings, when exposed to this partially purified fraction, developed epithelial cytopathology comparable to that seen during B. pertussis infection. Ciliary activity slowed and eventually ceased as ciliated cells were extruded from the ring, leaving an intact but mostly nonciliated epithelium. Further purification of this biological activity was achieved with preparative-scale high-voltage paper electrophoresis. Based on ninhydrin staining and the radioactive profile of material purified from radiolabeled B. pertussis cultures, four fractions were eluted from the paper by descending chromatography. Only component B caused a dose-dependent inhibition of cultured tracheal cell DNA synthesis and epithelial cytopathology in tracheal rings. Combination experiments also demonstrated enhanced inhibition by component B in the presence of component G (oxidized glutathione), a copurifying molecule from the growth medium. Amino acid analysis of component B revealed a composition of glutamic acid (five residues), alanine (five residues), glycine (two residues), cysteine (two residues), and diaminopimelic acid (one residue), as well as muramic acid and glucosamine.
AB - Cultured hamster trachea epithelial cells were selected as an in vitro model system to study Bordetella pertussis pathogenesis in the respiratory tract. DNA synthesis by serum-stimulated tracheal cells, in contrast to other cell types tested, was inhibited by the supernatant from log-phase B. pertussis broth cultures. A sensitive microassay with these tracheal cells permitted the development of a chromatographic purification scheme based on aggregation of the biological activity under salt-free conditions. The active fraction from this first stage of purification caused a dose-dependent inhibition of DNA synthesis without a similar effect on RNA or protein synthesis. Organ cultures of hamster tracheal rings, when exposed to this partially purified fraction, developed epithelial cytopathology comparable to that seen during B. pertussis infection. Ciliary activity slowed and eventually ceased as ciliated cells were extruded from the ring, leaving an intact but mostly nonciliated epithelium. Further purification of this biological activity was achieved with preparative-scale high-voltage paper electrophoresis. Based on ninhydrin staining and the radioactive profile of material purified from radiolabeled B. pertussis cultures, four fractions were eluted from the paper by descending chromatography. Only component B caused a dose-dependent inhibition of cultured tracheal cell DNA synthesis and epithelial cytopathology in tracheal rings. Combination experiments also demonstrated enhanced inhibition by component B in the presence of component G (oxidized glutathione), a copurifying molecule from the growth medium. Amino acid analysis of component B revealed a composition of glutamic acid (five residues), alanine (five residues), glycine (two residues), cysteine (two residues), and diaminopimelic acid (one residue), as well as muramic acid and glucosamine.
UR - http://www.scopus.com/inward/record.url?scp=0020071653&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0020071653&partnerID=8YFLogxK
U2 - 10.1128/iai.36.2.782-794.1982
DO - 10.1128/iai.36.2.782-794.1982
M3 - Article
C2 - 6177637
AN - SCOPUS:0020071653
VL - 36
SP - 782
EP - 794
JO - Infection and Immunity
JF - Infection and Immunity
SN - 0019-9567
IS - 2
ER -