The Expression And Secretion Mechanism Of Salmonella Pullorum Anti-inflammatory Effector Protein IpaJ And Its Function Of Mediating Host Immune Response

Salmonella enterica serovar Gallinarum biovar Pullorum(S.Pullorum)is a host-adapted pathogen that can cause pullorum disease,it mainly causes acute infection in chicks within three weeks of age.The mortality of S.Pullorum-infected newborn chicks is very high.Most adult chickens showed chronic or recessive infections,which are often ignored due to no clinical symptoms.But this infection can cause the vertical transmission of pathogens to their offspring through breeding eggs and reduce the hatching rate of breeding farms,and the pathogen can spread widely among their offsprings through horizontal transmission,which causes significant economic losses to the poultry breeding industry.Although the disease has been eradicated from commercial farms in most developed countries,it remains the predominant serotype of Salmonella in many developing countries.The uneven level of aquaculture development in China is closely associated with the high isolation rate of S.Pullorum in poultry farms.Besides,the emergence of multi-drug resistant strains also increasse the outbreaks of pullorum disease.Therefore,elucidation of the pathogenic mechanism of S.Pullorum can provide the theoretical basis for prevention and control of the disease.S.Enteritidis and S.Pullorum has genetic similarity,but they show completely different pathogenic characteristics:S.Enteritidis is a host-ranged serovar and causes self-limited acute gastroenteritis,while S.Pullorum is host-adapted and does not cause substantial inflammation in the chicken intestine.Compared with S.Enteritidis,S.Pullorum induces a decrease in transcription of pro-inflammatory cytokines and an increase in transcription of the antiinflammatory cytokines in macrophages.Recent studies have shown that S.Pullorum induce a Th2 biased immune response in contrast to the Th1 biased immune response induced by S.Enteritidis.The Th2 biased immune response can eradicate the extracellular bacteria,but the intracellular bacteria remained in the host to cause persistent infection.Genomic analysis speculates that the host adaptability and pathogenic characteristics of S.Pullorum are not only caused by genome degradation,but also jointly determined by new gene clusters obtained through horizontal transfer,such as the ipaJ gene specifically located in S.Pullorum pSPI12 plasmid.The virulence factor IpaJ is an important anti-inflammatory protein inhibiting host inflammation.However,the molecular mechanism for IpaJ expression and secretion and its anti-inflammation effects remains unclear.In this study,Tn-seq and DNA pull down techniques were used to identify the upstream factors that regulate the expression and secretion of IpaJ.We revealed that the transcriptional expression of IpaJ protein is directly regulated by transcription factor ItrA(ipaJ transcriptional regulator A,ItrA).Besides,we demonstrated that IpaJ is a T3SS-1 effector protein involved in bacterial infection.In S.Pullorum infected cells,IpaJ participates in the bacterial infection process and inhibits the inflammatory response of host cells.Further study indicated that IpaJ inhibited the activation of NF-κB and MAPK signaling pathways through its deubiquitin protease activity,thereby inhibits the host’s immune response.This study revealed that the expression and secretion mechanism of IpaJ as an anti-inflammatory effector protein of T3SS1 and demonstrated its mechanism of inhibiting host inflammatory response.This study also confirmed the host Th2 biased immune response induced by S.Pullorum.1.Screening and identification of transcriptional regulatory factors of S.Pullorum ipaJIn order to reveal the molecular regulation mechanism of IpaJ as a virulence factor,we performed the screening technology based on transposon insertion sequencing and the DNA pull down technology using the promoter of ipaJ as bait to identify the upstream factors regulating ipaJ expression.According to the screening results of Tn-seq,we found 94 positive regulators that may promote the expression of IpaJ and 4 repressors that may inhibit the expression of IpaJ.Subsequently,13 potential positive regulators were selected for construction of the mtant strains,and verified by detecting the expression level of IpaJ using Western blot analysis.It was found that HilC,YebC and HilA transcriptional factors related to T3SS-1 regulated the expression of IpaJ.The DNA pull down results showed that 35 regulatory factors may bind the ipaJ promoter region,and most of them were DeoR family and AraC family regulatory proteins.We selected 19 transcriptional factors encoding genes with high coverage for the construction of mutant strains.Deletion of SEN3597 caused the non-expression of IpaJ,indicating that the DeoR family transcriptional regulator SEN3597 is the main regulator of IpaJ expression,which was named ItrA(IpaJ transcription regulatory A,ItrA).In addition,we expressed and purified ItrA protein with His-tag for EMSA.The EMSA results showed that ItrA directly binded the promoter region of 50 bp upstream the ORF of ipaJ.Therefore,ItrA is a positive transcriptional regulator controling the expression of ipaJ.2.IpaJ is a T3SS-1 effector inhibiting host Th1 response and inflammationThe secretion level of IpaJ in different mutants was detected using TCA precipitation experiment.It was found that Salmonella pathogenicity island 1(SPI-1),hilA,and hilD(the main regulator of SPI-1)were the necessary factors