Functional region expression and its binding protein analysis of trimeric autotransporter adhesin in Actinobacillus pleuropneumoniae

Abstract:

[Objective] Actinobacillus pleuropneumoniae (APP) causes porcine pleuropneumonia, leading to great losses for the pig industry worldwide. Adhesion is the first step in the pathogenic process of APP. The newly discovered trimeric autotransporter adhesion (TAA) is an important pathogenic virulence factor of APP, and the 2 464 to 2 574 amino acids of TAA stalk region, named BD3, plays an important role in the bacterial adhesion. However, it is still unclear how APP combines with lung tissues. We express the stalk functional region of TAA, screen and analyze its binding protein in swine lung cells. [Methods] BD3 protein was expressed in prokaryotic expression system, purified, then incubated with swine lung tissue protein. The binding protein of BD3 was picked out by co-immunoprecipitation and analyzed by mass spectrometry. The cDNA library was constructed to get the sequence of the binding protein and then the bioinformatics method was done. [Results] Mass spectrometry analysis revealed that the peptide sequence of the binding protein, and the blast result in the database showed that Keratin 1 was the binding protein. cDNA library was constructed to know the sequence of swine Keratin 1. Bioinformatics analysis showed that low sequence homology of Keratin 1 existed between swine and human and it was a separate evolutionary branch, different from other keratins of swine or human, Keratin 1 had a transmembrane domain at 8 to 100 aa and the secondary structure mainly consisted of α-helix and β-fold. It contained α subunit of G protein at 82 to 362 aa and a TSP1 domain (thrombin-sensitive protein type I repeat domain) at 515 to 552 aa. [Conclusion] The finding of Keratin 1 that is in swine lung cells and binds with BD3 of TAA, lays a foundation for researching the mechanism of TAA adhesion to lung tissue, and has great significance to understand the pathogenic mechanism of APP specially colonized in lung tissues.