Exploring The Function And Pathogenic Mechanism Of SPI-19 In S.Pullorum

Salmonella enterica serovar Pullorum(S.Pullorum)is a host-specific pathogen,which can cause systemic infections in poultry.The high morbidity and mortality caused by the pathogen in chicks,and the persistent infection in adult chickens with weight loss,reduced egg production,diarrhea,reproductive tract damage,and abnormalities,has caused big threat to poultry industry.T6SS has been reported to be involved in invasion and colonization of bacteria in the host by inhibiting the growth of commercial bacteria in gut.However,the function and pathogenesis of T6SS encoded by SPI-19 of S.Pullorum remains unknown.This main purpose of this study is to explore the genetic composition,the function,and the pathogenic mechanism of SPI-19 in S.Pullorum.The SPI-19 deleted strain C79-13?SPI-19 was successfully constructed using ?-red homologous recombination to knock out this 43kb fragment.The SPI-19 was confirmed to promote invasion and colonization of S.Pullorum in host through in vivo and in vitro infection assays.But the T6SS was not related to inducing cytotoxicity to host cells and display antibacterial activity.In addition,measurement of cytokines expression levels and the production of ROS/RNS in the infected cells and chickens showed that SPI-19 contributed to bacterial induced immune evasion.Furthermore,transcriptome sequencing analysis revealed that deletion of SPI-19 caused the significant decreased expression of genes,including T3SS-related regulatory genes and effector genes,which was confirmed by qRT-PCR.1 The phenotypic effect caused by SPI-19 from S.PullorumIn this study,comparative sequence analysis was used to compare the homology of SPI-19 in S.Pullorum with that in S.Enteritidis and T6SS2 in APEC(avian pathogenic Escherichia coli).The results showed that the SPI-19 in S.Enteritidis is lack of most core genes encoding T6SS,while the gene of T6SS2 in APEC is highly homologous with SPI19 in S.Pullorum.It is speculated that the SPI-19 is related to host specificity of S.Pullorum as well as T6SS2 in APEC.In this section,the SPI-19 deleted strain S.Pullorum C79-13ASPI-19 was successfully constructed using ?-Red homologous recombination.The growth curve showed no significant difference was obtained between the wild-type and the mutant strain.The physiological and biochemical identification results showed only D-Mannose was affected by deletion of SPI-19.Bacterial infection to avian macrophages and epithelial cells demonstrated that the deletion of SPI-19 caused the significant decreased invasion and uptake in both cells.The bacterial load dynamic experiments in the internal organs of chicks revealed that in the liver and spleen,the number of bacteria in the wild-type-infected group was notably higher than that of the mutant group on the 1st and 3rd days post challenge.The bacteria were finally cleared till 21 days post challenge.In addition,the number of wild strains in ileum and cecum was significantly higher than that of the mutant strains at 28 days after infection.The above results demonstrated that the absence of SPI-19 caused the decreased colonization of S.Pullorum in host.However,with difference to T6SS in other bacteria,neither the wild-type strain nor the mutant strain has the antibacterial activity.And no significant difference of LDH production was detected between the cells infected with each strain.2 The pathogenesis of T6SS encoded by SPI-19 from S.PullorumIn order to reveal the expression conditions of T6SS,the qRT-PCR method was used to detect the expression level of T6SS genes during the invasion and survival in host cells.The result showed the T6SS was higher expressed during the infection process than cultured in LB medium,which implied that the T6SS displayed its function during the infection process.The detection of ROS/RNS released by bacterial infected macrophages at 2 h showed that the mutant induced more production of ROS/RNS than the wild type strain,which demonstrated that the SPI-19 could inhibit the secretion of NO and ROS to avoid the attack by host immune system.Furthermore,transcriptome sequencing analysis was performed to compare the gene expression profiles in the wild type and mutant strains.Analysis of down-regulated genes showed that most of T3SS-related regulatory genes and effector genes were affected by deletion of SPI-19,especially the genes located in T3SS1 region,which was confirmed by qRT-PCR analysis.The above results revealed the relationship between T6SS and T3SS,but the molecular mechanism remained to be studied in the future.Total RNA was extracted from HD-11 cells infected with C79-13 and C79-13?SPI-19,respectively.The qRT-PCR analysis of cytokines expression showed that the expression level of IL-18,IFN-y was increased significantly in the mutant-infected group,as well as IL-1?,IL-6,iNOS,and CXCLi-2.The result indicated that the SPI-19 was closely related to the host inflammatory response involved in clearing the pathogen.The S.Pullorum mainly induced Th2-biased immune response,and caused persistent infection in host through inhibition of Th1 immune response.In this section,we evaluated the effect of SPI-19 on the immune response induced by S.Pullorum in the host.The transcription of IFN-? in spleen was induced at 21 days post challenge in the group infected with the mutant strain.And the expression of IL-1? was significantly higher than that of wild-type strain on the 3rd,7th,and 28th day after infection,and the transcription level of IL-6 was notably higher than that of the wild-type strain on the 21st day.The inhibition of Th1 immune response by SPI-19 is beneficial to persistent infection of S.Pullorum in the host.