for IpaJ secretion,the absence of each of them prevented secretion of IpaJ into the culture medium or S.Pullorum-infected cells.Meanwhile,deletion of SPI-2 or ssrB(the master regulator of SPI-2)did not affect the normal secretion of IpaJ.This suggests that IpaJ is an effector protein of SPI-1-encoded III secretion system 1(T3SS-1).In addition,we measured the expression levels of HilA and IpaJ in S.Pullorum cultured at different growth stages.The results showed that IpaJ was highly expressed at the early stable phase of bacterial growth,which was consistent with the expression trend of HilA.To investigate the role of ipaJ located in the multi-copy plasmid pSPI12 prevalent in S.Pullorum,we established a method to eliminate the plasmid and constructed the plasmid-cured bacteria C79-13-ΔpSPI12 using the suicide vector pDM4.Briefly,a specific fragment from the plasmid was cloned into pDM4 vector and then transformed into C79-13 strain.After homologous recombination,the suicide vector was inserted into the plasmid.Upon induction by sucrose,the sacB gene in pDM4 was expressed to produce levansucrase,which can hydrolyse sucrose and glucose to fructose,resulting in the production of a toxic fructan that kills the bacteria having the pDM4-containing plasmid,while only bacteria that lost the plasmid could survive in sucrose.The complementary vector pBR322-ipaJ was introduced into C79-13-ΔpSPI12 and S.Enteritidis P125109(without ipaJ)to study the function of IpaJ.The chicken epithelial cell lines(LMH)and macrophages(HD-11,chMDM)were used as cell infection models to compare the invasive ability of the three strains(C79-13,C7913-ΔpSPI12 and C79-13ΔpSPI12::pipaJ).The results showed that deletion of ipaJ significantly reduced the ability of S.Pullorum to infect HD11,chMDM,and LMH cells.In addition,compared to the cells infected with the wild type and complementary strains,the transcriptional levels of Thl-type(INF-γ,IL-12α)and pro-inflammatory cytokines(CXCLi 1,CXCLi 2,IL-6,IL-1β and iNOS)mRNA were significantly up-regulated in HD11,chMDM,and chicken spleen cells infected with the ipaJ-deleted strain.Transformation of ipaJ into S.Enteritidis also reduced the expression of pro-inflammatory cytokines in infected macrophages or chicken spleen cells compared to the wild-type strain P125109 infected groups.Compared with the wild-type-and complementary strains-infected chickens,the bacteria in the C79-13-ΔpSPI12-infected group were rapidly removed from chicken livers and spleens.These results suggested that IpaJ mediates immune escape by inhibiting Thl response and inflammatory response of host cells as an T3SS-1 effector protein,which promotes the bacterial colonization and survival in chickens.3.IpaJ inhibits the activation of NF-κB signaling pathway through its deubiquitinase activityNuclear factor-kappa B(NF-κB)is considered to be the main regulatory factor of inflammation and is closely involved in the host’s immune response to pathogen infection.In order to verify whether IpaJ targets the NF-κB signaling pathway to inhibit the host inflammatory response,we found that IpaJ significantly inhibited the degradation of IκBα and p-IκBα in host cells induced by TNFα.Further studies showed that IpaJ blocked the activation of NF-κB signaling pathway induced by TNFα through inhibiting the ubiquitin degradation of IκBα.In order to systematically analyze the mechanism of IpaJ in the host,we found that IpaJ is involved in ubiquitin-mediated proteolysis,T cell receptor signaling pathway,natural killer cell-mediated cytotoxicity,apoptosis and other pathways based on proteomic analysis.IpaJ specifically reduced the ubiquitin-conjugating enzymes E2(UBE2E1,UBE2D2,UBE2B)in host cells,which may explain the inhibition of IκBα ubiquitination by IpaJ in Salmonellainfected cells.Through protein domain analysis,we found that the IpaJ in S.Pullorum is homologous to the IpaJ in Shigella.Both IpaJ proteins have a C39 family cysteine protease activity domain,consisting of C45,H184 and D197.GST-IpaJ protein was then expressed and purified for analysis of its protease activity.The GST-IpaJ could specifically cleave polyubiquitin chains linked to K48 and K63 in vitro,and it showed stronger cleavage ability to K63-linked polyubiquitin chains.These results suggested that IpaJ was a newly discovered deubiquitinase,which can block the activation of the NF-κB signaling pathway through inhibiting the ubiquitination degradation pathway of IκBα,thereby inhibiting the host inflammation.4.IpaJ regulates cell proliferation and T cell immune response by inhibiting the activation of MAPK signaling pathwayT3SS effectors usually have various targets in host cells.Therefor,in order to further study the effects of IpaJ on host signaling pathways during Salmonella infection,we performed the quantitative phosphoproteomic analysis of HeLa cells infected with S.Pullorum.The results showed that the phosphorylation levels of more than 300 proteins changed significantly after infection,representing 13%of all detected phosphorylated proteins.IpaJ displayed effects on host protein phosphorylation modification in S.Pullorum-infected cells,including cytoskeleton,transcription,signal transduction,cell cycle regulation.The cytoskeleton is the core targets of IpaJ-mediated cellular responses during bacterial infection,Western blot analysis revealed that IpaJ mainly down-regulated the phosphorylation levels of ERK and MEK proteins to inhibit the activation of MAPK pathway,and then regulated the cell growth,apoptosis,and T cell immune response in host cells.