Chromatographic separation and characterization of Pasteurella haemolytica cytotoxin.
Biochemical and immunologic properties of the cytotoxin (leukotoxin) produced by Pasteurella haemolytica were examined. Crude, bacteria-free supernatants from logarithmic phase P haemolytica were fractionated, using a series of column chromatographic techniques. Sequential anion exchange chromatography, gel-filtration chromatography, and chromatofocusing resulted in a cytotoxic substance (cytotoxin-C) of approximately 160 kilodaltons (kD), as determined by use of gel-filtration chromatography. Polyacrylamide-gel electrophoresis of cytotoxin-C yielded 3 protein bands with relative mobilities of 0.37, 0.42, and 0.63. On the basis of immunoblotting with a cytotoxin-neutralizing bovine immunoglobulin for antigen detection, the 2 low-mobility bands shared a strong region of immunogenicity. Using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, principal protein constituents of cytotoxin-C were found at 160, 66, 57, and 23 kD. Using immunoblotting with cytotoxin-neutralizing immunoglobulin, strong, distinct reactions with the 66- and 57-kD bands were detected. Immunization of rabbits and mice with cytotoxin-C resulted in sera that reacted strongly with cytotoxin-C in enzyme-linked immunosorbent assays and immunodiffusion assays. The major immunogenic proteins also were detected by use of immunoblotting with anticytotoxin-C sera from rabbits and mice. Postinoculation rabbit sera neutralized crude cytotoxin.
D000818:Animals; D000907:Antibodies, Bacterial / Q000096:biosynthesis; D000942:Antigens, Bacterial / Q000276:immunology; D001427:Bacterial Toxins / Q000032:analysis* / Q000276:immunology / Q000302:isolation & purification; D002417:Cattle; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003603:Cytotoxins / Q000032:analysis* / Q000276:immunology / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005098:Exotoxins / Q000032:analysis* / Q000276:immunology / Q000302:isolation & purification; D006863:Hydrogen-Ion Concentration; D005779:Immunodiffusion; D007158:Immunologic Techniques; D007525:Isoelectric Focusing; D051379:Mice; D008970:Molecular Weight; D010325:Pasteurella / Q000378:metabolism*; D011817:Rabbits
D000907:Antibodies, Bacterial; D000942:Antigens, Bacterial; D001427:Bacterial Toxins; D003603:Cytotoxins; D005098:Exotoxins; C018579:leukotoxin