Abstract:[Objective] To establish a more efficient knockout method for the serine protease coding gene (cp40) of Corynebacterium pseudotuberculosis and evaluate the role of this gene in the pathogenicity of C. pseudotuberculosis. [Methods] A vector pEC-cp40gRNA-HDarm, with guide RNA, upstream and downstream sequences flanking cp40 of C. pseudotuberculosis Xuanhan strain (XH02), and spacer, was constructed from pECXK99E. The recombinant vector pEC-cp40gRNA-HDarm was transferred into C. pseudotuberculosis competent cells carrying pCas9gRNA-ccdB to form the CRISPR/Cas9 gene editing system for the deletion of cp40. The roles of cp40 in the pathogenicity of C. pseudotuberculosis were evaluated by comparison of the colony morphology and growth curves between the cp40-deleted (Δcp40) strain and wild type (WT) strain, the viability and interleukin (IL)-1β secretion of J774A.1 macrophages infected with Δcp40 and WT in vitro, and the mortality and organ bacterial loads in mice infected with Δcp40 and WT in vivo. [Results] We successfully constructed the cp40-deleted strain XH02Δcp40 by using the established dual-plasmid CRISPR/Cas9 editing system. Compared with WT (XH02), XH02Δcp40 showed no obvious difference in the colony morphology or growth curve. However, the J774A.1 cells infected with XH02Δcp40 showed decreased lactate dehydrogenase (LDH) release (P=0.06) and propidium iodide (PI) staining ratio (P<0.01) compared with those infected with XH02. The mortality of XH02Δcp40-infected mice reduced by 50% and the bacterial loads in the liver and kidney of XH02Δcp40-infected mice significantly reduced compared with those of XH02-infected mice (P<0.001). [Conclusion] The CRISPR/Cas9 gene editing system established in this study can effectively delete cp40 of C. pseudotuberculosis. The results confirm that cp40 is a virulence-related gene, providing a foundation for subsequent research on the infection of C. pseudotuberculosis based on this gene.
