Purification and characterization of two Listeria ivanovii cytolysins, a sphingomyelinase C and a thiol-activated toxin (ivanolysin O).
The strong bizonal hemolysis on blood agar and the positive CAMP reaction with Rhodococcus equi denotes the production of two different cytolytic factors by Listeria ivanovii. One was characterized as a thiol-activated (SH) cytolysin of 61 kilodaltons and was termed ivanolysin O (ILO) since data suggested that it is different from listeriolysin O, the SH-cytolysin produced by Listeria monocytogenes. The other is a 27-kilodalton hemolytic sphingomyelinase C that was found to be the cytolytic factor responsible for the halo of incomplete hemolysis synergistically enhanced by R. equi exosubstances. When thiol-disulfide exchange affinity chromatography and gel filtration were applied to the purification of ILO from concentrated L. ivanovii culture supernatants, the copurification of the two cytolysins was observed. This phenomenon seems to be due to the formation of intermolecular disulfide bonds between ILO and the sphingomyelinase, since the latter was found to contain free SH groups, not essential for the activity. These SH groups could react with the single cysteine residue characteristically present in the SH-cytolysins, forming a dimeric cytolytic complex. The purification of ILO was achieved by a further gel filtration with a reducing agent (dithiothreitol) in the eluent. A method for the purification of the sphingomyelinase based on selective sequestration of ILO from the L. ivanovii concentrated culture supernatant by the SH cytolysin target molecule cholesterol and thiol-disulfide affinity chromatography is described.
Vazquez-Boland
J A
JA
Departamento de Patología Animal (Sanidad Animal), Facultad de Veterinaria, Universidad de León, Spain.
Dominguez
L
L
Rodriguez-Ferri
E F
EF
Suarez
G
G
D001427:Bacterial Toxins / Q000276:immunology / Q000302:isolation & purification*; D015153:Blotting, Western; D002784:Cholesterol / Q000378:metabolism; D003603:Cytotoxins / Q000276:immunology / Q000302:isolation & purification*; D008087:Listeria / Q000032:analysis*; D008970:Molecular Weight; D010727:Phosphoric Diester Hydrolases / Q000302:isolation & purification*; D013108:Sphingomyelin Phosphodiesterase / Q000276:immunology / Q000302:isolation & purification*; D013438:Sulfhydryl Compounds
D001427:Bacterial Toxins; D003603:Cytotoxins; D013438:Sulfhydryl Compounds; D002784:Cholesterol; D010727:Phosphoric Diester Hydrolases; D013108:Sphingomyelin Phosphodiesterase