Invasion plasmid antigen J (IpaJ) is a protein with cysteine protease activity that is present in Salmonella and Shigella species. Salmonella enterica serovar Pullorum uses IpaJ to inhibit the NF-κB pathway and the subsequent inflammatory response, resulting in bacterial survival in host macrophages. In the present study, we performed a DNA pull-down assay and EMSA and identified ItrA, a new DeoR family transcriptional regulator that could control the expression of IpaJ by directly binding to the promoter of ipaJ. The deletion of itrA inhibited the transcription of ipaJ in Salmonella. Tn-Seq revealed that two regulators of Salmonella pathogenicity island 1 (SPI-1), namely HilA and HilD, regulated the secretion of IpaJ. The deletion of hilA, hilD or SPI-1 inhibited the secretion of IpaJ in both cultured medium and Salmonella-infected cells. In contrast, the strain with the deletion of ssrB (an SPI-2 regulator-encoding gene) displayed normal IpaJ secretion, indicating that IpaJ is an effector of the SPI-1-encoded type III secretion system (T3SS1). To further demonstrate the role of IpaJ in host cells, we performed quantitative phosphoproteomics and compared the fold changes in signaling molecules in HeLa cells infected with wild-type S. Pullorum C79-13 with those in HeLa cells infected with the ipaJ-deleted strain C79-13ΔpSPI12. Both phosphoproteomics and Western blot analyses revealed that p-MEK and p-ERK molecules were increased in C79-13ΔpSPI12- and C79-13ΔpSPI12-pipaJ(C45A)-infected cells; and Co-IP assays demonstrated that IpaJ interacts with Ras to reduce its ubiquitination, indicating that IpaJ can inhibit the activation of the MAPK signaling pathway.

Salmonella enterica serovar Enteritidis is the most prevalent serotype that causes human infections worldwide. Consumption of S. Enteritidis-contaminated animal foods is a major source of human infections; however, eradicating bacteria from animals remains difficult. Therefore, it is necessary to develop new measures to prevent and control salmonellosis. Here, we used the outer-membrane vesicles (OMVs) of S. Enteritidis and assessed their protective efficacy and immune response in mice. Deletion of tolR in S. Enteritidis increased the production and size of OMVs compared to those in the wild type (WT) and ΔrfaQ strains. Intramuscular immunization with OMVs conferred greater protection than intraperitoneal and intranasal immunization. Moreover, OMVs extracted from both WT and ΔtolR strains provided an 83.3% protective rate in mice challenged with S. Enteritidis, which was higher than that provided by OMVs extracted from the ΔrfaQ strain. However, compared with OMVs from the ΔtolR strain, OMVs from WT and ΔrfaQ strains rapidly eradicated S. Enteritidis colonizing the liver, spleen, ileum, and cecum of BALB/c mice after immunization. Immunization with OMVs from each of the three strains induced humoral immune responses and showed no side effects on the growth of mice. Our study revealed that OMVs from various S. Enteritidis strains could be developed for use as subunit vaccine candidates against nontyphoidal Salmonella infections in mammals.

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Salmonella enterica subsp. enterica serovar 4,[5],12:i:- (Salmonella 4,[5],12:i:-) derived from S. Typhimurium has become the dominant serotype causing human salmonellosis. Replacement of the fljAB operon in S. Typhimurium by different resistance regions (RRs) can generate Salmonella 4,[5],12:i:- without expressing the second-phase flagellar antigen. However, the generation and evolution of RRs in Salmonella 4,[5],12:i:- across the world remain unknown. In the present study, except for the three reported RRs (RR1–RR3), five novel RRs (RR4–RR8) were identified in Chinese isolates from livestock and humans. The insertion of RR3 into the chromosomal hin-iroB site of S. Typhimurium produced RR3-S. Typhimurium as a primary intermediate strain. Following cis and/or trans intramolecular transpositions mediated by IS26 and/or IS1R elements, Salmonella 4,[5],12:i:- was produced by replacing the fljAB operon and/or its flanking sequences with RRs; the generation process was confirmed by the subcultivation of RR3-S. Typhimurium in vitro and in vivo. In addition, animal experiments revealed that RR3-Salmonella 4,[5],12:i:- displayed more efficient colonisation and survival abilities in mouse tissues than its parent strain, RR3-S. Typhimurium. The present study is the first to demonstrate the molecular mechanism underlying the origin and generation of Salmonella 4,[5],12:i:- from S. Typhimurium through complicated transpositions.