Molecular Mechanism Of Bile Salt Resistance Related Genes Screening From Listeria Monocytogenes

Listeria monocytogenes（Lm）is a Gram positive and facultative intracellular parasite caused zoonotic listeriosis with a high mortality rate.Lm infects host by contaminated food,during this process,it can tolerate pH changes in the gastrointestinal tract,which can also resist proteases,bile salts,and immune system.Among them,tolerance ability to bile salts in intestinal is a necessary factor for Lm to break through the intestinal barrier and infect host.Genes for resisting bile salt stress in Lm have been reported,while they are relatively limited.Therefore,this study discovered the genes related to bile salt tolerance through screening Lm XYSN transposon mutants,the strain was isolated from the sheep brain in a Listeria monocytogenes outbreak farm.We constructed the mutant strains,and detected their biological characteristics,such as growth rate,biofilm formation ability,and morphological change.In addition,the effects of screening genes on virulence were evaluated through the Caco-2 BBe cells and mouse.Finally,the regulatory network of LMxysn₀254 protein was explored by transcriptome sequencing and fluorescent quantitative PCR.Our study provided a mechanism for resistance to bile salt stress without reported of Lm.Moreover,our study made contribute to reveal the relationship between the high virulence and bile salt tolerance for Lm XYSN.1.Screening for genes related to bile salt tolerance in Listeria monocytogenesBile salts,as an amphoteric substance,can inhibit bacterial growth through promoting cell rupture,damaging macromolecules,and chelating metal ions,playing an important role in resisting Lm invasion of host.This study screened 30 mutant strains from 1041 Lm XYSN transposon mutants with the criterion of growth rate 50%lower than Lm XYSN under 0.2%bile salt-BHI medium.The transposon insertion genome sites were determined using two round PCR.It was found that protein functions were covering hypothetical proteins（37%）,transporters（20%）,regulatory factors（13%）and others by bioinformatics prediction analysis.2.Biological characteristics of bile salt resistance genes in Listeria monocytogenesThis study selected LMxysn₀254、LMxysn₁966、LMxysn₀808 and LMxysn₂902 to research their function,which were the number of 30 mutant strains.The deletion strains were constructed successfully through homologous recombination technology,and named as XYSNΔ0254,XYSNΔ1966,XYSNΔ0808,and XYSNΔ2902.The growth curve measurement results show that LMxysn₀254,LMxysn₀808 and LMxysn₁966 genes made contribute to bacterial growth,under 0.2%bile salt stress.We assayed the ability of biofilm formation to evaluate bile salt stress resistance,and these results as followed.LMxysn₀254 promoted biofilm formation in different temperature.XYSNΔ1966 decreased forming biofilm in 37℃ and 42℃,while this ability reduced only in 42℃ with the deletion of LMxysn₂902.In addition,observation of mutant strains morphology revealed the length of XYSNΔ0254 was significant increased.Above all,our results indicated that LMxysn₀254 and LMxysn₀808 were closely related to Lm growth,LMxysn₀254,LMxysn₁966 and LMxysn₂902 were involved in the biofilm formation under bile salt stress,which is beneficial to resist bile salt stress for Lm.We used human colon adenocarcinoma Caco-2 BBe cells as an in vitro model to investigate the role of bile salt resistance genes in adhesion and invasion for Lm.The result showed LMxysn₀254,LMxysn₁966 and LMxysn₀808 could enhance the adhesion ability to Caco-2 BBe cells for Lm.Then we used C57BL/6N to determine the colonization in organs,and the results as followed.LMxysn₀254,LMxysn₀808 and LMxysn₂902 were involved in colonization of ileum,in which the colonization in liver and gallbladder was significantly reduced with deletion of LMxysn₀254.LMxysn₀808 could also increase bacterial load of liver,spleen.These results suggested that four genes screened from transposon mutants were all related to virulence,among which LMxysn₀254 made most important contribution to bile salt resistant and infection host for Lm.3.The molecular mechanism of LMxysn₀254 involved in bile salt toleranceWe used transcriptome analysis to aligned and analyzed Lm XYSN and XYSNΔ0254 under BHI medium and 0.2%bile salt BHI medium for parsing molecular mechanism of LMxysn₀254 in bile salt resistance and high virulent.Comparing XYSNΔ0254 to Lm XYSN,RNA-seq showed that the genes encoding Clp family protease were significantly up-regulated,while virulence genes were significantly down-regulated such as hly,actA,plcA,and plcB.In addition,the bile salt resistant genes were all down-regulated in expression through qRT-PCR,such as bsh gene encoding bile salt lyase,regulatory genes sigB,brtA and prfA.Based on the above results,this study evaluated LMxysn₀254 regulatory network.Taken together,our study implied that LMxysn₀254 was closely related to bile salt resistance,and affected the expression of virulence genes.To investigate molecular mechanism of LMxysn₀254 involved in bile salt tolerance,we constructed the complete strain XYSNA0254::0254.Then we estimated biological characteristics of Lm XYSN,XYSNΔ0254 and XYSNΔ0254::0254.Most of bacteria could not divide normally and form longer chains with the deletion of LMxysn₀254 through scanning electron microscopy.Under bile salt stress,the growth curve of XYSNΔ0254 significant decreased,the biofilm formation ability dampened compared with wild type strain and LMxysn₀254 reversion strain.Additionally,we used C57BL/6N mice as an in vivo infection model to assess virulence of Lm XYSN,XYSNΔ0254,and XYSNΔ0254::0254.Our results showed that bacterial load of XYSNΔ0254 in gallbladder,spleen and ileum was significantly decreased comparing to Lm XYSN and complete strain,with fold change of 50fold,31-fold and 10-fold,respectively.Above these results,our study indicated that LMxysn₀254 made contribute to the process of bacterial division,reproduction,and biofilm formation,which was beneficial for the survival in bile salt environments for Lm XYSN.In addition,LMxysn₀254 promoted adhesion and invasion ability,and enhanced colonization ability in the intestine,gallbladder,and spleen for Lm.In summary,this study obtained 30 bile salt sensitive transposable mutants through screening transposon mutation library under bile salt stress.Then we researched functions of LMxysn₀254,LMxysn₁966,LMxysn₀808 and LMxysn₂902,based on gene location and bioinformatics analysis.Except for LMxysn₀808,three other genes enhanced biofilm formation under bile salt stress.Moreover,LMxysn₀254,LMxysn₀808 and LMxysn₂902 promoted colonization in intestine of mice for Lm.Among them LMxysn₀254 couled regulate the expression of virulence genes and genes related bile salt resistance.Meanwhile,LMxysn₀254 was conducive to divide normally,it also promoted the colonization intestine,gallbladder and spleen.In addition,this gene played an important role in regulating infection and pathogenicity for Lm.This study made contribution to reveal the molecular mechanism to bile salt resistance for Lm.