Screening And Evaluation Of The Attenuated Vaccine Andidate Genes From Type Ⅵ Secretion System Of Salmonella Enterica Serovar Pullorum

When Salmonella enterica serovar Pullorum(S.Pullorum)infects chicks,it can proliferate dramatically in the host and can be transmitted through feces,flight feathers and mutual contact,causing huge economic losses to poultry industry.The development of live attenuated vaccines that can induce both humoral immunity and cellular immunity is an effective method to control S.Pullorum infection in chickens.The application of modern genetic engineering technology to construct attenuated Salmonella vaccines needs to balance the virulence and immunogenicity of the attenuated Salmonella.Therefore,screening appropriate attenuated vaccine candidate genes is the key to the development of an attenuated vaccine.Our previous studies showed that the type Ⅵ secretion system(T6SS)encoded by S.Pullorum SPI-19 played an important role in bacterial infection and pathogenicity.In this study,in order to screen out the pathogenic genes and potential vaccine targets in SPI-19,the RNA-Seq was performed to detect the genes affected by deletion of SPI-19.Compared with the wild type strain,the genes encoding regulators and effectors of the type Ⅲ secretion system(T3SS)were significantly down-regulated in the SPI-19 deleted strain.However,deletion of clpV,vgrG and hcp2 encoding the structural protein of SPI-19/T6SS did not affect the expression and function of T3SS.Therefore,we conducted in-depth analysis of the genes located in SPI-19,and finally determined two genes,RS09150 and RS09155,encoding the proteins with the Sell repeat motif participate in the regulation of the T3SS expression and affect the bacterial infection in cells and host.Deletion of the genes caused the decreased virulence of the bacteria,but induced the cellular immune response of chickens,implying both genes could be the potential targets for attenuated vaccines of S.Pullorum.1.Effect analysis of clpV,vgrG,hcp2 in S.Pullorum SPI-19In this study,homologous recombination mediated by the suicide plasmid pDM4 was used to delete the clpV,vgrG and hcp2 gene to give rise to C79-13△clpV,C79-13ΔvgrG and C79-13Δhcp2 mutants.Then the avian macrophages HD-11 and epithelial cells LMH were used as cell infection models to determine the cell infection ability of S.Pullorum,the results show that C79-13ΔclpV,C79-13ΔvgrG,C79-13Δhcp2 showed enhanced ability to be untaked by macrophages,but C79-13△clpV and C79-13ΔvgrG showed decreased invasion to the epithelial cells.Besides,no difference was detected in the release of lactate dehydrogenase(LDH)in HD-11 infected by wild type strain and mutants,indicating that ClpV,VgrG and Hcp2 were not involved in inducing cytotoxicity and cell death.RNA-Seq was then performed to show that the deletion of SPI-19 significantly reduced the expression of T3SS compared with the wild type strain.The regulatory genes,structural genes and effector protein coding genes belonging to T3SS1 and T3SS2 were all significantly down-regulated in the SPI-19 deleted strain.However,there was no significant difference in the expression of T3SS genes in the three mutants compared with wild-type strain by qRT-PCR analysis,indicating the C1pV,Hcp2 and VgrG proteins did not affect the expression of T3SS.There may be other genes in SPI-19 that influence the ability of bacteria to infect host cells and colonize in the host.2 Functional analysis and immunity effect evaluation of RS09150 and RS09155 in SPI-19 of S.PullorumTwo regulatory genes RS09140 and RS09275 and two genes encoding Sell repeat motif proteins RS09150 and RS09155 were screened out for frrther study based on bioinformatics analysis.The corresponding deleted strains C79-13ΔRS09140,C79-3ΔRS09275,C79-13ΔRS09150 and C79-13ΔRS09155 was subjected to qRT-PCR analysis of T3SS genes.The results showed that deletion RS09140 or RS09275 did not affect the expression of T3SS gene,but the expression of the regulatory genes hilC,invF,ssrA,and ssrB of T3SS was significantly reduced in C79-13ΔRS09150 and C79-13ΔRS09155,and deletion of RS09150 also caused the decreased expression of hilA.The down-regulation of these regulatory genes would lead to the decreased expression of the entire T3SS,thereby affecting the bacterial invasion to the avian cells and colonization in the hostIn order to further study the effect of RS09150 and RS09155 on the pathogenicity of S.Pullorum,the bacterial infection ability of C79-13ΔRS09150 and C79-13ΔRS09155 to avian macrophages HD-11 and LMH epithelial cells and their colonization ability in chickens was evaluated.The bacterial infection results showed that deletion of RS09150 or RS09155 caused significantly decreased invasion ability of S.Pullorum to avian macrophages and epithelial cells.The survival of S.Pullorum in HD-11 cells showed that although the survival curve of mutants in macrophages are similar to the wild type strain,but the proliferation ability of the mutants was lower than the wild type from 3h post infection to 12 h,which is the point of maximum number of bacteria.The results revealed that deletion of RS09150 or RS09155 could reduce the bacterial invasion into cells by affecting the expression of T3SS1,and reduce the bacterial survival in cells by affecting the expression of T3SS2.The wild type strain,the mutants,and complementary strains were then used to infect 3-day-old Hailan white chicks,respectively.At 3 days post challenge(dpc),the bacterial load of C79-13ΔRS09150-infected chickens was significantly lowere that of chickens challenged with wild type strain.At 7 dpc and 14 dpc,the colonization ability of C79-1ΔRS09150 and C79-13ΔRS09155 in the ileum was lower than that of wild type strain.In the cecum,the bacterial load showed a slow decreasing trend,and at 28 dpc,but the amount of bacteria in the chickens infected with mutants was lower than that in the wild type strain infected group.In order to evaluate the RS09150 and RS09155 as potential target genes of attenuated vaccines,the cytokines expression levels were measured in the spleen of chickens after challenge.The results showed that the RS09150 and RS09155 mutants induced the chickens to produce more IL-1β,IL-6 and IFN-y than that of the wild type strain,indicating that the mutants can produce a strong Th1 response at the early stage of infection and promote the bacterial elimination,which is consistent with the bacterial load in the spleen.Besides,the survival number of chicks was also monitored within the 28 dpc,and the death of chickens was detected in the gourps challenged with the wild type strain or the complementary strain,while no death was detected in the groups challenged with mutant.Until 28 days after challenge,the survival rate of the group challenged with wild type strain was 70%,and the survival rate of the groups challenged with mutants was 100%.All of the results demonstrated that deletion of RS09150 or RS09155 not only reduced the virulence of S.Pullorum,but also induced T cell immune response,and promoted bacterial clearance.C79-13△RS09150 and C79-13ΔRS09155 has the potential to be develop as attenuated vaccines for pullorum disease.