Construction Of Attenuated Salmonella Lysis System Based On In Vivo Regulation Of Bacterial Type ? Secretion Protein

Recombinant bacterial vector vaccines,especially recombinant attenuated Salmonella vaccine?RASV?have been widely used as carriers to deliver protective antigens and nucleic acid vaccines because they can effectively stimulate the host to produce mucosal immune response,humoral immune response and cellular immune response.RASV is a kind of live bacterial vaccine,so the exogenous antigens or eukaryotic expression plasmids can not be effectively released,and the residual Salmonella strains in vivo may cause biological contamination.In order to solve the problems,based on the characteristic that peptidoglycan hydrolyzed proteins secreted by bacterial type VI secretion system,a series of bacterial lysis combinations regulated by in vivo inducible promoter were designed,and finally one RASV lysis system in vivo which was regulated by Mg²⁺was selected to deliver exogenous antigens.The type VI secretory system?T6SS?of Pseudomonas aeruginosa can secrete peptidoglycan hydrolytic factors:Tse1 and Tse3,which can destroy the bacterial cell wall and then causing bacterial lysis and death.In this study,three endogenous expression promoters were selected:Mg²⁺-limited promoter P_pagC,Fe²⁺-limited promoter P_{viu B} and constitutive expression promoter araC P_BADAD LacI-repressible P_trc.The above three promoters were infused into the upstream of the periplasmic localization signal peptide gene blass?SS1?or pelBss?SS2?which were infused into the upstream of the lysis gene tse1 or tse3 by overlapping PCR,respectively.Then these combinations were inserted into the p15A-T?Cm⁺?vectors by digestion and ligation,and the products were transformed into E.coli BL21?DE3?to express the lysis protein Tse1 or Tse3.Finally,the selected combinations of P_pagC-SS1/SS2-tse1 could effectively lyse the E.coli BL21?DE3?cells in vitro.In order to introduce the selected regulated lysis combinations into the genome of Salmonella,the P_pagC-SS1/SS2-tse1 combinations were inserted into the suicide plasmid vectors and then were transformed into E.coli?7212.The Salmonella mutants UK-1?pagL::P_pagC-SS1/SS2-tse1 were constructed by the method of homologous recombination.However,the mutants can not be lysed through the regulation of Mg²⁺.In order to solve the problem,the P_pagC-SS2-tse1 combination was inserted into an Asd⁺plasmid pYA3341 with high copy replicon to construct the lysis system.The results showed that the constructed pUC-Lysis plasmid achieved the controllable lysis phenotype in both the wild type Salmonella Typhimurium strain UK-1?asd and the attenuated Salmonella strain?9241.The induced strains were obviously inflated and deformed by observing under the microscope and the destruction of bacterial cell wall structure was observed under scanning electron microscope.In order to further verify the effect of lysis and antigens release,the constructed p15A-LacZ?Cm⁺?plasmids were transformed into the different serotypes Salmonella strains with control vectors or pUC-Lysis plasmids,respectively.Results showed that when strains were cultured in LB broth,the ratio of the amount of?-galactosidase released from the supernatant/the amount of total synthesis of?-galactosidase by bacteria in the control group and that in the experimental group were comparable.After transferring into N-minimal broth with low Mg²⁺concentration,there was no significant change happened in the control group,but the ratio of that in the experimental group increased significantly,indicating that the lysis plasmid can be induced by the limited Mg²⁺environment to achieve the lysis of Salmonella strains and the release of exogenous antigens.In order to verify the lysis effect of the RASV strains with the lysis plasmid in mice,seven-week-old mice were orally inoculated with the Salmonella strains UK-1?asd?pUC-Lysis?and?9241?pUC-Lysis?at a dose of 10⁹ CFU.Results showed that Salmonella strains were not detected in liver and spleen of mice after 11 days,and which were not detected in Peyer's patches after 15 days,indicating that the vaccine strains can be lysed after colonization in the deep tissues of mice.In order to verify the immune effect of the RASV strains to deliver protective antigens,the?-helix region of Streptococcus pneumoniae outer membrane protein PspA?rPspA Rx1?was cloned into the pUC-Lysis plasmid to yield the double regulated protein expression plasmid pUC-Lysis-PspA.Results showed that the?9241 strain with the pUC-Lysis-PspA plasmid still displayed a controllable lysis phenotype,and Western-blot could also detect the rPspA Rx1 protein.Seven-week-old mice were orally inoculated with the vaccine strain?9241?pUC-Lysis-PspA?at a dose of 10⁹ CFU,and the strong immune responses against the rPspA Rx1antigen and Salmonella outer membrane proteins?SOMPs?were generated in vivo.In summary,we constructed a new type of in vivo regulated Salmonella lysis system in this study.The Salmonella strains with that can normally grow in LB broth,but under lysis in N-minimal broth with low Mg²⁺concentration or in vivo,which achieved the effective release of protective antigens and biological epidemic prevention.