Research Of Virulence Factor In Listeria Monocytogenes Revealed By Comparative Genomic And Transcriptome Analysis

Listeria monocytogenes(LM)is a pathogen of zoonosis which caused Listeria(listeriosis)disease,resulting in septicaemia,meningitis,gastroenteritis and increasing mononuclear cell.It widely exists in various environments and L.monocytogenes bring the risk for human security.It has been a hot topic to explore the new virulence factors and to explore the regulation of virulence factors in the external environment.Thus,our research had four aims:(1)using Multilocus Sequence Typing(MLST)from the Pasteur MLST web data to survey the genetic diversity and distributive condition;(2)using Illumina pair-end sequencing technology to analyze the genome of epidemic isolate in L.monocytogenes.(3)Constructing a gene-knock mutant that lack the sig B gene to describe the phenogenetics responding under different temperatures,p H and water activities in L.monocytogenes.(4)using Illumina RNA-Hi Seq sequencing technology to explore the regulation mode of ?B in L.monocytogenes.Major research contents and the results were as follows:1.Survey of genetic diversity and distributive condition in Chinese L.monocytogenes isolatesOur research analyzed the genetic diversity and distributive condition by using the e BRUST and MEGA software.We obtained the epidemicity and distributive condition of L.monocytogenes isolates in China by analyzing 7 housekeeping genes in 54 L.monocytogenes isolates in China.In addition,we obtained the genetic diversity by using MEGA analysis of the nucleotide and peptide sequences.The results showed that CC155 was the most epidemic clonal complex(ST155,corresponding founders of CC155)which include ST155,ST705,ST806,ST311,ST712 and ST381.Phylogenetic tree based on nucleotide sequence showed that CC155 and CC7,CC121,CC9 and CC33 belong to the similarity of 100%.Phylogenetic tree based on peptide sequence showed that the homology between ST belonged to CC155 were higher than nucleotide sequence.It is concluded that the phylogenetic tree constructed by peptide can clearly explain the evolutionary relationship of strains.The results of serotype analysis showed that the ST strains belonged to CC155 were 1/2a,indicating that the prevalence strains were mostly pathogenic strains in China.Therefore,MLST website of L.monocytogenes in China provides a strong evidence to maintain the quality and safety of food and protect the public's health.2.Comparative genomic analysis of the epidemic ST Listeria monocytogenes in ChinaOur research obtained the genome of L.monocytogenes WaX12(ST155,serotype 1/2a)by using Illumina pair-end sequencing technology,which was isolated in Shanghai,China.Based on the 16 S r RNA analysis,the strains of L.monocytogenes Wa X12 and L.monocytogenes Finland 1998 were formed monophyletic groups.The Wa X12 genome sequence was reordered by Mauve to form one consecutive contig referring to L.monocytogenes Finland 1998 genome.Obviously,we can see three large insert districts differing in Finland 1998 genome.Additionally,this was first report to reveal that L.monocytogenes strain Wa X12 isolated from China contained five prophages.Prophage-1 contained virulence-associated factors(lma DCBA gene cluster).Prophage-2 had general structural modules of phages,which were common in Escherichia coli.Prophage-3 and prophage-4 had highly similar hits to Enterococcus phages,which contained Holin and phi-80 virulence factors respectively.Prophage-5 showed similarity in component composition and arrangement to Listeria bacteriophages A118 genome.A118 attachment site att P combined the corresponding att B in L.monocytogenes.We speculated these five prophage regions play vital roles in adaptation and virulence evolution of L.monocytogenes.The new sequence data in this study will provide a better and more complete insight into the variability and evolution of L.monocytogenes genome,and accelerate our understanding of the epidemiology,mechanisms of resistance and virulence of L.monocytogenes.3.Construction of L.monocytogenes Wa X12 delta sig B mutant and the analysis of growth kineticsThis research constructed the L.monocytogenes Wa X12 delta sigB mutant by using SOE PCR and homologous recombination.Both the upstream homologous arm and downstream homology arm was amplified by general PCR,and then the two arm were connected together to get the sig B missing fragment by SOE PCR.The Sig B missing gene fragment was then inserted into the shuttle vector p KSV7,to construct the recombinant shuttle plasmid p KSV7-?sig B.The resulting plasmid,p KSV7-?sig B was electroporated into the competent cell of Wa X12.The positive transformants which were amplificated one 847 bp fragment were identified by antibiotic medium.The positive transformants were subcultured for 20 generations in the pressure of chloramphenicol at 42?,to get the sig B deletion mutant.Then the sig B deletion mutant was identified by PCR amplification.The growth curve of Wa X12 and Wa X12-?sig B were fitted by modified Baranyi model from p H5 to p H9,as well as both growth kinetic parameters were analyzed.The results showed the ?sig B mutant was constructed correctly.The maximum exponential growth rate(?max),lag phase duration(?)and maximum population density(MPD-OD)of Wa X12-?sig B and Wa X12 had all significant difference(p<0.05)at p H5,but had no different significant at neutral and alkali.4.Comparative transcriptome analysis of the epidemic ST Listeria monocytogenes Wa X12In this study,we mapped the transcriptome of L.monocytogenes Wa X12 and its delta sig B mutant by RNA-Seq analyses and did comparison to elucidate the ?B-regulated genes and their expression level and functionality.Results showed the Wa X12 strain formed into 3,048 unigenes referring to EGD-e genome.In addition,the fructose and mannose metabolisms were the most significant difference(p<0.05)in metabolic pathways between the wild type and mutant.Comparative transcript analysis identified 123 positive and 17 negative genes under the control of ?B.These regulated genes mainly encoded the proteins related to virulence,energy,resistance and membrane.We used quantitative real-time PCR to verify the expression profiles of 7 different genes and confirmed the veracity of all the 140 regulated genes.Our research approaches and methodologies also provide a basis for future studies examining the effect of changing environmental conditions on the cellular response of bacterial cells.