| Salmonella enterica serovar Typhimurium(STM)is one of the most common serotypes of Salmonella,with a wide range of hosts,mainly causing gastroenteritis in humans and animals.Type III secretion system(T3SS)is a protein transport machinery present in many Gram-negative bacteria(such as STM),which functions to inject bacterial effector proteins into eukaryotic host cells to promote bacterial colonization and survive.T3 SS is regulated by various factors,such as small non-coding RNA(sRNA Gcv B)and its chaperone Hfq.At present,the research on bacterial T3 SS mainly focuses on its structure and mechanism of action,especially the mechanism of action between sRNA Gcv B and chaperone Hfq is rich in research data.Few reports have been reported on the regulation of STM T3SS-related genes by sRNA Gcv B and chaperone Hfq.To this end,this study took the STM LT2 standard strain as the research object,used the Red homologous recombination system to delete the STM T3SS-related gene and constructed a Lac Z gene fusion strain.Determination of the ability of film formation and migration,and analysis of the effect of STM T3SS-related gene deletion on the pathogenicity of the bacteria and the regulation of sRNA Gcv B and chaperone Hfq on STM T3SS-related genes,in order to develop high safety and Environmentally friendly attenuated vaccines lay a certain theoretical foundation.1.Construction of Salmonella typhimurium type III secretion system related gene deletion strainIn order to construct STM T3SS-related gene deletion strains,this study used the Red homologous recombination system to design specific primers containing the homology arms of the spa P,inv C,inv G,prg H,prg I,prg J or prg K genes and the plasmid p KD13 fragment,using the plasmid p KD13 As a template,amplify a fragment with homologous sequences of the target gene at both ends and contain the kanamycin resistance gene,and then introduce the fragment into the target bacteria containing the recombinase system by electroporation.After recombination,electrotransfer into plasmid p CP20 to eliminate kanamycin To achieve a traceless knockout of the spa P,inv C,inv G,prg H,prg I,prg J or prg K genes in Salmonella typhimurium;the deletion strains were screened by resistance plates,and a single colony was picked for PCR verification after purification.Results: After PCR detection,the positive transformants all amplified DNA fragments consistent with the expected fragment size(1 377 bp);after sequencing the amplified products,none of the deleted strains contained the original sequence of the target gene,indicating that the experiment was successful STM T3SS-related genes spa P,inv C,inv G,prg H,prg I,prg J and prg K deletion strains were constructed and named LT2 Δspa P,LT2 Δinv C,LT2 Δinv G,LT2 Δprg H,LT2 Δprg I,LT2 Δprg J and LT2 Δprg K,which lays the foundation for subsequent studies on the biological characteristics of deletion strains.2.Study on the biological characteristics of Salmonella typhimurium deletion strainIn order to explore some of the biological characteristics of STM T3SS-related gene deletion strains,this experiment carried out PCR detection,growth curve determination,bacterial colonization experiment,biofilm crystal violet staining observation,motility assay and animal pathogenicity experiment after strain passage.The genetic stability,growth characteristics,colonization ability,biofilm formation ability,migratory ability and pathogenic changes of the deleted strains were compared and analyzed.The results showed that the STM T3SS-related gene deletion strains could inherit stably,with little change in growth performance.Colonization ability,biofilm formation ability,movement ability and animal pathogenicity were all weakened to varying degrees.The above results show that the deletion of T3SS-related genes can reduce the pathogenicity of STM,which provides a new idea for the development of typhoid fever attenuated vaccine and the development of new antibacterial drugs.3.Analysis of the regulation of sRNA Gcv B and its chaperone Hfq on T3SS-related genesIn order to explore the regulation of sRNA Gcv B and its chaperone Hfq on T3SS-related genes,this study used λ-Red homologous recombinase and FLP recombinase system to construct LT2 Δspa P::lac Z,LT2 Δinv C::lac Z,LT2 Δinv G::lac Z,LT2 Δprg H::lac Z,LT2 Δprg I::lac Z,LT2 Δprg J::lac Z and LT2 Δprg K::lac Z gene fusion strains,and then the gcv B and hfq genes were transduced with P22 phage After the fusion strain was recombined to construct the recombinant deletion strain,q RT-PCR and β-galactosidase assay were used to detect the gene transcription and protein levels of spa P,inv C,inv G,prg H,prg I,prg J and prg K in the recombinant strains.At the gene level,Gcv B and Hfq have different regulatory effects on T3SS-related genes.At the gene level,both sRNA Gcv B can silence the expression of the above seven T3SS-related genes,while the chaperone Hfq can silence the expression of spa P,inv C and inv G genes,but can promote the expression of prg H,prg I,prg J and prg K genes.At the protein level,sRNA Gcv B can silence the expression of spa P,inv C,inv G,prg H and prg I related genes,but can promote the expression of prg J and prg K genes,while the regulatory effect of chaperone Hfq at the protein level is consistent with that at the gene level.These results indicate that Gcv B and Hfq have certain regulatory effects on the T3SS-related genes spa P,inv C,inv G,prg H,prg I,prg J and prg K,thereby affecting the pathogenicity of STM.This provides a theoretical basis for further revealing the mechanism of sRNA Gcv B and its chaperone Hfq in the infection of Salmonella typhimurium.In conclusion,this study successfully constructed a strain of Salmonella typhimurium with T3SS-related genes spa P,inv C,inv G,prg H,prg I,prg J or prg K deletion;T3SS-related genes spa P,inv C,inv G,prg H,prg I,prg J or prg K deletion It can reduce the pathogenicity of Salmonella typhimurium;it is speculated that sRNA Gcv B and its chaperone Hfq affect the pathogenicity of Salmonella typhimurium by regulating the expression of T3SS-related genes. |
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