Construction Of Mutant Strains And Biological Characteristics Study Of Two-component System RS08195/RS08200 In Listeria Monocytogenes

Listeria monocytogenes(Lm)is gram-positive saprophytic bacterium ubiquitously found in the environment.Meanwhile,Lm is an important food-borne pathogen and can cause listeriosis,especially in susceptible individuals.Lm is able to surmount a wide range of stress conditions including low temperatures and bile salt.Lm can form biofilm,which causes food contamination during food processing and storage.Bacterial two-component signal transduction systems(TCSs)are responsible for sensing environmental stimuli and regulating gene expressions to aid bacteria in adapting to arduous stress factors encountered in nature.The findings on TCSs would provide important information for understanding survival mechanism and pathogenesis in Lm.Lm strain LM201 was isolated from food stuff and sequenced by our laboratory.We found two adjacent genes,RS08195 and RS08200,encoded a pair of TCS in the genome of Lm by bioinformatics analyses.RS08195 encodes a response regulator(RR).RS08200 encodes a histidine kinase(HK).Up to now,the knowledge on this pair of TCS remains poor in Lm.In order to study the role of the TCS encoded by RS08195/RS08200 for survival and virulence of Lm,we constructed the RS08195/RS08200 in-frame deletion mutant S6004.The biological characteristics in S6004 were detected.RNA-sequences for S6004 and parent LM201 were performed.The following was the major results in this study.1.Bioinformatics analysis for RS08195/RS08200By conserved domain analysis,we found that RS08195 encodes a response regulator(RR),which constituted REC and ANTAR domain.REC domain is a phosphorylation state of the receiver phosphoryl group at the N end of RR.ANTAR domain at the C end of RR belongs to AmiR_NasR subfamily.RS08200 encodes a histidine kinase(HK)with the conserved domain HATPase_c,but the end of N has no transmembrane helical region.By amino acid sequence alignment analysis,we found that the identity of RS08195 in LM201 was 100% with the same strain and same serotype Lm F2365 RS05195,as well the same strain and diffrenent serotype Lm EGD-e lmo1172.The identity of LM201 RS08200 with Lm F2365 RS05900 and Lm EGD-e lmo1173 is respectively 100% and 97%.lmo1172/lmo1173 encoded EutVW,so the TCS encoded by RS08195/RS08200 were named EutVW in this study.2.Construction of RS08195/RS08200 deletion mutant and complement strainGenome of Listeria monocytogenes isolate LM201 was used as template to amplify the left and right homologous arms of RS08195/RS08200 gene.The homologous arm over-lap fragment was ligated to the temperature-sensitive shuttle vector pHT304-ts by BamHI and KpnI,obtained the recombinant plasmid p5002.p5002 was transfected into LM201 by electroporation,and screened the RS08195/RS08200 deletion mutant under the pressure of temperature and resistance.The mutant was named S6004.RS08195/RS08200 gene was amplified to construct complement strain.RS08195/RS08200 was ligated into pHT304 and obtained recombinant plasmid p5003.p5003 was electrotransformed into the mutant S6004 and obtained the complement strain S6006.The expression of RS08195/RS08200 was verified by reverse transcription PCR.3.Detection of biological characteristics for mutant S6004Compared with the parent LM201,the growth rate,conventional biochemical reaction and the tolerance of O2 of mutant S6004 had no significant difference.The hemolysis titer of parent LM201 was 2~4,and the mutant S6004 was 2~6.Deletion of the RS08195/RS08200 in LM201 resulted in a biofilm deficient phenotype and the proliferation ability in milk at 37℃ was significantly reduced(P <0.05).The results of cell adhesion test,invasion test,macrophage phagocytosis and nematode virulence assays indicated that the mutant S6004 had no significant difference compared with the parent LM201.The results of mice LD50 showed that the LD50 of LM201 was 9.31×10~4 CFU and S6004 was 2.36×10~5 CFU.The data statistical analysis indicated that no difference was found between S6004 and LM201(t<2.2281).The ΔRS08195/RS08200 had significantly reduced bacterial loads in spleen at 24 hours after infection compared with the parent strain LM201(P<0.05),but in liver,the bacterial loads had no significant difference.4.RNA-Seq analysisCompared with the parent LM201,the mutant S6004 had 115 genes expression up-regulated,and the expression of 98 genes was down-regulated.Bifferential expression analyses showed that eut G located in eut operon and AgrC of the HK of AgrAC,a pair of TCS,were down-expressed.dltB,dltC and dltD regulated by VirR were down-expressed,while the expression of PrfA-dependant hly,inlA and inl H were up-regulated.GO enrichment results showed differential expression gene were mainly enriched in molecular function term.RS08195 and agrC were enriched to some same GO terms.These results showed that the TCS encoed by RS08195/RS08200 is connected with AgrAC.KEGG-Pathway enrichment results showed that differential expression genes were mainly enriched in amino acid metabolic pathways and nutrition metabolic pathways.Three down-regulated gene agrA,agrB and frdA were enriched to TCS pathway.agrA and agrB were involved in the regulation process of AgrAC.AgrAC have been previously shown to regulate biofilm formation.frdA involves in the regulation of TCS related to nitrogen metabolism.These results showed that the TCS encoded by RS08195/RS08200 are connected with Agr and FrdA.