Construction Of A PCR Method Targeting IpaJ Gene To Identify S.Pullorum And Mechanism Of IpaJ Inhibiting The Activation Of NF-κB Pathway

S.Pullorum is the causative pathogen of pullorum disease,which leads to high morbidity and mortality rate within 20-day-old chickens,but rarely causes severe clinical disease in adult birds.S.Pullorum causes infection by vertical transmission and horizonal transmission.The outbreak of pollurom disease causes heavy economic losses in China and other developing countries.Rapid method developed to detect S.Pullorum has great significance for effective prevention and control of pullorum disease.Additionaly,in order to understand the pathogenesis of S.Pullorum and find new therapeutic measures to the disease,it is essential to reveal the function of S.Pullorum effector proteins.1.Construction of a PCR method targeting ipaJ gene to identify S.PullorumTraditional methods for identification of S.enterica rely on biochemical reactions and serotyping,which are accurate but time-consuming to complete whole procedure.In this study,we developed a one-step PCR method targeting the specific ipaJ gene harbored by pSPI12 plasmid to identify S.Pullorum.Among the 650 of S.Pullorum strains isolated during 1962 to 2016 around China,644 strains identified are ipaJ positive,accounting to a detection rate of 99.08%.Six strains were ipaJ negative because pSPI12 was non-existent in these strains according to the results of whole genome sequencing analysis.The detection method showed no cross-reaction with other S.enterica serotypes including S.Gallinarum,which show close genetic relationship with S.Pullorum.Thus,this method could directly differentiate S.Gallinarum from S.Pullorum without biochemical identification.The limit of detection for this PCR method was as low as 90 fg/μL or 102 CFU/μL.In addition,this method applied to detect Salmonella isolated from the chicken farm and the results were consistent with what we obtained from traditional methods.In conclusion,all of the features showed that this PCR method is rapid,accurate and convenient for efficient identification of S.Pullorum.2.Mechanism of IpaJ inhibiting the activation of NF-κB signaling pathwayEffector proteins secreted by Salmonella often play a crucial role in modulating host signal transduction and cellular processes to benefit the survival of bacteria in host cells.In this study,we demonstrated that the IpaJ protein from S.Pullorum significantly inhibits the activation of the key pro-inflammatory transcription factor NF-κB induced by TNFα,IL-1β and LPS.IpaJ also inhibits the NF-κB pathway during Salmonella infection.IpaJ harbors the catalytic cysteine(C),histidine(H)and aspartate(D)residues required for its function.As predicted by this alignment,alanine substitutions at C90,H230 or D242 abolished the ability of IpaJ to inhibit activation of host NF-κB signal pathway.Compared with S.Pullorum wild type(WT)strain,ApSPI12 strain infection led to increased IκB degradation and ubiquitination in infected cells,but IpaJ did not interfere with the phosphorylation of IκB induced by TNFa.Moreover,IpaJ is efficient for inhibition of NF-κB translocation to the nucleus and ultimately interferes with the secretion of pro-inflammatory cytokines IL-1β,IL-6 and IL-8 in infected HeLa cells.Interestingly,IpaJ from S.Pullorum also contributes to fragmentation of Golgi apparatus as well as IpaJ expressed in S.flexneri,also the IpaJ from S.flexneri has the ability to inhibit the activation of NF-κB pathway.In addition,the deficiency of SPI1 or SPI2 did not interfere with the expression and secretion of IpaJ protein.It demonstrated that the expression and secretion of IpaJ be not regulated by T3SS.It is the first time that bacterial effector IpaJ from S.Pullorum has been suggested to have such function.