Construction of pSPI12-cured Salmonella enterica serovar Pullorum and identification of IpaJ as an immune response modulator

In Salmonella, plasmids participate in many pathways involved in virulence, metabolism, and antibiotic resistance. To investigate the function of the ipaJ gene in a multi-copy plasmid pSPI12 prevalent in Salmonella enterica serovar Pullorum (S. Pullorum), we established a method to eliminate the plasmid and constructed the plasmid-cured bacteria C79-13-ΔpSPI12 by using the suicide vector pDM4. Briefly, a 500 bp fragment ipaJU from pSPI12 was cloned into pDM4 and transformed into S. Pullorum C79-13 by conjugative transfer. After homologous recombination, the suicide vector was inserted into pSPI12 to produce pSPI12-pDM4-ipaJU. Induction of the expression of the sacB gene in the suicide vector killed the bacteria harbouring plasmid, while the progeny losing the plasmid survived in the plate with sucrose. The plasmid-cured strain showed extremely decreased ability to infect chicken macrophage HD11 cells and LMH hepatic epithelial cells compared to wild type strain and complementary strain carrying ipaJ. Additionally, IFN-γ mRNA levels were up-regulated in HD11 cells or chicken spleens infected by plasmid-cured strain, but no difference was detected in IL-4 among the three strains. Transforming ipaJ into S. Enteritidis also decreased expression of proinflammatory cytokines in infected macrophages or chicken spleens compared to wild type strain. These results suggest that the ipaJ gene in pSPI12 is involved in S. Pullorum infection and that IpaJ protein modulates immune response.