A 24-kDa cloned zinc metalloprotease from Actinobacillus pleuropneumoniae is common to all serotypes and cleaves actin in vitro.
Actinobacillus pleuropneumoniae causes pleuropneumonia in swine. This bacterium secretes proteases that degrade porcine hemoglobin and IgA in vitro. To further characterize A. pleuropneumoniae proteases, we constructed a genomic library expressed in Escherichia coli DH5alpha, and selected a clone that showed proteolytic activity. The recombinant plasmid carries an 800-base pair A. pleuropneumoniae gene sequence that.codes for a 24-kDa polypeptide. A 350-base pair PstI fragment from the sequence hybridized at high stringency with DNA from 12 serotypes of A. pleuropneumoniae, but not with DNA from Actinobacillus suis, Haemophilus parasuis, Pasteurella haemolytica, Pasteurella multocida A or D, or E. coli DH5alpha, thus showing specificity for A. pleuropneumoniae. The expressed polypeptide was recognized as an antigen by convalescent-phase pig sera. Furthermore, a polyclonal antiserum developed against the purified polypeptide recognized an A. pleuropneumoniae oligomeric protein in both crude-extract and cell-free culture media. This recombinant polypeptide cleaved azocoll, gelatin, and actin. Inhibition of the proteolytic activity by diethylpyrocarbonate suggests that this polypeptide is a zinc metalloprotease.
García-Cuéllar
C
C
Departamento de Biología Celular, Centro de Investigación y de Estudios Avanzados del IPN, México DF, México.
Montañez
C
C
Tenorio
V
V
Reyes-Esparza
J
J
Durán
M J
MJ
Negrete
E
E
Guerrero
A
A
de la Garza
M
M
D000189:Actinobacillus Infections / Q000473:pathology*; D016977:Actinobacillus pleuropneumoniae / Q000145:classification / Q000201:enzymology* / Q000235:genetics; D000199:Actins / Q000378:metabolism; D000818:Animals; D003001:Cloning, Molecular; D004269:DNA, Bacterial; D015723:Gene Library; D008666:Metalloendopeptidases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010957:Plasmids; D012703:Serotyping; D013552:Swine; D013553:Swine Diseases / Q000382:microbiology*; D015032:Zinc
D000199:Actins; D004269:DNA, Bacterial; D008666:Metalloendopeptidases; D015032:Zinc