Effects Of Regulatory Proteins RtsB And Fur On The Virulence Of Salmonella Typhimurium

Salmonella is an important zoonotic pathogen,which is an intracellular bacterium that can infect various animals and cause a variety of diseases in mammals and poultry(such as chicken typhoid,porcine paratyphoid,chicken paratyphoid,etc.).Moreover,it is a leading etiological agent associated with foodborne illness outbreaks worldwide,which commonly result in heavy economic losses globally.This study was carried out to explore the effects of regulatory proteins RtsB and Fur on the pathogenity of Salmonella typhimurium.The mutants and complemented strains were constructed,and their biological characteristics were compared with those of wild-type strain.Moreover,the virulence of mutant strains was determined,which will contribute to understand the pathogenic mechanism of S.typhimurium1.Construction and characterization of the rtsB gene mutant strain of S.typhimuriumThe sequence analysis showed that the rtsB gene of S.typhimurium encodes LuxR family regulatory protein,which has a typical helix-turn-helix DNA binding motif.We constructed the rtsB gene mutant strain and complemented strain of S.typhimurium SAT52 using the Red recombination system and complementary plasmid to analyze the effects of regulatory protein RtsB on the biological characteristics and pathogenicity of S.typhimurium.Then we determined the growth rate,motility,biofilm formation,cells adhesion and invasion ability,and pathogenicity of the mutant,complemented strain and their parental strain.The results showed that the deletion of the rtsB gene did not affect the growth rate of S.typhimurium SAT52.However,the mutant strain showed significantly enhanced motility and decreased biofilm formation ability.Compared with the wild-type strain,the capacities adhering to Hela cells and survival in RAW264.7 cells were significantly decreased for the mutant strain.The results of animal experiment showed that the virulence of mutant strain?rtsB is significantly milder than that of wild-type strain.These data indicated that regulator RtsB plays an important role in the pathogenicity of S.typhimurium,which would help us to comprehensive understand the pathogenic mechanism of S.typhimurium.2.Construction and characterization of the fur gene mutant strain of S.typhimuriumThe ferric uptake regulator(Fur)is involved in the survival and virulence of bacteria by regulating the related gene expressions.To clarify the effects of Fur on the biological characteristics and pathogenicity of S.typhimurium,the fur gene mutant strain and complemented strain of S.typhimurium SAT52 were constructed using the Red recombination system and complementary plasmid.Then,the biological characteristics and pathogenity of mutant,wild-type and complemented strains were compared.The results showed that inactivation of fur led to a slightly decreased growth rate of S.typhimurium SAT52,but significant enhanced the interbacterial competition activity.The cell and animal infection assays indicated that Fur contributed to the intramacrophage survival in RAW264.7 cells and systemic persistance in mice,which facilitates the infection and virulence of S.typhimurium.These results indicated that Fur was involved in the pathogenesis of S.typhimurium,which provided a basis for elucidating the pathogenic mechanism of S.typhimurium.3.Molecular mechanism of Ferric uptake regulator(Fur)facilitating the virulence of S.typhimurium by regulating type VI secretion System(T6SS)Bacteria strictly regulate the expression of type VI secretion system(T6SS)to promote the interbacterial competition capacity and virulence.However,the regulation of the Samonella pathogenicity island 6(SPI-6)T6SS in S.typhimurium is not well understood.We constructed the clpV gene mutant,complemented and fur and clpV dual-gene mutant strains of S.typhimurium SAT52 using the Red recombination system and complementary plasmid.The regulation and interaction of Fur with the T6SS were determined by qRT-PCR,Western blotting,?-galactosidase assays,electrophoretic mobility shift assay(EMSA),DNase I footprint test and point mutation technique.Then,the effect of Fur mediated T6SS regulation on the biological characteristics and pathogenicity of S,typhimurium was analyzed.The results showed that T6SS core gene clpV expression is upregulated during infection and under iron-depleted conditions.We also found that Fur repressed the expression of clp V in S.typhimurium under iron-rich conditions.Furthermore,EMSA,DNaseI footprint analysis and point mutation analysis revealed that Fur protein bound directly to the clpV promoter region and regulated its expression.We also observed that relieving the Fur-mediated repression of clpV contributed to the interbacterial competition activity and virulence of S.typhimurium.These findings provide insights to understand the direct regulation of Fur on the expression and functional activity of SPI-6 T6SS in S.typhimurium,thus contributing to elucidate the adaptability and virulence mechanism of bacteria.