The contribution of urease activity to the pathogenesis of Actinobacillus pleuropneumoniae infection in pigs

Biochemical analyses of the urease from the serotype 1 strain, CM5, of Actinobacillus pleuropneumoniae revealed that it should be active in the respiratory tract of pigs where levels of available urea are low. In order to investigate the possible role(s) of urease activity in virulence of A. pleuropneumoniae infection, the genes responsible for urease expression were cloned and sequenced, and urease-negative mutants were generated by transposon mutagenesis. The urease gene cluster contained typical structural (ureABC) and accessory genes (ureEFGD ), separated by a unique ORF (ureX) of unknown function. These genes were sufficient for urease activity when cloned into Escherichia coli, though the level of urease activity expressed by the minimal subclone was greatly reduced compared to that of the wild-type A. pleuropneumoniae.;Analysis of 19 urease-negative transposon mutants revealed that additional genes are required for urease activity in A. pleuropneumoniae. Eleven of the 19 mutants contained insertions within the first 4 genes of a putative nickel and cobalt transport operon (cbiKLMQO). The CbiM and CbiQ proteins share sequence similarity with cobalt transport membrane proteins, and CbiO shares sequence similarity with cobalt transport ATP-binding proteins. Addition of high concentrations of NiCl2greatly increased the level of urease activity expressed by the minimal urease clone, whereas an E. coli clone containing a 15 kbp insert including the putative transport operon together with the urease gene cluster did not require addition of NiCl2 for high urease activity. The presence of a truncated lysR homologue (apuR upstream of the transport operon affected expression of urease activity in clones containing opposite orientations of the 15 kbp insert, indicating that expression of the transport operon, and possibly the urease cluster, may be transcriptionally regulated by the product of this gene. An additional ORF (utp) was identified between the transport operon and the urease cluster. The predicted polypeptide encoded by this gene shares a high degree of sequence similarity with mammalian urea transport proteins, although its function in A. pleuropneumoniae remains to be determined.;The contribution of urease activity to the pathogenesis of porcine pleuropneumonia was assessed using two different urease-negative mutants. There was no difference in virulence when a cbiK::Tn10 mutant was compared to the parental strain at a high dose of infection (106 cfu/ml). However, neither the cbiK::Tn10 nor a ureG::Tn10 mutant (which contained an unmapped second insertion) caused any disease in pigs exposed to a low dose challenge (103 cfu/ml). Under the same conditions, urease-positive A. pleuropneumonide was recovered from the lungs of 8 of 20 pigs challenged with strain CM5. The results of these studies support the hypothesis that urease activity contributes to the pathogenesis of A. pleuropneumoniae infection in pigs.