Purification and characterization of a protease from Actinobacillus pleuropneumoniae serotype 1, an antigen common to all the serotypes.
A high molecular-mass proteolytic enzyme of Actinobacillus pleuropneumoniae serotype 1, was purified from culture supernatants (CSN) by using DEAE-cellulose and sepharose-4B-gelatin chromatography. In 10% SDS-polyacrylamide gels copolymerized with porcine gelatin, the protease showed a single band of activity of > 200 kDa. However, minor molecular-mass proteolytic bands were observed when the protease was electrophoresed in the presence of either 5% beta-mercaptoethanol, 50 mM dithiothreitol, or 0.25 M urea. Furthermore, when the > 200-kDa purified protein was passed through a sucrose gradient, several bands with proteolytic activity were found: 62, 90, 190, and 540 kDa. The proteolytic activity was increased in the presence of calcium or zinc and was not affected after being heated at 90 degrees C for 5 min. Proteolytic activities were also observed in CSN from all A. pleuropneumoniae serotypes and biotypes. The purified protease hydrolyzed porcine IgA and IgG in vitro. In addition, by immunoblot the protease was recognized by serum of naturally infected pigs with serotypes 1 and 5, and by serum of pigs experimentally infected with serotypes 1, 2, 8, or 9. Serum of a pig vaccinated with CSN of a serotype 3 strain also recognized the protease, but not sera of pigs vaccinated with a bacterin (serotype 1). Proteins from CSN of all the serotypes, which were precipitated with 70% (NH4)2SO4, were recognized by a polyclonal antibody raised against the purified protease. Taken together these results indicate that an antigenic protease is produced in vivo by all the serotypes of A. pleuropneumoniae. The results indicate that proteases could have a role in the disease and in the immune response of pigs infected with A. pleuropneumoniae.
Departamento de Biología Celular, Centro de Investigación y de Estudios Avanzados del IPN, México D.F., Mexico.
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E
Negrete-Abascal
V R
VR
Tenorio
A L
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Guerrero
R M
RM
García
M E
ME
Reyes
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M
de la Garza
D016977:Actinobacillus pleuropneumoniae / Q000145:classification / Q000201:enzymology*; D000818:Animals; D002846:Chromatography, Affinity; D002848:Chromatography, DEAE-Cellulose; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D005612:Freeze Drying; D007070:Immunoglobulin A / Q000378:metabolism; D007074:Immunoglobulin G / Q000378:metabolism; D007700:Kinetics; D008970:Molecular Weight; D012703:Serotyping; D013379:Substrate Specificity; D013552:Swine; D014462:Ultrafiltration
D007070:Immunoglobulin A; D007074:Immunoglobulin G; D010450:Endopeptidases