Construction of EspP2 Deletion Strain

The EspP2 deletion strain was constructed and screened as previously described [46 (link)]. Briefly, primers P1/P2 and P3/P4 were used to amplify, respectively, the 620-bp upstream and the 611-bp downstream homologous regions of EspP2 from G. parasuis SC1401 genomic DNA. Next, primers P5/P6 were used to amplify a 935-bp kanamycin resistance cassette from pKD4. Subsequently, the three fragments were purified using a Qiaquick spin column kit (Qiagen, Hilden, Germany) and ligated into restriction sites BamH I and Xho I of linearized pK18mobSacB, resulting in the vector pK18-EspP2. A natural transformation method was used to convert pK18-EspP2 into G. parasuis SC1401, as previously described [46 (link)]. After 36 h incubation, the resultant kanamycin-resistant transformants were grown to large-scale culture in TSB++ supplemented with Kan, in order to be further identified by PCR. Of note, we previously tested the natural transformation efficiency of several standard and local strains of G. parasuis and found that SC1401 had the highest efficiency. We therefore chose it as the wild-type strain for this study and from there constructed the EspP2 deletion strain.