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 bacte rium secretes proteases that degrade porcine hemoglobin and IgA in vitro. T o further characterize A. pleuropneumoniae proteases, we constructed a geno mic library expressed in Escherichia coli DH5 alpha, and selected a clone t hat showed proteolytic activity. The recombinant plasmid carries an 800-bas e pair A. pleuropneumoniae gene sequence that codes for a 24-kDa polypeptid e. A 350-base pair PstI fragment from the sequence hybridized at high strin gency with DNA from 12 serotypes of A. pleuropneumoniae, but not with DNA f rom Actinobacillus suis, Haemophilus parasuis, Pasteurella haemolytica, Pas teurella multocida A or D, or E. coli DH5 alpha, thus showing specificity f or A. pleuropneumoniae. The expressed polypeptide was recognized as an anti gen by convalescent-phase pig sera. Furthermore, a polyclonal antiserum dev eloped against the purified polypeptide recognized an A. pleuropneumoniae o ligomeric protein in both crude-extract and cell-free culture media. This r ecombinant polypeptide cleaved azocoll, gelatin, and actin. Inhibition of t he proteolytic activity by diethylpyrocarbonate suggests that this polypept ide is a zinc metalloprotease.