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

In <i>Salmonella</i>, plasmids participate in many pathways involved in virulence, metabolism, and antibiotic resistance. To investigate the function of the <i>ipaJ</i> gene in a multi-copy plasmid pSPI12 prevalent in <i>Salmonella enterica</i> serovar Pullorum (<i>S</i>. 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 <i>ipaJU</i> from pSPI12 was cloned into pDM4 and transformed into <i>S.</i> Pullorum C79-13 by conjugative transfer. After homologous recombination, the suicide vector was inserted into pSPI12 to produce pSPI12-pDM4-<i>ipaJU</i>. Induction of the expression of the <i>sacB</i> 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 <i>ipaJ</i>. Additionally, IFN-<i>γ</i> 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 <i>ipaJ</i> into <i>S.</i> Enteritidis also decreased expression of proinflammatory cytokines in infected macrophages or chicken spleens compared to wild type strain. These results suggest that the <i>ipaJ</i> gene in pSPI12 is involved in <i>S.</i> Pullorum infection and that IpaJ protein modulates immune response.