Construct Of SsrAB,hilA,hilD-deficient In Salmonella Enteritidis And Characteristic Of Mutants

Salmonella is an important pathogen that causes Salmonellosis in human beings and many kinds of animals in clinical.The genes that relate to the virulence of Salmonella compose areas known as Salmonella Pathogenicity Islands(SPI).It is report that SPI1 and SPI2 play important roles in Salmonella invasion and colonization respectively.And some study shows that ssrAB gene can regulate the expression of SPI2,hilA induce the SPI1,whereas hilD regulate both SPI1 and SPI2 through ssrAB and hilA.But the function in Salmonella virulence of ssrAB,hilA and hilD is unclear.The pathogenicity of ssrAB-,hilAor hil D-deficient is unknown as well.This study takes Salmonella enteritidis as object,make single,double,and triple deletions on ssrAB,hilA and hilD though homologous recombination technology(renamed as ΔssrAB,ΔhilA,ΔhilD,ΔssrAB hilA,ΔhilA hilD,ΔssrABhilD and Δssr ABhilAhilD).Then we study the differences of the growth ratio,biofilm formation,median lethal dose to mice,colonization ratio in murine intestine,the adhesion ability on HeLa cell,and the ability of provoke host to product cytokines(IL-1,IL-18 and IFN-γ)between mutants and wild type strain.It shows that there is no significant difference in growth ratio between deletions and wild type strain.The biofilm formation of mutants is as well as wild type except hilD and ssrAB,triple deficient is significant lower than other strains(P<0.05).Animal test shows that the virulence of ΔssrAB,ΔhilA and ΔhilD is lower than wild type in statistical analysis.The media lethal does of triple mutants ΔssrABhilAhilD is the highest in all strains(P<0.05).Besides,the structure of intestinal villi recovered in mutants group,all the mutants can provoke the cytokines too.Plus the cell adhesion assay and colonization assay shows that,cell adhesion ofΔhilA and ΔssrABhilAhilD is significant lower than any other strains,it may shows that hilA plays an important role in Salmonella invasion,however,the colonization of ΔssrAB is significant lower than other strains,ssrAB takes part in Salmonella colonization.Our works shows that we construct the single,double and triple deficient of ssrAB,hilA and hilD genes through molecular technology,and further more,the virulence of ΔssrAB,ΔhilA,ΔhilD,ΔssrAB hilA,and ΔssrABhilAhilD is significant lower than wild strain,and the IL-1,IL-18 and IFN-γcytokin they provoke is not significant than wild type,they might be taken as Salmonella attenuated vaccines or attenuated vectors.