Regulation Of Salmonella Typhimurium H1ld On SsrAB

Salmonella is an important zoonotic pathogen,it can infect comprehensive hosts including human beings,poultry,canine,swine,murine,and so on.The clinlcal symptoms of Salmonelosis are emesia,dearrhea and fervescence.The pathogenicity factors includ flagella,pili and the gene that loading on Salmonella pathogenicity Island(SPI).About 39 SPIs have been reported for the moment.These SPIs interacted eatch other is the important factors of the pathogenicity of Salmonella.SPI-1 and SPI-2 are necessary for Salmonella infection,invasion and colonization in the host.SPI-1 mainly mediated Salmonella infection,while SPI-2 is the key to Salmonella colonization and escaping immunity.Both of them encoded Type III secretion systems(T3SS),which secreting virulence proteins into the host cells.A lot of researches said that HilD which loaded on the SPI-1 not only regulated the SPI-1,but also regulated SPI-2.The binary regulator SsrAB regulated SPI-2.However,the effection of HilD on SPI-2 has not been reported so far,in addition,how the HilD mediated ssrAB has also not been determined yet in vivo.In this study,Salmonella typhimurium was used as the object,Caenorhabditis elegans(C.Elegans)was used as model host.The homologous recombination method was used to merge the gfp gene in ssrAB gene in Salmonella wild strains,the hilD mutant strain and the hilD complementaty strain respectively.In order to ensure the preciseness of the experiment,the pcDNA-3.1(+)plasmid was transformed into Salmonella wild strain and gfp fusion strain by electroporation,then been feded by Caenorhabditis elegans.The fluorescence intensity of GFP was determined by fluorescence microscopy in vivo and in vitro.The pET30a-hilD vector was constructed,then transferred into BL21 strain to express and purify HilD protein,then,the binding region of HilD to ssrAB was analyzed by Electrophoretic Mobility Shift Assay(EMSA).Our results said that,the gfp fusion strains were constructed successfully which were named as S.T(ssrAB-gfp),S.T(?hilD+ssrAB-gfp),S.T(?hilD+ssrAB-gfp +pcDNA3.1-hilD),respectively.gfp fusion strains show green fluorescence in vitro and in vivo,the fluorescence intensity of the strains range from strong to weak was S.T(ssrAB-gfp+pcDNA3.1)?S.T(?hilD + ssrAB-gfp +pcDNA3.1-hilD),S.T(?hilD+ssrAB-gfp +pcDNA3.1),the control group S.T(pcDNA3.1)has not shown fluorescence.However,the luminescence intensity of hilD-complemention strain was significantly higher than that of hilD-mutation strain.HilD was expressed and purificated.Acoording to the EMSAresults,HilD could be bound to ssrAB and that the binding of the target protein to ssrAB was specific,and the binding area was positioned in +412 to +430.The results showed that Salmonella typhimurium wild strain,hilD-mutation strain,hilD-complementation strain were fused gfp gene into ssrAB and translated the plasmid of pcDNA-3.1(+),respectively.In vivo and in vitro,HilD had a positive effection on ssrAB,HilD could be interacted in +412 to +430 of ssrA.